title: Combined Ventilation Of Two Subjects With A Single Mechanical Ventilator Using A New Medical Device: An In Vitro Study

abstract:
INTRODUCTIONThe SARS-CoV2 pandemic has created a sudden lack of ventilators. DuplicAR(R) is a novel device that allows simultaneous and independent ventilation of two subjects with a single ventilator. The aims of this study are: a) to determine the efficacy of DuplicAR(R) to independently regulate the peak and positive-end expiratory pressures in each subject, both under pressure-controlled ventilation and volume-controlled ventilation, and b) to determine the ventilation mode in which DuplicAR(R) presents the best performance and safety.

MATERIALS AND METHODSTwo test lungs are connected to a single ventilator using DuplicAR(R). Three experimental stages are established: 1) two identical subjects, 2) two subjects with the same weight but different lung compliance, and 3) two subjects with different weight and lung compliance. In each stage, the test lungs are ventilated in two ventilation modes. The positive-end expiratory pressure requirements are increased successively in one of the subjects. The goal is to achieve a tidal volume of 7 ml/kg for each subject in all different stages through manipulation of the ventilator and the DuplicAR(R) controllers.

RESULTSDuplicAR(R) allows adequate ventilation of two subjects with different weight and/or lung compliance and/or PEEP requirements. This is achieved by adjusting the total tidal volume for both subjects (in volume-controlled ventilation) or the highest peak pressure needed (in pressure-controlled ventilation) along with the basal positive-end expiratory pressure on the ventilator, and simultaneously manipulating the DuplicAR(R) controllers to decrease the tidal volume or the peak pressure in the subject that needs less and/or to increase the positive-end expiratory pressure in the subject that needs more. While ventilatory goals can be achieved in any of the ventilation modes, DuplicAR(R) performs better in pressure-controlled ventilation, as changes experienced in the variables of one subject do not modify the other one.

CONCLUSIONSDuplicAR(R) is an effective tool to manage the peak inspiratory pressure and the positive-end expiratory pressure independently in two subjects connected to a single ventilator. The driving pressure can be adjusted to meet the requirements of subjects with different weight and lung compliance. Pressure-controlled ventilation has advantages over volume-controlled ventilation and is therefore the recommended ventilation mode.

category: bioengineering


title: Multiplexed Imaging Analysis of the Tumor-Immune Microenvironment Reveals Predictors of Outcome in Triple-Negative Breast Cancer

abstract: Triple-negative breast cancer, the poorest-prognosis breast cancer subtype, lacks clinically approved biomarkers for patient risk stratification and treatment management. Prior literature has shown that interrogation of the tumor-immune microenvironment may be a promising approach for the discovery of methods to fill these gaps. Recently developed high-dimensional tissue imaging technology, such as multiplexed ion beam imaging, provide spatial context to protein expression in the microenvironment, allowing in-depth characterization of cellular processes. We demonstrate that profiling the functional proteins involved in cell-to-cell interactions in the microenvironment can predict recurrence and overall survival. We highlight the immunological relevance of the immunoregulatory proteins PD-1, PD-L1, IDO, and Lag3 by tying interactions involving them to recurrence and survival. Multivariate analysis reveals that our methods provide additional prognostic information compared to clinical variables. In this work, we present a computational pipeline for the examination of the tumor-immune microenvironment using multiplexed ion-beam imaging that produces interpretable results, and is generalizable to other cancer types.

category: cancer biology


title: Mapping cellular subpopulations within triple negative breast cancer tumors provides a tool for cancer sensitization to radiotherapy

abstract:
Triple negative breast cancer (TNBC) is an aggressive type of cancer that is known to be resistant to radiotherapy (RT). Evidence is accumulating that is indicative of the plasticity of TNBC, where one cancer subtype switches to another in response to various treatments, including RT. In this study we aim to overcome tumor resistance by designing TNBC-sensitizing targeted therapies that exploit the plasticity occurring due to radiation exposure. Using single cell analysis of molecular changes occurring in irradiated TNBC tumors, we identified two initially undetected distinct subpopulations, represented by overexpressed Her2 and cMet, expanding post-RT and persisting in surviving tumors. Using murine cancer models and patient-derived TNBC tumors, we showed that only simultaneous targeting of Her2 and cMet was successful in sensitizing TNBC to RT and preventing its regrowth. The strategy presented herein holds the potential to be broadly applicable in clinical use.

HighlightsO_LISensitization of TNBC to radiotherapy (RT) is a clinically unmet need
C_LIO_LISingle cell strategy creates a precise map of subpopulations expanding post-RT
C_LIO_LIEvolution of intra-tumor heterogeneity is turned into a therapeutic advantage
C_LIO_LISimultaneous targeting of expanding subpopulations sensitizes TNBC to radiotherapy
C_LI


title: Interplay between intrinsic reprogramming potential and microenvironment controls neuroblastoma cell plasticity and identity

abstract: Two cell identities, noradrenergic and mesenchymal, have been characterized in neuroblastoma cell lines according to their epigenetic landscapes relying on specific circuitries of transcription factors. Yet, their relationship and relative contribution in patient tumors remain poorly defined. Here, we demonstrate that the knock-out of GATA3, but not of PHOX2A or PHOX2B, in noradrenergic cells induces a mesenchymal phenotype. Our results document spontaneous plasticity in several models between both identities and show that plasticity relies on epigenetic reprogramming. We demonstrate that an in vivo microenvironment provides a powerful pressure towards a noradrenergic identity for these models. Consistently, tumor cells with a mesenchymal identity are not detected in a series of PDX models. Further study of the intra-tumor noradrenergic heterogeneity reveals two distinct cell populations exhibiting features of chromaffin-like or sympathoblast-like cells. This work emphasizes that both external cues of the environment and intrinsic factors control plasticity and cell identity in neuroblastoma.


title: Structural Brain Correlates of Creative Personality

abstract: Creative individuals and their enigmatic personalities have always been a subject of fascination. The current study explored the neuroanatomical basis of creative personality using voxel-based morphometry. The sample comprised of two groups - Creative (CR) group (professional creative artists) and matched controls with no demonstrated artistic creativity (NC) with 20 participants in each group, in the age range of 20-40 years, right-handed, and had minimum average intelligence (IQ > 90). Participants in CR were selected using the creativity achievement questionnaire, creativity was assessed using the Wallach & Kogan test of creativity, and personality was administered using NEO-FFI. Results indicated significantly higher openness to new experiences in CR which positively correlates with the right middle frontal gyrus. An increased grey matter volume in the inferior frontal gyrus, anterior cingulate gyrus in CR, pointing towards the integration of cognitive and imaginative processes that might be implicated in creative personality.

category: neuroscience


title: Neural correlates of orbital telorism

abstract: Orbital telorism, the interocular distance, is a clinically informative and in extremes is considered a minor physical anomaly. While its extremes, hypo- and hypertelorism, have been linked to disorders often related to cognitive ability, little is known about the neural correlates of normal variation of telorism within the general population. We derived measures of orbital telorism from cranial magnetic resonance imaging (MRI) by calculating the distance between the eyeball center of gravity in two population-based datasets (N=5,653, N=29,824, Mean age 64.66, 63.75 years). This measure was found to be related grey matter tissue density within numerous regions of the brain, including, but surprisingly not limited to, the frontal regions, in both positive and negative directions. Additionally, telorism was related to several cognitive functions, such as Perdue Pegboard test (Beta, P-value, (CI95%) -0.02, 1.63x10-7(-0.03;-0.01)) and fluid intelligence (0.02, 4.75x10-06 (0.01:0.02)), with some relationships driven by individuals with a smaller orbital telorism. This is reflective of the higher prevalence of hypo-telorism in developmental disorders, specifically those that accompany lower cognitive lower functioning. This study suggests, despite previous links only made in clinical extremes, that orbital telorism holds some relation to structural brain development and cognitive function in the general population. This relationship is likely driven by shared developmental periods.


title: A mammalian methylation array for profiling methylation levels at conserved sequences

abstract: Infinium methylation arrays are not available for the vast majority of non-human mammals. Moreover, even if species-specific arrays were available, probe differences between them would confound cross-species comparisons. To address these challenges, we developed the mammalian methylation array, a single custom array that measures 36k CpGs that are well conserved across mammalian species. We designed a set of probes on the array that can tolerate specific cross-species mutations. We annotate the array in over 200 species and report CpG island status and chromatin states in select species. Calibration experiments demonstrate the high fidelity in humans, rats, and mice. The mammalian methylation array has several strengths: it applies to all mammalian species even those that have not yet been sequenced, it provides deep coverage of conserved cytosines facilitating the development epigenetic biomarkers, and it increases the probability that biological insights gained in one species will translate to others.

category: bioinformatics


title: Using dual-calibrated functional MRI to map brain oxygen supply and consumption in multiple sclerosis

abstract: Evidence suggests that cerebrovascular function and oxygen consumption are altered in multiple sclerosis (MS). Here, we quantified the vascular and oxygen metabolic MRI burden in patients with MS (PwMS) and assessed the relationship between these MRI measures of and metrics of damage and disability. In PwMS and in matched healthy volunteers, we applied a newly developed dual-calibrated fMRI method of acquisition and analysis to map grey matter (GM) cerebral blood flow (CBF), oxygen extraction fraction (OEF), cerebral metabolic rate of oxygen consumption (CMRO2) and effective oxygen diffusivity of the capillary network (DC). We also quantified physical and cognitive function in PwMS and controls. There was no significant difference in GM volume between 22 PwMS and 20 healthy controls (p=0.302). Significant differences in CBF (PwMS vs. controls: 44.91 {+/-} 6.10 vs. 48.90 {+/-} 5.87 ml/100g/min, p=0.010), CMRO2 (117.69 {+/-} 17.31 vs. 136.49 {+/-} 14.48 mol/100g/min p<0.001) and DC (2.70 {+/-} 0.51 vs. 3.18 {+/-} 0.41 mol/100g/mmHg/min, p=0.002) were observed in the PwMS. No significant between-group differences were observed for OEF (PwMS vs. controls: 0.38 {+/-} 0.09 vs. 0.39 {+/-} 0.02, p=0.358). Regional analysis showed widespread reductions in CMRO2 and DC for PwMS compared to healthy volunteers. There was a significant correlation between physiological measures and T2 lesion volume, but no association with current clinical disability. Our findings demonstrate concurrent reductions in oxygen supply and consumption in the absence of an alteration in oxygen extraction that may be indicative of a reduced demand for oxygen (O2), an impaired transfer of O2 from capillaries to mitochondria, and/or a reduced ability to utilise O2 that is available at the mitochondria. With no between-group differences in GM volume, our results suggest that changes in brain physiology may precede MRI-detectable GM loss and thus may be one of the pathological drivers of neurodegeneration and disease progression.


title: EdU incorporation to assess cell proliferation and drug susceptibility for the brain-eating amoeba, Naegleria fowleri

abstract: Naegleria fowleri is a pathogenic free-living amoeba that is commonly found in warm, freshwater and can cause a rapidly fulminant disease known as primary amoebic meningoencephalitis (PAM). New drugs are urgently needed to treat PAM, as the fatality rate is >97%. Until recently, few advances have been made in the discovery of new drugs for N. fowleri and one drawback is the lack of validated tools and methods to enhance drug discovery and diagnostics research. In this study we aimed to validate alternative methods to assess cell proliferation that are commonly used for other cell types and develop a novel drug screening assay to evaluate drug efficacy on N. fowleri replication. EdU (5-ethynyl-2'-deoxyuridine) is a pyrimidine analog of thymidine that can be used as a quantitative endpoint for cell proliferation. EdU incorporation is detected via a copper catalyzed click reaction with an Alexa Fluor linked azide. EdU incorporation in replicating N. fowleri was validated using fluorescence microscopy and quantitative methods for assessing EdU incorporation were developed by using an imaging flow cytometer. Currently used PAM therapeutics inhibited N. fowleri replication and EdU incorporation in vitro. EdA (5'ethynyl-2'-deoxyadenosine), an adenine analog, also was incorporated by N. fowleri, but was more cytotoxic than EdU. In summary, EdU incorporation could be used as a complimentary method for drug discovery for these neglected pathogens.

category: microbiology


title: Creating wounds in cell monolayers using micro-jets

abstract: Many wound-healing assays are used in cell biology and biomedicine; they are often labor intensive and/or require specialized and costly equipment. We describe a contactless method to create wounds with any imaginable 2D pattern in cell monolayers using micro-jets of either media or an immiscible and biocompatible fluorocarbon (i.e., FC40). We also combine this with another method that allows automation and multiplexing using standard Petri dishes. A dish is filled with a thin film of media overlaid with FC40, and the two liquids reshaped into an array of microchambers in minutes. Each chamber in such a grid is isolated from others by fluid walls of FC40. Cells are now added, allowed to grow into a monolayer, and wounds created using the microjets; then, healing is monitored by microscopy. As arrays of chambers can be made using the media and Petri dishes familiar to biologists, and as dishes fit seamlessly into their incubators, microscopes, and workflows, we anticipate this assay will find wide application in biomedicine.


title: Fibrinolysis influences SARS-CoV-2 infection in ciliated cells

abstract: Rapid spread of COVID-19 has caused an unprecedented pandemic worldwide, and an inserted furin site in SARS-CoV-2 spike protein (S) may account for increased transmissibility. Plasmin, and other host proteases, may cleave the furin site of SARS-CoV-2 S protein and {gamma} subunits of epithelial sodium channels ({gamma} ENaC), resulting in an increment in virus infectivity and channel activity. As for the importance of ENaC in the regulation of airway surface and alveolar fluid homeostasis, whether SARS-CoV-2 will share and strengthen the cleavage network with ENaC proteins at the single-cell level is urgently worthy of consideration. To address this issue, we analyzed single-cell RNA sequence (scRNA-seq) datasets, and found the PLAU (encoding urokinase plasminogen activator), SCNN1G ({gamma}ENaC), and ACE2 (SARS-CoV-2 receptor) were co-expressed in alveolar epithelial, basal, club, and ciliated epithelial cells. The relative expression level of PLAU, TMPRSS2, and ACE2 were significantly upregulated in severe COVID-19 patients and SARS-CoV-2 infected cell lines using Seurat and DESeq2 R packages. Moreover, the increments in PLAU, FURIN, TMPRSS2, and ACE2 were predominately observed in different epithelial cells and leukocytes. Accordingly, SARS-CoV-2 may share and strengthen the ENaC fibrinolytic proteases network in ACE2 positive airway and alveolar epithelial cells, which may expedite virus infusion into the susceptible cells and bring about ENaC associated edematous respiratory condition.


title: Vimentin binds to SARS-CoV-2 spike protein and antibodies targeting extracellular vimentin block in vitro uptake of SARS-CoV-2 virus-like particles

abstract: Infection of human cells by pathogens, including SARS-CoV-2, typically proceeds by cell surface binding to a crucial receptor. In the case of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2) has been identified as a necessary receptor, but not all ACE2-expressing cells are equally infected, suggesting that other extracellular factors are involved in host cell invasion by SARS-CoV-2. Vimentin is an intermediate filament protein that is increasingly recognized as being present on the extracellular surface of a subset of cell types, where it can bind to and facilitate pathogens cellular uptake. Here, we present evidence that extracellular vimentin might act as a critical component of the SARS-CoV-2 spike protein-ACE2 complex in mediating SARS-CoV-2 cell entry. We demonstrate direct binding between vimentin and SARS-CoV-2 pseudovirus coated with the SARS-CoV-2 spike protein and show that antibodies against vimentin block in vitro SARS-CoV-2 pseudovirus infection of ACE2-expressing cells. Our results suggest new therapeutic strategies for preventing and slowing SARS-CoV-2 infection, focusing on targeting cell host surface vimentin.

category: biophysics


title: Extracellular vimentin as a target against SARS- CoV-2 host cell invasion


title: Cellular, molecular, and therapeutic characterization of pilocarpine-induced temporal lobe epilepsy.

abstract: We probed a transcriptomic dataset of pilocarpine-induced TLE using various ontological, machine-learning, and systems-biology approaches. We showed that, underneath the complex and penetrant changes, moderate-to-subtle upregulated homeostatic and downregulated synaptic changes associated with the dentate gyrus and hippocampal subfields could not only predict TLE but various other forms of epilepsy. At the cellular level, pyramidal neurons and interneurons showed disparate changes, whereas the proportion of non-neuronal cells increased steadily. A probabilistic Bayesian network demonstrated an aberrant and oscillating physiological interaction between oligodendrocytes and interneurons in driving seizures. Validating the Bayesian inference, we showed that the cell types driving the seizures were associated with known antiepileptic and epileptic drugs. These findings provide predictive biomarkers of epilepsy, insights into the cellular connections and causal changes associated with TLE and a drug discovery method focusing on these events.

category: systems biology


title: Prime editing enables precise genome editing in mouse liver and retina

abstract: Prime editing can induce any small-sized genetic change without donor DNA or double strand breaks. However, it has not been investigated whether prime editing is possible in postnatal animals. Here we delivered prime editors 2 and 3 into a mouse model of hereditary tyrosinemia, a genetic liver disease, using hydrodynamic injection, which corrected the disease-causing mutation and rescued the phenotype. We also achieved prime editing in the retina and retina pigment epithelium in wild-type mice by delivering prime editor 3 using trans-splicing adeno-associated virus. Deep sequencing showed that unintended edits at or near the target site or off-target effects were not detectable except for low levels (0% to 1.2%) of indels when PE3, but not PE2, was used. Our study suggests that precise, prime editor-mediated genome editing is possible in somatic cells of adult animals.


title: Towards the restoration of ancient hominid craniofacial anatomy: Chimpanzee morphology reveals covariation between craniometrics and facial soft tissue thickness

abstract: In modern humans, facial soft tissue thicknesses have been shown to covary with craniometric dimensions. However, to date it has not been confirmed whether these relationships are shared with non-human apes. In this study, we analyze these relationships in chimpanzees (Pan troglodytes) with the aim of producing regression models for approximating facial soft tissue thicknesses in Plio-Pleistocene hominid individuals. Using CT scans of 19 subjects, 637 soft tissue, and 349 craniometric measurements, statistically significant multiple regression models were established for 26 points on the face and head. Examination of regression model validity resulted in minimal differences between observed and predicted soft tissue thickness values. Assessment of interspecies compatibility using a bonobo (Pan paniscus) and modern human (Homo sapiens) subject resulted in minimal differences for the bonobo but large differences for the modern human. These results clearly show that (1) soft tissue thicknesses covary with craniometric dimensions in P. troglodytes, (2) confirms that such covariation is uniformly present in both extant Homo and Pan species, and (3) suggests that chimp-derived regression models have interspecies compatibility with hominids who have similar craniometric dimensions to P. troglodytes. As the craniometric dimensions of early hominids, such as South African australopithecines, are more similar to P. troglodytes than those of H. sapiens, chimpanzee-derived regression models may be used for approximating their craniofacial anatomy. It is hoped that the results of the present study and the reference dataset for facial soft tissue thicknesses of chimpanzees it provides will encourage further research into this topic.

category: zoology


title: Methylation by multiple type I restriction modification systems avoids influencing gene regulation in uropathogenic Escherichia coli

abstract:
DNA methylation is a common epigenetic mark that influences transcriptional regulation, and therefore cellular phenotype, across all domains of life, extending also to bacterial virulence. Both orphan methyltransferases and those from restriction modification systems (RMSs) have been co-opted to regulate virulence epigenetically in many bacteria. However, the potential regulatory role of DNA methylation mediated by archetypal Type I systems in Escherichia coli has never been studied. We demonstrated that removal of DNA methylated mediated by three different Escherichia coli Type I RMSs in three distinct E. coli strains had no detectable effect on gene expression or growth in a screen of 1190 conditions. Additionally, deletion of the Type I RMS EcoUTI in UTI89, a prototypical cystitis strain of E. coli, which led to loss of methylation at >750 sites across the genome, had no detectable effect on virulence in a murine model of ascending urinary tract infection (UTI). Finally, introduction of two heterologous Type I RMSs into UTI89 also resulted in no detectable change in gene expression or growth phenotypes. These results stand in sharp contrast with many reports of RMSs regulating gene expression in other bacteria, leading us to propose the concept of "regulation avoidance" for these E. coli Type I RMSs. We hypothesize that regulation avoidance is a consequence of evolutionary adaptation of both the RMSs and the E. coli genome. Our results provide a clear and (currently) rare example of regulation avoidance for Type I RMSs in multiple strains of E. coli, further study of which may provide deeper insights into the evolution of gene regulation and horizontal gene transfer.

Author summaryDNA methylation is perhaps the most common epigenetic modification, and it is commonly associated with gene regulation (in nearly all organisms) and virulence (particularly well studied in bacteria). Regarding bacterial virulence, the current DNA methylation literature has focused primarily on orphan methyltransferases or phasevariable restriction modification systems (RMSs). Interestingly, no reports have studied the potential regulatory role of the first RMS discovered, the Type I RMS EcoKI. We used transcriptomics, Phenotype Microarrays, and a murine model of urinary tract infection to screen for functional consequences due to Type I methylation in three unrelated strains of E. coli. Remarkably, we found zero evidence for any epigenetic regulation mediated by these Type I RMSs. Thus, these Type I RMSs appear to function exclusively in host defense against incoming DNA (the canonical function of RMSs), while the methylation status of many hundreds of the corresponding recognition sites has no detectable impact on gene expression or any phenotypes. This led us to the concept of "regulation avoidance" by such DNA methyltransferases, which contrasts with the current literature on bacterial epigenetics. Our study hints at the existence of an entire class of regulation avoidant systems, which provides new perspectives on methylation-mediated gene regulation and bacterial genome evolution.


title: fmr1 mutation interacts with sensory experience to alter the early development of behavior and sensory coding in zebrafish

abstract: While Autism Spectrum Disorders (ASDs) are developmental in origin little is known about how they affect the early development of behavior and sensory coding, or how this is modulated by the sensory environment. The most common inherited form of autism is Fragile X syndrome, caused by a mutation in FMR1. Here we show that zebrafish fmr1-/- mutant larvae raised in a naturalistic visual environment display early deficits in hunting behavior, tectal map development, tectal network properties and decoding of spatial stimuli. However when given a choice they preferred an environment with reduced visual stimulation, and rearing them in this environment improved these metrics. Older fmr1-/- fish showed differences in social behavior, spending more time observing a conspecific, but responding more slowly to social cues. Together these results help reveal how fmr1-/- changes the early development of vertebrate brain function, and how manipulating the environment could potentially help reduce these changes.


title: EFFICACY OF DISTRIBUTED TRAINING DEPENDS ON THE DORSO-LATERAL STRIATUM

abstract: Distributed training has long been known to lead to more robust memory formation as compared to massed training. Here we demonstrate that distributed and massed training differentially engage the dorsolateral and dorsomedial striatum and optogenetic priming of dorsolateral striatum can artificially increase the robustness of massed training to the level of distributed training, identifying a novel therapeutic avenue for memory enhancement.


title: Rat ultrasonic vocalizations and novelty-induced social and non-social investigation behavior in a seminatural environment

abstract: Although rats are known to emit ultrasonic vocalizations (USVs), it remains unclear whether these calls serve an auditory communication purpose. For USVs to be part of communication, the vocal signals will need to be a transfer of information between two or more conspecifics, and with the possibility to induce changes in the behavior of the recipient. Therefore, the aim of our study was to investigate the role of USVs in adult rats social and non-social investigation strategies when introduced into a large novel environment with unfamiliar conspecifics. We quantified a wide range of social and non-social behaviors in the seminatural environment, which could be affected by subtle signals, including USVs. We found that during the first hour in the seminatural environment the ability to vocalize did not affect how quickly adult rats met each other, their overall social investigation behavior, their passive social behavior nor their aggressive behavior. Furthermore, the non-social exploratory behaviors and behaviors reflecting anxiety/stress-like states were also unaffected. These results demonstrated that a disability to vocalize did not result in significant disadvantages (or changes) compared to intact conspecifics regarding social and non-social behaviors. This suggests that other (multi)sensory cues are more relevant in social interactions than USVs.


title: Differential detection of Tomato mosaic virus (ToMV) and Tomato brown rugose fruit virus (ToBRFV) using CRISPR-Cas12

abstract: CRISPR/Cas12-based detection is a novel approach for efficient, sequence-specific identification of viruses. Here we adopt the use of CRISPR/Cas12a to identify the Tomato brown rugose fruit virus (ToBRFV), a new and emerging Tobamovirus causing substantial damage to the global tomato industry. Specific guide RNAs (gRNAs) were designed to detect either ToBRFV or the closely related Tomato mosaic virus (ToMV). This technology enabled the differential detection of ToBRFV and ToMV. Sensitivity assays revealed that viruses can be detected from 15-30 ng of RT-PCR product, and that specific detection could be achieved from a mix of ToMV and ToBRFV. In addition, we show that this method enabled the identification of ToBRFV in samples collected from commercial greenhouses. These results demonstrate a new method for species-specific detection of plant viruses. This could provide a platform for the development of efficient and user-friendly ways to distinguish between closely related strains and resistance-breaking pathogens.

category: molecular biology


title: Antibody Humanization: Intra and Inter VH-VL Non-bond Energy Sort Best Humanized Antibody

abstract: Humanization of non-human derived antibody is necessary to reduce the immunogenicity of antibody drug. In recent years, computer aided antibody humanization became an efficient and rapid routine process. We report here a new computational humanization pipeline which includes CDR grafting onto the crystal structures of homologous antibody and CDR grafted humanized antibody structures. Then, the intra and inter VH-VL binding energies are calculated and sorted for optimized humanized antibody. The resulted humanized antibodies are ranked and compared to experimental dataset, which confirms the validity of humanized antibody design by intra and inter VH-VL binding energy.


title: Optimized antibody humanization by intra and inter VH-VL binding energy sorting


title: Large scale measurement of protein synthesis rates over the diurnal cycle reveal pathway specific regulation of translation

abstract:
Plants have a diurnal separation of metabolic fluxes and a need for differential maintenance of protein machinery in the day and night. To directly assess aggregate protein translation and degradation for specific proteins and to estimate the ATP investment involved, the individual rates of synthesis and degradation of hundreds of different proteins were measured in Arabidopsis thaliana rosettes. This quantification of translation control through incorporation of heavy hydrogen into newly synthesised protein confirmed that most protein synthesis occurs during the day hours (~3:1 day:night), but revealed it was highly divergent across functional categories. Proteins involved in photosynthesis, especially components of the light harvesting complexes, were synthesized much faster in the day (~10:1), while the protein components of carbon metabolism and vesicle trafficking were translated at similar rates day or night. Comparison of aggregate translation with a range of comparable studies using polysomal loading of transcripts or transcript abundance suggests that translational control is a major contributor to effective gene expression over the diurnal cycle. Diurnal protein degradation rate was observed to be tightly coordinated with protein synthesis rate as few leaf proteins changed in abundance despite reduced translation rates during the night. The direct, quantitative and aggregate analysis of protein synthesis provides an integration of transcriptional, post-transcriptional and translational control of leaf proteome homeostasis and an opportunity to assess the ATP investments involved. The data reveals how the pausing of photosystem synthesis and degradation at night allows the redirection of a decreased energy budget to a selective night-time maintenance schedule.

One sentence summaryAdvances in collection and processing of stable isotope incorporation data has allowed characterisation of differential protein synthesis rates over the diurnal cycle which reveal both global and specific translational regulation programs of core biochemical pathways.

category: plant biology


title: TRPM2 ion channels steer neutrophils towards a source of hydrogen peroxide

abstract: Neutrophils must navigate accurately towards pathogens in order to destroy invaders and thus defend our bodies against infection. Here we show that hydrogen peroxide, a potent neutrophil chemoattractant, guides chemotaxis by activating calcium-permeable TRPM2 ion channels and thus generating an intracellular leading-edge calcium "pulse". The thermal sensitivity of TRPM2 activation means that chemotaxis towards hydrogen peroxide is strongly promoted by small temperature elevations, suggesting that an important function of fever may be to enhance neutrophil chemotaxis by facilitating calcium influx through TRPM2. Chemotaxis towards conventional chemoattractants such as LPS, CXCL2 and C5a does not depend on TRPM2 but is driven in a similar way by leading-edge calcium pulses. Other proposed initiators of neutrophil movement, such as PI3K, Rac and lyn, influence chemotaxis by modulating the amplitude of calcium pulses. We propose that intracellular leading-edge calcium pulses are universal drivers of the motile machinery involved in neutrophil chemotaxis.

category: immunology


title: Loss of mutually protective effects between osteoclasts and chondrocytes in damaged joints drives osteoclast-mediated cartilage degradation via matrix metalloproteinases

abstract: Osteoclasts are large multinucleated cells that resorb bone to regulate bone remodelling during skeletal maintenance and development. It is overlooked that osteoclasts also digest cartilage during this process, as well as in degradative conditions including osteoarthritis, rheumatoid arthritis and primary bone sarcomas such as giant cell tumour of bone. This study explores the poorly understood mechanisms behind the interaction between osteoclasts and cartilage. Morphologically, osteoclasts differentiated on acellular human cartilage formed multinucleated cells expressing characteristic osteoclast marker genes (e.g. CTSK, MMP9) and proteins (TRAP, VNR) that visibly damaged the cartilage surface by SEM, but without the formation of resorption pits. Osteoclasts caused increased glycosaminoglycan (GAG) release from acellular and cellular human cartilage that was dependent on direct contact with the substrate. Direct co-culture with chondrocytes during osteoclast differentiation increased the number of large osteoclasts formed. When osteoclasts were cultured on dentine, direct co-culture with chondrocytes inhibited osteoclast formation and reduced basal degradation of cartilage. This suggests a mutually protective effect on their  native tissue between bone-resident osteoclasts and chondrocytes, that is reversed when the joint structure breaks down and osteoclasts are in contact with non-native substrates. Mechanistically, osteoclast-mediated cartilage degradation was inhibited by the pan-MMP inhibitor GM6001 and by TIMP1, indicative of a role for soluble MMPs. RNA sequencing and RT-qPCR analysis identified MMP8 as overexpressed in osteoclasts differentiated on cartilage versus dentine, while MMP9 was the most highly expressed MMP on both substrates. Inhibition of either MMP8 or MMP9 by siRNA in mature osteoclasts reduced GAG release, confirming their involvement in cartilage degradation. Immunohistochemical expression of MMP8 and MMP9 was evident in osteoclasts in osteosarcoma tissue sections. Understanding and controlling the activity of osteoclasts might represent a new therapeutic approach for pathologies characterized by cartilage degeneration and presents an attractive target for further research.

category: cell biology


title: Structured illumination microscopy artifacts caused by illumination scattering

abstract: Despite its wide application in live-cell super-resolution (SR) imaging, structured illumination microscopy (SIM) suffers from aberrations caused by various sources. Although artifacts generated from inaccurate reconstruction parameter estimation and noise amplification can be minimized, aberrations due to the scattering of excitation light on samples have rarely been investigated. In this paper, by simulating multiple subcellular structure with the distinct refractive index (RI) from water, we study how different thicknesses of this subcellular structure scatter incident light on its optical path of SIM excitation. Because aberrant interference light aggravates with the increase in sample thickness, the reconstruction of the 2D-SIM SR image degraded with the change of focus along the axial axis. Therefore, this work may guide the future development of algorithms to suppress SIM artifacts caused by scattering in thick samples.


title: Production and assessment of monoclonal antibodies against the SpyCas9 protein of Streptococcus pyogenes

abstract: The biotechnological applications of the programmable DNA-cleaving enzymes known as clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) continue to expand from agricultural trait development to therapeutic gene therapies. The Cas9 CRISPR system isolated from Streptococcus pyogenes (SpyCas9) is classified as class II, which is popular due to high efficiency and robustness. Antibodies that specifically detect SpyCas9 could expand the applicability of this enzyme. Here, we report on the development of monoclonal antibodies against SpyCas9. Four hybridoma cells were selected for their expression of anti-SpyCas9. Hybridoma supernatant contained reactive and highly specific antibodies to SpyCas9. Anti-SpyCas9 antibody was purified and was non-selective against six other bacterial Cas9 proteins. SpyCas9 protein could be detected in HEK293T cells in decreasing amounts over a 48 hour period, indicating the antibody could be used to detect residual levels of SpyCas9 remaining following cell treatment with a CRISPR/SpyCas9 system. The anti-SpyCas9 antibody may help researchers facilitate Cas9 for further study for the use of techniques such as enzyme-linked immunosorbent assays (ELISAs), western blot, immunoprecipitation, and immunohistochemical staining.


title: Long term consumption of green tea EGCG enhances healthspan and lifespan in mice bymitigating multiple aspects of cellular senescence in mitotic and post-mitotic tissues, gutdysbiosis and immunosenescence

abstract: Cellular senescence is emerging as the causal nexus of aging, and its potential modulators present an effective strategy to counter age-related morbidity. The current study profiled the extent of cellular senescence in different organs of mice at four different time-points of lifespan, and explored the influence of epigallocatechin gallate (EGCG) consumption in impacting multiple aspects of aging biology. We report that adipose and intestinal tissues are highly vulnerable to cellular senescence as evident by age-associated increase in DNA damage response, activation of cell cycle inhibitors (p53/p21) and induction of SASP (p38MAPK/NF-{kappa}B/Cox-2). Further, a distinct modulation of nutrient signaling pathway mediators (AMPK/Akt/SIRT3 and 5), and a decrease in autophagy effectors was also observed in aging animals. Systemic inflamm-aging markers (TNF-/IL-l{beta}) and splenic CD4/CD8 ratio increased with age, while NK cell population decreased. Metagenomic analyses revealed age-related decrease in the diversity of microbial species while an increase in the abundance of various pathogenic bacterial genera was also observed. Long term EGCG consumption enhanced lifespan of animals by attenuating markers of DNA damage, cell cycle inhibitors and SASP in adipose, intestine and liver tissue. Mechanistically, EGCG inhibited the activation of AMPK and Akt and enhanced mitochondrial SIRT3 and SIRT5 expression, as well as autophagic response in adipose and intestinal tissues. Systemic presence of inflamm-aging markers decreased while expression of T cell immune response regulator CD69 increased in EGCG fed animals. EGCG also improved age-related decrease in the diversity of microbial species and suppressed the growth of pathogenic microbes. In short, our results provide compelling evidence that post-mitotic adipose tissue is a major site of cellular senescence and SASP activation, and that chronic EGCG consumption can influence several aspects of aging and senescence resulting in improved organismal healthspan and lifespan.


title: Chronic neuronal excitation leads to homeostatic suppression of structural long-term potentiation

abstract: Synaptic plasticity is long-lasting changes in synaptic currents and structure. When neurons are exposed to signals that induce aberrant neuronal excitation, they increase the threshold for the induction of synaptic plasticity, called homeostatic plasticity. To further understand the homeostatic regulation of synaptic plasticity and its molecular mechanisms, we investigated glutamate uncaging/photoactivatable (pa)CaMKII-dependent sLTP induction in hippocampal CA1 neurons after chronic neuronal excitation by GABAA receptor antagonists. The neuronal excitation suppressed the glutamate uncaging-evoked Ca2+ influx and failed to induce sLTP. Single-spine optogenetic stimulation using paCaMKII also failed to induce sLTP, suggesting that CaMKII downstream signaling is impaired in response to chronic neuronal excitation. Furthermore, while the inhibition of Ca2+ influx was protein synthesis-independent, paCaMKII-induced sLTP depended on it. Our findings demonstrate that chronic neuronal excitation suppresses sLTP in two independent ways (i.e., the inhibitions of Ca2+ influx and CaMKII downstream signaling), which may contribute to the robust neuronal protection in excitable environments.


title: Functional development of a V3-specific broadly neutralizing antibody isolated from a case of HIV superinfection

abstract: Stimulating broadly neutralizing antibodies (bnAbs) directly from germline remains a barrier for HIV vaccines. HIV superinfection elicits bnAbs more frequently than single infection, providing clues of how to elicit such responses. We used longitudinal antibody sequencing and structural studies to characterize bnAb development from a superinfection case. Mature bnAb QA013.2 bound both initial transmitted and superinfecting virus, but its inferred naive bound only the superinfecting strain and was not neutralizing. QA013.2 requires residues spanning FWRH1-CDRH1 to attain breadth, which is uncommon for V3-specific bnAbs. A 4.15 [A] cryo-EM structure of QA013.2 bound to heterologous native-like trimer showed recognition of V3 signatures (N301, N332, and GDIR). Antigenic profiling revealed that viral escape was achieved not only by changes in the structurally-defined epitope, but also by mutations in V1. These results highlight shared and distinct properties of QA013.2 relative to other V3-specific bnAbs in the setting of sequential, diverse antigenic variants.


title: Time dependent proinflammatory responses shape virus interference during coinfections of influenza A virus and influenza D virus

abstract:
Both influenza A virus (IAV) and influenza D virus (IDV) are enzootic in pigs. IAV causes approximately 100% morbidity with low mortality, whereas IDV leads to only mild respiratory diseases in pigs. In this study, we performed a series of coinfection experiments in vitro and in vivo to understand how IAV and IDV interact and cause pathogenesis during coinfection. Results showed that IAV inhibited IDV replication when infecting swine tracheal epithelial cells (STEC) with IAV 24- or 48-hours prior to IDV inoculation, and that IDV suppressed IAV replication when IDV preceded IAV inoculation by 48 hours. Virus interference was not identified during simultaneous IAV/IDV infections or with 6 hours between the two viral infections, regardless of their order. The interference pattern at 24- and 48-hours correlated with proinflammatory responses induced by the first infection, which was about 24-hours slower for IDV than IAV. The viruses did not interfere with each other if both infected the cells before proinflammatory responses were induced. Coinfection in pigs further demonstrated that IAV interfered both viral shedding and virus replication of IDV, especially in the upper respiratory tract. Clinically, coinfection of IDV and IAV did not show significant enhancement of disease pathogenesis, compared with the pigs infected with IAV alone. In summary, this study suggests that interference during coinfection of IAV and IDV is primarily due to the proinflammatory response and is therefore dependent on the time between infection, and the order of infection.

ImportanceBoth IAV and IDV are enzootic in pigs, and feral pigs have a higher risk for both IAV and IDV exposures than IDV exposure alone. This study suggests that in coinfection with IAV and IDV either virus can interfere with the replication of the other virus by stimulating proinflammatory responses; however, the proinflammatory response was 24 hours slower for IDV than IAV. In vitro there was no interference during simultaneous coinfection, regardless of infection order. Coinfection of IDV and IAV in pigs did not show enhanced pathogenesis, compared with those infected only with IAV. This study can facilitate our understanding of virus epidemiology and pathogenesis associated with IAV and IDV coinfection.


title: Graph Signal Processing on protein residue networks helps in studying its biophysical properties

abstract: Understanding the physical and chemical properties of proteins is vital, and many efforts have been made to study the emergent properties of the macro-molecules as a combination of long chains of amino acids. Here, we present a graph signal processing based approach to model the biophysical property of proteins. For each protein inter-residue proximity-based network is used as basis graph and the respective amino acid properties are used as node-signals. Signals on nodes are decomposed on networks Laplacian eigenbasis using graph Fourier transformations. We found that the intensity in low-frequency components of graph signals of residue features could be used to model few biophysical properties of proteins. Specifically, using our approach, we could model protein folding-rate, globularity and fraction of alpha-helices and beta-sheets. Our approach also allows amalgamation of different types of chemical and graph theoretic properties of residue to be used together in a multi-variable regression model to predict biophysical properties.


title: Fast and Robust 2D Inverse Laplace Transformation of Single-Molecule Fluorescence Lifetime Data

abstract:
Fluorescence spectroscopy at the single-molecule scale has been indispensable for studying conformational dynamics and rare states of biological macromolecules. Single-molecule 2D-fluorescence lifetime correlation spectroscopy (sm-2D-FLCS) is an emerging technique that holds great promise for the study of protein and nucleic acid dynamics as it 1) resolves conformational dynamics using a single chromophore, 2) measures forward and reverse transitions independently, and 3) has a dynamic window ranging from microseconds to seconds. However, the calculation of a 2D fluorescence relaxation spectrum requires an inverse Laplace transition (ILT), which is an ill-conditioned inversion that must be estimated numerically through a regularized minimization. The current methods for performing ILTs of fluorescence relaxation can be computationally inefficient, sensitive to noise corruption, and difficult to implement. Here, we adopt an approach developed for NMR spectroscopy (T1-T2 relaxometry) to perform 1D and 2D-ILTs on single-molecule fluorescence spectroscopy data using singular-valued decomposition and Tikhonov regularization. This approach provides fast, robust, and easy to implement Laplace inversions of single-molecule fluorescence data.

Significance StatementInverse Laplace transformations are a powerful approach for analyzing relaxation data. The inversion computes a relaxation rate spectrum from experimentally measured temporal relaxation, circumventing the need to choose appropriate fitting functions. They are routinely performed in NMR spectroscopy and are becoming increasing used in single-molecule fluorescence experiments. However, as Laplace inversions are ill-conditioned transformations, they must be estimated from regularization algorithms that are often computationally costly and difficult to implement. In this work, we adopt an algorithm first developed for NMR relaxometry to provide fast, robust, and easy to implement 1D and 2D inverse Laplace transformations on single-molecule fluorescence data.


title: Elucidating biosynthetic pathway of piperine using comparative transcriptome analysis of leaves, root, and spike in Piper longum L.

abstract: Piper longum (Pipli; Piperaceae) is an important spice valued for its pungent alkaloids, especially piperine. Albeit, its importance, the mechanism of piperine biosynthesis is still poorly understood. The Next Generation Sequencing (NGS) for P. longum leaves, root and spikes was performed using Illumina platform, which generated 16901456, 54993496 and 22900035, respectively of high quality reads. In de novo assembly P. longum 173381 numbers of transcripts were analyzed. Analysis of transcriptome data from leaf, root and spike showed gene families that were involved in the biosynthetic pathway of piperine and other secondary metabolites. To validate differential expression of the identified genes, 27 genes were randomly selected to confirm the expression level by quantitative real time PCR (qRT-PCR) based on the up regulation and down regulation of differentially expressed genes obtained through comparative transcriptome analysis of leaves and spike of P. longum. With the help of UniProt database the function of all characterized genes was generated.


title: Testosterone induces a conditioned place preference to the nest of a monogamous mouse under field conditions

abstract: Transient increases in testosterone (T-pulses) occur after social interactions in males of various vertebrate species, but the functions of T-pulses are poorly understood. Under laboratory conditions, the rewarding nature of T-pulses induces conditioned place preferences (CPPs), but what are the effects in a complex field environment? We present the first evidence that T-pulses administered to males at their nest site in the wild increased time spent at the nest regardless of pup presence in the monogamous, biparental, and territorial California mouse (Peromyscus californicus). Female partners of the T-males, in turn, spent less time at the nest. Independent of treatment, mice produced more ultrasonic vocalizations (USVs) when alone, but T-mice produced more USVs than controls. T-males produced USVs with a smaller bandwidth that likely traveled farther. Our combined results provide compelling evidence that T-pulses can significantly shift the behavioral focus and location of individuals in a complex field setting.

category: ecology


title: Nonlinear collision between anisotropic propagating waves in mouse somatosensory cortex

abstract: How does cellular organization shape the spatio-temporal patterns of activity in the cortex while processing sensory information? After measuring the propagation of activity in the mouse primary somatosensory cortex (S1) in response to single whisker deflections with Voltage Sensitive Dye (VSD) imaging, we developed a two dimensional mean field model of S1. We observed that, for strong enough excitatory cortical interactions, whisker deflections generate a propagating wave in S1. We developed an inversion method that reconstructs model parameters from VSD data, revealing that a spatially heterogeneous organization of synaptic strengths between pyramidal neurons in S1 is likely to be responsible for the anisotropic spatio-temporal patterns of activity measured experimentally. Finally, we report that two consecutive stimuli activating different spatial locations in S1 generate two waves which collide sub-linearly. In the model, such sub-linear interaction is explained by a lower sensitivity to external perturbations of neural networks during activated states.


title: Intricate regulation of ribosome biogenesis genes in response to mTORC1 signaling

abstract: Genes encoding ribosomal proteins are repressed in response to inhibition of mTORC1. In Saccharomyces cerevisiae, this involves dissociation of the activator Ifh1p in a process that depends on Utp22p, a protein that also functions in pre-rRNA processing. Ifh1p has a paralog, Crf1p, which can mediate mTORC1 inhibition by acting as a repressor. Ifh1p and Crf1p derive from a common ancestor, which may have acted as both an activator and a repressor. We report here that UTP22 and RRP7, which encodes another pre-rRNA processing factor, are controlled by mTORC1; both gene promoters are bound by Ifh1p, which dissociates on mTORC1 inhibition. Notably, Crf1p acts as an activator as evidenced by reduced expression in a crf1{Delta} strain. By contrast, Crf1p is required to repress expression of HMO1, which encodes a cofactor involved in communicating mTORC1 activity to target genes. Our data therefore indicate that Crf1p exhibits the dual repressor/activator functions of the Ifh1p-Crf1p ancestor.


title: Dia1 Coordinates Differentiation and Cell Sorting in a Stratified Epithelium

abstract:
Although implicated in adhesion, few studies address how actin assembly factors guide cell positioning in multicellular tissue. The formin, Dia1, localizes to the proliferative basal layer of epidermis. In organotypic cultures, Dia1 depletion reduced basal cell density and resulted in stratified tissue with disorganized differentiation and proliferative markers. Since crowding induces differentiation in epidermal tissue, we hypothesized that Dia1 allows cells to reach densities amenable to differentiation prior to stratification. Consistent with this hypothesis, forced crowding of Dia1-deficient cells rescued transcriptional abnormalities. Dia1 promotes rapid growth of lateral adhesions, a behavior consistent with the ability of cells to remain monolayered when crowded. In aggregation assays, cells sorted into distinct layers based on Dia1 expression status. These results suggested that as basal cells proliferate, reintegration and packing of Dia1-positive daughter cells is favored while Dia1-negative cells tend to delaminate to a suprabasal compartment. These data demonstrate how formin expression patterns play a crucial role in constructing distinct domains within stratified epithelia.

SummaryHarmon et al demonstrate that differential expression of an actin nucleator, the formin, Dia1, drives cell sorting and maintains distinct morphological domains within an epithelial tissue. This illuminates the possible utility of evolving a large formin family in orchestrating the compartmentalization and differentiation of complex tissues.


title: Genetic Diversity of Dianthus Plants Revealed by SRAP Markers

abstract: Dianthus is a valuable genetic resource for flower breeding. However, study on the genetic similarity of Dianthus plants is rare. In this work, 20 pairs of SRAP markers were used to analyze genetic diversity of 44 Dianthus plants, including 13 lines of wild Dianthus chinensis L., 7 lines of wild Dianthus superbus L. and 24 commercial varieties including Dianthus caryophyllus L., Dianthus plumarius L. and Dianthus barbatus L.. Results showed that precise interspecific and intraspecific genetic diversity was provided in Dianthus plants by using 20 pairs of SRAP molecular markers. The interspecific genetic diversity of Dianthus plants was much abundant and the intraspecific genetic difference of wild Dianthus species was related to their geographical distribution and habitats. In this work, theoretical basis and technical support were provided for crossbreeding and molecular mechanism research of Dianthus plants.

category: genetics


title: ERK signaling licenses SKN-1A/NRF1 for proteasome production and proteasomal stress resistance

abstract: The ubiquitin-proteasome system is vital for cell growth and homeostasis, but for most cancers proteasomal inhibition has not been effective as a therapy. Normal and cancer cells adapt to proteasomal stress through an evolutionarily conserved recovery response, in which the transcription factor NRF1 upregulates proteasome subunit genes. Starting with a C. elegans screen to identify regulators of the recovery response, here we show that this response depends upon phosphorylation of NRF1 on a single residue by the growth factor-activated kinase ERK1/2. Inhibition of this phosphorylation impairs NRF1 nuclear localization and proteasome gene activation, sensitizes C. elegans and cancer cells to proteasomal stress, and synergizes with proteasome inhibition to retard human melanoma growth in vivo in a mouse model. The evolutionarily conserved ERK1/2-NRF1 axis couples proteasome production to growth signaling, and represents a promising new strategy for expanding the range and efficacy of proteasomal inhibition therapy in cancer.


title: Serial passage of the human probiotic 	E. coli Nissle 1917 in an insect host leads to changed bacterial phenotypes

abstract: Probiotics are living microorganisms that are increasingly and successfully used for the therapy of various diseases. The most common use of probiotics is the therapeutic and preventive application for gastrointestinal disorders. The probiotic Escherichia coli strain Nissle 1917 (EcN) has been proven to effectively prevent and alleviate intestinal diseases, including various types of inflammatory bowel disease. Despite the widespread medical application of EcN, the underlying mechanisms of its protective effect remain elusive. The present work aimed to establish an insect model system to enable further research on the modes of action of EcN and the dynamics of adaptation to a novel host organism. Using a long-term serial passage approach, we orally introduced EcN to the host, the red flour beetle Tribolium castaneum. After multiple cycles of intestinal colonization in beetle larvae, several attributes of the passaged replicate lines were assessed. We observed phenotypic changes in growth and motility but no genetic changes in the lines after passaging through the host and its flour environment. One of the EcN lines exposed to the host displayed peculiar morphological and physiological characteristics showing that serial passage of EcN can generate differential phenotypes.


title: OPA1 Deletion in Brown Adipose Tissue Improves Thermoregulation and Systemic Metabolism via FGF21

abstract: Adrenergic stimulation of brown adipocytes alters mitochondrial dynamics, including proteolytic processing of the mitochondrial fusion protein optic atrophy 1 (OPA1). However, direct mechanisms linking OPA1 to brown adipose tissue (BAT) physiology are incompletely understood. By deleting OPA1 selectively in BAT (OPA1 BAT KO), we demonstrate that OPA1 is required for cold-induced thermogenesis. Unexpectedly, OPA1 deficiency induced fibroblast growth factor 21 (FGF21) as a BATokine in an activating transcription factor 4 (ATF4)- dependent manner. BAT-derived FGF21 mediates an adaptive response in OPA1 BAT KO mice, by inducing browning of white adipose tissue (WAT), increasing resting metabolic rates, and improving thermoregulation. However, FGF21 does not mediate the resistance to diet-induced obesity observed in these animals. These findings reveal a requirement for OPA1 in BAT thermogenesis, and uncovers a homeostatic mechanism of BAT-mediated metabolic protection governed by an ATF4-FGF21 axis, that is activated independently of BAT thermogenic function.

category: physiology


title: Single-molecule super-resolution imaging of T-cell plasma membrane CD4 redistribution upon HIV-1 binding

abstract: The first step of cellular entry for the human immunodeficiency virus type-1 (HIV-1) occurs through the binding of its envelope protein (Env) with the plasma membrane receptor CD4 and co-receptor CCR5 or CXCR4 on susceptible cells, primarily CD4+ T cells and macrophages. Although there is considerable knowledge of the molecular interactions between Env and host cell receptors that lead to successful fusion, the precise way in which HIV-1 receptors redistribute to sites of virus binding at the nanoscale remains unknown. Here, we quantitatively examine changes in the nanoscale organisation of CD4 on the surface of CD4+ T cells following HIV-1 binding. Using single-molecule super-resolution imaging, we show that CD4 molecules are distributed mostly as either individual molecules or small clusters of up to 4 molecules. Following virus binding, we observe a local 3-to-10-fold increase in cluster diameter and molecule number for virus-associated CD4 clusters. Moreover, a similar but smaller magnitude reorganisation of CD4 was also observed with recombinant gp120. For the first time, our results quantify the nanoscale CD4 reorganisation triggered by HIV-1 on host cells. Our quantitative approach provides a robust methodology for characterising the nanoscale organisation of plasma membrane receptors in general with the potential to link spatial organisation to function.


title: Capturing the complexity of topologically associating domains through multi-feature optimization

abstract: The three-dimensional structure of human chromosomes is tied to gene regulation and replication timing, but there is still a lack of consensus on the computational and biological definitions for chromosomal substructures such as topologically associating domains (TADs). TADs are described and identified by various computational properties leading to different TAD sets with varying compatibility with biological properties such as boundary occupancy of structural proteins. We unify many of these computational and biological targets into one algorithmic framework that jointly maximizes several computational TAD definitions and optimizes TAD selection for a quantifiable biological property. Using this framework, we explore the variability of TAD sets optimized for six different desirable properties of TAD sets: high occupancy of CTCF, RAD21, and H3K36me3 at boundaries, reproducibility between replicates, high intra- vs inter-TAD difference in contact frequencies, and many CTCF binding sites at boundaries. The compatibility of these biological targets varies by cell type, and our results suggest that these properties are better reflected as subpopulations or families of TADs rather than a singular TAD set fitting all TAD definitions and properties. We explore the properties that produce similar TAD sets (reproducibility and inter- vs intra-TAD difference, for example) and those that lead to very different TADs (such as CTCF binding sites and inter- vs intra-TAD contact frequency difference).

category: genomics


title: The evolution of cold nociception in drosophilid larvae and identification of a neural basis for cold acclimation

abstract:
Cold temperatures can be fatal to insects, but many species have evolved the ability to cold acclimate, thereby increasing their cold tolerance. While there is a growing body of knowledge concerning the mechanisms underlying cold tolerance, relatively little is known concerning how insects sense noxious cold (cold nociception), or how cold nociception might function in cold tolerance. It has been previously shown that Drosophila melanogaster larvae perform highly stereotyped, cold-evoked behaviors under the control of noxious cold-sensing neurons (nociceptors) innervating the barrier epidermis. In the present study, we first sought to describe cold-nociceptive behavior among 11 drosophilid species with differing cold tolerances and from differing climates. Behavioral analyses revealed that the predominant cold-evoked response among drosophilid larvae is a head-to-tail contraction (CT) behavior, which is likely inherited from a common ancestor. However, despite lack of phylogenetic signal (suggesting trait lability), the CT behavior was transient and there was no clear evidence that cold sensitivity was related to thermal environment; collectively this suggests that the behavior might not be adaptive. We therefore sought to uncover an alternative way that cold nociception might be protective. Using a combination of cold-shock assays, optogenetics, electrophysiology, and methods to genetically disrupt neural transmission, we demonstrate that cold sensing neurons in Drosophila melanogaster (Class III nociceptors) are sensitized by and critical to cold acclimation. Moreover, we demonstrate that cold acclimation can be optogenetically-evoked, sans cold. Collectively, these findings reveal that cold nociception constitutes a peripheral neural basis for Drosophila larval cold acclimation.

Significance StatementMany insects adapt to cold in response to developmental exposure to cool temperatures. While there is a growing body of knowledge concerning the mechanisms underlying cold tolerance, it is unknown how sensory neurons might contribute. Here, we show that noxious cold sensing (cold nociception) is widely present among drosophilid larvae, and that cold-sensing neurons (Class III cold nociceptors) are necessary and sufficient drivers of cold acclimation. This suggests that cold acclimation has, at least in part, a neural basis.


title: Why extinction estimates from extant phylogenies are so often zero

abstract: Time-calibrated phylogenies comprising only extant lineages are widely used to estimate historical speciation and extinction rates. Such extinction rate estimates have long been controversial as many phylogenetic studies report zero extinction in many taxa, a finding in conflict with the fossil record. To date, the causes of this widely observed discrepancy remain unresolved. Here we provide a novel and simple explanation for these "zero-inflated" extinction rate estimates, based on the recent discovery that there exist many alternative "congruent" diversification scenarios that cannot possibly be distinguished on the sole basis of extant timetrees. Consequently, estimation methods tend to converge to some scenario congruent to (i.e., statistically indistinguishable from) the true diversification scenario, but not necessarily to the true diversification scenario itself. This congruent scenario may in principle exhibit negative extinction rates, a biologically meaningless but mathematically feasible situation, in which case estimators will tend to hit and stick to the boundary estimate of zero extinction. To test this explanation, we estimated extinction rates using maximum likelihood for a set of simulated trees and for 121 empirical trees, while either allowing or preventing negative extinction rates. We find that the existence of congruence classes and imposed bounds on extinction rates can explain the zero-inflation of previous extinction rate estimates, even for large trees (1000 tips) and in the absence of any detectable model violations. Not only do our results likely resolve a long-standing mystery in phylogenetics, they demonstrate that model congruencies can have severe consequences in practice.

category: evolutionary biology


title: Large-scale quantification of human osteocyte lacunar morphological biomarkers as assessed by ultra-high-resolution desktop micro-computed tomography

abstract:
Ultra-high-resolution imaging of the osteocyte lacuno-canalicular network (LCN) three-dimensionally (3D) in a high-throughput fashion has greatly improved the morphological knowledge about the constituent structures - positioning them as potential biomarkers. Technologies such as serial focused ion beam/scanning electron microscopy (FIB/SEM) and confocal scanning laser microscopy (CLSM) can image in extremely high resolution, yet only capture a small number of lacunae. Synchrotron radiation computed tomography (SR-CT) can image with both high resolution and high throughput but has a limited availability. Desktop micro-computed tomography (micro-CT) provides an attractive balance: high-throughput imaging on the micron level without the restrictions of SR-CT availability. Over the past decade, desktop micro-CT has been used to image osteocyte lacunae in a variety of animals, yet few studies have employed it to image human lacunae using clinical biopsies.

In this study, accuracy, reproducibility, and sensitivity of large-scale quantification of human osteocyte lacunar morphometries were assessed by ultra-high-resolution desktop micro-computed tomography. For this purpose, thirty-one transiliac human bone biopsies containing trabecular and cortical regions were imaged using ultra-high-resolution desktop micro-CT at a nominal isotropic voxel resolution of 1.2m. The resulting 3D images were segmented, component labeled, and the following morphometric parameters of 7.71 million lacunae were measured: Lacunar number (Lc.N), density (Lc.N/BV), porosity (Lc.TV/BV), volume (Lc.V), surface area (Lc.S), surface area to volume ratio (Lc.S/Lc.V), stretch (Lc.St), oblateness (Lc.Ob), sphericity (Lc.Sr), equancy (Lc.Eq), and angle (Lc.{theta}).

Accuracy was quantified by comparing automated lacunar identification to manual identification. Mean true positive rate (TPR), false positive rate (FPR), and false negative rate (FNR) were 89.0%, 3.4%, and 11.0%, respectively. Regarding the reproducibility of lacunar morphometry from repeated measurements, precision errors were low (0.2 - 3.0%) and intraclass correlation coefficients were high (0.960 - 0.999). Significant differences between cortical and trabecular regions (p<0.001) existed for Lc.N/BV, Lc.TV/BV, local lacunar surface area (<Lc.S>), and local lacunar volume (<Lc.V>), all of which demonstrate the sensitivity of the method and are possible biomarker candidates. This study provides the foundation required for future large-scale morphometric studies using ultra-high-resolution desktop micro-CT and high-throughput analysis of millions of osteocyte lacunae in human bone samples. Furthermore, the validation of this technology for imaging of human lacunar properties establishes the quality and reliability required for the accurate, precise, and sensitive assessment of osteocyte morphometry in clinical bone biopsies.

category: bioengineering


title: Seroprevalence and associated risk factors of Toxoplasma gondii infections among pregnant women in Makassar, Indonesia

abstract: The protozoan parasite, Toxoplasma gondii is estimated to infect one-third of the worlds population. Infection in pregnant women can cause severe conditions for their babies. Until now, there is no data regarding Toxoplasma infection from Makassar pregnant mothers. This study aims to obtain information on Toxoplasma specific antibodies and to measure the risk factor associate with parasite infection. This cross-sectional study conducted in 9 of 47 primary health centres (Puskesmas) in Makassar. Blood samples and questionnaires were collected from 184 pregnant women aged 15-42 years old from September to October 2020. ELISA technique was used to examine the IgG and IgM antibodies. Univariable and multivariable analyses were carried out to measure factors that independently associate with Toxoplasma antibody positivity. Our result showed the range of Toxoplasma IgM and IgG are 0.06-1.01 and 0.09-3.01, respectively. While no one of our participants has an acute Toxoplasma gondii infection (IgM positive), we found 32,6% pregnant mothers are exposed to parasite (positive IgG). Contact with cats [OR(95CI): 8.04 (3.09-20.87)], consume chicken satay [OR(95CI): 7.46(3.06-18.21)] and consume un-boiled water [OR(95CI): 5.23(1.57-17.38)] are independently associate with positive Toxoplasma IgG antibody. Based on the result, we conclude that pregnant women in Makassar are exposed to T. gondii and the cyst of parasite contaminates food and water in Makassar.


title: Distinct roles and actions of PDI family enzymes in catalysis of nascent-chain disulfide formation

abstract: The mammalian endoplasmic reticulum (ER) harbors more than 20 members of the protein disulfide isomerase (PDI) family that act to maintain proteostasis. Herein, we developed an in vitro system for directly monitoring PDI- or ERp46-catalyzed disulfide bond formation in ribosome-associated nascent chains (RNC) of human serum albumin. The results indicated that ERp46 more efficiently introduced disulfide bonds into nascent chains with short segments exposed outside the ribosome exit site than PDI. Single-molecule analysis by high-speed atomic force microscopy further revealed that PDI binds nascent chains persistently, forming a stable face-to-face homodimer, whereas ERp46 binds for a shorter time in monomeric form, indicating their different mechanisms for substrate recognition and disulfide bond introduction. Similarly to ERp46, a PDI mutant with an occluded substrate-binding pocket displayed shorter-time RNC binding and higher efficiency in disulfide introduction than wild-type PDI. Altogether, ERp46 serves as a more potent disulfide introducer especially during the early stages of translation, whereas PDI can catalyze disulfide formation in RNC when longer nascent chains emerge out from ribosome.

category: biochemistry


title: MAVERICC: Efficient marker-free rescue of vaccinia virus recombinants by in vitro CRISPR-Cas9 engineering

abstract:
Vaccinia virus (VACV)-based vectors are in extensive use as vaccines and cancer immunotherapies. VACV engineering has traditionally relied on homologous recombination between a parental viral genome and a transgene-bearing transfer plasmid, a highly inefficient process that necessitates the use of a selection or screening marker to isolate recombinants. Recent extensions of this approach have sought to enhance the recovery of transgene-bearing viruses through the use of CRISPR-Cas9 engineering to cleave the viral genome in infected cells. However, these methods do not completely eliminate the generation of WT viral progeny and thus continue to require multiple rounds of viral propagation and plaque purification. Here, we describe MAVERICC (marker-free vaccinia virus engineering of recombinants through in vitro CRISPR/Cas9 cleavage), a new strategy to engineer recombinant VACVs in a manner that overcomes current limitations. MAVERICC also leverages the CRISPR/Cas9 system but requires no markers and yields essentially pure preparations of the desired recombinants in a single step. We used this approach to rapidly introduce point mutations, insertions, and deletions at multiple locations in the VACV genome, both singly and in combination. The efficiency and versatility of MAVERICC make it an ideal choice for generating mutants and mutant libraries at arbitrarily selected locations in the viral genome to build complex VACV vectors, effect vector improvements, and facilitate the study of poxvirus biology.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/425447v1_ufig1.gif" ALT="Figure 1">
View larger version (19K):
org.highwire.dtl.DTLVardef@17151cborg.highwire.dtl.DTLVardef@b0f480org.highwire.dtl.DTLVardef@184166corg.highwire.dtl.DTLVardef@dc177d_HPS_FORMAT_FIGEXP  M_FIG C_FIG Overview of MAVERICC. Conceptual overview of the approach outlined in this manuscript. To make VACV recombinants, the parental virus is first purified and vDNA is isolated with phenol:chloroform extraction. This purified vDNA is then treated with Cas9 enzyme and sgRNAs that are directed to a specific locus in the VACV genome. The cleaved vDNA is then transfected into FWPV-infected BSC-40 cells along with a transfer amplicon containing an insertion or mutation of interest flanked by homologous sequences. Recombination is allowed to occur for 5-7 days, during which time the cleaved vDNA is healed by the transfer amplicon, thus editing the VACV genome, and packaged into infectious viral particles. Individual plaques are grown up and rVACVs are isolated after a single round of plaque purification. Image was created with Biorender.com.


title: Transient response of basal ganglia network in healthy and low-dopamine state

abstract:
The basal ganglia (BG) are crucial for a variety of motor and cognitive functions. Changes induced by persistent low-dopamine (e.g. in Parkinsons disease), result in aberrant changes in steady-state population activity ({beta}-band oscillations) and transient response of the BG. Typically, brief cortical stimulation results in a triphasic response in the substantia nigra pars reticulata (SNr, output of the BG). The properties of the triphasic responses are shaped by dopamine levels. While it is relatively well understood how changes in BG result in aberrant steady state activity, it is not clear which BG interactions are crucial for the aberrant transient responses in the BG. Moreover, it is also not clear whether the same or different mechanisms underlie the aberrant changes in steady-state activity and aberrant transient response. Here we used numerical simulations of a network model of BG to identify the key factors that determine the shape of the transient responses. We show that an aberrant transient response of the SNr in low-dopamine state, involves changes in both, the direct pathway and the recurrent interactions within the globus pallidus externa (GPe) and between GPe and sub-thalamic nucleus. We found that the connections from D2-type spiny projection neurons to GPe are most crucial in shaping the transient response and by restoring them to their healthy level, we could restore the shape of transient response even in low-dopamine state. Finally, we show that the changes in BG that result in aberrant transient response are also sufficient to generate pathological oscillatory activity.

Significance statementTo understand how changes induced by low-dopamine (e.g. in Parkinsons disease, PD) affect basal ganglia (BG) function, we need to identify the factors that determine the shape of BG responses to brief cortical stimuli. We show that transient response of the BG is also affected by recurrent interactions within the subnuclei of the BG, and not just feedforward pathways. We found that input and local connectivity within the globus pallidus externa are crucial for shaping the transient response. We also show that the same network changes may underlie both, pathological {beta}-band oscillations and aberrant transient responses. Our results highlight the importance of the recurrent connectivity within the BG and provide a coherent view of emergence of pathological activity in PD.

category: neuroscience


title: Neural stem cells alter nucleocytoplasmic partitioning and accumulate nuclear polyadenylated transcripts during quiescence

abstract: Quiescence is a cellular state characterised by reversible cell-cycle arrest and diminished biosynthetic activity that protects against environmental insults, replicative exhaustion and proliferation-induced mutations1. Entry into and exit from this state controls development, maintenance and repair of tissues plus, in the adult central nervous system, generation of new neurons and thus cognition and mood2-4. Cancer stem cells too can undergo quiescence, which confers them resistance to current therapies5, 6. Despite clinical relevance, quiescence is poorly understood and is defined functionally given lack of molecular markers. Decrease of the most resource-intensive cellular process of protein synthesis is a feature of quiescence, controlled across species and cell types by inhibition of the Target of Rapamycin (TOR) pathway1, 7. Here, we combine Drosophila genetics and a mammalian model to show that altered nucleocytoplasmic partitioning and nuclear accumulation of polyadenylated RNAs are novel evolutionarily conserved hallmarks of quiescence regulation. Furthermore, nuclear accumulation of messenger RNA (mRNA) in quiescent NSCs (qNSCs) largely predicts protein downregulation, accounting for uncoupling between transcriptome and proteome in quiescence. These mechanisms provide a previously unappreciated regulatory layer to reducing protein synthesis in quiescent cells, whilst priming them for reactivation in response to appropriate cues.

category: developmental biology


title: Environmental enrichment delays the development of stereotypic behavior and reduces variability in behavioral experiments using California mice (Peromyscus californicus).

abstract: Domesticated mice and rats have shown to be powerful model systems for biomedical research, but there are cases in which the biology of species is a poor match for the hypotheses under study. The California mouse (Peromyscus californicus) has unique physiological and behavioral traits and has emerged as a powerful model for studying sex differences in the biology of psychiatric disease, which is particularly relevant considering the new NIH guidelines that require the inclusion of sex as a biological variable. Despite its growing role in preclinical research, there is a lack of studies assessing species-specific housing needs, which presents a challenge for research facilities seeking to ensure good welfare and obtaining high-quality experimental data. Indeed, captive California mice present a high prevalence of stereotypic backflipping behavior, a common consequence of suboptimal housing and a potential source of experimental outcome variability. Using three different cage systems, the present studies show that increasing housing space as well as social and environmental complexity can delay the development of stereotypic behavior in male and female California mice. Critically, this reduction in stereotypy is accompanied by increased effect sizes of stress in an established model for social anxiety. These results suggest that increased cage size and enrichment could enhance welfare in California mice while simultaneously increasing the quality of behavioral experiments.

category: animal behavior and cognition


title: West Nile virus detection in horses in three Brazilian states.

abstract: We report genetic evidence of WNV circulation from southern and northeastern Brazilian states isolated from equine red blood cells. In the northeastern state the tenth human case was also detected, presenting neuroinvasive disease compatible with WNV infection. Our analyses demonstrate that much is still unknown on the virus local epidemiology. We advocate for a shift to active surveillance, to ensure adequate control for future epidemics with spill-over potential to humans.


title: Genomic signatures of UV resistance evolution in Escherichia coli depend on the growth phase during exposure

abstract: Physiological states can determine the ability of organisms to handle stress. Does this mean that the same selection pressure will lead to different evolutionary outcomes, depending on the organisms physiological state? If yes, what will be the genomic signatures of such adaptation(s)? We used experimental evolution in Escherichia coli followed by whole-genome whole-population sequencing to investigate these questions. The sensitivity of Escherichia coli to ultraviolet (UV) radiation depends on the growth phase during which it experiences the radiation. We evolved replicate E. coli populations under two different conditions of UV exposures, namely exposure during the lag and the exponential growth phases. Initially, the UV sensitivity of the ancestor was greater during the exponential phase than the lag phase. However, at the end of 100 cycles of exposure, UV resistance evolved to similar extents in both treatments. Genome analysis showed that mutations in genes involved in DNA repair, cell membrane structure and RNA polymerase were common in both treatments. However, different functional groups were found mutated in populations experiencing lag and exponential UV treatment. In the former, genes involved in transcriptional and translational regulations and cellular transport were mutated, whereas the latter treatment showed mutations in genes involved in signal transduction and cell adhesion. Interestingly, the treatments showed no phenotypic differences in a number of novel environments. Taken together, these results suggest that selection pressures at different physiological stages can lead to differences in the genomic signatures of adaptation, which need not necessarily translate into observable phenotypic differences.


title: Fast and Accurate Influenza Forecasting in the United States with Inferno

abstract: Infectious disease forecasting is an emerging field and has the potential to improve public health through anticipatory resource allocation, situational awareness, and mitigation planning. By way of exploring and operationalizing disease forecasting, the U.S. Centers for Disease Control and Prevention (CDC) has hosted FluSight since the 2013/14 flu season, an annual flu forecasting challenge. Since FluSights onset, forecasters have developed and improved forecasting models in an effort to provide more timely, reliable, and accurate information about the likely progression of the outbreak. While improving the predictive performance of these forecasting models is often the primary objective, it is also important for a forecasting model to run quickly, facilitating further model development, improvement, and scalability. In this vein I introduce Inferno, a fast and accurate flu forecasting model inspired by Dante, the top performing model in the 2018/19 FluSight challenge. When compared to all models that participated in FluSight 2018/19, Inferno would have placed 2nd in both the national and state challenges, behind only Dante. Inferno, however, runs in minutes and is trivially parallelizable, while Dante takes hours to run, representing a significant operational improvement with minimal impact to performance. A future consideration for forecasting competitions like FluSight will be how to encourage improvements to secondarily important properties of forecasting models, such as runtime, generalizability, and interpretability.

category: bioinformatics


title: Physically asymmetric division of the C. elegans zygote ensures invariably successful embryogenesis

abstract: Asymmetric divisions that yield daughter cells of different sizes are frequent during early embryogenesis, but the importance of such a physical difference for successful development remains poorly understood. Here, we investigated this question using the first division of C. elegans embryos, which yields a large AB cell and a small P1 cell. We equalized AB and P1 sizes using acute genetic inactivation or optogenetic manipulation of the spindle positioning protein LIN-5. We uncovered that only some embryos tolerated equalization, and that there was a size asymmetry threshold for viability. Cell lineage analysis of equalized embryos revealed an array of defects, including faster cell cycle progression in P1 descendants, as well as defects in cell positioning, division orientation and cell fate. Moreover, equalized embryos were more susceptible to external compression. Overall, we conclude that unequal first cleavage is essential for invariably successful embryonic development of C. elegans.


title: AusTraits: a curated plant trait database for the Australian flora

abstract: We introduce the AusTraits database - a compilation of measurements of plant traits for taxa in the Australian flora (hereafter AusTraits). AusTraits synthesises data on 375 traits across 29230 taxa from field campaigns, published literature, taxonomic monographs, and individual taxa descriptions. Traits vary in scope from physiological measures of performance (e.g. photosynthetic gas exchange, water-use efficiency) to morphological parameters (e.g. leaf area, seed mass, plant height) which link to aspects of ecological variation. AusTraits contains curated and harmonised individual-, species- and genus-level observations coupled to, where available, contextual information on site properties. This data descriptor provides information on version 2.1.0 of AusTraits which contains data for 937243 trait-by-taxa combinations. We envision AusTraits as an ongoing collaborative initiative for easily archiving and sharing trait data to increase our collective understanding of the Australian flora.


title: A syntenin-deficient microenvironment educates AML for aggressiveness

abstract:
In acute myeloid leukemia (AML), the stromal microenvironment plays a prominent role in promoting tumor cell survival and progression. Although widely explored, the crosstalk between leukemic and stromal cells remains poorly understood. Syntenin, a multi-domain PDZ protein, controls both the trafficking and signaling of key molecules involved in intercellular communication. Therefore, we aimed to clarify the role of environmental syntenin in the progression of AML. By in vivo approaches in syngeneic mice, we demonstrate that a syntenin-deficient environment reprograms AML blasts to survive independently of the stroma. Up-regulation of EEF1A2 in the blasts controls this gain of cell survival. Furthermore, using ex vivo co-culture systems, we show that syntenin-deficient bone marrow stromal cells (BMSC) enhance the survival of different types of AML cells, including patient samples, and suffice to educate syngeneic AML, recapitulating micro-environmental effects observed in vivo. We establish that syntenin-deficiency causes an increase of eIF5A and autophagy-related factors in BMSC, and provide evidence that the inhibition of autophagy prevents syntenin-deficient BMSC to stimulate AML survival. Altogether, these findings indicate that host-syntenin in the BM microenvironment acts as a repressor of AML aggressiveness.

Key points- A syntenin-deficient host reprograms AML blasts, enhancing total protein synthesis and cell survival pathways
- Autophagy in the syntenin-deficient microenvironment is responsible for the gain of AML cell survival

category: cancer biology


title: Compensating complete loss of signal recognition particle during co-translational protein targeting by the translation speed and accuracy

abstract: Signal recognition particle (SRP) is critical for delivering co-translational proteins to the bacterial inner membrane. Previously, we identified SRP suppressors in Escherichia coli that inhibit translation initiation and elongation, which provided insights into the mechanism of bypassing the requirement of SRP. Suppressor mutations tended to be located in regions that govern protein translation under evolutionary pressure. To test this hypothesis, we re-executed the suppressor screening of SRP. Here we isolated a novel SRP suppressor mutation located in the Shine-Dalgarno sequence of the S10 operon, which partially offset the targeting defects of SRP-dependent proteins. We found that the suppressor mutation decreased the protein translation rate, which extended the time window of protein targeting. This increased the possibility of the correct localization of inner membrane proteins. Furthermore, the fidelity of translation was decreased in suppressor cells, suggesting that the quality control of translation was inactivated to provide an advantage in tolerating toxicity caused by the loss of SRP. Our results demonstrated that the inefficient protein targeting due to SRP deletion can be rescued through modulating translational speed and accuracy.

category: microbiology


title: Phosphorylation of the selective autophagy receptor TAX1BP1 by canonical and noncanonical IκB kinases promotes its lysosomal localization and clearance of MAVS aggregates

abstract:
TAX1BP1 is a selective autophagy receptor which inhibits NF-{kappa}B and RIG-I-like receptor (RLR) signaling to prevent excessive inflammation and maintain homeostasis. Selective autophagy receptors such as p62/SQSTM1 and OPTN are phosphorylated by the noncanonical I{kappa}B kinase TBK1 to stimulate their selective autophagy function. However, it is unknown if TAX1BP1 is regulated by TBK1 or other kinases under basal conditions or during RNA virus infection. Here, we found that the noncanonical I{kappa}B kinases TBK1 and IKKi phosphorylate TAX1BP1 to regulate its basal turnover, whereas the canonical I{kappa}B kinase IKK and the core autophagy factor ATG9 play essential roles in RNA virus-mediated TAX1BP1 autophagosomal degradation. TAX1BP1 phosphorylation by canonical and noncanonical I{kappa}B kinases promotes its localization to lysosomes resulting in its degradation. Furthermore, TAX1BP1 plays a critical role in the clearance of MAVS aggregates, and phosphorylation of TAX1BP1 augments its MAVS aggrephagy function. Together, our data support a model whereby I{kappa}B kinases license TAX1BP1 selective autophagy function to inhibit MAVS and RLR signaling.

Author SummaryThe RIG-I-like receptor (RLR) pathway induces type I interferon (IFN) and proinflammatory cytokines in response to RNA virus infection. MAVS is a mitochondrial adaptor protein in the RLR pathway that forms prion-like aggregates upon activation; however, how MAVS aggregates are cleared to restore homeostasis is unclear. Autophagy is a lysosomal degradation pathway important for the clearance of potentially cytotoxic protein aggregates that could induce inflammation and/or cell death. TAX1BP1 is a selective autophagy receptor that inhibits RLR signaling, but the precise mechanisms remain unknown. Here, we found that TAX1BP1 is a substrate for multi-site phosphorylation by canonical and noncanonical I{kappa}B kinases which triggered its lysosomal localization and degradation. We also found that TAX1BP1 was critical for the clearance of MAVS aggregates in a phosphorylation-dependent manner. Overall, our data suggest that phosphorylation serves a key regulatory function for TAX1BP1 to inhibit RLR signaling.


title: SLTRiP induces long lasting and protective T-cell memory response

abstract: Major developments have been made in the past many years to characterize and explore potential vaccine candidates that can induce host immune responses against parasite. These advances were based on the fact that the induction of host immune responses could effectively target parasite at different stages of its life cycle and thus, abrogate Plasmodium infections. The role of T-cells against malaria comes from initial studies on rodents showing these cells could inhibit parasite development during pre-erythrocytic stages. Since then, the importance of the cellular immune responses against malaria has been increasingly emphasized, especially for vaccine development against pre-erythrocytic stages. Previous work in our laboratory has confirmed that SLTRiP confers protection against the pre-erythrocytic stage of Plasmodium growth in rodents. Here we report that the protection is mainly due to cell mediated immune responses and PbSLTRiP specific cellular memory responses could be efficiently recalled in mice challenged with P. berghei parasites even after a year following immunization. Our results thereby, highlight the role of the T cell response involved in protection. Characterization of T-cells by intracellular cytokine staining (ICS) revealed that the induced T cells were polyfunctional and involved in secretion of pro-inflammatory cytokines which mediate anti-parasitic activity. The findings contribute to our understanding of the immunological mechanisms underlying the protective vaccines.


title: Hypertrophied human adipocyte spheroids as in vitro model of weight gain and adipose tissue dysfunction

abstract: The rise in obesity prevalence has created an urgent need for new and improved methods to study human adipocytes and the pathogenic effects of weight gain in vitro. Despite numerous studies showing the advantages of culturing adipocyte progenitors as 3D structures, the majority continue using traditional 2D cultures which result in small, multilocular adipocytes with poor representability. We hypothesized that providing differentiating pre-adipocytes with a vascular growth niche would mimic in vivo adipogenesis and improve the differentiation process. Here we present a simple, easily applicable culture protocol that allows for the differentiation and culturing of human adipocytes with a more unilocular morphology and larger lipid droplets than previous protocols. We moreover offer a protocol for inducing adipocyte enlargement in vitro, resulting in larger lipid droplets and development of several key features of adipocyte dysfunction, including altered adipokine secretions and impaired lipolysis. Taken together, our hypertrophied human adipocyte spheroids offer an improved culture system for studying the cellular and molecular mechanisms causing metabolic dysfunction and inflammation during weight gain.


title: Gradual differentiation uncoupled from cell cycle exit generates heterogeneity in the epidermal stem cell layer.

abstract:
High turnover tissues continually lose specialized cells that are replaced by stem cell activity. In the adult mammalian epidermis, it is unclear how molecularly heterogenous stem/progenitor cell populations fit into the complete trajectory of epidermal differentiation. We show that differentiation, from commitment to exit from the stem cell layer, is a multi-day process wherein cells transit through a continuum of transcriptional changes. Differentiation-committed cells remain capable of dividing to produce daughter cells fated to further differentiate, demonstrating that differentiation is uncoupled from cell cycle exit. These cell divisions are not required as part of an obligate transit amplifying program but instead protect density in the stem cell layer. Thus, instead of distinct contributions from multiple progenitors, a continuous gradual differentiation process fuels homeostatic epidermal turnover.

One sentence summaryHeterogeneity in the epidermal stem cell layer reflects a gradual differentiation program that is uncoupled from the loss of proliferative capacity.