title: scTour: a deep learning architecture for robust inference and accurate prediction of cellular dynamics

abstract: Despite the continued efforts to computationally dissect developmental processes using single-cell genomics, a batch-unaffected tool that is able to both infer and predict the underlying dynamics is lacking. Here, I present scTour, a novel deep learning architecture to perform robust inference and accurate prediction of the cellular dynamics in diverse processes. For inference, scTour can efficiently and simultaneously estimate the developmental pseudotime, intronic read-independent vector field, and transcriptomic latent space under a single, integrated framework. For prediction, scTour can precisely reconstruct the underlying dynamics of unseen cellular states or an independent dataset agnostic to the model. Of note, both the inference and prediction are invariant to batch effects. scTours functionalities are successfully applied to a variety of biological processes from 16 datasets such as cell differentiation, reprogramming and zonation, providing a comprehensive infrastructure to investigate the cellular mechanisms underpinning development in an efficient manner.


title: Metastatic potential in clonal melanoma cells is driven by a rare, early-invading subpopulation

abstract: Metastasis occurs when tumor cells leave the primary tumor site and disseminate to distal organs. Most cells remain in the primary tumor, however, the circumstances that allow certain cells to drive this dissemination remain unclear. Here, we show that rare, highly invasive melanoma cells can appear even within clonal cell lines due to non-genetic fluctuations. These differences were intrinsic to the cells independent of their external context, and were marked by transiently high levels of SEMA3C expression. The invasive subpopulation drove dissemination of tumor cells to distal locations in a mouse model of melanoma. The transcription factor NKX2.2 emerged as a regulator of the proportion of invasive cells. There was an overall tradeoff between proliferation and invasion in single cells. Our results suggest that phenotypes like metastasis can be the result of intrinsic differences between single cells.

category: cancer biology


title: Immunological Synapse Dynamics Induced by Bispecific T-cell Engagers Predict Clinical Pharmacodynamics and Tumor Evolution across Anatomical Sites

abstract: Bispecific T cell engagers (BiTEs) engage T cells for anti-tumor activity. Despite early successes such as blinatumomab, the therapeutic potential of BiTEs for cancer immunotherapy remains limited by low response rates and high potential for relapse. Formation of the immunological synapse (IS) is principal to BiTE pharmacology and BiTE-directed lysis of tumor cells by T cells. Unfortunately, our knowledge of BiTE-induced IS formation dynamics and its relation to treatment resistance remains rudimentary. This work develops an integrated experimental and theoretical approach to investigate IS formation dynamics induced by BiTEs under diverse conditions. Our theoretical models recapitulate experimental observations by defining IS formation as spatiotemporally coordinated events driven by molecular and cellular interactions. These models adequately predicted tumor-killing profiles in patients and revealed a trajectory of antigen escape and tumor evolution across anatomical sites, particularly highlighting the role of the bone marrow in disease relapse. Our work yields implications for developing more effective cancer immunotherapies.

category: pharmacology and toxicology


title: A systems biology approach to disentangle the direct and indirect effects of global transcription factors on gene expression in Escherichia coli

abstract: Delineating the pleiotropic effects of global transcriptional factors (TFs) is critical in understanding the system-wide regulatory response in a particular environment. Currently, with the availability of genome-wide TF binding and gene expression data for Escherichia coli, several gene targets can be assigned to the global TFs albeit inconsistently. Here, using a systematic integrated approach with emphasis on metabolism, we characterize and quantify the direct effects as well as the growth-rate mediated indirect effects of global TFs using deletion mutants of FNR, ArcA, and IHF regulators under glucose fermentative condition. Apart from the previous known metabolite-TF interactions, we predicted with high confidence several novel interactions arising from the allosteric effects of the intracellular metabolites. Our combined experimental and compendium level computational analyses revealed the co-expressed genes regulated by these TFs as well as the coordination of the direct and indirect target gene expressions in the context of the economy of intracellular metabolites. Besides, we extended our analysis to combinatorial deletions of these TFs to determine their cross-talk effects as well as conserved patterns of regulatory interactions. Overall, this study leverages the fundamentals of TF-driven regulation which could serve as a better template for deciphering mechanisms underlying complex phenotypes.

category: systems biology


title: Organelle-selective click labeling coupled with flow cytometry allows high-throughput CRISPR screening of genes involved in phosphatidylcholine metabolism

abstract: Lipids comprise biomembranes and are involved in many crucial cell functions. While cellular lipid synthesis and transport appear to be governed by intricate protein networks, the whole scheme is insufficiently understood. Although functional genome-wide screening should contribute to deciphering the regulatory networks of lipid metabolism, technical challenges remain - especially for high-throughput readouts of lipid phenotypes. Here, we coupled organelle-selective click labeling of phosphatidylcholine (PC) with flow cytometry-based CRISPR screening technologies to convert organellar PC phenotypes into a simple fluorescence readout for genome-wide screening. This technique, named O-ClickFC, was successfully applied in genome-scale CRISPR-knockout screens to identify previously reported genes associated with PC synthesis (PCYT1A, ACACA), vesicular membrane trafficking (SEC23B, RAB5C), and non-vesicular transport (PITPNB, STARD7). Moreover, this work revealed previously uncharacterized roles of FLVCR1 as a new choline transporter; CHEK1 as a post-translational regulator of the PC-synthetic pathway, and TMEM30A as responsible for translocation of PC to the outside of the plasma membrane bilayer. These findings demonstrate the versatility of O-ClickFC as an unprecedented platform for genetic dissection of cellular lipid metabolism.

category: cell biology


title: Green Lacewing Chrysoperla rufilabris (Neuroptera: Chrysopidae) is a potential biological agent for controlling crapemyrtle bark scale (Hemiptera: Eriococcidae)

abstract: Crapemyrtle bark scale (CMBS; Acanthococcus lagerstroemiae), an invasive sap-sucking hemipteran, has spread across 16 U.S. states. Infestation of CMBS negatively impacts the flowering of crapemyrtles and reduces the aesthetic quality of ornamental plants. The widespread use of soil-applied neonicotinoid insecticides to repress the CMBS infestation could threaten important beneficial insects; therefore, using natural enemies to control CMBS is greatly needed. This study evaluated larval green lacewing (Chrysoperla rufilabris) as a biocontrol agent of CMBS. Predatory behavior of the larval C. rufilabris upon CMBS was documented under a stereomicroscope using infested crapemyrtle samples collected from different locations in College Station. Predation potential of C. rufilabris upon CMBS eggs and foraging performance using Y-maze assay were both investigated in laboratory conditions. Results confirmed that larval C. rufilabris preyed on CMBS nymphs, eggs, and adult females. The evaluation of predation potential results showed that the number of CMBS eggs consumed in 24 hours by 3rd instar C. rufilabris (176.4 {+/-}6.9) was significantly higher than by 2nd instar (151.5{+/-}6.6) and by 1st instar (11.8{+/-}1.3). The foraging performance results showed that larval C. rufilabris could target CMBS under dark, indicating that some cues associated with olfactory response were likely involved when preying on CMBS. This study is the first report that validated C. rufilabris as a natural predator of CMBS and its potential as a biological agent to control CMBS. Future investigation about the olfactory response of larval C. rufilabris to CMBS would benefit the development of environmental-friendly strategies to control CMBS spread.

category: ecology


title: Comparative analysis of temperature preference behavior and effects of temperature on daily behavior in eleven Drosophila species

abstract: Temperature is one of the most critical environmental factors that influence various biological processes. Species distributed in different temperature regions are considered to have different optimal temperatures for daily life activities. However, how organisms have acquired various features to cope with particular temperature environments remains to be elucidated. In this study, we have systematically analyzed the temperature preference behavior and effects of temperatures on daily locomotor activity and sleep in 11 Drosophila species. We also investigated the function of antennae in the temperature preference behavior of these species. We found that, (1) an optimum temperature for daily locomotor activity and sleep of each species approximately matches with temperatures it frequently encounters in its habitat, (2) effects of temperature on locomotor activity and sleep are diverse among species, but each species maintains its daily activity and sleep pattern even at different temperatures, and (3) each species has a unique temperature preference behavior, and the contribution of antennae to this behavior is diverse among species. These results suggest that Drosophila species inhabiting different climatic environments have acquired species-specific temperature response systems according to their life strategies. This study provides fundamental information for understanding the mechanisms underlying their temperature adaptation and lifestyle diversification.

category: evolutionary biology


title: Highly parallelized laboratory evolution of wine yeasts for enhanced metabolic phenotypes

abstract: Adaptive Laboratory Evolution (ALE) of microbes can improve the efficiency of sustainable industrial processes important to the global economy, but chance and genetic background effects often lead to suboptimal outcomes. Here we report an ALE platform to circumvent these flaws through parallelized clonal evolution at an unprecedented scale. Using this platform, we clonally evolved 104 yeast populations in parallel from many strains for eight desired wine production traits. Expansions of both ALE replicates and lineage numbers broadened the evolutionary search spectrum and increased the chances of evolving improved wine yeasts unencumbered by unwanted side effects. ALE gains often coincided with distinct aneuploidies and the emergence of semi-predictable side effects that were characteristic of each selection niche. Many high performing ALE strains retained their desired traits upon transfer to industrial conditions and produced high quality wine. Overall, our ALE platform brings evolutionary engineering into the realm of high throughput science and opens opportunities for rapidly optimizing microbes for use in many industrial sectors which otherwise could take many years to accomplish.

category: bioengineering


category: molecular biology


title: gr Predictor: a Deep-Learning Model for Predicting the Hydration Structures around Proteins

abstract:
Among the factors affecting biological processes such as protein folding and ligand binding, hydration, which is represented by a three-dimensional water-site-distribution-function around the protein, is crucial. The typical methods for computing the distribution functions, including molecular dynamics simulations and the three-dimensional reference interaction site model (3D-RISM) theory, require a long computation time from hours to tens of hours. Here, we propose a deep-learning model rapidly estimating the distribution functions around proteins obtained by the 3D-RISM theory from the protein 3D structure. The distribution functions predicted using our deep-learning model are in good agreement with those obtained by the 3D-RISM theory. Particularly, the coefficient of determination between the distribution function obtained by the deep-learning model and that obtained using the 3D-RISM theory is approximately 0.98. Furthermore, using a graphics processing unit (GPU), the calculation by the deep learning model is completed in less than one minute, more than 2 orders of magnitude faster than the calculation time of 3D-RISM theory. Therefore, our deep learning model provides a practical and efficient way to calculate the three-dimensional water-site-distribution-functions. The program called "gr Predictor" is available under the GNU General Public License from https://github.com/YoshidomeGroup-Hydration/gr-predictor.

Table of Contents graphic

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category: biophysics


title: Roles of Clamp Closing and Allosteric Effects of Discriminator and Upstream Interactions on Downstream Elements in Stabilizing an E. coli RNA Polymerase-Promoter Open Complex

abstract: E. coli RNA polymerase (2{beta}{beta}{omega}{sigma}70) forms stable open complexes (OC) at {lambda}PR and T7A1 promoters at 37 {degrees}C at similar rates but with very different lifetimes. To probe the origins of these differences, we determine OC lifetimes for full-length (FL) and downstream-truncated (DT) {lambda}PR and T7A1 promoter variants with eight combinations of discriminators and upstream elements. We find the discriminator is the major determinant of OC lifetime, while upstream elements modulate the discriminator effect. The very different lifetimes of these stable OC arise primarily from differences in [Formula], the free energy change for converting I2 (the initial open intermediate) to stable OC. [Formula] is twice as favorable for {lambda}PR-discriminator variants as for T7A1-discriminator variants. Truncation at +6 (DT+6) eliminates most differences in [Formula], destabilizing {lambda}PR-discriminator variants without affecting T7A1-discriminator variants. All DT+6 OC are much more stable and longer-lived than I2. Urea greatly destabilizes OC, primarily affecting RNAP interactions with +6 and upstream DNA. From these findings we deduce that stabilization of I2 involves closing the {beta}-clamp with extensive coupled folding to contact the region of promoter DNA from the transcription start-site to +6. Lifetime differences between DT+6, DT+12 and FL promoters allow dissection of contributions to OC stability from interactions of regions of the downstream duplex with downstream mobile elements (DME; {beta}-lobe, {beta}-clamp, {beta}-jaw). We propose an allosteric network by which differences in discriminator and -10 sequence are sensed by {sigma}1.2 and {beta}-gate loop and transmitted to {sigma}1.1 and {beta}-lobe to affect DME-duplex interactions that determine OC lifetime.


title: Benchmarking the Widely Used Structure-based Binding Affinity Predictors on the Spike-ACE2 Deep Mutational Interaction Set

abstract: Since the start of COVID-19 pandemic, a huge effort has been devoted to understanding the Spike-ACE2 recognition mechanism. To this end, two recent deep mutational scanning studies traced the impact of all possible mutations/variants across the Spike-ACE2 interface. Expanding on these studies, we benchmarked four widely used structure-based binding affinity predictors (FoldX, EvoEF1, MutaBind2, SSIPe) and two naive predictors (HADDOCK, UEP) on the variant Spike-ACE2 deep mutational interaction set. Among these approaches, FoldX ranked first with a 64% success rate, followed by EvoEF1 with a 57% accuracy. Upon performing residue-based analyses, we revealed critical algorithmic biases, especially in ranking mutations with increasing/decreasing hydrophobicity/volume. We also showed that the approaches using evolutionary-based terms in their affinity predictions classify most mutations as affinity depleting. These observations suggest plenty of room to improve the conventional affinity predictors for predicting the binding affinity change of the viral host-pathogen system SARS-CoV-2-ACE2. To aid the improvement of the available approaches, we provide our mutant models, together with our benchmarking data at https://github.com/CSB-KaracaLab/ace2-rbd-point-mutation-benchmark

category: bioinformatics


title: In Silico transcriptional analysis of asymptomatic and severe COVID-19 patients reveals the susceptibility of severe patients to other comorbidities and non-viral pathological conditions

abstract: COVID-19 is a severe respiratory disease caused by SARS-CoV-2, a novel human coronavirus. The host response to SARS-CoV-2 infection is not clearly understood. Patients infected with SARS-CoV-2 exhibit heterogeneous intensity of symptoms, i.e., asymptomatic, mild, and severe. Moreover, effects on organs also vary from person to person. These heterogeneous responses pose pragmatic hurdles for implementing appropriate therapy and management of COVID-19 patients. Post-COVID complications pose another major challenge in managing the health of these patients. Thus, understanding the impact of disease severity at the molecular level is vital to delineate the precise host response and management. In the current study, we performed a comprehensive transcriptomics analysis of publicly available seven asymptomatic and eight severe COVID-19 patients. Exploratory data analysis using Principal Component Analysis (PCA) showed the distinct clusters of asymptomatic and severe patients. Subsequently, the differential gene expression analysis using DESeq2 identified 1,224 significantly upregulated genes (logFC>= 1.5, p-adjusted value <0.05) and 268 significantly downregulated genes (logFC<= -1.5, p-adjusted value <0.05) in severe samples in comparison to asymptomatic samples. Eventually, Gene Set Enrichment Analysis (GSEA) of upregulated genes revealed significant enrichment of terms, i.e., anti-viral and anti-inflammatory pathways, secondary infections, Iron homeostasis, anemia, cardiac-related, etc. Gene set enrichment analysis of downregulated genes indicates lipid metabolism, adaptive immune response, translation, recurrent respiratory infections, heme-biosynthetic pathways, etc. In summary, severe COVID-19 patients are more susceptible to other health issues/concerns, non-viral pathogenic infections, atherosclerosis, autoinflammatory diseases, anemia, male infertility, etc. And eventually, these findings provide insight into the precise therapeutic management of severe COVID-19 patients and efficient disease management.


title: Persistent post-COVID-19 smell loss is associated with inflammatory infiltration and altered olfactory epithelial gene expression

abstract: Most human subjects infected by SARS-CoV-2 report an acute alteration in their sense of smell, and more than 25% of COVID patients report lasting olfactory dysfunction. While animal studies and human autopsy tissues have suggested mechanisms underlying acute loss of smell, the pathophysiology that underlies persistent smell loss remains unclear. Here we combine objective measurements of smell loss in patients suffering from post-acute sequelae of SARS-CoV-2 infection (PASC) with single cell sequencing and histology of the olfactory epithelium (OE). This approach reveals that the OE of patients with persistent smell loss harbors a diffuse infiltrate of T cells expressing interferon-gamma; gene expression in sustentacular cells appears to reflect a response to inflammatory signaling, which is accompanied by a reduction in the number of olfactory sensory neurons relative to support cells. These data identify a persistent epithelial inflammatory process associated with PASC, and suggests mechanisms through which this T cell-mediated inflammation alters the sense of smell.

category: neuroscience


title: The SARS-CoV-2 Omicron BA.1 spike G446S potentiates HLA-A*24:02-restricted T cell immunity

abstract: Although the Omicron variant of the SARS-CoV-2 virus is resistant to neutralizing antibodies, it retains susceptibility to cellular immunity. Here, we characterized vaccine-induced T cells specific for various SARS-CoV-2 variants and identified HLA-A*24:02-restricted CD8+ T cells that strongly suppressed Omicron BA.1 replication. Mutagenesis analyses revealed that a G446S mutation, located just outside the N-terminus of the cognate epitope, augmented TCR recognition of this variant. In contrast, no enhanced suppression of replication was observed against cells infected with the prototype, Omicron BA.2, and Delta variants that express G446. The enhancing effect of the G446S mutation was lost when target cells were treated with inhibitors of tripeptidyl peptidase II, a protein that mediates antigen processing. These results demonstrate that the G446S mutation in the Omicron BA.1 variant affects antigen processing/presentation and potentiates antiviral activity by vaccine-induced T cells, leading to enhanced T cell immunity towards emerging variants.

category: immunology


title: Ocular tropism of SARS-CoV-2 with retinal inflammation through neuronal invasion in animal models

abstract:
Although ocular manifestations are commonly reported in patients with coronavirus disease 2019 (COVID-19), there is currently no consensus on ocular tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To investigate this, we infected K18-hACE2 mice with SARS-CoV-2 using various routes. We observed ocular manifestation and retinal inflammation with cytokine production in the eyes of intranasally (IN) infected mice. An intratracheal (IT) injection resulted in virus spread from the lungs to the brain and eyes via trigeminal and optic nerves. Ocular and neuronal invasion were confirmed by an intracerebral (IC) infection. Notably, eye-dropped (ED) virus did not infect the lungs and was undetectable with time. Using infectious SARS-CoV-2-mCherry clones, we demonstrated the ocular and neurotropic distribution of the virus in vivo by a fluorescence-imaging system. Evidence for the ocular tropic and neuroinvasive characteristics of SARS-CoV-2 was confirmed in wild-type Syrian hamsters. Our data provides further understanding of the viral transmission; SARS-CoV-2 clinical characteristics; and COVID-19 control procedures.

SummarySARS-CoV-2 can spread from the respiratory tract to the brain and eyes via trigeminal and optic nerves in animal models. This ocular tropism of SARS-CoV-2 through neuronal invasion likely causes ocular manifestation and retinal inflammation.

Graphical Abstract

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category: microbiology


title: Antibody escape and cryptic cross-domain stabilization in the SARS CoV-2 Omicron spike protein

abstract: The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the repeated emergence of variants of concern. The Omicron variant has two dominant sub-lineages, BA.1 and BA.2, each with unprecedented numbers of nonsynonymous and indel spike protein mutations: 33 and 29, respectively. Some of these mutations individually increase transmissibility and enhance immune evasion, but their interactions within the Omicron mutational background is unknown. We characterize the molecular effects of all Omicron spike mutations on expression, human ACE2 receptor affinity, and neutralizing antibody recognition. We show that key mutations enable escape from neutralizing antibodies at a variety of epitopes. Stabilizing mutations in the N-terminal and S2 domains of the spike protein compensate for destabilizing mutations in the receptor binding domain, thereby enabling the record number of mutations in Omicron sub-lineages. Taken together, our results provide a comprehensive account of the mutational effects in the Omicron spike protein and illuminate previously unknown mechanisms of how the N-terminal domain can compensate for destabilizing mutations within the more evolutionarily constrained RBD.


title: Reassessing the helix bundle crossing model for gating in a non-selective ion channel

abstract: A critical part of ion channel function is the ability to open and close in response to stimuli, and thus conduct ions in a regulated fashion. While X-ray diffraction studies of ion channels suggested a general steric gating mechanism located at the helix bundle crossing (HBC), recent functional studies on several channels indicate that the helix bundle crossing is open even in closed, non-conductive channels. Two NaK channel variants were crystallized in very different, open and closed conformations and served as an important model of the HBC gating hypothesis. However, neither of these NaK variants are conductive in liposomes unless phenylalanine 92 is mutated to alanine (F92A). Here we use NMR to probe distances at near-atomic resolution of the two NaK variants in lipid bicelles. We demonstrate that in contrast to the crystal structures, both NaK variants are in a fully open conformation, akin to the well known MthK channel structure were the HBC is widely open. Further inquiry into the gating mechanism suggests that the selectivity filter and pore helix are coupled to the M2 helix below and undergo changes in structure when F92 is mutated. Overall, our data shows that NaK exhibits coupling between the selectivity filter and HBC similar to K+ channels and has a more complex gating mechanism than previously thought.

category: biochemistry


title: A chip-based array for high-resolution fluorescence characterization of free-standing horizontal lipid membranes under voltage clamp

abstract: Optical techniques, such as fluorescence microscopy, are of great value in characterizing the structural dynamics of membranes and membrane proteins. A particular challenge is to combine high-resolution optical measurements with high-resolution voltage clamp electrical recordings providing direct information on e.g. single ion channel gating and/or membrane capacitance. Here, we report on a novel chip-based array device which facilitates optical access with water or oil-immersion objectives of high numerical aperture to horizontal free-standing lipid membranes while conrolling membrane voltage and recroding currents using micropatterned Ag/AgCl-electrodes. We demonstrate both wide-field and confocal imaging, as well as time-resolved single photon counting on free-standing membranes spanning sub-picoliter cavities are demonstrated while electrical signals, including single channel activity, are simultaneously acquired. This optically addressable microelectrode cavity array will allow combined electrical-optical studies of membranes and membrane proteins to be performed as a routine experiment.


title: Coisolation of peptide pairs for peptide identification and MS/MS-based quantification

abstract: SILAC-based metabolic labeling is a widely adopted proteomics approach that enables quantitative comparisons among a variety of experimental conditions. Despite its quantitative capacity, SILAC experiments analyzed with data dependent acquisition (DDA) do not fully leverage peptide pair information for identification and suffer from undersampling compared to label-free proteomic experiments. Herein, we developed a data dependent acquisition strategy that coisolates and fragments SILAC peptide pairs and uses y-ions for their relative quantification. To facilitate the analysis of this type of data, we adapted the Comet sequence database search engine to make use of SILAC peptide paired fragments and developed a tool to annotate and quantify MS/MS spectra of coisolated SILAC pairs. In an initial feasibility experiment, this peptide pair coisolation approach generally improved expectation scores compared to the traditional DDA approach. Fragment ion quantification performed similarly well to precursor quantification in the MS1 and achieved more quantifications. Lastly, our method enables reliable MS/MS quantification of SILAC proteome mixtures with overlapping isotopic distributions, which are difficult to deconvolute in MS1-based quantification. This study demonstrates the initial feasibility of the coisolation approach. Coupling this approach with intelligent acquisition strategies has the potential to improve SILAC peptide sampling and quantification.


title: Localization, induction, and cellular effects of tau-phospho-threonine 217

abstract:
IntroductionTau phosphorylation at T217 is a promising AD biomarker, but its functional consequences were unknown.

MethodsHuman brain and cultured mouse neurons were analyzed by immunoblotting and immunofluorescence for total tau, taupT217, taupT181, taupT231 and taupS396/pS404. dSTORM super resolution microscopy was used to localize taupT217 in cultured neurons. EGFP-tau was expressed in fibroblasts as wild type and T217E pseudo-phosphorylated tau, and fluorescence recovery after photobleaching (FRAP) reported tau turnover rates on microtubules.

ResultsIn brain, taupT217 appears in neurons at Braak stages I-II, becomes more prevalent later and co-localizes partially with other phospho-tau epitopes. In cultured neurons taupT217, is increased by extracellular tau oligomers (xcTauOs), and is associated with developing post-synaptic sites. FRAP recovery was fastest for EGFP-tauT217E.

ConclusionTaupT217 increases in brain as AD progresses and is induced by xcTauOs. Post-synaptic taupT217 suggests a role for T217 phosphorylation in synapse impairment. T217 phosphorylation reduces taus affinity for microtubules.


title: Fleeting Effects of Incentives: Adult Age Differences in ERP Measures of Motivated Attention

abstract: Reward-based motivation is associated with transient and sustained dopaminergic activity and with modulatory effects on attention and cognitive control. Age-related changes in the dopamine system are well documented, but little is known about age differences in the temporal dynamics of motivational influences on cognitive functions. The current study examined the effects of financial incentives on visual attention using event-related potentials (ERPs). Participants (26 younger, aged 18-33; 24 older, aged 65-95) completed an incentivized flanker task in which trial-level incentive cues signaled the availability of performance-contingent reward, and subsequent alerting cues signaled the onset of the flanker target. ERP components of interest included incentive-cue P2 and CNV, alerting-cue N1, target N1, and target P3. Transient effects of incentives were assessed by comparing ERP amplitudes across incentive and non-incentive trials from mixed-incentive blocks. Younger adults showed transient effects of incentives on all components, whereas older adults showed effects for incentive-cue P2 and alerting-cue N1 only. Sustained effects of incentives were assessed by comparing ERP amplitudes across non-incentive trials from mixed-incentive blocks and non-incentive trials from pure non-incentive blocks. Both age groups showed sustained effects of incentives on cue-locked ERPs, but only younger adults showed sustained effects on target-locked ERPs. Reaction-time patterns mirrored the ERP findings, in that younger adults showed greater incentive-based modulation than older adults. Overall, these findings suggest that both transient and sustained effects of incentives on visual attention are more fleeting for older than younger adults, consistent with widespread alterations in dopaminergic neuromodulation in aging.


title: Neural instructive signals for associative cerebellar learning

abstract: Supervised learning depends on instructive signals that shape the output of neural circuits to support learned changes in behavior. Climbing fiber inputs to the cerebellar cortex represent one of the strongest candidates in the vertebrate brain for conveying neural instructive signals. However, recent studies have shown that Purkinje cell stimulation can also drive cerebellar learning, and the relative importance of these two neuron types in providing instructive signals for cerebellum-dependent behaviors remains unresolved. Here we used cell-type specific perturbations of climbing fibers, Purkinje cells, and other cerebellar circuit elements to systematically evaluate their contributions to delay eyeblink conditioning. Our findings reveal that while optogenetic stimulation of either climbing fibers or Purkinje cells can substitute for a sensory unconditioned stimulus, subtle reductions in climbing fiber signaling prevent learning entirely. We conclude that climbing fibers and corresponding Purkinje cell complex-spike events provide essential instructive signals for associative cerebellar learning.


title: Pyochelin biotransformation shapes bacterial competition

abstract:
It has become evident that numerous infections are polymicrobial and Pseudomonas aeruginosa and Staphylococcus aureus are among the most frequently isolated co-infecting bacterial species. We applied mass spectrometry guided interaction studies to determine how chemical interaction shapes the fitness and community structure during co-infection of these two pathogens. We demonstrate that S. aureus is equipped with an elegant mechanism, inactivation of pyochelin via the yet uncharacterized methyltransferase Spm (staphylococcal pyochelin methyltransferase), to increase its survival during in vivo competition with P. aeruginosa. Methylation of pyochelin abolishes the siderophore activity of pyochelin and significantly lowers pyochelin-mediated intracellular ROS production in S. aureus. In a murine wound co-infection model, a S. aureus mutant unable to metyhlate pyochelin shows significant lower fitness compared to its parental strain.

One-Sentence SummaryStaphylococcus aureus inactivates pyochelin to increase its survival during in vivo competition with Pseudomonas aeruginosa.


title: The DNA replication initiation protein DnaD is recruited to a specific strand of the Bacillus subtilis chromosome origin

abstract: Genome replication is a fundamental biological activity shared by all organisms. Chromosomal replication proceeds bidirectionally from origins, requiring the loading of two helicases, one for each replisome. The molecular mechanisms for helicase loading at bacterial chromosome origins (oriC) are unclear. Here we investigated the essential DNA replication initiation protein DnaD in the model organism Bacillus subtilis. A set of DnaD residues required for ssDNA binding was identified, and photo-crosslinking revealed that this ssDNA binding region interacts preferentially with one strand of oriC. Biochemical and genetic data support the model that DnaD recognizes a new single-stranded DNA (ssDNA) motif located in oriC (DnaD Recognition Element, "DRE"). Considered with cryo-electron microscopy (cryo-EM) imaging of full length DnaD, we propose that the location of the DRE within the oriC orchestrates strand-specific recruitment of helicase to achieve bidirectional DNA replication. These findings significantly advance our mechanistic understanding of bidirectional replication from a bacterial chromosome origin.


title: SirA inhibits the essential DnaA:DnaD interaction to block helicase recruitment during Bacillus subtilis sporulation

abstract: Bidirectional DNA replication from a chromosome origin requires the asymmetric loading of two helicases, one for each replisome. Our understanding of the molecular mechanisms underpinning helicase loading at bacterial chromosome origins is incomplete. Here we report both positive and negative mechanisms for directing helicase recruitment in the model organism Bacillus subtilis. Systematic characterization of the essential initiation protein DnaD revealed distinct protein interfaces required for homo-oligomerization, interaction with the master initiator protein DnaA, and interaction with the helicase co-loader protein DnaB. Informed by these properties of DnaD, we went on to find that the developmentally expressed repressor of DNA replication initiation, SirA, blocks the interaction between DnaD with DnaA, thereby inhibiting helicase recruitment to the origin during sporulation. These results advance our understanding of the mechanisms underpinning DNA replication initiation in B. subtilis, as well as guiding the search for essential cellular activities to target for antimicrobial drug design.


title: Lithium-induced ciliary lengthening sparks Arp2/3 complex-dependent endocytosis

abstract: Ciliary length is highly regulated across cell types, but this tight regulation can be disrupted by lithium, which causes ciliary elongation across cell types and organisms. Here, we use the powerful ciliary model Chlamydomonas reinhardtii to investigate the mechanism behind lithium-induced ciliary elongation. Protein synthesis is not required for lengthening, and the target of lithium is GSK3, which has substrates that can influence membrane dynamics. Further, in addition to elongation of the microtubule core, ciliary assembly requires a supply of ciliary membrane. To test if the membrane for ciliary lengthening could be from the Golgi or the cell body plasma membrane, we treated cells with either Brefeldin A or Dynasore respectively. Cilia were able to elongate normally with Brefeldin treatment, but Dynasore treatment resulted in defective lengthening. Genetic or acute chemical perturbation of the Arp2/3 complex, which is required for endocytosis in these cells, blocks lithium-induces ciliary lengthening. Finally, we looked at filamentous actin in lithium-treated cells and found an increase in Arp2/3 complex-and endocytosis-dependent puncta near the base of cilia. Blocking endocytosis by inhibiting the Arp2/3 complex or dynamin, confirmed by visual loss of endocytic structures, prevents lithium-induced ciliary elongation. We previously reported that endocytosis was required for early ciliary assembly from zero length, and here, we demonstrate that endocytosis is also required for ciliary elongation from steady state length. Thus, we hypothesize that lithium-induced ciliary elongation occurs through a mechanism that involves a supply of additional ciliary membrane through endocytosis.


title: Extracellular matrix assembly stress drives Drosophila central nervous system morphogenesis

abstract: The forces controlling tissue morphogenesis are attributed to cellular-driven activities and any role for extracellular matrix (ECM) is assumed to be passive. However, all polymer networks, including ECM, can theoretically develop autonomous stresses during their assembly. Here we examine the morphogenetic function of an ECM prior to reaching homeostatic equilibrium by analyzing de novo ECM assembly during Drosophila ventral nerve cord (VNC) condensation. Asymmetric VNC shortening and a rapid decrease in surface area correlate with exponential assembly of Collagen-IV (Col4) surrounding the tissue. Concomitantly, a transient developmentally-induced Col4 gradient leads to coherent long-range flow of ECM, which equilibrates the Col4 network. Finite element analysis and perturbation of Col4 network formation through the generation of dominant Col4-truncations that affect assembly, reveals that VNC morphodynamics is driven by a sudden increase in ECM-driven surface tension. These data highlight that ECM assembly stress and associated network instabilities can actively participate in tissue morphogenesis.

category: developmental biology


title: Genome-wide detection of human variants that disrupt intronic branchpoints

abstract: Pre-mRNA splicing is initiated with the recognition of a single-nucleotide intronic branchpoint (BP) within a BP motif by spliceosome elements. Fifty-six rare variants in 44 human genes have been reported to alter splicing and cause disease by disrupting BP. However, until now, no computational approach has been available to efficiently detect such variants in next-generation sequencing (NGS) data. We established a comprehensive human genome-wide BP database by integrating existing BP data, and by generating new BP data from RNA-seq of lariat debranching enzyme DBR1-mutated patients and from machine-learning predictions. We in-depth characterize multiple features of BP in major and minor introns, and find that BP and BP-2 (two-nucleotides upstream of BP) positions exhibit a lower rate of variation in human populations and higher evolutionary conservation than the intronic background, whilst being comparable to the exonic background. We develop BPHunter as a genome-wide computational approach to systematically and efficiently detect intronic variants that may disrupt BP recognition in NGS data. BPHunter retrospectively identifies 48 of the 56 known pathogenic BP mutations in which we summarize a strategy for prioritizing BP mutation candidates, and the remaining 8 all create AG dinucleotides between BP and acceptor site which is probably the reason for mis-splicing. We demonstrate the utility of BPHunter prospectively by using it to identify a novel germline heterozygous BP variant of STAT2 in a patient with critical COVID-19 pneumonia, and a novel somatic intronic 59-nucleotide deletion of ITPKB in a lymphoma patient, both of which we validate experimentally. BPHunter is publicly available from https://hgidsoft.rockefeller.edu/BPHunter and https://github.com/casanova-lab/BPHunter.

category: genetics


title: Homing pigeon (Columba livia) dominance hierarchies are stable over time and resistant to perturbation

abstract: Dominance hierarchies are a social dynamic common in many species, which help balance the costs and benefits of social living. Which individuals in a group occupy high ranking positions is influenced by a multitude of different intrinsic and/or extrinsic factors. While homing pigeons (Columba livia) have been a model system for their navigational abilities and flight dynamics, less is known about their dominance hierarchies and general social behaviour when not in flight. Here we document the dominance hierarchies in two closed populations of homing pigeons, and investigate the physiological factors associated with dominance rank, including body size, metabolic rate, personality, and iridescent plumage % reflectance. Measurements of body size and resting metabolic rate all positively correlated with dominance rank in accordance with the performance model of energetic management. Iridescent plumage % reflectance was negatively correlated with rank, suggesting a potential cost of dominance. Attempts to alter hierarchy structure through manipulations of body mass and feather iridescence were ineffective which hierarchy dynamics remaining stable throughout the perturbations and across measurement sessions.

category: animal behavior and cognition


title: Sequence dependencies and biophysical features both govern cleavage of diverse cut-sites by HIV protease

abstract: The infectivity of HIV-1 requires its protease cleave multiple cut-sites with low sequence similarity. The diversity of cleavage sites has made it challenging to investigate the underlying sequence properties that determine binding and turnover of substrates by PR. We engineered a mutational scanning approach utilizing yeast display, flow cytometry, and deep sequencing to systematically measure the impacts of all individual amino acid changes at 12 positions in three different cut-sites (MA/CA, NC/p1, and p1/p6). The resulting fitness landscapes revealed common physical features that underlie cutting of all three cut-sites at the amino acid positions closest to the scissile bond. In contrast, positions more than two amino acids away from the scissile bond exhibited a strong dependence on the sequence background of the rest of the cut-site. We observed multiple amino acid changes in cut-sites that led to faster cleavage rates, including a preference for negative charge five and six amino acids away from the scissile bond at locations where the surface of protease is positively charged. Analysis of individual cut sites using full-length matrix-capsid proteins indicate that long-distance sequence context can contribute to cutting efficiency such that analyses of peptides or shorter engineered constructs including those in this work should be considered carefully. This work provides a framework for understanding how diverse substrates interact with HIV-1 protease and can be extended to investigate other viral proteases with similar properties.


title: Flexible control of representational dynamics in a disinhibition-based model of decision making

abstract: Current models utilize two primary circuit motifs to replicate neurobiological decision making. Recurrent gain control implements normalization-driven relative value coding, while recurrent excitation and competitive inhibition together implement winner-take-all (WTA) dynamics. Despite evidence for concurrent valuation and selection computations in individual brain regions, existing models focus selectively on either normalization or WTA dynamics and how both arise in a single circuit architecture is unknown. Here we show that a novel hybrid motif unifies both normalized representation and WTA competition, with dynamic control of circuit state governed by local disinhibition. In addition to capturing empirical psychometric and chronometric data, the model produces persistent activity consistent with working memory. Furthermore, disinhibition provides a simple mechanism for flexible top-down control of network states, enabling the circuit to capture diverse task-dependent neural dynamics. These results suggest a new biologically plausible mechanism for decision making and emphasize the importance of local disinhibition in neural processing.


title: HNRNPH1 destabilizes the G-quadruplex structures formed by G-rich RNA sequences that regulate the alternative splicing of an oncogenic fusion transcript

abstract: In the presence of physiological monovalent cations, thousands of RNA G-rich sequences can form parallel G-quadruplexes (G4s) unless RNA-binding proteins inhibit, destabilize, or resolve the formation of such secondary RNA structures. Here, we have used a disease-relevant model system to investigate the biophysical properties of the RNA-binding protein HNRNPH1s interaction with G-rich sequences. We demonstrate the importance of two EWSR1-exon 8 G-rich regions in mediating the exclusion of this exon from the oncogenic EWS-FLI1 transcripts expressed in a subset of Ewing sarcomas, using complementary analysis of tumor data, long-read sequencing, and minigene studies. We determined that HNRNPH1 binds the EWSR1-exon 8 G-rich sequences with low nM affinities irrespective of whether in a non-G4 or G4 state but exhibits different kinetics depending on RNA structure. Specifically, HNRNPH1 associates and dissociates from G4-folded RNA faster than the identical sequences in a non-G4 state. Importantly, we demonstrate using gel shift and spectroscopic assays that HNRNPH1, particularly the qRRM1-qRRM2 domains, destabilizes the G4s formed by the EWSR1-exon 8 G-rich sequences in a non-catalytic fashion. Our results indicate that HNRNPH1s binding of G-rich sequences favors the accumulation of RNA in a non-G4 state and that this contributes to its regulation of RNA processing.


title: Trophoblast and blood vessel organoid cultures recapitulate the role of WNT2B in promoting intravillous vascularization in human intrauterine and ectopic pregnancy

abstract: Tubal ectopic pregnancy (TEP), a pregnancy complication caused by aberrant implantation in fallopian tubes, accounts for 9-13% pregnancy-related deaths. The lack of models for human TEP hampers the understanding of its pathological mechanisms. Here, we employed multiple models to investigate the crosstalk between human trophoblast development and intravillous vascularization. We found that the severity of TEP, the size of placental villi, and the depth of trophoblast invasion are correlated with the extent of intravillous vascularization. We identified a key pro-angiogenic factor secreted by trophoblasts, WNT2B, that promotes villous vasculogenesis, angiogenesis, and vascular network expansion. In an organoid coculture model consisting of trophoblast organoids and blood vessel organoids, knockdown of WNT2B in trophoblast organoids compromises their pro-angiogenic effect on the development of blood vessel organoids. These organoid-based models reveal an important role for WNT-mediated angiogenesis in pregnancies and could be employed to investigate the commutations between trophoblasts and endothelial/endothelial progenitor cells.


title: Severe drought events trigger long-term salt marsh dieback in the Southeastern and Gulf of Mexico USA

abstract: The frequency of salt marsh dieback events has been increasing in the last 25 years with unknown consequences to the resilience of the ecosystem to accelerated sea level rise (SLR). Sudden dieback events triggered by extreme climatic events were previously thought to recover naturally within a few months to years. We provide evidence that about 15% of total marsh area has not revegetated 10- and 20-years after dieback events in two case studies in the Southeast and Gulf of Mexico USA, respectively. In both case studies, dieback onset coincided with severe drought events. In West Ashley, SC, dieback areas were associated with lower elevation zones in the interior of the marsh. Restoration by grass planting was shown to be effective in the South Carolina urban marsh, indicating that the stressors that caused dieback may be overcome despite that most of dieback areas have not recovered naturally. A mechanistic modelling approach was used to predict the resilience of the South Carolina salt marsh to accelerated SLR under different scenarios of primary productivity, ecological management, and rates of SLR. Predictions indicate that a highly productive restored marsh (2000 g m-2 y-1) would persist at a moderate SLR rate of 60 cm 100 y-1, whereas a non-restored mudflat would rapidly lose all of its elevation capital. Further, at an elevated SLR rate of 122 cm 100 y-1, most likely an extreme climate change scenario, even highly productive ecosystems augmented by sediment placement would not keep pace with SLR. Thus, both climate change adaptation and mitigation actions are urgently needed to preserve highly valuable present-day marsh ecosystems close to urban environments.


title: Transcriptional dynamics of transposable elements in the type I IFN response in Myotis lucifugus cells

abstract:
BackgroundBats are a major reservoir of zoonotic viruses, and there has been growing interest in characterizing bat-specific features of innate immunity and inflammation. Recent studies have revealed bat-specific adaptations affecting interferon (IFN) signaling and IFN- stimulated genes (ISGs), but we still have a limited understanding of the genetic mechanisms that have shaped the evolution of bat immunity. Here we investigated the transcriptional and epigenetic dynamics of transposable elements (TEs) during the type I IFN response in little brown bat (Myotis lucifugus) primary embryonic fibroblast cells, using RNA-seq and CUT&RUN.

ResultsWe found multiple bat-specific TEs that undergo both locus-specific and family-level transcriptional induction in response to IFN. Our transcriptome reassembly identified multiple ISGs that have acquired novel exons from bat-specific TEs, including NLRC5, SLNF5 and a previously unannotated isoform of the IFITM2 gene. We also identified examples of TE-derived regulatory elements, but did not find strong evidence supporting genome-wide epigenetic activation of TEs in response to IFN.

ConclusionCollectively, our study uncovers numerous TE-derived transcripts, proteins, and alternative isoforms that are induced by IFN in Myotis lucifugus cells, highlighting candidate loci that may contribute to bat-specific immune function.


title: genomeSidekick: a user-friendly epigenomics data analysis tool

abstract: Recent advances in epigenomics measurements have resulted in a preponderance of genomic sequencing datasets that require focused analyses to discover mechanisms governing biological processes. In addition, multiple epigenomics experiments are typically performed within the same study, thereby increasing the complexity and difficulty of making meaningful inferences from large datasets. One gap in the sequencing data analysis pipeline is the availability of tools to efficiently browse genomic data for scientists that do not have bioinformatics training. To bridge this gap, we developed genomeSidekick, a graphical user interface written in R that allows researchers to perform bespoke analyses on their transcriptomic and chromatin accessibility or chromatin immunoprecipitation data without the need for command line tools. Importantly, genomeSidekick outputs lists of up- and downregulated genes or chromatin features with differential accessibility or occupancy; visualizes  omics data using interactive volcano plots; performs Gene Ontology analyses locally; and queries PubMed for selected gene candidates for further evaluation. Outputs can be saved using the user interface and the code underlying genomeSidekick can be edited for custom analyses. In summary, genomeSidekick brings wet lab scientists and bioinformaticians into a shared fluency with the end goal of driving mechanistic discovery.

category: genomics


title: Transcriptome-guided annotation and functional classification of long non-coding RNAs in Arabidopsis thaliana

abstract: Long non-coding RNAs (lncRNAs) are a prominent class of eukaryotic regulatory genes. Despite the numerous available transcriptomic datasets, the annotation of plant lncRNAs remains based on dated annotations that have been historically carried over. We present a substantially improved annotation of Arabidopsis thaliana lncRNAs, generated by integrating 224 transcriptomes in multiple tissues, conditions, and developmental stages. We annotate 6764 lncRNA genes, including 3772 that are novel. We characterize their tissue expression patterns and find 1425 lncRNAs are co-expressed with coding genes, with enriched functional categories such as chloroplast organization, photosynthesis, RNA regulation, transcription, and root development. This improved transcription-guided annotation constitutes a valuable resource for studying lncRNAs and the biological processes they may regulate.


title: Phertilizer: growing a clonal tree from ultra-low coverage single-cell DNA sequencing data of tumors

abstract:
MotivationEmerging ultra-low coverage single-cell DNA sequencing (scDNA-seq) technologies have enabled high resolution evolutionary studies of copy number aberrations (CNAs) within tumors. While these sequencing technologies are well suited for identifying CNAs due to the uniformity of sequencing coverage, the sparsity of coverage poses challenges for the study of single-nucleotide variants (SNVs). In order to maximize the utility of increasingly available ultra-low coverage scDNA-seq data and obtain a comprehensive understanding of tumor evolution, it is important to simultaneously analyze the evolution of SNVs alongside CNAs from the same set of tumor cells.

ResultsWe present Phertilizer, a method to infer a clonal tree, capturing both SNV and CNA evolution, from ultra-low coverage single-cell DNA sequencing of a tumor. Based on a probabilistic model, our method recursively partitions the data by identifying key evolutionary events in the history of the tumor. We demonstrate the performance of Phertilizer on simulated data as well as on two real datasets, finding that Phertilizer effectively utilizes the copy-number signal inherent in the data to more accurately uncover clonal structure and genotypes compared to previous methods.

Availabilityhttps://github.com/elkebir-group/phertilizer

Contact{idoia,melkebir}@illinois.edu

Supplementary informationSupplementary data are available at Bioinformatics online.


title: MetaRelSubNetVis: Referenceable network visualizations based on integrated patient data with group-wise comparison

abstract:
MotivationNetworks are a common data structure to describe relations among biological entities. Enriched with information to specify the entities or their connections, they provide a solid foundation for data-dependent visualization. When such annotations overlap, for example in a protein-protein interaction network that is enriched with patient-specific expressions, visualization is reliant on user interaction. Thereby, effective and reliable exchange of visualization parameters between collaborators is crucial to the communication within workflows.

ResultsHere, we introduce MetaRelSubNetVis, a web-based tool that allows users to interactively apply group-wise visualizations to networks augmented with patient data. Our application can visually reflect patient-specific attributes for single patients or in a comparative context. Furthermore, we improved upon the exchange of network visualizations by providing unambiguous links that result in the same visual markup. Our work provides new prospects in interacting with and collaborating on network data, especially with respect to the exchange and integration of network visualizations.

Contactflorian.auer@informatik.uni-augsburg.de


title: Sirtuin gene isoforms and genomic sequences of mouse and humans: Divergence across species

abstract:
BackgroundThere are seven sirtuin genes in the mammalian genome. Each sirtuin gene contains multiple exons, and is likely to undergo alternative splicing, thereby increasing sirtuin gene diversity. Since the alternatively spliced isoforms tend to increase with advancing age, it is important to study the effect of sequence change on isoform function. Additionally, the divergence of isoform patterns between human and mouse will help us to properly interpret the findings from animal models, especially in age-related studies. Recently, more than 20 human sirtuin isoforms have been identified, but whether the mouse genome might have similar isoforms remains incompletely established.

MethodsThe mRNA, protein and genomic DNA sequences of mouse sirtuin genes, as well as the transcription factor binding sites, including that of SRF, were analyzed. A cellular stress model with serum deprivation and restoration was used to reflect the blood supply and nutrients level changes in the ischemia and reperfusion condition, and the expression of sirtuin isoform was assessed.

ResultsHere, we report the identification of 15 mouse sirtuin isoforms, of which over half have not been previously reported. Exon skipping was the main event, which led to domain losses in the nuclear localization signal, nucleolar localization signal, and/or the mitochondrial targeting sequence among sirtuin isoforms. Among 7 sirtuin genes, 6 mouse sirtuin genes had different exon numbers versus that of human sirtuin genes. Only the sirtuin-2 gene had the same number of exons in both human and mouse. However, there were differences in the sirtuin-2 gene isoforms and the regulatory domains between the two species. The expression of sirtuin gene isoforms under serum stress was also different.

ConclusionsAlternative splicing increases both sirtuin transcriptome and proteome diversity. However, the sirtuin isoforms were not well conserved between human and mouse, which should be taken into consideration when extrapolating animal studies for human physiology and pathology. Our results will help to elucidate the role of sirtuin genes in the regulation of cellular stress response, including ischemia and reperfusion. We propose that the existence of the CArG and CArG-like sequences in sirtuin genes may imply a role for SRF in the sirtuin family transcriptional regulation.


title: A coupled atrioventricular-aortic setup for in-vitro hemodynamic study of the systemic circulation: Design, Fabrication, and Physiological relevancy

abstract: In-vitro models of the systemic circulation have gained a lot of interest for fundamental understanding of cardiovascular dynamics and for applied hemodynamic research. In this study, we introduce a physiologically accurate in-vitro hydraulic setup that models the hemodynamics of the coupled atrioventricular-aortic system. This unique experimental simulator has three major components: 1) an arterial system consisting of a human-scale artificial aorta along with the main branches, 2) an artificial left ventricle (LV) sac connected to a programmable piston-in-cylinder pump for simulating cardiac contraction and relaxation, and 3) an artificial left atrium (LA). The setup is designed in such a way that the basal LV is directly connected to the aortic root via an aortic valve, and to the LA via an artificial mitral valve. As a result, two-way hemodynamic couplings can be achieved for studying the effects that the LV, aorta, and LA have on each other. The collected pressure and flow measurements from this setup demonstrate a remarkable correspondence to clinical hemodynamics. We also investigate the physiological relevancies of isolated effects on cardiovascular hemodynamics of various major global parameters found in the circulatory system, including LV contractility, LV preload, heart rate, aortic compliance, and peripheral resistance. Subsequent control over such parameters ultimately captures physiological hemodynamic effects of LV systolic dysfunction, preload (cardiac) diseases, and afterload (arterial) diseases. The detailed design and fabrication of the proposed setup is also provided.


title: GeneticsMakie.jl: A versatile and scalable toolkit for visualizing locus-level genetic and genomic data

abstract: With the continued deluge of results from genome-wide association and functional genomic studies, it has become increasingly imperative to quickly combine and visualize different layers of genetic and genomic data within a given locus to facilitate exploratory and integrative data analyses. While several tools have been developed to visualize locus-level genetic results, the limited speed, scalability, and flexibility of current approaches remains a significant bottleneck. Here, we present a Julia package for high-performance genetics and genomics-related data visualization that enables fast, simultaneous plotting of hundreds of association results along with multiple relevant genomic annotations. Leveraging the powerful plotting and layout utilities from Makie.jl facilitates the customization and extensibility of every component of a plot, enabling generation of publication-ready figures.


title: Modeling COVID-19 disease biology to identify drug treatment candidates

abstract: Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, there are a limited number of effective treatments. A variety of drugs that have been approved for other diseases are being tested for the treatment of COVID-19, and thus far only remdesevir, dexamethasone, baricitinib, tofacitinib, tocilizumab, and sarilumab have been recommended by the National Institutes of Health (NIH) COVID-19 Treatment Guidelines Panel for the therapeutic management of hospitalized adults with COVID-19. Using a disease biology modeling approach, we constructed a protein-protein interactome network based on COVID-19- associated genes/proteins described in research literature together with known protein-protein interactions in epithelial cells. Phenotype and disease enrichment analysis of the COVID-19 disease biology model demonstrated strong statistical enrichments consistent with patients clinical presentation. The model was used to interrogate host biological response induced by SARS-CoV-2 and identify COVID-19 drug treatment candidates that may inform on drugs currently being evaluated or provide insight into possible targets for potential new therapeutic agents. We focused on cancer drugs as they are often used to control inflammation, inhibit cell division, and modulate the host microenvironment to control the disease. From the top 30 COVID-19 drug candidates, twelve have a role as an antineoplastic agent, seven of which are approved for human use. Altogether, nearly 40% of the drugs identified by our model have been identified by others for COVID-19 clinical trials. Disease biology modeling incorporating disease-associated genes/proteins discussed in the research literature together with known molecular interactions in relevant cell types is a useful method to better understand disease biology and identify potentially effective therapeutic interventions.


title: Quantitative modeling demonstrates format-invariant representations of mathematical problems in the brain

abstract: Mathematical thoughts can be described in either symbolic form or natural language. Previous studies have reported that activation overlaps exist for these two types of mathematical problems, but it is unclear whether they are based on similar brain representations. Furthermore, quantitative modeling of mathematical problem solving has yet to be attempted. In the present study, eight subjects were subjected to 3 h of functional magnetic resonance experiments involving math word and math expression problems, and a read word condition without any calculations was used as a control. To evaluate the brain representations of mathematical problems quantitatively, we constructed voxel-wise encoding models. Both intra-and cross-format encoding modeling significantly predicted brain activity in the bilateral intraparietal sulcus (IPS), even after subtraction of the control condition. Representational similarity analysis and principal component analysis revealed that mathematical problems with different formats had similar cortical organization in the bilateral IPS. These findings support the idea that our mathematical thoughts are represented in the brain in a format-invariant manner.


title: Synaptotagmin 9 modulates spontaneous neurotransmitter release in striatal neurons by regulating substance P secretion

abstract: Synaptotagmin 9 (SYT9) is a tandem C2-domain Ca2+ sensor for exocytosis in neuroendocrine cells; its function in neurons remains unclear. Here, we show that SYT9 does not trigger rapid synaptic vesicle exocytosis in cultured cortical, hippocampal, or striatal neurons; rather, synaptotagmin 1 (SYT1) fulfills this function. SYT9 can regulate evoked synaptic vesicle exocytosis, but only when massively over-expressed. In striatal neurons, loss of SYT9 reduced the rate of spontaneous miniature neurotransmitter release events (minis). To delve into the underlying mechanism, we localized SYT9 in striatal neurons and found that it was targeted to dense core vesicles, where it regulated the release of substance P (SP), a neuropeptide known to modulate mini frequency. Exogenous SP, but not other striatal peptide hormones, completely rescued the Syt9 KO mini phenotype. Biochemical experiments revealed that Ca2+-binding to the C2A domain of SYT9 serves to regulate membrane fusion in vitro, and mutations that disrupt this activity abolished the ability of SYT9 to regulate minis in striatal neurons. We conclude that SYT9 indirectly regulates synaptic transmission in striatal neurons by controlling SP release.


title: Sequence characterization of the complete chloroplast genome of Eupatorium fortunei

abstract: Eupatorium fortunei has been utilized as herbal medicine to cure various diseases in various regions of the world especially China, Korea, and other Asian countries. The available literature shows Eupatorium fortunei contain anti-metastatic, anti-angiogenic, anti-bacterial, and anti-oxidant, along with compounds to cure human cancer. In present study, Eupatorium fortunei was used as an experimental material, and its genome was sequenced based on high-throughput sequencing technology. We assembled the complete chloroplast genome of Eupatorium fortunei and obtained NCBI accession number (OK545755). We analyzed chloroplast genome structure of Eupatorium fortunei and systematic analysis was conducted, including the size of the entire genome sequence, genome volume, large single-copy (LSC), small single-copy (SSC), inverted repeat region (IR)-LSC/SSC boundaries, etc. The obtained results showed that chloroplast genome of Eupatorium fortunei is a typical tetrad structure with a total length of 152,401 bp and a sum of 133 annotated genes, including eight ribosomal RNA (rRNA)-encoding genes, 37 transfer RNA (tRNA)-encoding genes, 87 protein-encoding genes, and one pseudo-gene. A sum of 29 Single Sequence Repeats (SSR)s were identified for mono-nucleotide, di-nucleotide and tri-nucleotide microsatellites with mono-nucleotides in majority. The phylogenetic analysis revealed that Eupatorium fortunei is more closely related to Litothamnus nitidus and belongs to the same branch. The genome assembly and analysis of the chloroplast genome of Eupatorium fortunei provides more accurate evidence for further taxonomic identification and localization of Asteraceae/Compositae plant.


title: NuRD independent Mi-2 activity represses ectopic gene expression during neuronal maturation.

abstract: During neuronal development, extensive changes to chromatin states occur to regulate lineage-specific gene expression. The molecular factors underlying the repression of non-neuronal genes in differentiated neurons are poorly characterised. The Mi2/NuRD complex is a multiprotein complex with nucleosome remodelling and histone deacetylase activity. Whilst NuRD has previously been implicated in the development of nervous system tissues the precise nature of the gene expression programmes that it coordinates are ill-defined. Furthermore, evidence from several species suggests that Mi-2 may be incorporated into multiple complexes that may not possess histone deacetylase activity. Here, we show that Mi-2 activity is required for suppressing the ectopic expression of germline genes in neurons independently of HDAC1/NuRD, whilst components of the NuRD complex including Mi-2 regulate neural gene expression to ensure proper development of the larval nervous system. We find that Mi-2 binding in the genome is dynamic during neuronal maturation and Mi-2 mediated repression of ectopic gene expression is restricted to the early stages of neuronal development, indicating that Mi-2/NuRD is required for establishing stable neuronal transcriptomes during the early stages of neuronal differentiation.


title: Bleaching-resistant single-molecule fluorescence and FRET monitoring based on fluorophore exchange via transient DNA binding

abstract: Photobleaching of fluorescent probes limits the observation span of a typical single molecule fluorescence measurement and hinders simultaneous observation of dynamics across timescales. Here, we present a general strategy to circumvent photobleaching by replenishing fluorescent probes throughout the experiment via transient binding of fluorescently labelled single-stranded DNAs to complementary target DNA strands attached to the target molecule. We show our strategy allows observation of near-continuous single-molecule fluorescence for more than an hour, a timescale two orders of magnitude longer than the photobleaching time under our conditions. We also show our method is adaptable to FRET and study the conformational dynamics of DNA Holliday Junctions for extended periods. By adjusting the temporal resolution and observation span, we envision to capture the conformational dynamics of proteins and nucleic acids over wide range of timescales.


title: Biofilms preserve transmissibility of a multi-drug resistance plasmid

abstract: Self-transmissible multidrug resistance (MDR) plasmids are a major health concern because they can spread antibiotic resistance to pathogens. Even though most pathogens form biofilms, little is known about how MDR plasmids persist and evolve in biofilms. We hypothesize that (i) biofilms act as refugia of MDR plasmids by retaining them in the absence of antibiotics longer than well-mixed planktonic populations, and that (ii) the evolutionary trajectories that account for the improvement of plasmid persistence over time differ between biofilms and planktonic populations. In this study, we evolved Acinetobacter baumannii with an MDR plasmid in biofilm and planktonic populations with and without antibiotic selection. In the absence of selection biofilm populations were better able to maintain the MDR plasmid than planktonic populations. In planktonic populations plasmid persistence improved rapidly but was accompanied by a loss of genes required for the horizonal transfer of plasmids. In contrast, in biofilms most plasmids retained their transfer genes, but on average plasmid persistence improved less over time. Our results showed that biofilms can act as refugia of MDR plasmids and favor the horizontal mode of plasmid transfer, which has important implications for the spread of MDR.


title: Route of oxytetracycline administration differentially impacts the growth and gut microbiome of pigs co-infected with Bordetella bronchiseptica and Pasteurella multocida

abstract:
Along with judicious antibiotic use, there is great interest in how the dose regimen of an antibiotic affects the animal gut microbiota. This study evaluated the impact of experimental respiratory infection alone or respiratory infection followed by oxytetracycline (oxytet) treatment on the animals health and its fecal microbiome. Piglets of approximately three weeks-of-age were separated into four groups (n=20 per group). One group remained non-infected and administered non-medicated feed and the other three groups were infected with Bordetella bronchiseptica (day 0) and Pasteurella multocida (day 4), with one group receiving non- medicated feed and the remaining two groups receiving oxytetr starting on day 7 by injection or in-feed (day 7-14). Infection with B. bronchiseptica and P. multocida negatively impacted piglet growth and induced mild pneumonia. Infection alone had minimal effect on the fecal microbiota community. When oxytet was administered either by injection or in-feed to treat the respiratory infections, both routes had minimal effect on clearing B. bronchiseptica and P. multocida in the animal. However, both routes appeared to limit lung lesion severity, and injected oxytet reduced the negative impact of infection on weight gain. Both routes had limited impact on the animals overall gut microbiome, including relative abundances of bacterial taxa and antibiotic resistance genes tet32, tetW, and aph2. Overall, oxytet administered by either route did not clear the respiratory infection, but oxytet administration minimized the negative health impacts of infection and had minor impact on the pig gut microbiome.

ImportanceEfforts to address antibiotic resistance calls for improved antibiotic stewardship, including considering antibiotic administration route. While our previous study found in-feed oxytet had greater impact on the gut microbiome of healthy piglets than injected oxytet, it remained unknown if oxytet treatments would have the same impact on the microbiota of infected piglets. We evaluated the impact of respiratory infection alone or respiratory infection followed by oxytet treatment on the animals health and their gut microbiome profile. Respiratory infection negatively affected piglets health, but infection alone had minimal impact on the gut community. When oxytet was administered either in-feed or by injection to treat the respiratory infection, neither route of administration led to the clearance of the respiratory pathogens. However, oxytet minimized the negative health impacts of infection, and had minor impact on the pig gut microbiome. These findings are informative for disease management in food animals while integrating antibiotic stewardship practices.


title: The yeast claudin Dcv1 is important for demarcating the front domain of pheromone-responding yeast cells

abstract:
Cell polarization in response to chemical gradients is important in development and homeostasis across eukaryota. Chemosensing cells orient toward or away from gradient sources by polarizing along a front-rear axis. Using the chemotropic mating response of the budding yeast S. cerevisiae as a model of environmentally-induced cell polarization, we found that Dcv1, a claudin homolog, is a determinant of front-rear polarity. Although Dcv1 localized uniformly on the plasma membrane (PM){dagger} of vegetative cells, it was confined to the rear of cells responding to pheromone, away from the pheromone receptor and mating projection. Deletion of DCV1 conferred mislocalization of sensory, polarity, and trafficking proteins, as well as the PM lipids ergosterol, phosphatidylinositol-4,5-bisphosphate (PIP2), and phosphatidylserine (PS). These phenotypes correlated with defects in pheromone-gradient tracking and cell fusion. We propose that the novel claudin-like and rear-domain protein, Dcv1, demarcates the mating-specific front domain primarily by restricting PM lipid distribution. Consistent with this hypothesis, a mutation that blocks ergosterol biosynthesis partially phenocopies dcv1{Delta}.

Summary statementThe yeast claudin Dcv1 facilitates the proper localization of plasma membrane lipids and proteins that are required for front-rear polarity, efficient chemotropism, and cell fusion.


title: Sperm-mediated regulation of region-specific responses in the oviduct during establishment of pregnancy in mice

abstract: The oviduct comprises 4 main regions: infundibulum (oocyte pick-up), ampulla (fertilization), isthmus (sperm capacitation and reservoir, preimplantation embryonic development), and uterotubal junction (UTJ; sperm and embryo transport). Mounting evidence in livestock and rodents suggest that gametes alter gene expression in secretory and ciliated epithelial cells of the oviduct. To elucidate whether adaptive interactions between the oviduct and gamete/embryo exist, we performed bulk RNA-sequencing on oviductal tissues collected from infundibulum+ampulla (IA) or isthmus+UTJ (IU) at various developmental stages (0.5, 1.5, 2.5-, and 3.5-days post coitus (dpc)) in mice. Samples were also collected during days 0.5, 1.5, 2.5, and 3.5 of pseudopregnancy (dpp). We found a strong region (IA vs. IU)-specific expression of large clusters of genes. The transition from 0.5 dpc to other pregnancy timepoints induces large sets of differentially expressed genes (DEGs) in pregnancy and pseudopregnancy in both IA and IU regions. Specifically, genes involved in pro-inflammatory responses were detected in both IU and IA regions. The presence of sperm at 0.5 dpc induces DEGs involved in pro-inflammatory responses in the IU region with an enrichment of biological processes for inflammatory cytokines, macrophage, and neutrophil recruitment. Additionally, DEGs are enriched in mitogen-activated protein kinase (MAPK) pathways along with genes in the Dusp family, Map3k8, Il1b, and Il1r2, among others. However, at 1.5 dpc we observed a strong shift to an anti-inflammatory condition in the IU region. These observations were absent in 0.5 and 1.5 dpp, suggesting that the DEGs observed for those inflammatory responses during pregnancy were likely induced by the presence of sperm. scRNA-seq analysis revealed that the inflammatory responsive genes were likely produced by secretory epithelial cells, compared to other cell types in the oviduct. In addition, multiple DEGs involved in pyruvate and glycolysis were enriched in the IU region, which could provide metabolic support for developing embryos. Lastly, we have also identified that there were cells that express immune markers in the oviduct, indicating that the oviduct is an immuno-dynamic tissue. In conclusion, our findings indicate that the oviduct is adaptive and responsive to the presence of sperm and embryos in a spatiotemporal manner. In this report, we intend to disseminate our findings on the transcriptional profiles during different stages of pregnancy. The complete study and validation at the protein level are currently underway and will be updated as soon as the data are available.


title: A multi-continental analysis of the responses of freshwater food webs to climate and land use change

abstract:
O_LIFood web responses to environmental change are not straightforward to understand as they occur through an intricate arrangement of direct and indirect effects. Although previous investigations have advanced knowledge on freshwater food web structure, we must better understand the intricate relationships between the main drivers of environmental change and trophic networks in lentic and lotic ecosystems.
C_LIO_LIWe compiled multicontinental data to investigate how climate and land use change are related to the structure of freshwater food webs, considering the inherent differences in lentic and lotic ecosystems. We analyzed the direct and indirect relationships between land use intensity, and temperature and precipitation temporal trends, and food webs using multi-group structural equation modeling.
C_LIO_LIThe strength and direction of the relationships between climate, land use, and food webs varied considerably among lentic and lotic ecosystems, but most indicated indirect effects through the number of links in the network. While network connectance both increased and decreased with land use and climate change, the number of trophic levels decreased with land use intensity and maximum temperature and increased with increasing precipitation. Omnivory increased with land use intensity in both ecosystems but was negatively related to changes on maximum temperature in lake food webs.
C_LIO_LIEven though food webs are expected to become more connected in face of disturbances, and our work supported this regarding local warming, the negative relationships between network connectance and land use intensification suggests that food webs become more specialized at disturbed sites. On the other hand, reduction in the number of trophic levels indicates the loss of top consumers in face of warming and increasing land use intensity.
C_LIO_LIThe response of food webs in both lentic and lotic ecosystems to climate and land use change occurred mostly through changes in species interactions. Our results indicate that the intensification of land use makes food webs more specialized, with less trophic levels. Also, inherent aspects of freshwater ecosystems seemed to play a major role in the way food webs respond to disturbance and must be considered to fully understand and predict the effects of global changes on freshwater biodiversity.
C_LI


title: Men in menopause? Experimental verification of the mate choice theory of menopause with Drosophila melanogaster

abstract: Menopause is an unusual fertility reducing trait and it goes against natural selection. Group selection-based theories, such as the Grandmother hypothesis can, in principle, explain the evolution of menopause, but they do not explain how a fertility reducing mutation became fixed in populations. A population genetics theory of menopause, Mate Choice Theory, explains the evolution of menopause resulting from asymmetric mating involving older males and younger females leading to accumulation and neutral evolution of infertility causing mutations in older females. In this study we tested the mate choice theory using Drosophila melanogaster. Long term laboratory experiments involving asymmetric mating between young females and old males and vise-versa, were conducted over 40 generations and measurement of fitness parameters affecting female survival, fertility, fecundity, and male survival, fertility and testis length and width, were measured. The results showed, as expected, that younger females mated with older males produced infertile females with fewer ovarioles, fewer matured eggs, and fewer offspring; and younger males mated with older females produced sterile males with thinner testis compared to the control. The outcome of the experiment was symmetrical in that it works irrespective of sex, making both sexes, males and females, menopausal depending on which sex is deprived of reproduction in older age. These results show that change in mates behavior, such as preferential mating, can have profound impact on womens health and could provide a basis for understanding reproductive senescence with age in both aging females and males.


title: A flexible modeling and inference framework for estimating variant effect sizes from GWAS summary statistics

abstract: Genome-wide association studies (GWAS) have highlighted that almost any trait is affected by many variants of relatively small effect. On one hand this presents a challenge for inferring the effect of any single variant as the signal-to-noise ratio is high for variants of small effect. This challenge is compounded when combining information across many variants in polygenic scores for predicting trait values. On the other hand, the large number of contributing variants provides an opportunity to learn about the average behavior of variants encoded in the distribution of variant effect sizes. Many approaches have looked at aspects of this problem, but no method has unified the inference of the effects of individual variants with the inference of the distribution of effect sizes while requiring only GWAS summary statistics and properly accounting for linkage disequilibrium between variants. Here we present a flexible, unifying framework that combines information across variants to infer a distribution of effect sizes and uses this distribution to improve the estimation of the effects of individual variants. We also develop a variational inference (VI) scheme to perform efficient inference under this framework. We show this framework is useful by constructing polygenic scores (PGSs) that outperform the state-of-the-art. Our modeling framework easily extends to jointly inferring effect sizes across multiple cohorts, where we show that building PGSs using additional cohorts of differing ancestries improves predictive accuracy and portability. We also investigate the inferred distributions of effect sizes across many traits and find that these distributions have effect sizes ranging over multiple orders of magnitude, in contrast to the assumptions implicit in many commonly-used statistical genetics methods.


title: Ultrafast and interpretable single-cell 3D genome analysis with Fast-Higashi

abstract: Single-cell Hi-C (scHi-C) technologies can probe three-dimensional (3D) genome structures in single cells and their cell-to-cell variability. However, existing scHi-C analysis methods are hindered by the data quality and the complex 3D genome patterns. The lack of computational scalability and interpretability poses further challenges for large-scale scHi-C analysis. Here, we introduce Fast-Higashi, an ultrafast and interpretable method based on tensor decomposition that can jointly identify cell identities and chromatin meta-interactions. Fast-Higashi is able to simultaneously model multiple tensors with unmatched features of different sizes. A new partial random walk with restart (Partial RWR) algorithm in Fast-Higashi efficiently mitigates data sparseness. Extensive evaluations on real scHi-C datasets demonstrate the advantage of Fast-Higashi over existing methods for embedding, leading to improved delineation of rare cell types and better reconstruction of developmental trajectories. Fast-Higashi can directly infer chromatin meta-interactions, identify 3D genome features that define distinct cell types, and help elucidate cell type-specific connections between genome structure and function. Moreover, Fast-Higashi can be generalized to incorporate other single-cell omics data. Fast-Higashi provides a highly efficient and interpretable scHi-C analysis solution that is applicable to a broad range of biological contexts.


title: Stress fibers orient traction forces on micropatterns: A hybrid cellular Potts model study

abstract:
Adhering cells exert traction forces on the underlying substrate. We numerically investigate the intimate relation between traction forces, the structure of the actin cytoskeleton, and the shape of cells adhering to adhesive micropatterned substrates. By combining the Cellular Potts Model with a model of cytoskeletal contractility, we reproduce prominent anisotropic features in previously published experimental data on fibroblasts, endothelial cells, and epithelial cells on adhesive micropatterned substrates. Our work highlights the role of cytoskeletal anisotropy in the generation of cellular traction forces, and provides a computational strategy for investigating stress fiber anisotropy in dynamical and multicellular settings.

Author summaryCells that make up multicellular life perform a variety of mechanical tasks such as pulling on surrounding tissue to close a wound. The mechanisms by which cells perform these tasks are, however, incompletely understood. In order to better understand how they generate forces on their environment, cells are often studied in vitro on compliant substrates, which deform under the so called "traction forces" exerted by the cells. Mathematical models complement these experimental approaches because they help to interpret the experimental data, but most models for traction forces on adhesive substrates assume that cells contract isotropically, i.e., they do not contract in a specific direction. However, many cell types contain organized structures of stress fibers - strong contracting cables inside the cell - that enable cells to exert forces on their environment in specific directions only. Here we present a computational model that predicts both the orientations of these stress fibers as well as the forces that cells exert on the substrates. Our model reproduces both the orientations and magnitudes of previously reported experimental traction forces, and could serve as a starting point for exploring mechanical interactions in multicellular settings.


title: Examining the efficacy of localised gemcitabine therapy for the treatment of pancreatic cancer using a hybrid agent-based model

abstract:
The prognosis for pancreatic ductal adenocarcinoma (PDAC) patients has not significantly improved in the past 3 decades, highlighting the need for more effective treatment approaches. Poor patient outcomes and lack of response to therapy can be attributed, in part, to the dense, fibrotic nature of PDAC tumours, which impedes the uptake of systemically administered drugs. Wet-spun alginate fibres loaded with the chemotherapeutic agent gemcitabine have been developed as a potential tool for overcoming the physical and biological barriers presented by the PDAC tumour microenvironment and deliver high concentrations of drug to the tumour directly over an extended period of time. While exciting, the practicality, safety, and effectiveness of these devices in a clinical setting requires further investigation. Furthermore, an in-depth assessment of the drug-release rate from these devices needs to be undertaken to determine whether an optimal release profile exists. Using a hybrid computational model (agent-based model and partial differential equation system), we developed a simulation of pancreatic tumour growth and response to treatment with gemcitabine loaded alginate fibres. The model was calibrated using in vitro and in vivo data and simulated using a finite volume method discretization. We then used the model to compare different intratumoural implantation protocols and gemcitabine-release rates. In our model, the primary driver of pancreatic tumour growth was the rate of tumour cell division and degree of extracellular matrix deposition. We were able to demonstrate that intratumoural placement of gemcitabine loaded fibres was more effective than peritumoural placement. Additionally, we found that an exponential gemcitabine release rate would improve the tumour response to fibres placed peritumourally. Altogether, the model developed here is a tool that can be used to investigate other drug delivery devices to improve the arsenal of treatments available for PDAC and other difficult-to-treat cancers in the future.

Author SummaryPancreatic cancer has a dismal prognosis with a median survival of 3-5 months for untreated disease. The treatment of pancreatic cancer is challenging due to the dense nature of pancreatic tumours which impedes retention of drug at the tumour site. As such, systemic administration of chemotherapies, such as gemcitabine, has a limited efficacy. To overcome this, sustained-release devices have been proposed. These devices are injected locally and release drug slowly over time, providing a concentrated local, sustained drug concentration. To investigate the possible efficacy of these devices, we developed a mathematical model that would allow us to probe treatment perturbations in silico. We modelled the individual cancer cells and their growth and death from gemcitabine loaded into the sustained delivery devices. Our platform allows future investigations for these devices to be run in silico so that we may better understand the forms of the drug release-profile that are necessary for optimal treatment.

category: cancer biology


title: High-dimensional and spatial analysis reveals immune landscape dependent progression in cutaneous squamous cell carcinoma

abstract:
PurposeThe tumour immune microenvironment impacts the biological behaviour of the tumour but its effect on clinical outcomes in head and neck cutaneous squamous cell carcinomas (HNcSCC) is largely unknown.

Experimental DesignWe compared the immune milieu of high-risk HNcSCC that never progressed to metastasis with those that metastasised using multi-parameter imaging mass cytometry. The cohort included both immunosuppressed patients (IS) and patients with an absence of clinical immune-suppression (ACIS). Spatial analyses were used to identify cellular interactions that were associated with tumour behaviour.

ResultsNon-progressing primary HNcSCC were characterised by higher CD8+ and CD4+ T cell responses, including numerically increased Regulatory T cells. By contrast, primary lesions from HNcSCC patients who progressed were largely devoid of T cells with lower numbers of innate immune cells and increased expression of checkpoint receptors and in the metastatic lesions were characterised by an accumulation of B cells. Spatial analysis reveals multiple cellular interactions associated with non-progressing primary tumours that were distinct in primary tumours of disease progressing patients. Cellular regional analysis of the tumour microenvironment also shows squamous cell-enriched tumour regions associated with primary non-progressing tumours.

ConclusionsEffective responses from both CD8+ and CD4+ T cells in the tumour microenvironment are essential for immune control of primary HNcSCC. Our findings indicate that the early events that shape the immune responses in primary tumours dictate progression and disease outcomes in HNcSCC.

Translational RelevanceThe ability to predict metastatic tumour progression at the time of initial diagnosis of primary HNcSCC could tailor personalised medical care including disease surveillance strategies and identifying patients who will benefit most from adjuvant therapy.

One Sentence SummaryThe immune landscape of high-risk cutaneous squamous cell carcinoma differs in tumours that never progress compared to those that progress to metastasis.

category: pathology


title: The role of environmental calcium in the extreme acid tolerance of northern banjo frog (Limnodynastes terraereginae) larvae

abstract:
Many aquatically respiring animals inhabiting low pH waters can suffer acute inhibition of ion uptake and loss of branchial (gill) epithelial integrity, culminating in a fatal, rapid loss of body Na+. Environmental calcium levels ([Ca2+]e) are pivotal in maintaining branchial junction integrity, with supplemental Ca2+ reversing the negative effects of low pH in some animals. Tolerance of some naturally acidic environments by aquatic animals is further complicated by low [Ca2+]e, yet many of these environments are surprisingly biodiverse. How these animals overcome the combined damaging actions of low pH and low environmental Ca2+ remains unknown. Here, we examined the effects of [Ca2+]e on the response to low pH in larvae of the highly acid tolerant frog Limnodynastes terraereginae. Acute exposure to low pH water in the presence of low [Ca2+]e increased net Na+ efflux. Provision of additional [Ca2+]e reduced net Na+ efflux, but the effect was saturable. Acclimation to both low and high [Ca2+]e improved the resistance of larvae to Na+ efflux at low pH. Inhibition of apical Ca2+ uptake by ruthenium red resulted in an abrupt loss of tolerance to low pH in larvae acclimated to low pH water. Acclimation to acidic water increased branchial gene expression of the intracellular Ca2+ transport protein calbindin, consistent with a role for increased transcellular Ca2+ trafficking in the tolerance of acidic water. This study confirmed the physiological challenge of low [Ca2+]e on branchial integrity in acidic waters and highlighted a potential role for maintenance of transcellular Ca2+ uptake in the acid tolerance of L. terraereginae.

Summary statementTolerance of naturally acidic, dilute, and soft waters by larvae of the frog Limnodynastes terraereginae involves adaptations to the branchial calcium transport pathway which protects intercellular junctions against damage.

category: physiology