title: The relationships between structural organization, material properties, and loading conditions and the risk of fracture and fracture location in the femur

abstract: Increased risk of skeletal fractures due to bone mass loss is a major public health problem resulting in significant morbidity and mortality, particularly in the case of hip fractures. Current clinical methods based on two-dimensional measures of bone mineral density (areal BMD or aBMD) are often unable to identify individuals at risk of fracture. The underlying hypothesis of this study was that combinations of femur structural traits are different for those femurs that suffer a fragility fracture within the proximal region of the femur and those that sustain a fracture in either the subtrochanteric or midshaft region of the femur, resulting in an "atypical femur fracture". Accordingly, the objective of this study was to determine the effects of varying combinations of structural traits, material properties, and loading conditions on femur stress response and the location of stress response variation using a validated parametric finite element model. Statistical shape and trait modelling of the femur was used to describe variability in the structural organization of a set of femurs in an efficient manner and the resulting description of structural variability was exploited to investigate how different mechanisms of fracture might occur, whether in the proximal region or in the subtrochanteric and midshaft region. In combination with parameters describing loading condition and material property variation, variation in structural organization is associated with regional increases in maximum principal stress and the percentage of bone expected to damage, and these increases are likely associated with increased fracture risk. The results of this study indicate that there are multiple pathways and combinations of descriptor variation that may result in increased fracture risk and that these pathways can lead to fracture in any region of the femur under both overload conditions, such as with sideways fall loading, and stance loading, which due to the repetitive nature may lead to the accumulation of fatigue damage within the bone and further impair bone condition and increased susceptibility to fracture.


title: Self-supervised contrastive learning for integrative single cell RNA-seq data analysis

abstract: Single-cell RNA-sequencing (scRNA-seq) has become a powerful tool to reveal the complex biological diversity and heterogeneity among cell populations. However, the technical noise and bias of the technology still have negative impacts on the downstream analysis. Here, we present a self-supervised Contrastive LEArning framework for scRNA-seq (CLEAR) profile representation and the downstream analysis. CLEAR overcomes the heterogeneity of the experimental data with a specifically designed representation learning task and thus can handle batch effects and dropout events. In the task, the deep learning model learns to pull together the representations of similar cells while pushing apart distinct cells, without manual labeling. It achieves superior performance on a broad range of fundamental tasks, including clustering, visualization, dropout correction, batch effect removal, and pseudo-time inference. The proposed method successfully identifies and illustrates inflammatory-related mechanisms in a COVID-19 disease study with 43,695 single cells from peripheral blood mononuclear cells. Further experiments to process a million-scale single-cell dataset demonstrate the scalability of CLEAR. This scalable method generates effective scRNA-seq data representation while eliminating technical noise, and it will serve as a general computational framework for single-cell data analysis.

category: bioinformatics


title: Transcriptomic Analysis of Human Skin Wound Healing and Rejuvenation Following Ablative Fractional Laser Treatment

abstract: Ablative fractional laser treatment is considered the gold standard for skin rejuvenation. In order to understand how fractional laser works to rejuvenate skin, we performed microarray profiling on skin biopsies to identify temporal and dose-response changes in gene expression following fractional laser treatment. The backs of 14 women were treated with ablative fractional laser (Fraxel(R)) and 4 mm punch biopsies were collected from an untreated site and at the treated sites 1, 3, 7, 14, 21 and 28 days after the single treatment. In addition, in order to understand the effect that multiple fractional laser treatments have on skin rejuvenation, several sites were treated sequentially with either 1, 2, 3, or 4 treatments (with 28 days between treatments) followed by the collection of 4 mm punch biopsies. RNA was extracted from the biopsies, analyzed using Affymetrix U219 chips and gene expression was compared between untreated and treated sites. We observed dramatic changes in gene expression as early as 1 day after fractional laser treatment with changes remaining elevated even after 1 month. Analysis of individual genes demonstrated significant and time related changes in inflammatory, epidermal, and dermal genes, with dermal genes linked to extracellular matrix formation changing at later time points following fractional laser treatment. When comparing the age-related changes in skin gene expression to those induced by fractional laser, it was observed that fractional laser treatment reverses many of the changes in the aging gene expression. Finally, multiple fractional laser treatments resulted in continued changes in gene expression, with many genes either differentially regulated or continuously upregulated with increasing number of treatments, indicating that maximal skin rejuvenation requires multiple fractional laser treatments. In conclusion, fractional laser treatment of skin activates several biological processes involved in wound healing and tissue regeneration, all of which significantly contribute to the rejuvenating effect of fractional laser treatment on aged skin.


title: CASowary: CRISPR-Cas13 guide RNA predictor for transcript depletion

abstract: Recent discovery of the gene editing system -CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) associated proteins (Cas), has resulted in its widespread use for improved understanding of a variety of biological systems. Cas13, a lesser studied Cas protein, has been repurposed to allow for efficient and precise editing of RNA molecules. The Cas13 system utilizes base complementarity between a crRNA/sgRNA (crispr RNA or single guide RNA) and a target RNA transcript, to preferentially bind to only the target transcript. Unlike targeting the upstream regulatory regions of protein coding genes on the genome, the transcriptome is significantly more redundant, leading to many transcripts having wide stretches of identical nucleotide sequences. Transcripts also exhibit complex three-dimensional structures and interact with an array of RBPs (RNA Binding Proteins), both of which further limit the scope of effective target sequences. As a result, there currently exists no method to predict whether a specific sgRNA will effectively knockdown a transcript. Here we present a novel machine learning and computational tool, CASowary, to predict the efficacy of a sgRNA. We used publicly available RNA knockdown data from Cas13 characterization experiments for 555 sgRNAs targeting the transcriptome in HEK293 cells, in conjunction with transcriptome-wide protein occupancy information on RNA. Our model utilizes a Decision Tree architecture with a set of 112 sequence and target availability features, to classify sgRNA efficacy into one of four classes, based upon expected level of target transcript knockdown. After accounting for noise in the training data set, the noise-normalized accuracy exceeds 70%. Additionally, highly effective sgRNA predictions have been experimentally validated using an independent RNA targeting Cas system -CIRTS, confirming the robustness and reproducibility of our models sgRNA predictions. Utilizing transcriptome wide protein occupancy map generated using POP-seq in Hela cells against publicly available protein-RNA interaction map in Hek293 cells, we show that CASowary can predict high quality guides for numerous transcripts in a cell line specific manner. Application of CASowary to whole transcriptomes should enable rapid deployment of CRISPR/Cas13 systems, facilitating the development of therapeutic interventions linked with aberrations in RNA regulatory processes.


title: Directed yeast genome evolution by controlled introduction of trans-chromosomic structural variations

abstract: Naturally occurring structural variations (SVs) are a considerable source of genomic variation and can reshape chromosomes 3D architecture. The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system has been proved to generate random SVs to impact phenotypes and thus constitutes powerful drivers of directed genome evolution. However, how to reveal the molecular mechanism insights into the interactions between phenotypes and complex SVs, especially inversions and translocations, has so far remained challenging. In this study, we develop a SV-prone yeast strain by using SCRaMbLE with two synthetic chromosomes, synV and synX. An heterologous biosynthesis pathway allowing a high throughput screen for increased yield of astaxanthin is used as readout and a proof of concept for the application of SV in industry. We report here that complex SVs, including a pericentric inversion and a trans-chromosomes translocation between synV and synX, result in two neochromosomes and a 2.7-fold yield of astaxanthin. We demonstrated that inversion and inversion reshaped chromosomes 3D architecture and led to large reorganization of the genetic information nearby the breakpoint of the SVs along the chromosomes. Specifically, the pericentric inversion increased the expression of STE18 and the trans-chromosomic translocation increased the expression of RPS5 and MCM22, which contributed to higher astaxanthin yield. We also used the model learned from the aforementioned random screen and successfully harnessed the precise introduction of trans-chromosomes translocation and pericentric inversions by rational design. Overall, our work provides an effective tool to not only accelerate the directed genome evolution but also reveal mechanistic insight of complex SVs for altering phenotypes.

category: synthetic biology


title: A mutation in the pmrD gene of Shigella flexneri abrogates functional PhoPQ-PmrD-PmrAB signaling and polymyxin B resistance

abstract: Shigella spp. are the causative agent of bacillary dysentery, a major cause of food-borne morbidity and mortality worldwide. These organisms are recently evolved, polyphyletic pathovar of E. coli, and since their divergence they have undergone multiple cases of gene gain and gene loss and understanding how gene inactivation events alter bacterial behaviour represents an important objective to be better able to understand how virulence and other phenotypes are affected. Here, we identify a frameshift mutation in the pmrD gene of S. flexneri that although it would be predicted to make a functional, full-length protein, no such production occurs, likely due to the non-optimal spacing between the translational initiation site and the Shine-Dalgarno sequence. We show that this loss severs the normal connection between the PhoPQ two-component regulatory system and the PmrAB two-component regulatory system, abrogating low Mg2+ mediated cationic antimicrobial peptide and polymyxin B resistance, while maintaining normal PmrAB-mediated polymyxin B resistance. In contrast, S. sonnei maintains a functional PmrD protein and canonical signaling through this regulatory network. This species specific gene loss suggests that S. flexneri and S. sonnei have evolved different regulatory responses to changing environmental conditions.

category: microbiology


title: Deep Learning Image Classification of Red Blood Cell Deformability

abstract: Red blood cells (RBCs) must be highly deformable to transit through the microvasculature to deliver oxygen to tissues. The loss of RBC deformability resulting from pathology, natural aging, or storage in blood bags can impede the proper function of these cells. A variety of methods have been developed to measure RBC deformability, but these methods require specialized equipment, long measurement time, and highly skilled personnel. To address this challenge, we investigated whether a machine learning approach could be applied to determine donor RBC deformability using single cell microscope images. We used the microfluidic ratchet device to sort RBCs based on deformability. Sorted cells are then imaged and used to train a deep learning model to classify RBCs based on deformability. This model correctly predicted deformability of individual RBCs with 84 {+/-} 11% accuracy averaged across ten donors. Using this model to score the deformability of RBC samples were accurate to within 4.4 {+/-} 2.5% of the value obtained using the microfluidic ratchet device. While machine learning methods are frequently developed to automate human image analysis, our study is remarkable in showing that deep learning of single cell microscopy images could be used to measure RBC deformability, a property not normally measurable by imaging. Measuring RBC deformability by imaging is also desirable because it can be performed rapidly using a standard microscopy system, potentially enabling RBC deformability studies to be performed as part of routine clinical assessments.

category: bioengineering


title: Determination of best Raman spectroscopy spatial offsets for transcutaneous bone quality assessments in human hands

abstract: Spatially offset Raman spectroscopy (SORS) is able to detect bone signal transcutaneously and could assist in predicting bone fracture risk. Criteria for optimal source-detector offsets for transcutaneous human measurements, however, are not well-established. Although larger offsets yield a higher percentage of bone signal, the absolute amount of bone signal decreases. Spectral unmixing into bone, adipose, and non-adipose components was employed to quantify changes in bone signal to noise ratio across a range of offsets, and optimal offsets for phalanx and metacarpal measurements were determined. The bone signal to noise ratio was maximized at offsets ranging from 4-6 mm.


title: HyperChIP for identifying hypervariable signals across ChIP/ATAC-seq samples

abstract: With the reduction in sequencing costs, studies become prevalent that profile the chromatin landscape for tens or even hundreds of human individuals by using ChIP/ATAC-seq techniques. Identifying genomic regions with hypervariable ChIP/ATAC-seq signals across given samples is essential for such studies. In particular, the hypervariable regions (HVRs) across tumors from different patients indicate their heterogeneity and can contribute to revealing potential cancer subtypes and the associated epigenetic markers. We present HyperChIP as the first complete statistical tool for the task. HyperChIP uses scaled variances that account for the mean-variance dependence to rank genomic regions, and it increases the statistical power by diminishing the influence of true HVRs on model fitting. Applying it to a large pan-cancer ATAC-seq data set, we found that the identified HVRs not only provided a solid basis to uncover the underlying similarity structure among the involved tumor samples, but also led to the identification of transcription factors pertaining to the similarity structure when coupled with a motif-scanning analysis.


title: Understanding SARS-CoV-2 budding through molecular dynamics simulations of M and E protein complexes

abstract: SARS-CoV-2 and other coronaviruses pose major threats to global health, yet computational efforts to understand them have largely overlooked the process of budding, a key part of the coronavirus life cycle. When expressed together, coronavirus M and E proteins are sufficient to facilitate budding into the ER-Golgi intermediate compartment (ERGIC). To help elucidate budding, we ran atomistic molecular dynamics (MD) simulations using the Feig laboratorys refined structural models of the SARS-CoV-2 M protein dimer and E protein pentamer. Our MD simulations consisted of M protein dimers and E protein pentamers in patches of membrane. By examining where these proteins induced membrane curvature in silico, we obtained insights around how the budding process may occur. Multiple M protein dimers acted together to induce global membrane curvature through protein-lipid interactions while E protein pentamers kept the membrane planar. These results could eventually help guide development of antiviral therapeutics which inhibit coronavirus budding.

category: biophysics


title: Elucidation of SARS-CoV-2 budding mechanisms through molecular dynamics simulations of M and E protein complexes


title: Size of the cell nucleus and its effect on the chromatin structure in living cells.

abstract:
DNA-architectural proteins play a major role in organization of chromosomal DNA in living cells by packaging it into chromatin, whose spatial conformation is determined by an intricate interplay between the DNA-binding properties of architectural proteins and physical constraints applied to the DNA by a tight nuclear space. Yet, the exact effects of the cell nucleus size on DNA-protein interactions and chromatin structure currently remain obscure. Furthermore, there is even no clear understanding of molecular mechanisms responsible for the nucleus size regulation in living cells. To find answers to these questions, we developed a general theoretical framework based on a combination of polymer field theory and transfer-matrix calculations, which showed that the nucleus size is mainly determined by the difference between the surface tensions of the nuclear envelope and the endoplasmic reticulum membrane as well as the osmotic pressure exerted by cytosolic macromolecules on the nucleus. In addition, the model demonstrated that the cell nucleus functions as a piezoelectric element, changing its electrostatic potential in a size-dependent manner. This effect has been found to have a profound impact on stability of nucleosomes, revealing a previously unknown link between the nucleus size and chromatin structure. Overall, our study provides new insights into the molecular mechanisms responsible for regulation of the cell nucleus size, as well as the potential role of nuclear organization in shaping the cell response to environmental cues.

SIGNIFICANCE STATEMENTThe cell nucleus plays a central role in the life of eukaryotic cells, providing the highest level of control of intracellular processes. Depending on the stage of the cell cycle and / or surrounding environment, the size of the cell nucleus may undergo changes that are believed to cause chromatin reorganization, affecting gene transcription. At present, however, there is no clear understanding of the molecular mechanisms that may be responsible for such regulation, whose exact effect on chromatin structure remains unclear. In this study, by developing an advanced computational approach, we explore these issues from a physical perspective, revealing previously unknown mechanisms contributing to organization of the cell nucleus and chromatin.


title: Nucleus size and its effect on the chromatin structure in living cells.


title: Molecular mechanism of the anti-lung cancer effect of Jin Ning Fang based on network pharmacology and experimental verification

abstract:
BackgroundJin Ning Fang (JNF) is widely used as an adjuvant therapy for lung cancer. However, its molecular mechanism against lung cancer is still unclear.

MethodsThe chemical compounds JNF were screened from the TCMSP database and its target proteins were then predicted. The genes related to lung cancer were collected from the CTD and DisGeNET databases. Next, targets were integrated with disease-related genes to obtain candidate genes. Functional enrichment and protein-protein interaction (PPI) analysis were also performed, followed by construction of pharmacological network. Meanwhile, Autodock was used to assess the affinity between targets and compound. Finally, the anti-cancer effect of JNF on lung cancer cells was detected and some predicted key genes was validated by using real-time PCR.

ResultsTwenty-five overlapping targets were obtained, and pathway analysis showed that JNF might exert its anti-cancer function by regulating some biological pathways, such as apoptosis pathway. PPI and pharmacological network revealed several core targets (such as AKT1, AR, and ESR1) and three compounds (quercetin, calcium carbonate, and beta-sitosterol). Then, beta-sitosterol had a high affinity with AKT1, AR, and ESR1. Further in vitro experiments confirmed that JNF could inhibit proliferation and promote apoptosis of A549 cells. The expression of FDPS, PIM1, VCAM1, SLC29A1, NQO1, and ESR1 were significantly decreased, while mRNA level of AR and ANPEP were markedly increased after JNF treatment.

ConclusionJNF may exert anti-lung cancer effect through multiple targets and pathways, and identified genes may be used as potential biomarkers for diagnosis and treatment of lung cancer.

category: cancer biology


title: Thalamic deep brain stimulation as a paradigm to reduce consciousness: implications for cortico-striatal dynamics, absence epilepsy and consciousness studies

abstract:
Anesthetic manipulations provide much-needed causal evidence for neural correlates of consciousness, but non-specific drug effects complicate their interpretation. Evidence suggests that thalamic deep brain stimulation (DBS) can either increase or decrease consciousness, depending on the stimulation target and parameters. The putative role of the central lateral thalamus (CL) in consciousness makes it an ideal DBS target to manipulate circuit-level mechanisms in cortico-striato-thalamic (CST) systems, thereby influencing consciousness and related processes. We used multi-microelectrode DBS targeted to CL in macaques while recording from frontal, parietal, and striatal regions. DBS induced episodes reminiscent of absence epilepsy, here termed absence-like activity (ALA), with decreased behavior and vacant staring coinciding with low-frequency oscillations. DBS modulated ALA likelihood in a frequency-specific manner. ALA events corresponded to decreases in measures of neural complexity (entropy) and integration ({Phi}*), an index of consciousness, and substantial changes to communication in CST circuits. During ALA, power spectral density and coherence at low frequencies increased across CST circuits, especially in thalamo-parietal and cortico-striatal pathways. Decreased consciousness and neural integration corresponded to shifts in cortico-striatal network configurations that dissociated parietal and subcortical structures. Overall, the features of ALA and implicated networks were similar to those of absence epilepsy. As this same multi-microelectrode DBS method - but at different stimulation frequencies - can also increase consciousness in anesthetized macaques, it can be used to flexibly address questions of consciousness with limited confounds, as well as inform clinical investigations of absence epilepsy and other consciousness disorders.

SIGNIFICANCEWe use tailored, multi-microelectrode thalamic deep brain stimulation to reversibly decrease consciousness for otherwise healthy, wakeful animals in a stimulation frequency-dependent manner. This represents a bidirectional mechanism for controlling consciousness, as the same method can increase consciousness under certain conditions. Theories of consciousness debate the relative contribution of parietal and frontal lobes, and largely ignore subcortical contributions. In this study, changes in consciousness predominantly involve changes in subcortical and parietal regions, implying that they contribute more to consciousness than frontal regions. Further, decreases in consciousness (indexed by {Phi}*) coincide with decreased movement, staring, and low-frequency activity in the EEG, similar to absence epilepsy. Thus, the systems-level mechanisms for decreased consciousness in this study have broader clinical implications for absence epilepsy.

category: neuroscience


title: The olfactory gating of visual preferences to human skin and colors in mosquitoes

abstract: Mosquitoes track odors, locate hosts, and find mates visually. The color of a food resource, such as a flower or warm-blooded host, can be dominated by long wavelengths of the visible light spectrum (green to red for humans) and is likely important for object recognition and localization. However, little is known about the hues that attract mosquitoes or how odor affects mosquito visual search behaviors. We used a real-time 3D tracking system and wind tunnel that allowed careful control of the olfactory and visual environment to quantify the behavior of more than 1.3 million mosquito trajectories. We found that CO2 induces a strong attraction to specific hues, including those that humans perceive as cyan, orange, and red. Sensitivity to orange and red correlates with mosquitoes strong attraction to the color spectrum of human skin, which is dominated by these wavelengths. Attraction was eliminated by filtering the orange and red bands from the skin color spectrum and by introducing mutations targeting specific long-wavelength opsins or CO2 detection. Collectively, our results show that odor is critical for mosquitoes wavelength preferences and that the mosquito visual system is a promising target for inhibiting their attraction to human hosts.


title: Quantification of within patient Staphylococcus aureus phenotypic heterogeneity as a proxy for presence of persisters across clinical presentations

abstract:
BackgroundDifficult-to-treat infections caused by antibiotic susceptible strains have been linked with the occurrence of persisters. Persisters are a subpopulation of dormant bacteria that tolerate antibiotic exposure despite lacking genetic resistance. They can be identified phenotypically upon plating on nutrient agar because of their altered growth dynamics, resulting in colony size heterogeneity. The occurrence of within-patient bacterial phenotypic heterogeneity in various infections and clinical determinants of persister formation remain unknown.

MethodsWe plated bacteria derived from 132 patient-samples of difficult-to-treat infections directly on nutrient-rich agar and monitored colony growth by time-lapse imaging. Of these, we retained 36 Staphylococcus aureus mono-cultures for further analysis. We investigated clinical factors potentially associated with increased colony growth-delay with regression analyses. Additionally, we corroborated the clinical findings using in vitro grown static biofilms, exposed to distinct antibiotics.

ResultsThe extent of phenotypic heterogeneity of patient-derived S. aureus varied substantially between patients. Increased heterogeneity coincided with increased median growth-delay. Multivariable regression showed that rifampicin treatment was significantly associated with increased median growth-delay. S. aureus grown in biofilms and exposed to high concentrations of rifampicin or a combination of rifampicin with either clindamycin or levofloxacin exhibited prolonged growth-delay, correlating with a strain-dependent increase in antibiotic tolerance.

ConclusionsUpon direct cultivation on nutrient-rich agar, S. aureus from difficult-to-treat infections commonly exhibited colony size heterogeneity. This was due to heterogeneous delays in growth resumption, with delays larger than two days in the most extreme cases. Since bacteria in a dormant state are tolerant to antibiotics, the observation of large growth-delays might have direct clinical implications. Future studies are needed to assess the potential of bacterial phenotypic heterogeneity quantification for staphylococcal infections prognosis.


title: Spores and extracellular matrix components impart molecular order in Bacillus subtilis biofilms.

abstract:
Biofilms are multicellular microbial communities that encase themselves in an extracellular matrix (ECM) of secreted biopolymers and attach to surfaces and interfaces. Bacterial biofilms are detrimental in hospital and industrial settings, but they can be beneficial in agricultural contexts. An essential property of biofilms that grants them with increased survival relative to planktonic cells is phenotypic heterogeneity; the division of the biofilm population into functionally distinct subgroups of cells. Phenotypic heterogeneity in biofilms can be traced to the cellular level, however, the molecular structures and elemental distribution across whole biofilms as well as possible linkages between them remain unexplored. Mapping X-ray diffraction (XRD) across intact biofilms in time and space, we revealed the dominant structural features in Bacillus subtilis biofilms, stemming from matrix components, spores and water. By simultaneously following the X-ray fluorescence (XRF) signal of biofilms and isolated matrix components, we discovered that the ECM preferentially binds calcium ions over other metal ions, specifically, zinc, manganese and iron. These ions, remaining free to flow below macroscopic wrinkles that act as water channels, eventually accumulate and lead to sporulation. The possible link between ECM properties, regulation of metal ion distribution and sporulation across whole intact biofilms unravels the importance of molecular-level heterogeneity in shaping biofilm physiology and development.

Significance StatementBiofilms are multicellular soft microbial communities that are able to colonize synthetic surfaces as well as living organisms. To survive sudden environmental changes and efficiently share their common resources, cells in a biofilm divide into subgroups with distinct functions, leading to phenotypic heterogeneity. Here, by studying intact biofilms by synchrotron X-ray diffraction and fluorescence, we revealed correlations between biofilm macroscopic architectural heterogeneity and the spatio-temporal distribution of extracellular matrix, spores, water and metal ions. Our findings demonstrate that biofilm heterogeneity is not only affected by local genetic expression and cellular differentiation, but also by passive effects resulting from the physicochemical properties of the molecules secreted by the cells, leading to differential distribution of nutrients that propagates through macroscopic length scales.


title: Extracellular matrix and spores impart structural hierarchy and compositional heterogeneity in Bacillus subtilis biofilms


title: Multiscale X-ray study of Bacillus subtilis biofilms reveals interlinked structural hierarchy and elemental heterogeneity


title: Histone Variant macroH2A1 Spatially and Functionally Organizes Human Papillomavirus Replication Foci in the Productive Stage of Infection

abstract:
The life cycle of HPV depends on keratinocyte differentiation as the virus modulates and takes advantage of cellular pathways to replicate its genome and assemble viral particles in differentiated cells. Viral genomes are amplified in nuclear replication foci in differentiated keratinocytes, and DNA repair factors from the DNA damage response signaling pathway are recruited to replicate viral DNA. The HPV genome is associated with cellular histones at all stages of the infectious cycle, and here we show the histone variant macroH2A1 is bound to the HPV genome and enriched in viral replication foci in differentiated cells. MacroH2A1 isoforms play important roles in cellular transcriptional repression, double strand break repair, and replication stress. The viral E8^E2 protein also binds to the HPV genome and inhibits viral replication and gene expression by recruiting NCoR/SMRT complexes. We show here that E8^E2 and SMRT also localize within replication foci, though not through direct interaction with macroH2A1. Conversely, transcription complexes containing RNA polymerase II and Brd4 are located on the surface of the foci. Foci generated with an HPV16 E8^E2 mutant genome are not enriched for SMRT or macroH2A1 but contain transcriptional complexes throughout the foci. We demonstrate that macroH2A1 promotes viral late transcription and propose that it does so by spatially separating replication and transcription activities within replication foci.

IMPORTANCEHuman papillomaviruses are small DNA viruses that cause chronic infection of cutaneous and mucosal epithelium. In some cases, persistent infection with HPV can result in cancer, and 5% human cancers are the result of HPV infection. In differentiated cells, HPV amplifies viral DNA in nuclear replication factories and transcribes late mRNAs to produce capsid proteins. However, very little is known about the spatial organization of these activities in the nucleus. Here we show that repressive viral and cellular factors localize within the foci to supress viral transcription, while active transcription takes place on the surface. The cellular histone variant macroH2A1 is important for this spatial organization.


title: Uncoupling of the Diurnal Growth Program by Artificial Genome Relaxation in Synechocystis sp. PCC 6803

abstract: In cyanobacteria DNA supercoiling varies over the diurnal light/dark cycle and is integrated with temporal programs of transcription and replication. We manipulated DNA supercoiling in Synechocystis sp. PCC 6803 by CRISPRi-based knock-down of gyrase subunits and overexpression of topoisomerase I (TopoI), and characterized the phenotypes. Cell division was blocked, most likely due to inhibition of genomic but not plasmid DNA replication. Cell growth continued to 4-5x of the wildtype cell volume, and metabolic flux was redirected towards glycogen in the TopoI overexpression strain. TopoI induction initially lead to down-regulation of GC-rich and up-regulation of AT-rich genes. The response quickly bifurcated and four diurnal co-expression cohorts (dawn, noon, dusk and night) all responded differently, in part with a circadian ({approx} 24 h) pattern. A GC-rich region - 50 bp of transcription start sites is differentially enriched in these four cohorts. We suggest a model where energy- and gyrase-gated transcription of growth genes at the dark/light transition (dawn) generates DNA supercoiling which then facilitates DNA replication and initiates the diurnal transcriptome program.


title: Anaerobic biodegradation of chloroform and dichloromethane with a Dehalobacter enrichment culture

abstract:
Chloroform (CF) and dichloromethane (DCM) are among the more commonly identified chlorinated aliphatic compounds found in contaminated soil and groundwater. Complete dechlorination of CF has been reported under anaerobic conditions by microbes that respire CF to DCM and others that biodegrade DCM. The objectives of this study were to ascertain if a commercially available bioaugmentation enrichment culture (KB-1(R) Plus) uses an oxidative or fermentative pathway for biodegradation of DCM; and to determine if the products from DCM biodegradation can support organohalide respiration of CF to DCM in the absence of an exogenous electron donor. In various treatments with the KB-1(R) Plus culture to which 14C-CF was added, the predominant product was 14CO2, indicating that oxidation is the predominant pathway for DCM. Recovery of 14C-DCM when biodegradation was still in progress confirmed that CF first undergoes reductive dechlorination to DCM. 14C-labeled organic acids, including acetate and propionate, were also recovered, suggesting that synthesis of organic acids provides a sink for the electron equivalents from oxidation of DCM. When the culture was washed to remove organic acids from prior additions of exogenous electron donor and only CF and DCM were added, the culture completely dechlorinated CF. The total amount of DCM added was not sufficient to provide the electron equivalents needed to reduce CF to DCM. Thus, the additional reducing power came via the DCM generated from CF reduction. Nevertheless, the rate of CF consumption was considerably slower in comparison to treatments that received an exogenous electron donor.

IMPORTANCEChloroform (CF) and dichloromethane (DCM) are among the more commonly identified chlorinated aliphatic compounds found in contaminated soil and groundwater. One way to address this problem is to add microbes to the subsurface that can biodegrade these compounds. While microbes are known that can accomplish this task, less is known about the pathways used under anaerobic conditions. Some use an oxidative pathway, resulting mainly in carbon dioxide. Others use a fermentative pathway, resulting in formation of organic acids. In this study, a commercially available bioaugmentation enrichment culture (KB-1(R) Plus) was evaluated using carbon-14 labelled chloroform. The main product formed was carbon dioxide, indicating the use of an oxidative pathway. The reducing power gained from oxidation was shown to support reductive dechlorination of CF to DCM. The results demonstrate the potential to achieve full dechlorination of CF and DCM to nonhazardous products that are difficult to identify in the field.


title: Parental origin of Gsα inactivation differentially affects bone remodeling in a mouse model of Albright hereditary osteodystrophy

abstract: Albright hereditary osteodystrophy (AHO) is caused by heterozygous inactivation of GNAS, a complex locus that encodes the alpha-stimulatory subunit of GPCRs (Gs) in addition to NESP55 and XLs due to alternative first exons. AHO skeletal manifestations include brachydactyly, brachymetacarpia, compromised adult stature, and subcutaneous ossifications. AHO patients with maternally-inherited GNAS mutations develop pseudohypoparathyroidism type 1A (PHP1A) with resistance to multiple hormones that mediate their actions through GPCRs requiring Gs (eg., PTH, TSH, GHRH, calcitonin) and severe obesity. Paternally-inherited GNAS mutations cause pseudopseudohypoparathyroidism (PPHP), in which patients have AHO skeletal features but do not develop hormonal resistance or marked obesity. These differences between PHP1A and PPHP are caused by tissue-specific reduction of paternal Gs expression. Previous reports in mice have shown loss of Gs causes osteopenia due to impaired osteoblast number and function and suggest AHO patients could display evidence of reduced bone mineral density (BMD). However, we previously demonstrated PHP1A patients display normal-increased BMD measurements without any correlation to body mass index or serum PTH. Due to these observed differences between PHP1A and PPHP, we utilized our laboratorys AHO mouse model to address whether Gs heterozygous inactivation by the targeted disruption of exon 1 of Gnas differentially affects bone remodeling based on the parental inheritance of the mutation. Mice with paternally-inherited (GnasE1+/-p) and maternally-inherited (GnasE1+/-m) mutations displayed reductions in osteoblasts along the bone surface compared to wildtype. GnasE1+/-p mice displayed reduced cortical and trabecular bone parameters due to impaired bone formation and excessive bone resorption. GnasE1+/-m mice however displayed enhanced bone parameters due to increased osteoblast activity and normal bone resorption. These distinctions in bone remodeling between GnasE1+/-p and GnasE1+/-m mice appear to be secondary to changes in the bone microenvironment driven by calcitonin-resistance within GnasE1+/-m osteoclasts and therefore warrant further studies into understanding how Gs influences osteoblast-osteoclast coupling interactions.

category: cell biology


title: Disordered climate threatens short-distance migrants

abstract: Global climate change has led to a warmer world, changing the migratory and breeding behaviors of many species, and short-distance migrants may benefit from climate change. With climate change leading to an increasingly disordered climate, we show here that a disordered spring climate disturbs the migration and breeding of a short-distance anadromous fish. In 2020, on the Qinghai-Tibetan Plateau, an abnormally low temperature in April delayed the migration rhythm of Gymnocypris przewalskii by nearly 10 days, while the gonadal development rhythm of the breeding population was almost normal. The phenology mismatch decreased the migrating populations by 30-70%, reducing the larval flux by nearly 80%. This case reveals that for short-distance migrants, different phenologies within the same species respond to disordered climates differently, which leads to phenology mismatches and then threatens the species. Along with increasing local extreme weather and climate events, short-distance migrants need more attention and conservation actions.

category: ecology


title: Detection and characterization of replication origins defined by DNA polymerase epsilon

abstract:
BackgroundAlthough the process of DNA replication is highly conserved the location of origins of replication (ORI) may vary from one tissue to the next or one round of replication to the next in eukaryotes, suggesting flexibility in the choice of locations to initiate replication. Lists of human ORI therefore vary widely in number and location and there are no methods available to compare them.

ResultsHere we report the genome-wide localization of ORI in POLE-mutated human tumors using whole genome sequencing data. Mutations accumulated after many rounds of replication of unsynchronized dividing cell populations in tumors allow to identify constitutive origins, which we show are shared with high-fidelity between individuals and tumor types. Using a Smith-Waterman-like dynamic programming approach, we compared replication origin positions obtained from multiple different methods. The comparison allowed us to define a consensus set of replication origins, identified consistently by multiple ORI detection methods.

ConclusionsMany DNA features co-localized with the consensus set of ORI, including chromatin loop anchors, G-quadruplexes, S/MARs and CpGs. Among all features, the H2A.Z histone exhibited the most significant association. Our results show that mutation-based detection of replication origins is a viable approach to determining their location and associated sequence features.

category: genomics


title: AlphaTims: Indexing trapped ion mobility spectrometry - time of flight data for fast and easy accession and visualization

abstract:
High resolution mass spectrometry-based proteomics generates large amounts of data, even in the standard liquid chromatography (LC) - tandem mass spectrometry configuration. Adding an ion mobility dimension vastly increases the acquired data volume, challenging both analytical processing pipelines and especially data exploration by scientists. This has necessitated data aggregation, effectively discarding much of the information present in these rich data sets. Taking trapped ion mobility spectrometry (TIMS) on a quadrupole time-of-flight platform (Q-TOF) as an example, we developed an efficient indexing scheme that represents all data points as detector arrival times on scales of minutes (LC), milliseconds (TIMS) and microseconds (TOF). In our open source AlphaTims package, data are indexed, accessed and visualized by a combination of tools of the scientific Python ecosystem. We interpret unprocessed data as a sparse 4D matrix and use just-in-time compilation to machine code with Numba, accelerating our computational procedures by several orders of magnitude while keeping to familiar indexing and slicing notations. For samples with more than six billion detector events, a modern laptop can load and index raw data in about a minute. Loading is even faster when AlphaTims has already saved indexed data in a HDF5 file, a portable scientific standard used in extremely large-scale data acquisition. Subsequently, data accession along any dimension and interactive visualization happen in milliseconds. We have found AlphaTims to be a key enabling tool to explore high dimensional LC-TIMS-QTOF data and have made it freely available as an open-source Python package with a stand-alone graphical user interface at https://github.com/MannLabs/alphatims or as part of the AlphaPept  ecosystem.

HighlightsO_LIEasy visualization and fast accession of LC-TIMS-QTOF data
C_LIO_LIFreely available graphical user interface, command-line interface and Python module on Windows, Linux and macOS.
C_LI


title: 3D Convolutional Neural Networks for Classification of Alzheimer's and Parkinson's Disease with T1-Weighted Brain MRI

abstract: Parkinsons disease (PD) and Alzheimers disease (AD) are progressive neurodegenerative disorders that affect millions of people worldwide. In this work, we propose a deep learning approach to classify these diseases based on 3D T1-weighted brain MRI. We analyzed several datasets including the Parkinsons Progression Markers Initiative (PPMI), an independent dataset from the University of Pennsylvania School of Medicine (UPenn), the Alzheimers Disease Neuroimaging Initiative (ADNI), and the Open Access Series of Imaging Studies (OASIS) dataset. The UPenn and OASIS datasets were used as independent test sets to evaluate the model performance during inference. We also implemented a random forest classifier as a baseline model by extracting key radiomics features from the same T1-weighted MRI scans. The proposed 3D convolutional neural network (CNN) model was trained from scratch for the classification tasks. For AD classification, the 3D CNN model achieved an ROC-AUC of 0.878 on the ADNI test set and an average ROC-AUC of 0.789 on the OASIS dataset. For PD classification, the proposed 3D CNN model achieved an ROC-AUC of 0.667 on the PPMI test set and an average ROC-AUC of 0.743 on the UPenn dataset. Model performance was largely maintained when using only 25% of the training dataset. The 3D CNN outperformed the random forest classifier for both the PD and AD tasks. The 3D CNN also generalized better on unseen MRI data from different imaging centers. These approaches show promise for screening of PD and AD patients using only T1-weighted brain MRI, which is relatively widely available. This model with additional validation could also be used to help differentiate between challenging cases of AD and PD when they present with similarly subtle motor and non-motor symptoms.


title: Nitrate-regulated growth processes involve activation of gibberellin pathway

abstract: Nitrate, one of the main nitrogen (N) sources for crops, acts as a nutrient and key signaling molecule coordinating gene expression, metabolism and various growth processes throughout the plant life cycle. It is widely accepted that nitrate-triggered developmental programs cooperate with hormone synthesis and transport, to finely adapt plant architecture to N availability. Here, we report that nitrate, acting through its signaling pathway, promotes growth in Arabidopsis and wheat, in part by modulating the accumulation of gibberellin (GA)-regulated DELLA growth repressors. We show that nitrate reduces the abundance of DELLAs by increasing GA contents through activation of GA metabolism gene expression. Consistently, the growth restraint conferred by nitrate deficiency is partially rescued in global-DELLA mutant that lacks all DELLAs. At the cellular level, we show that nitrate enhances both cell proliferation and elongation in a DELLA-dependent and -independent manner, respectively. Our findings establish a connection between nitrate and GA signaling pathways that allow plants to adapt their growth to nitrate availability.

category: plant biology


title: CD8+ T Cell Metabolic Rewiring Defined by Single-Cell RNA-Sequencing Identifies a Critical Role of ASNS Expression Dynamics in T Cell Differentiation

abstract: Cytotoxic T cells dynamically rewire their metabolism during the course of an immune response. While T-cell metabolism has been extensively studied at phenotypic endpoints of activation and differentiation, the underlying dynamics remain largely elusive. Here, we leverage on single-cell RNA-sequencing (scRNA-seq) measurements of in vitro activated and differentiated CD8+ T cells cultured in physiological media to resolve these metabolic dynamics. We find that our scRNA-seq analysis identifies most metabolic changes previously defined in in vivo experiments, such as a rewiring from an oxidative to an anabolism-promoting metabolic program during activation to an effector state, which is later reverted upon memory polarization. Importantly, our scRNA-seq data further provide a dynamic description of these changes. In this sense, our data predict a differential time-dependent reliance of CD8+ T cells on the synthesis versus uptake of various non-essential amino acids during T-cell activation, which we corroborate with additional functional in vitro experiments. We further exploit our scRNA-seq data to identify metabolic genes that could potentially dictate the outcome of T-cell differentiation, by ranking them based on their expression dynamics. Among the highest-ranked hits, we find asparagine synthetase (Asns), whose expression sharply peaks for effector CD8+ T cells and further decays towards memory polarization. We then confirm that these in vitro Asns expression dynamics are representative of an in vivo situation in a mouse model of viral infection. Moreover, we find that disrupting these expression dynamics in vitro, by depleting asparagine from the culture media, delays central-memory polarization. Accordingly, we find that preventing the decay of ASNS by stable overexpression at the protein level in vivo leads to a significant increase in effector CD8+ T-cell expansion, and a concomitant decrease in central-memory formation, in a mouse model of viral infection. This shows that ASNS expression dynamics dictate the fate of CD8+ T-cell differentiation. In conclusion, we provide a resource of dynamic expression changes during CD8+ T-cell activation and differentiation that is expected to increase our understanding of the dynamic metabolic requirements of T cells progressing along the immune response cascade.

category: immunology


title: Single-cell imaging of Pseudomonas reveals dynamic polar accumulation of the extracellular iron-scavenger pyoverdin

abstract:
Pyoverdin is a water-soluble metal-chelator synthesized by members of the genus Pseudomonas and used for the acquisition of insoluble ferric iron. Although freely diffusible in aqueous environments, preferential dissemination of pyoverdin among adjacent cells, fine-tuning of intracellular siderophore concentrations, and fitness advantages to pyoverdin-producing versus nonproducing cells, indicate control of location and release. Here, using time-lapse fluorescence microscopy to track single cells in growing microcolonies of Pseudomonas fluorescens SBW25, we show accumulation of pyoverdin at cell poles. Accumulation is induced by arrest of cell division, is achieved by cross-feeding in pyoverdin-nonproducing mutants, is independent of cell shape, and is reversible. Furthermore, it occurs in multi-species communities. Analysis of the performance of pyoverdin-producing and nonproducing cells under conditions promoting polar localization shows an advantage to accumulation on resumption of growth after stress. While the genetic basis of polarization remains unclear, evaluation of deletion mutants of pyoverdin transporters (opmQ, fpvA) establishes non-involvement of these candidate loci. Examination of pyoverdin polar accumulation in a model community and in a range of laboratory and natural species of Pseudomonas, including P. aeruginosa PAO1 and P. putida KT2440, confirms that the phenotype is characteristic of Pseudomonas.

SignificanceBacteria secrete extracellular products that enable nutrients to be obtained from the environment. A secreted product of relevance for medicine, agriculture and biotechnology is the iron-chelating siderophore, pyoverdin, which is produced by members of the genus Pseudomonas. By analyzing the behavior of single cells we show that on cessation of cell division, pyoverdin localizes to cell poles, but is then released to the environment prior to resumption of cell growth. Of particular significance is the ecological relevance of this behavior: cells that accumulate the siderophore resume growth with minimal delay. Our study reveals a hitherto unrecognized dimension to the biology of Pseudomonas that may prove central to understanding the broader ecological and physiological relevance of pyoverdin.


title: To bin or not to bin: analyzing single-cell growth data

abstract: Collection of high-throughput data has become prevalent in biology. Large datasets allow the use of statistical constructs such as binning and linear regression to quantify relationships between variables and hypothesize underlying biological mechanisms based on it. We discuss several such examples in relation to single-cell data and cellular growth. In particular, we show instances where what appears to be ordinary use of these statistical methods leads to incorrect conclusions such as growth being non-exponential as opposed to exponential and vice versa. We propose that the data analysis and its interpretation should be done in the context of a generative model, if possible. In this way, the statistical methods can be validated either analytically or against synthetic data generated via the use of the model, leading to a consistent method for inferring biological mechanisms from data. On applying the validated methods of data analysis to infer cellular growth on our experimental data, we find the growth of length in E. coli to be non-exponential. Our analysis shows that in the later stages of the cell cycle the growth rate is faster than exponential.


title: Mutated in Colorectal Cancer (MCC): a centrosomal protein that relocalizes to the ncMTOC during intestinal cell differentiation

abstract: Mutated in Colorectal Cancer (MCC) encodes a coiled-coil protein implicated, as its name suggests, in the pathogenesis of hereditary human colon cancer. To date, however, the contributions of MCC to intestinal homeostasis remain unclear. Here, we examine the subcellular localization of MCC, both at the mRNA and protein levels, in the adult intestinal epithelium. Our findings reveal that Mcc transcripts are restricted to proliferating crypt cells, including Lgr5+ stem cells, and that Mcc protein is distinctly associated with the centrosome in these cells. Upon intestinal cellular differentiation, Mcc is redeployed to the non-centrosomal microtubule organizing center (ncMTOC) at the apical domain of villus cells. Using intestinal organoids, we show that the shuttling of the Mcc protein depends on phosphorylation by Casein Kinases 1{delta}/{varepsilon}, which are critical modulators of WNT signaling. Together, our findings support a putative role for MCC in establishing and maintaining the cellular architecture of the intestinal epithelium as a component of both the centrosome and ncMTOC.


title: Heterogeneity and dynamics of ERK and Akt activation by G protein-coupled receptors depend on the activated heterotrimeric G proteins

abstract:
Kinases are fundamental regulators of cellular functions and play key roles in GPCR-mediated signaling pathways. Kinase activities are generally inferred from cell lysates, hiding the heterogeneity of the individual cellular responses to extracellular stimuli. Here, we study the dynamics and heterogeneity of ERK and Akt in genetically identical cells in response to activation of endogenously expressed GPCRs. We use kinase translocation reporters, high-content imaging, automated segmentation and clustering methods to assess cell-to-cell signaling heterogeneity. We observed ligand-concentration dependent response kinetics to histamine, a2-adrenergic, and S1P receptor stimulation that varied between cells. By using G protein inhibitors, we observed that Gq mediated the ERK and Akt responses to histamine. In contrast, Gi was necessary for ERK and Akt activation in response to 2-adrenergic receptor activation. ERK and Akt were also strongly activated by S1P, showing high heterogeneity at the single cell level, especially for ERK. In all cases, the cellular heterogeneity was not explained by distinct pre-stimulation levels or saturation of the measured response. Cluster analysis of time-series derived from 68,000 cells obtained under the different conditions revealed several distinct populations of cells that display similar response dynamics. The single-cell ERK responses to histamine and brimonidine showed remarkably similar dynamics, despite the activation of different heterotrimeric G proteins. In contrast, the ERK response dynamics to S1P showed high heterogeneity, which was reduced by the inhibition of Gi. To conclude, we have set up an imaging and analysis strategy that reveals substantial cell-to-cell heterogeneity in kinase activity driven by GPCRs.

O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: Extracellular mechanical forces drive endocardial cell volume decrease during cardiac valve morphogenesis

abstract: Organ morphogenesis involves dynamic changes of tissue properties at the cellular scale. In addition, cells need to adapt to their mechanical environment through mechanosensitive pathways. How mechanical cues influence cell behaviors during morphogenesis, however, remains poorly understood. Here we studied the influence of mechanical forces during the formation of the atrioventricular canal (AVC) where cardiac valves develop. We show that in zebrafish the AVC forms within a zone of tissue convergence between the atrium and the ventricle which is associated with increased activation of the actomyosin meshwork and endocardial cell orientation changes. We demonstrate that tissue convergence occurs with a major reduction of endocardial cell volume triggered by mechanical forces and the mechanosensitive channels TRPP2/TRPV4. In addition, we show that the extracellular matrix component hyaluronic acid controls cell volume changes. Together, our data suggest that cell volume change is a key cellular feature activated by mechanical forces during cardiovascular morphogenesis. This work further unravels how mechanical forces and extracellular matrix can influence tissue remodeling in developing organs.

category: developmental biology


title: The effective family size of immigrant founders predicts their long-term demographic outcome: from Quebec settlers to their 20th-century descendants

abstract: Human evolution involves population splits, size fluctuations, founder effects, and admixture. Population history reconstruction based on genetic diversity data routinely relies on simple demographic models while projecting the past. No specific demographic assumptions are needed to understand the genetic structure of the founder population of Quebec. Because genealogy and genetics are intimately related, we used descending genealogies of this population to pursue the fate of its founder lineages. Maternal and paternal lines reflect the transmission of mtDNA and the Y-chromosome, respectively. We followed their transmission in real-time, from the 17th century down to its 20th-century population. We counted the number of married children of immigrants (i.e., their effective family size, EFS), estimated the proportion of successful immigrants in terms of their survival ratio, and assessed net growth rates and extinction. Likewise, we evaluated the same parameters for their Quebec-born descendants. The survival ratio of the first immigrants was the highest and declined over time in association with the decreasing immigrants EFS. Parents with high EFS left plentiful married progeny, putting EFS as the most important variable determining the parental demographic success throughout time for generations ahead. The 17th and 18th-century immigrants bear the most remarkable demographic and genetic impact on the 20th-century population of Quebec. Lessons learned from Quebec genealogies can teach us about the consequences of founder effects and migrations through real peoples history. The effective family size of immigrant founders predicts their long-term demographic outcome.

category: evolutionary biology


title: The role of trade-offs and feedbacks in shaping integrated plasticity, behavioral syndromes, and behavioral correlations

abstract: Despite a decade and a half of intensive research effort, we still have little understanding of why behavioral responses are frequently correlated. This gap in our understanding stems from the fact that much of the work looking into behavioral correlations has focused on among-individual correlations, i.e. "behavioral syndromes". Unfortunately, this focus on syndromes ignores the fact that behavioral correlations are, instead, primarily due to within-individual correlations


title: Development of an ASO therapy for Angelman syndrome by targeting an evolutionarily conserved region at the start of the UBE3A-AS transcript

abstract:
Angelman syndrome is a devastating neurogenetic disorder for which there is currently no effective treatment. It is caused by mutations or epimutations affecting the expression or function of the maternally inherited allele of the ubiquitin-protein ligase E3A (UBE3A) gene. The paternal UBE3A allele is imprinted in neurons of the central nervous system (CNS) by the UBE3A antisense (UBE3A-AS) transcript, which represents the distal end of the SNHG14 transcription unit. Reactivating the expression of the paternal UBE3A allele in the CNS has long been pursued as a therapeutic option for Angelman syndrome. Here, we designed and optimized antisense oligonucleotides (ASO) targeting an evolutionarily conserved region demarcating the start of the human UBE3A-AS transcript and show that ASOs targeting this region can reverse imprinting of UBE3A in cultured Angelman syndrome neurons and throughout the CNS of a non-human primate model. Findings from this study advanced the first investigational molecular therapy for Angelman syndrome into clinical development (ClinicalTrials.gov, NCT04259281).

SUMMARYHere, we describe the preclinical studies supporting the first investigational molecular therapy for Angelman syndrome to advance into clinical development (ClinicalTrials.gov, NCT04259281).

category: genetics


title: A novel anti-influenza combined therapy assessed by single cell RNA-sequencing

abstract: Influenza makes millions of people ill every year, placing a large burden on the healthcare system and the economy. To develop a novel treatment against influenza, we combined virucidal sialylated cyclodextrins with interferon lambda and demonstrated, in human airway epithelia, that the two compounds inhibit the replication of a clinical H1N1 strain more efficiently when administered together rather than alone. We investigated the mechanism of action of the combined treatment by single cell RNA sequencing analysis and found that both the single and combined treatments impair viral replication to different extents across distinct epithelial cell types. We also showed that each cell type comprises multiple sub-types, whose proportions are altered by H1N1 infection, and assess the ability of the treatments to restore them. To the best of our knowledge this is the first study investigating the effectiveness of an antiviral therapy by transcriptomic studies at the single cell level.


title: Evolution of binding preferences among whole-genome duplicated transcription factors

abstract: Throughout evolution, new transcription factors (TFs) emerge by gene duplication, promoting growth and rewiring of transcriptional networks. How TF duplicates diverge is known for only a few studied cases. To provide a genome-scale view, we considered the 35% of budding yeast TFs, classified as whole-genome duplication (WGD)-retained paralogs. Using high-resolution profiling, we find that ~60% of paralogs evolved differential binding preferences. We show that this divergence results primarily from variations outside the DNA binding domains (DBDs), while DBD preferences remain largely conserved. Analysis of non-WGD orthologs revealed that ancestral preferences are unevenly split between duplicates, while new targets are acquired preferentially by the least conserved paralog (biased sub/neo-functionalization). Dimer-forming paralogs evolved mostly one-sided dependency, while other paralogs interacted through low-magnitude DNA-binding competition that minimized paralog interference. We discuss the implications of our findings for the evolutionary design of transcriptional networks.


title: Analysis of long and short enhancers in melanoma cell states

abstract: Understanding how enhancers drive cell type specificity and efficiently identifying them is essential for the development of innovative therapeutic strategies. In melanoma, the melanocytic (MEL) and the mesenchymal-like (MES) states present themselves with different responses to therapy, making the identification of specific enhancers highly relevant. Using massively parallel reporter assay (MPRA) in a panel of patient-derived melanoma lines (MM lines), we set to identify and decipher melanoma enhancers by first focusing on regions with state specific H3K27 acetylation close to differentially expressed genes. A more in-depth evaluation of those regions was then pursued by investigating the activity of ATAC-seq peaks found therein along with a full tiling of the acetylated regions with 190 bp sequences. Activity was observed in more than 60% of the selected regions and we were able to precisely locate the active regions within ATAC-seq peaks. Comparison of sequence content with activity, using the deep learning model DeepMEL2, revealed that AP-1 alone is responsible for the MES enhancer activity, while SOX and MITF both influence MEL enhancer activity with SOX being required to achieve high levels of activity. Overall, our MPRA assays shed light on the relationship between long and short sequences in terms of their sequence content, enhancer activity, and specificity as reporters across melanoma cell states.


title: Ribosomal protein L23 drives the metastasis of hepatocellular carcinoma via upregulating MMP9

abstract: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths globally and tumor metastasis is one of the major causes of high mortality. To identify novel molecules contributing to HCC metastasis is critical to understanding the underlining mechanism of cancer metastasis. Here, combining the analyze based on published database and liver tissues from HCC patients, we identified that RNA binding protein L23 (RPL23) as a tumor metastasis driver in HCC. RPL23 was elevated in HCC and closely related to poor clinical outcomes. Furthermore, RPL23 depletion inhibited HCC cell proliferation, migration and invasion, while RPL23 overexpression promoted HCC cell metastasis. Mechanistically, RPL23 positively regulated MMP9 expression by stabilizing its mRNA. And increased MMP9 is involved in RPL23-mediated HCC metastasis. Importantly, RPL23 silencing reduced tumor growth and metastasis in vivo. In summary, we identified that RPL23 play an important role in HCC metastasis in an MMP9-dependent manner and may be a novel potential therapeutic target for HCC tumorigenesis and metastasis.


title: Functional and comparative genomics reveals conserved noncoding sequences in the nitrogen-fixing clade

abstract: Nitrogen is one of the most inaccessible plant nutrients, but certain species have overcome this limitation by establishing symbiotic interactions with nitrogen-fixing bacteria in the root nodule. This root nodule symbiosis (RNS) is restricted to species within a single clade of angiosperms, suggesting a critical evolutionary event at the base of this clade, which has not yet been determined. While genes implicated in the RNS are present in most plant species (nodulating or not), gene sequence conservation alone does not imply functional conservation - developmental or phenotypic differences can arise from variation in the regulation of transcription. To identify putative regulatory sequences implicated in the evolution of RNS, we aligned the genomes of 25 species capable of nodulation. We detected 3,091 conserved noncoding sequences (CNS) in the nitrogen-fixing clade that are absent from outgroup species. Functional analysis revealed that chromatin accessibility of 452 CNS significantly correlates with the differential regulation of genes responding to lipo-chitooligosaccharides in Medicago truncatula. These included 38 CNS in proximity to 19 known genes involved in RNS. Five such regions are upstream of MtCRE1, Cytokinin Response Element 1, required to activate a suite of downstream transcription factors necessary for nodulation in M. truncatula. Genetic complementation of a Mtcre1 mutant showed a significant association between nodulation and the presence of these CNS, when they are driving the expression of a functional copy of MtCRE1. Conserved noncoding sequences, therefore, may be required for the regulation of genes controlling the root nodule symbiosis in M. truncatula.


title: Optogenetic actuator/biosensor circuits for large-scale interrogation of ERK dynamics identify sources of MAPK signaling robustness.

abstract: Combining single-cell measurements of ERK activity dynamics with perturbations provides insights into the MAPK network topology. We built circuits consisting of an optogenetic actuator to activate MAPK signaling and an ERK biosensor to measure single-cell ERK dynamics. This allowed us to conduct RNAi screens to investigate the role of 50 MAPK proteins in ERK dynamics. We found that the MAPK network is robust against most node perturbations. We observed that the ERK-RAF and the ERK-RSK2-SOS negative feedbacks operate simultaneously to regulate ERK dynamics. Bypassing the RSK2-mediated feedback, either by direct optogenetic activation of RAS, or by RSK2 perturbation, sensitized ERK dynamics to further perturbations. Similarly, targeting this feedback in a human ErbB2-dependent oncogenic signaling model increased the efficiency of a MEK inhibitor. The RSK2-mediated feedback is thus important for the ability of the MAPK network to produce consistent ERK outputs and its perturbation can enhance the efficiency of MAPK inhibitors.

category: systems biology


title: Optogenetic actuator/ERK biosensor circuits identify MAPK network nodes that shape ERK dynamics


title: One year later: longitudinal effects of flexible school start times on teenage sleep and subjective psychological outcomes

abstract:
Early school times fundamentally clash with the late sleep of teenagers. This mismatch results in chronic sleep deprivation, which poses acute and long-term health risks and impairs students learning. Despite conclusive evidence that delaying school times has immediate benefits for sleep, the long-term effects on sleep are unresolved due to a shortage of longitudinal data. Here, we studied whether a flexible school start system, with the daily choice of an 8AM or 08:50AM-start, allowed secondary school students to improve their sleep and psychological functioning in a longitudinal pre-post design over exactly 1 year. Based on 2 waves, each with 6-9 weeks of daily sleep diary, we found that students maintained their 1-hour-sleep gain on days with later starts, both longitudinally (n=28) and cross-sectionally (n=79). This sleep gain was independent of chronotype and frequency of later starts but differed between genders. Girls were more successful in keeping early sleep onsets despite later sleep offsets, whereas boys delayed their onsets and thus had reduced sleep gains after 1 year. Students also reported psychological benefits (n=93), increased sleep quality and reduced alarm-driven waking on later school days. Despite these benefits on later schooldays, overall sleep duration was not extended in the flexible system. This was likely due to the persistently low uptake of the late-start option. If uptake can be further promoted, the flexible system is an appealing alternative to a fixed delay of school starts owing to possible circadian advantages (speculatively through prevention of phase-delays) and psychological mechanisms (e.g. sense of control).

Significance statementTeenage sleep becomes progressively later during adolescence but school starts do not accommodate this shifted sleep window. This mismatch results in chronic sleep deprivation in teenagers worldwide, which is a pervasive public health concern. Delaying school starts could counteract this misalignment if students keep sleep onsets stable. However, few studies have investigated long-term effects of delayed starts on sleep. We observed here that students slept persistently longer and better when school started later in a flexible start system. Girls were especially successful in keeping stable onsets. Students also benefitted psychologically and liked the flexible system despite only a modest uptake of later starts. The flexible system is an interesting alternative to a fixed delay and should receive more scientific attention.


title: Electrophysiological measures of visual suppression

abstract: In the early visual system, suppression occurs between neurons representing different stimulus properties. This includes features such as orientation (cross-orientation suppression), eye-of-origin (interocular suppression) and spatial location (surround suppression), which are thought to involve distinct anatomical pathways. We asked if these separate routes to suppression can be differentiated by their pattern of gain control on the contrast response function measured in human participants using steady-state electroencephalography. Changes in contrast gain shift the contrast response function laterally, whereas changes in response gain scale the function vertically. We used a Bayesian hierarchical model to summarise the evidence for each type of gain control. A computational meta-analysis of 16 previous studies found the most evidence for contrast gain effects with overlaid masks, but no clear evidence favouring either response gain or contrast gain for other mask types. We then conducted two new experiments, comparing suppression from four mask types (monocular and dichoptic overlay masks, and aligned and orthogonal surround masks) on responses to sine wave grating patches flickering at 5Hz. At the occipital pole, there was strong evidence for contrast gain effects in all four mask types at the first harmonic frequency (5Hz). Suppression generally became stronger at more lateral electrode sites, but there was little evidence of response gain effects. At the second harmonic frequency (10Hz) suppression was stronger overall, and involved both contrast and response gain effects. Although suppression from different mask types involves distinct anatomical pathways, gain control processes appear to serve a common purpose, which we suggest might be to suppress less reliable inputs.


title: Steady-state measures of visual suppression


title: Gliding motility of a uranium tolerant Bacteroidetes bacterium Chryseobacterium sp. strain PMSZPI: Insights into the architecture of spreading colonies

abstract:
Uranium tolerant soil bacterium Chryseobacterium sp. strain PMSZPI moved over solid agar surfaces by gliding motility thereby forming spreading colonies which is a hallmark of members of Bacteroidetes phylum. PMSZPI genome harbored orthologs of all the gld and spr genes considered as core bacteroidetes gliding motility genes of which gldK, gldL, gldM, and gldN were co-transcribed. Here, we present the intriguing interplay between gliding motility and cellular organization in PMSZPI spreading colonies. While nutrient deficiency enhanced colony spreading, high agar concentrations and presence of motility inhibitor like 5-hydroxyindole reduced the spreading. A detailed in situ structural analysis of spreading colonies revealed closely packed cells forming multiple layers at center of colony while the edges showed clusters of cells periodically arranged in hexagonal lattices interconnected with each other. The cell migration within the colony was visualized as branched structures wherein the cells were buried within extracellular matrix giving rise to  fern like patterns. PMSZPI colonies exhibited strong iridescence that showed correlation with gliding motility. Presence of uranium reduced motility and iridescence and induced biofilm formation. This is a first report of gliding motility and iridescence in a bacterium from uranium enriched environment that could be of significant interest from an ecological perspective.

Originality-Significance StatementThis work provides the first description of the gliding motility and iridescence or structural coloration in a Bacteroidetes soil bacterium from uranium enriched environment. The periodic arrangement of the cell population in the spreading colonies achieved through gliding motility resulted in bright structural coloration of the colonies when illuminated. The study describes the exogenous factors including nutrition, substrate, presence of uranium influencing the motility and iridescence of the bacterium. The highly organized cell population in the gliding and iridescent bacterium may have conferred survival advantage in metal/uranium enriched ecosystem.


title: Proximity labeling identifies LOTUS domain proteins that promote the formation of perinuclear germ granules in C. elegans

abstract: The germline produces gametes that transmit genetic and epigenetic information to the next generation. Maintenance of germ cells and development of gametes require germ granules--well-conserved membraneless and RNA-rich organelles. The composition of germ granules is elusive owing to their dynamic nature and their exclusive expression in the germline. Using C. elegans germ granule, called P granule, as a model system, we employed a proximity-based labeling method in combination with mass spectrometry to comprehensively define its protein components. This set of experiments identified over 200 proteins, many of which contain intrinsically disordered regions. An RNAi-based screen identified factors that are essential for P granule assembly, notably EGGD-1 and EGGD-2, two previously uncharacterized LOTUS-domain proteins. Loss of eggd-1 and eggd-2 results in separation of P granules from nuclear envelope, germline atrophy and reduced fertility. We show that intrinsically disordered regions of EGGD-1 are required to anchor EGGD-1 to the nuclear periphery while its LOTUS domains are required to promote perinuclear localization of P granules. Together, our work expands the repertoire of P granule constituents and provides new insights into the role of LOTUS-domain proteins in germ granule organization.


title: Phytoplankton exudates provide full nutrition to a subset of accompanying heterotrophic bacteria via carbon, nitrogen and phosphorus allocation

abstract: Marine bacteria rely on phytoplankton exudates as carbon sources (DOCp). Yet, it is unclear to what extent phytoplankton exudates also provide nutrients such as phytoplankton-derived N and P (DONp, DOPp). We address these questions by exudate addition experiments from the ubiquitous pico-cyanobacterium Prochlorococcus to contrasting ecosystems in the Eastern Mediterranean - a coastal and an open-ocean, oligotrophic station. Nutrient addition did not lower the incorporation of exudate DONp, nor did it reduce alkaline phosphatase activity, suggesting that bacterial communities are able to exclusively cover their nitrogen and phosphorus demands with organic forms provided by phytoplankton exudates. Approximately half of the cells in each ecosystem took up detectable amounts of Prochlorococcus-derived C and N, yet based on 16S rRNA sequencing different bacterial genera were responsible for the observed exudate utilization patterns. In the coastal community, several phylotypes of Aureimarina, Psychrosphaera and Glaciecola responded positively to the addition of phytoplankton exudates, whereas phylotypes of Pseudoalteromonas increased and dominated the open ocean communities. Together, our results strongly indicate that phytoplankton exudates provide coastal and open-ocean bacterial communities with organic carbon, nitrogen and phosphorus, and that phytoplankton exudate serve a full-fledged meal for specific members of the accompanying bacterial community in the nutrient-poor eastern Mediterranean.


title: Modelling porcine reproductive and respiratory syndrome virus dynamics to quantify the contribution of multiple modes of transmission: between-farm animal and vehicle movements, farm-to-farm proximity, feed ingredients, and re-break

abstract: Accounting for multiple modes of livestock disease dissemination in epidemiological models remains a challenge. We developed and calibrated a mathematical model for transmission of porcine reproductive and respiratory syndrome virus (PRRSV), tailored to fit nine modes of between-farm transmission pathways including: farm-to-farm proximity (local transmission), contact network of batches of pigs transferred between farms (pig movements), re-break probabilities for farms with previous PRRSV outbreaks, with the addition of four different contact networks of transportation vehicles (vehicles to transport pigs to farms, pigs to markets, feed and crew) and the amount of animal by-products within feed ingredients (e.g. animal fat or meat and bone meal). The model was calibrated on weekly PRRSV outbreaks data. We assessed the role of each transmission pathway considering the dynamics of specific types of production (i.e., sow, nursery). Although our results estimated that the networks formed by transportation vehicles were more densely connected than the network of pigs transported between-farms, pig movements and farm proximity were the main PRRSV transmission routes regardless of farm types. Among the four vehicle networks, vehicles transporting pigs to farms explained a large proportion of infections, sow = 20.9%; nursery = 15%; and finisher = 20.6%. The animal by-products showed a limited association with PRRSV outbreaks through descriptive analysis, and our model results showed that the contribution of animal fat contributed only 2.5% and meat and bone meal only 0.03% of the infected sow farms. Our work demonstrated the contribution of multiple routes of PRRSV dissemination, which has not been deeply explored before. It also provides strong evidence to support the need for cautious, measured PRRSV control strategies for transportation vehicles and further research for feed by-products modeling. Finally, this study provides valuable information and opportunities for the swine industry to focus effort on the most relevant modes of PRRSV between-farm transmission.


title: Modelling porcine reproductive and respiratory syndrome virus dissemination dynamics to quantifythe contribution of multiple modes of transmission: between-farm pig and vehicle movements, farmto-farm proximity, feed ingredients, and re-break


title: Modelling and assessing additional transmission routes for porcine reproductive and respiratory syndrome virus: vehicle movements and feed ingredients


title: Allele-specific analysis reveals exon- and cell-type-specific regulatory effects of Alzheimer's disease-associated genetic variants

abstract: Elucidating regulatory effects of Alzheimers disease (AD)-associated genetic variants is critical for unraveling their causal pathways and understanding the pathology. However, their cell-type-specific regulatory mechanisms in the brain remain largely unclear. Here, we conducted an analysis of allele-specific expression quantitative trait loci (aseQTLs) for 33 AD-associated variants in four brain regions and seven cell types using ~3000 bulk RNA-seq samples and >0.25 million single nuclei. We develop a flexible framework using a hierarchical Poisson mixed model unifying samples in both allelic and genotype-level expression data. We identified 24 AD-associated variants (~73%) that are allele-specific eQTLs (aseQTLs) in at least one brain region. Multiple aseQTLs are region-dependent or exon-specific, such as rs2093760 with CR1, rs7982 with CLU, and rs3865444 with CD33. Notably, the APOE {varepsilon}4 variant reduces APOE expression across all regions, even in healthy controls. In pinpointing the cell types responsible for the observed region-level aseQTLs, we found rs2093760 as an aseQTL of CR1 in oligodendrocytes but not in microglia. Many AD-associated variants are aseQTLs in microglia or monocytes of immune-related genes, including HLA-DQB1, HLA-DQA2, CD33, FCER1G, MS4A6A, SPI1, and BIN1, highlighting the regulatory role of AD-associated variants in the immune response. These findings provide further insights into potential causal pathways and cell types mediating the effects of the AD-associated variants.


title: A locus conferring tolerance to Theileria infection in African cattle

abstract:
East Coast fever, a tick-borne cattle disease caused by the Theileria parva parasite, is among the biggest natural killers of cattle in East Africa, leading to over 1 million deaths annually. Here we report on the genetic analysis of a cohort of Boran cattle demonstrating heritable tolerance to infection by T. parva (h2 = 0.65, s.e. 0.57). Through a linkage analysis we identify a 6 Mb genomic region on Bos taurus chromosome 15 that is significantly associated with survival outcome following T. parva exposure. Testing this locus in an independent cohort of animals replicates this association with survival following T. parva infection. A stop gained polymorphism in this region was found to be highly associated with survival across both related and unrelated animals, with only one of the 20 homozygote carriers (T/T) of this change succumbing to the disease in contrast to 44 out of 97 animals homozygote for the reference allele (C/C). Consequently, we present a genetic locus linked to tolerance of one of Africas most important cattle diseases, raising the promise of marker-assisted selection for cattle that are less susceptible to infection by T. parva.

Author SummaryMore than a million cattle die of East Coast fever in Africa each year, the impact of which disproportionately falls onto low-income, smallholder farmers. The lack of a widely accessible vaccine, heavy reliance on chemicals to control the tick vector and inadequate drug treatments means that new approaches for controlling the disease are urgently required. Through a genetic study of an extended pedigree of Boran cattle that are more than three times less likely to succumb to the disease than matched controls, we identify a region on chromosome 15 of the cattle genome associated with a high level of tolerance to the disease. We show that a variant in this region is also associated with survival in an independent cohort, and is linked to rates of cell expansion during infection. This genetic variant can therefore support marker-assisted selection, allowing farmers to breed tolerant cattle and offers a route to introduce this beneficial DNA to non-native breeds, enabling reduced disease incidence and increased productivity, which would be of benefit to millions of rural smallholder farmers across Africa.


title: Structural basis of Ty3 retrotransposon integration at RNA Polymerase III-transcribed genes

abstract:
Retrotransposons are endogenous elements that have the ability to mobilise their DNA and integrate at different locations in the host genome. In budding yeast, the Ty3 retrotransposon integrates with an exquisite specificity in a narrow window upstream of RNA Polymerase III-transcribed genes, such as the genes of transfer RNAs, representing a paradigm for specific targeted integration.

Here we present the cryo-EM reconstruction at 4.0 [A]-resolution of an active Ty3 strand-transfer complex (Ty3 intasome) caught in the act of integrating onto a specific tRNA gene bound to the RNA Polymerase III general transcription factor TFIIIB, which is required for Ty3 specific targeting.

The structure unravels the molecular mechanisms underlying Ty3 integration specificity at RNA Polymerase III-transcribed genes and sheds light into the architecture of a retrotransposon integration machinery during the process of strand transfer at a genomic locus. The Ty3 intasome establishes contacts with a region of the TATA-binding protein (TBP), a subunit of TFIIIB, which is blocked by the ubiquitous transcription regulator negative cofactor 2 (NC2) in RNA Pol II-transcribed genes.

A previously unrecognised chromodomain of the Ty3 integrase mediates non- canonical interactions with TFIIIB and the tRNA gene itself, defining with extreme precision the position of the integration site. Surprisingly, Ty3 retrotransposon tethering to TFIIIB topologically resembles LEDGF/p75 transcription factor targeting by HIV retrovirus, highlighting mechanisms of convergent evolution by unrelated mobile elements and host organisms.

The Ty3 intasome-TFIIIB-tRNA promoter complex presented here represents a detailed molecular snapshot of a general transcription factors co-option by a mobile element, resulting in harmless integration into the host genome.


title: Isolating the Sources of Pipeline-Variability in Group-Level Task-fMRI results

abstract: While the development of tools and techniques has broadened our horizons for comprehending the complexities of the human brain, a growing body of research has highlighted the pitfalls of such methodological plurality. In a recent study, we found that the choice of software package used to run the analysis pipeline can have a considerable impact on the final group-level results of a task-fMRI investigation (Bowring et al., 2019, BMN). Here we revisit our work, seeking to identify the stages of the pipeline where the greatest variation between analysis software is induced. We carry out further analyses on the three datasets evaluated in BMN, employing a common processing strategy across parts of the analysis workflow and then utilizing procedures from three software packages (AFNI, FSL and SPM) across the remaining steps of the pipeline. We use quantitative methods to compare the statistical maps and isolate the main stages of the workflow where the three packages diverge. Across all datasets, we find that variation between the packages results is largely attributable to a handful of individual analysis stages, and that these sources of variability were heterogeneous across the datasets (e.g. choice of first-level signal model had the most impact for the ds000001 dataset, while first-level noise model was more influential for ds000109 dataset). We also observe areas of the analysis workflow where changing the software package causes minimal differences in the final results, finding that the group-level results were largely unaffected by which software package is used to model the low-frequency fMRI drifts.


title: Lytic transglycosylases mitigate periplasmic crowding by degrading soluble cell wall turnover products

abstract: The peptidoglycan cell wall is a predominant structure of bacteria, determining cell shape and supporting survival in diverse conditions. Peptidoglycan is dynamic and requires regulated synthesis of new material, remodeling, and turnover - or autolysis - of old material. Despite exploitation of peptidoglycan synthesis as an antibiotic target, we lack a fundamental understanding of how peptidoglycan synthesis and autolysis intersect to maintain the cell wall. Here, we uncover a critical physiological role for a widely misunderstood class of autolytic enzymes, lytic transglycosylases (LTGs). We demonstrate that LTG activity is essential to survival by contributing to periplasmic processes upstream and independent of peptidoglycan recycling. Defects accumulate in Vibrio cholerae LTG mutants due to generally inadequate LTG activity, rather than absence of specific enzymes, and essential LTG activities are likely independent of protein-protein interactions, as heterologous expression of a non-native LTG rescues growth of a conditionally LTG-null mutant. Lastly, we demonstrate that soluble, uncrosslinked, endopeptidase-dependent peptidoglycan chains, also detected in the wild-type, are enriched in LTG mutants, and that LTG mutants are hypersusceptible to the production of diverse periplasmic polymers. Collectively, our results suggest that LTGs prevent toxic crowding of the periplasm with synthesis-derived peptidoglycan polymers and contrary to prevailing models, that this autolytic function can be temporally separate from peptidoglycan synthesis.


title: Anti-arthralgic activity of the n-hexane extract (HTp) of yellow oleander seeds;Thevetia peruviana (Pers.) K. Schum

abstract: Thevetia peruviana (Pers.) K.Schum. (Apocynaceae) seeds are known to possess cardioactive glycosides such as thevetin A, thevetin B, nerifolin etc. They are also used locally for general pain relief for which there is no scientific evidence to our knowledge. Arthralgia is regarded generally as pain without inflammation. It is endemic in the society and sufferers continue to imbibe pain relieving drugs in their tons all over the world. Analgesic activity test was carried out using the formalin-induced pain models, at 0.1g, 0.2g and 0.3g/kg doses of n-hexane extracts of Thevetia peruviana seeds (HTp) in Wistar mice. Diclofenac was used as positive control. Acute toxicity test was carried out at doses of 1000, 2500 and 5000 mg/kg weight of test subject. It was observed that HTp at concentrations of 0.1g, 0.2g and 0.3g/kg showed significant analgesic effect at compared to the control. The percentage inhibition observed was 29.60%, 44.80% and 50.72% for the early pain phase and 100% for the late pain phase respectively, indicating HTps NSAID-like property. HTp showed the highest percentage inhibition at 300 mg/kg (50.72 %) and significant (P<0.005) pain reduction. HTp did not produce any toxicity up to a dose of 5000 mg/kg weight which is very interesting as the seeds are known for their toxicity due to the cardiac glycoside presence. The results of the study suggest that HTp does indeed relieve pain significantly in a dose dependent manner, thus justifying its use in management of arthralgia.

category: pharmacology and toxicology


title: Sex differences in age- and episodic memory-related effects on task-based functional connectivity

abstract: Aging is associated with episodic memory decline and changes in functional brain connectivity. Understanding whether and how biological sex influences age- and memory performance-related functional connectivity has important theoretical and clinical implications for our understanding of brain and cognitive aging. Yet, little is known about the effect of sex on neurocognitive aging. Here, we scanned 161 healthy adults between 19-76 yrs of age in an event-related functional magnetic resonance imaging (fMRI) study of face-location spatial context memory. Adults were scanned while performing easy and difficult versions of the task at both encoding and retrieval. We used multivariate whole-brain partial least squares (PLS) connectivity to test the hypothesis that there are sex differences in age- and episodic memory performance-related functional connectivity. We examined how individual differences in age and retrieval accuracy correlated with task-related connectivity. We then repeated this analysis after disaggregating the data by self-reported sex. We found that increased encoding and retrieval-related connectivity within the dorsal attention network (DAN), and between DAN and frontoparietal network (FPN) and visual networks, was positively correlated to retrieval accuracy and negatively correlated with age in both sexes. We also observed sex differences in age- and performance-related functional connectivity: i) greater between-network integration was apparent at both levels of task difficulty in women only, and ii) increased DAN-DMN connectivity with age was observed in men and was correlated with poorer memory performance. Therefore, the neural correlates of age-related episodic memory decline differ in women and men and has important theoretical and clinical implications for the cognitive neuroscience of memory, aging and dementia prevention.


title: Age- and episodic memory-related differences in task-based functional connectivity in women and men


title: Site-specific recognition of SARS-CoV-2 nsp1 protein with a tailored titanium dioxide nanoparticle

abstract: The ongoing world-wide Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) pandemic shows the need for new sensing and therapeutic means against the CoV viruses. The SARS-CoV-2 nsp1 protein is important, both for replication and pathogenesis, making it an attractive target for intervention. In recent years nanoparticles have been shown to interact with peptides, ranging in size from single amino acids up to proteins. These nanoparticles can be tailor-made with specific functions and properties including bioavailability. To the best of our knowledge, in this study we show for the first time that a tailored titanium oxide nanoparticle interacts specifically with a unique site of the full-length SARS-CoV-2 nsp1 protein. This can be developed potentially into a tool for selective control of viral protein functions.

category: molecular biology


title: ANTH domains within CALM, HIP1R, and Sla2 recognize ubiquitin internalization signals.

abstract: Ubiquitin (Ub) serves as a signal for clathrin-mediated endocytosis (CME) by engaging Ub-binding proteins with the internalization apparatus. Ub is a versatile internalization signal because it can be added to a wide variety of membrane proteins, expanding the capacity of cells to use a variety of regulatory mechanisms to specify the conditions under which a particular protein will be internalized. Several candidate adaptors that can recognize ubiquitinated membrane proteins have been identified that work in endocytic processes that are both clathrin-dependent and independent. These include Epsin and Eps15, which bind and help sort Ub-cargo into internalization sites. Here we identify additional components of the endocytosis apparatus that bind Ub. The N-terminal ANTH domains found in CALM, AP180, HIP1R and yeast Sla2 all bind monoubiquitin with {micro}M affinity. ANTH domains belong to a larger superfamily of domains including ENTH and VHS domains, many of which have Ub-binding regions outside of their VHS/ENTH/ANTH domains that enable them to mediate Ub-dependent sorting events throughout the cell. Solution NMR studies combined with a crystal structure of the CALM ANTH domain in a complex with Ub show that Ub binds to a C-terminal region of the ANTH domain that is not present in ENTH domains. Combined loss of Ub-binding by ANTH-domain proteins and other Ub-binding domains within the internalization apparatus of yeast caused defects in the Ub-dependent internalization of the GPCR Ste2 but had no effect on internalization of Ste2 via other internalization signals. These studies define new components of the internalization machinery that work collectively with Epsin and Eps15 to specify recognition of Ub as an internalization signal.