title: Emergence of Heartbeat Frailty in Advanced Age: Perspectives from Life-Long EKG Recordings in Mice

abstract:
SAN failure, aka sick-sinus syndrome, which features sinus bradycardia, SAN impulse pauses, and irregularity of RR interval rhythms are manifestations of SAN cell dysfunction that increases exponentially with advanced age, i.e., SAN frailty. Abnormalities in intrinsic RR interval variability may be the earliest signatures of SAN cell dysfunction leading to SAN frailty in late life. We measured RR interval variability within EKG timeseries prior to and during double autonomic blockade in long-lived C57/BL6 mice at 3 month intervals from 6 months of age until the end of life.

Long-lived mice (those that achieved the median cohort lifespan of 24 months and beyond) displayed relatively minor changes in intrinsic RR interval variability prior to 21 months of age. Between 21 and 30 months of age, marked changes in intrinsic RR interval variability signatures in time, frequency, non-linear, and fragmentation domains result in a marked increase in the mean intrinsic RR interval. The effects of autonomic input partially compensated for the prolongation of the mean RR interval by impacting the age-associated deterioration in the RR interval variability signatures toward a youthful pattern. Cross-sectional analyses of other subsets of mice at ages at or beyond the median life span of our longitudinal cohort demonstrated increased non-cardiac, constitutional, whole body frailty, a decrease in energetic efficiency, and an increase in respiratory exchange ratio. In this context, we interpret the progressive increase in intrinsic RR interval variability beyond 21 months of age to be an indication of heartbeat frailty.

category: physiology


title: Multimodal cues displayed by submissive rats promote prosocial choices by dominants

abstract: Animals often display prosocial behaviours, performing actions that benefit others. Although prosociality is essential for social bonding and cooperation, we still know very little about how animals integrate behavioural cues from those in need to make decisions that increase their wellbeing. To address this question, we used a two-choice task where rats can provide rewards to a conspecific in the absence of self-benefit, and interrogated which conditions promote prosociality by manipulating the social context of the interacting animals. While sex or degree of familiarity did not affect prosocial choices in rats, social hierarchy revealed to be a potent modulator, with dominant decision-makers showing faster emergence and higher levels of prosocial choices towards their submissive cage-mates. Leveraging quantitative analysis of multimodal social dynamics prior to choice, we identified that pairs with dominant decision-makers exhibited more proximal interactions in social distance. Interestingly, these more coordinated interactions were driven by submissive animals that modulated their position and movement towards their dominants and increased 50kHz vocalisation rate when their partners were going to behave selfishly. This display of multimodal cues by submissive animals while signalling need promoted social saliency and a faster emergence of prosocial choices from dominant rats. Multivariate analysis highlighted non-canonical body language as the main information dominants use on a trial-by-trial basis to learn that their actions have effects on others. Our results provide a refined understanding of the behavioural dynamics that rats use for action-selection upon perception of socially relevant cues and navigate social decision-making.

category: neuroscience


title: Unifying Gene Expression Complexity and Cellular Operational Efficiency: Application of the Locality and Caching Principles via a Cell-to-Computer Analogy

abstract: Gene expression is time-consuming, and the delay from transcription activation to produced proteins is sequentially longer from bacteria to yeast and to humans. How human cells overcome the delay and attain operational efficiency, i.e., quick proteomic response to signals, is not well understood. The computer has endured the same system latency issue due to much slower information retrieval (hard drive (HD) to memory and to central processing unit (CPU)) than CPU execution, and mitigated it via efficient memory management, namely, the spatiotemporal locality principles that control specialized user programs and the permanent caching of core system functions - the operating system (OS) kernel. In this study, we unified gene expression and HD-memory-CPU information flow as instances of the Shannon information theory, that support the respective system operations and both consist of three components: information storage, the execution/decoding step, and the channel for the dynamic storage-to-execution information flow; the gene expression machinery and their regulators, and the OS kernel, were deemed as the respective channels. This abstraction prompted a multi-omic comparative analysis, leading to three observations. First, the temporal locality principle explains the mRNA stabilization-by-translation regulatory mechanism and controls specialized cellular functions. Second, the caching principle explains cytoplasmic mRNA sequestration and the defiance of the locality principle by highly sequestered mRNAs. Third, strikingly, in both systems, the caching principle controls the information channels; similar to permanent caching of OS kernel, basic gene expression machinery and their regulators are the top most sequestered mRNAs. In short, the locality and the caching principles act in the cells to control specialized and core cellular functions, respectively. The results close the knowledge gap between, and thus unify, transcriptome regulation and cellular operational latency mitigation.

category: systems biology


title: Unifying Transcriptome Regulation and Cellular Operational Efficiency: Application of the Locality and Caching Principles via a Cell-to-Computer Analogy


title: Benchmarking taxonomic classifiers with Illumina and Nanopore sequence data for clinical metagenomic diagnostic applications

abstract:
Culture-independent metagenomic detection of microbial species has the potential to provide rapid and precise real-time diagnostic results. However, it is potentially limited by sequencing and classification errors. We use simulated and real-world data to benchmark rates of species misclassification using 100 reference genomes for each of ten common bloodstream pathogens and six frequent blood culture contaminants (n=1600). Simulating both with and without sequencing error for both the Illumina and Oxford Nanopore platforms, we evaluated commonly used classification tools including Kraken2, Bracken, and Centrifuge, utilising mini (8GB) and standard (30-50GB) databases. Bracken with the standard database performed best, the median percentage of reads across both sequencing platforms identified correctly to the species level was 98.46% (IQR 93.0:99.3) [range 57.1:100]. For Kraken2 with a mini database, a commonly used combination, median species-level identification was 79.3% (IQR 39.1:88.8) [range 11.2:100]. Classification performance varied by species, with E. coli being more challenging to classify correctly (59.4% to 96.4% reads with correct species, varying by tool used). By filtering out shorter Nanopore reads (<3500bp) we found performance similar or superior to Illumina sequencing, despite higher sequencing error rates. Misclassification was more common when the misclassified species had a higher average nucleotide identity to the true species. Our findings highlight taxonomic misclassification of sequencing data occurs and varies by sequencing and analysis workflow. This "bioinformatic contamination" should be accounted for in metagenomic pipelines to ensure accurate results that can support clinical decision making.

ImportanceMetagenomics may transform clinical microbiology by enabling more rapid species detection in a potentially unbiased manner and reducing reliance on culture-based approaches. However, it is still limited by ongoing challenges such as sequencing and classification software errors. In this study, we use simulated and real-world data to define the intrinsic rates of species misclassification that occur using Illumina and Oxford Nanopore sequencing platforms with commonly used taxonomic classification tools and databases. We quantify the extent of "bioinformatic contamination" arising from the classification process. This enables us to identify the best performing tools that maximize classification accuracy, and to suggest how taxonomic misclassification can be formally accounted for in clinical diagnostic workflows. Specifically, we specify thresholds for identifying or excluding polymicrobial infections in metagenomic samples, based on rates of misclassification of similar species, which might have clinical implications when treating infection.

category: microbiology


title: Cell- and non-cell-autonomous ARF3 coordinates meristem proliferation and organ patterning in Arabidopsis

abstract: In cell-cell communication, non-cell-autonomous transcription factors play vital roles in controlling plant stem cell fate. We previously reported that AUXIN RESPONSE FACTOR 3 (ARF3), a member of the ARF family with critical roles in floral meristem maintenance and determinacy, has a distinct accumulation pattern that differs from the expression domain of its encoding gene in the shoot apical meristem (SAM). However, the biological meaning of this difference is obscure. Here, we demonstrate that ARF3 expression is mainly activated at the periphery of the SAM by auxin, where ARF3 cell-autonomously regulates the expression of meristem-organ boundary-specific genes, such as CUP-SHAPED COTYLEDON1-3 (CUC1-3), BLADE ON PETIOLE1-2 (BOP1-2) and TARGETS UNDER ETTIN CONTROL3 (TEC3) to determine organ patterning. We also show that ARF3 is translocated into the organizing center, where it represses cytokinin activity and WUSCHEL expression to regulate meristem activity non-cell-autonomously. Therefore, ARF3 acts as a molecular link that mediates the interaction of auxin and cytokinin signaling in the SAM while coordinating the balance between meristem maintenance and organogenesis. Our findings reveal an ARF3-mediated coordination mechanism through cell-cell communication in dynamic SAM maintenance.

category: developmental biology


title: Ancestral acetylcholine receptor β-subunit forms homopentamers that prime before opening spontaneously

abstract: Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from two -subunits, and one each of the {beta}-, {delta}-, and {varepsilon}-subunits. To form functional channels, the subunits must assemble with one another in a precise stoichiometry and arrangement. Despite being different, the four subunits share a common ancestor that is presumed to have formed homopentamers. The extent to which the properties of the modern-day receptor result from its subunit complexity is unknown. Here we show that a reconstructed ancestral muscle-type {beta}-subunit can form homopentameric ion channels. These homopentamers open spontaneously and display single-channel hallmarks of muscle-type acetylcholine receptor activity. Our findings demonstrate that signature features of muscle-type acetylcholine receptor function are independent of agonist, and do not necessitate the complex heteropentameric architecture of the modern-day receptor.

category: biophysics


title: PVT1, a YAP1 dependent stress responsive lncRNA drives ovarian cancer metastasis and chemoresistance

abstract:
Metastatic growth of ovarian cancer cells into the peritoneal cavity requires adaptation to various cellular stress factors to facilitate cell survival and growth. Here we demonstrate the role of PVT1, one such stress induced long non-coding RNA, in ovarian cancer growth and metastasis. PVT1 is an amplified and overexpressed lncRNA in ovarian cancer with strong predictive value for survival and response to targeted therapeutics. We find that expression of PVT1 is regulated by ovarian tumor cells in response to cellular stress, particularly loss of cell-cell contacts and changes in matrix rigidity occurring in a YAP1 dependent manner. Induction of PVT1 promotes tumor cell survival, growth, and migration. Conversely, reducing PVT1 levels robustly abrogates metastatic behavior and tumor cell dissemination in cell lines and syngeneic transplantation models in vivo. We find that reducing PVT1 causes widespread transcriptome changes leading to alterations in cellular stress response and metabolic pathways including doxorubicin metabolism, which directly impacts chemosensitivity. Together, these findings implicate PVT1 as a promising therapeutic target to suppress metastasis and avoid chemoresistance in ovarian cancer.

SUMMARY BLURBPVT1 is a stress-responsive IncRNA regulated by YAP1, leading to increased ovarian cancer metastasis and chemoresistance, making PVT1 a promising therapeutic target.

category: cancer biology


title: PFTK1 kinase regulates axogenesis during development via RhoA activation.

abstract: PFTK1/Eip63E is a member of the Cyclin-dependent kinases (CDKs) family and plays an important role in normal cell cycle progression. Eip63E expresses primarily in postnatal and adult nervous system in Drosophila melanogaster but its role in CNS development remains unknown. We sought to understand the function of Eip63E in the CNS by studying the fly ventral nerve cord during development. Our results demonstrate that Eip63E regulates axogenesis in neurons and its deficiency leads to neuronal defects. Functional interaction studies performed using the same system identify an interaction between Eip63E and the small GTPase Rho1. Furthermore, deficiency of Eip63E homolog in mice, PFTK1, in a newly generated PFTK1 knockout mice results in increased axonal outgrowth confirming that the developmental defects observed in the fly model are due to defects in axogenesis. Importantly, RhoA phosphorylation and activity is affected by PFTK1 in primary neuronal cultures. We here report that GDP bound inactive RhoA is a substrate of PFTK1 and PFTK1 phosphorylation maintains RhoA activity. In conclusion, our work establishes an unreported role of PFTK1 in axon development mediated by phosphorylation and activation of GDP-bound RhoA. The results presented add to our understanding of the role of Cdks in the maintenance of RhoA mediated axon growth and its impact on CNS development and axonal regeneration.


title: Cryo-EM Structure of Adeno-associated virus-4 at 2.2 	A resolution

abstract: Adeno-associated virus (AAV) is the vector of choice for several approved gene therapy treatments and is the basis for many ongoing clinical trials. Various strains of AAV exist (referred to as serotypes), each with their own transfection characteristics. Here, we present a high-resolution cryo-electron microscopy structure (2.2 [A]) for AAV serotype 4 (AAV4). The receptor responsible for transduction of the AAV4 clade of AAV viruses (including AAV11, 12 and rh32.33) is unknown. Other AAVs interact with the same cell receptor, Adeno-associated virus receptor (AAVR), in one of two different ways. AAV5-like viruses interact exclusively with the polycystic kidney disease-like [PKD]-1 domain of AAVR while most other AAVs interact primarily with the PKD2 domain. A comparison of the present AAV4 structure with prior corresponding structures of AAV5, AAV2 and AAV1 in complex with AAVR, provides a foundation for understanding why the AAV4-like clade is unable to interact with either PKD1 or PKD2. The conformation of the AAV4 capsid in variable regions I, III, IV and V on the viral surface appears to be sufficiently different from AAV2 to ablate binding with PKD2. Differences between AAV4 and AAV5 in variable region VII appear sufficient to exclude binding with PKD1.


title: Complement facilitates developmental microglial pruning of astrocyte and vascular networks

abstract: Microglia, the resident immune cell of the central nervous system (CNS), play a pivotal role in facilitating neurovascular development through mechanisms that are not fully understood. This current work resolves a previously unknown role for microglia in facilitating the developmental pruning of the astrocytic template resulting in a spatially organized retinal superficial vascular bed. Mechanistically, our study identified that local microglial expression of complement (C)3 and C3aR is necessary for the regulation of astrocyte patterning and vascular growth during retinal development. Ablation of retinal microglia and deficiency of C3 or C3aR reduced developmental pruning and clearance of astrocytic bodies leading to increased astrocyte density, altered vascular patterning, and elevated gene expression levels of extracellular matrix proteins involved in vascular growth during retinal development. These data demonstrate that C3/C3aR signaling is an important checkpoint required for the finetuning of vascular density during neuroretinal development.


title: Alkaloid avoidance in poison frog tadpoles

abstract:
Animals show a spectrum of avoidance-tolerance to foods containing toxic secondary metabolites. However, this spectrum has not been evaluated in animals that may actively seek out these compounds as a chemical defense. Poison frogs sequester toxic and unpalatable alkaloids from their diet, and in some species, tadpoles are exposed to these toxins before the development of their skin granular glands, which are used for toxin compartmentalization. Here, we examined the effects of the alkaloid decahydroquinoline (DHQ) in tadpoles of the Mimetic poison frog, Ranitomeya imitator, using alkaloid supplemented food. We found that although their survival is lowered by the alkaloid, their development is only mildly affected, with no evident effects on their growth. Furthermore, locomotor activity and feeding behavior was altered in the first week of DHQ treatment, probably in part through nicotinic blockade. However, after two weeks, tadpoles learned to avoid the alkaloid by visiting the food area only when necessary to eat. Our results suggest that poison frogs navigate the avoidance-tolerance spectrum during the development of their sequestration machinery.

SummaryAnimals avoid consuming toxic foods or have anti-toxin machinery to avoid food poisoning. Adult poison frogs eat toxic insects and store the toxins in their skin glands. Some poison frog species even feed their tadpoles with toxins to protect them from predation at the risk of poisoning them. In this study, we observed that toxic food did not affect the development of tadpoles because they quickly learned to eat just enough to grow without getting poisoned. Our results indicate that poison frogs use diverse ways to avoid food poisoning during development.

category: animal behavior and cognition


title: A Systematic, Complexity-Reduction Approach to Dissect Microbiome: the Kombucha Tea Microbiome as an Example

abstract: One defining goal of microbiome research is to uncover mechanistic causation that dictates the emergence of structural and functional traits of microbiomes. However, the extraordinary degree of ecosystem complexity has hampered the realization of the goal. Here we developed a systematic, complexity-reducing strategy to mechanistically elucidate the compositional and metabolic characteristics of microbiome by using the kombucha tea microbiome as an example. The strategy centered around a two-species core that was abstracted from but recapitulated the native counterpart. The core was convergent in its composition, coordinated on temporal metabolic patterns, and capable for pellicle formation. Controlled fermentations uncovered the drivers of these characteristics, which were also demonstrated translatable to provide insights into the properties of communities with increased complexity and altered conditions. This work unravels the pattern and process underlying the kombucha tea microbiome, providing a potential conceptual framework for mechanistic investigation of microbiome behaviors.


title: Accounting for motion in fMRI: What part of the spectrum are we characterizing in autism spectrum disorder?

abstract: The exclusion of high-motion participants can reduce the impact of motion in functional Magnetic Resonance Imaging (fMRI) data. However, the exclusion of high-motion participants may change the distribution of clinically relevant variables in the study sample, and the resulting sample may not be representative of the population. Our goals are two-fold: 1) to document the biases introduced by common motion exclusion practices in functional connectivity research and 2) to introduce a framework to address these biases by treating excluded scans as a missing data problem. We use a study of autism spectrum disorder in children without an intellectual disability to illustrate the problem and the potential solution. We aggregated data from 545 children (8-13 years old) who participated in resting-state fMRI studies at Kennedy Krieger Institute (173 autistic and 372 typically developing) between 2007 and 2020. We found that autistic children were more likely to be excluded than typically developing children, with 28.5% and 16.1% of autistic and typically developing children excluded, respectively, using a lenient criterion and 81.0% and 60.1% with a stricter criterion. The resulting sample of autistic children with usable data tended to be older, have milder social deficits, better motor control, and higher intellectual ability than the original sample. These measures were also related to functional connectivity strength among children with usable data. This suggests that the generalizability of previous studies reporting naive analyses (i.e., based only on participants with usable data) may be limited by the selection of older children with less severe clinical profiles because these children are better able to remain still during an rs-fMRI scan. We adapt doubly robust targeted minimum loss based estimation with an ensemble of machine learning algorithms to address these data losses and the resulting biases. The proposed approach selects more edges that differ in functional connectivity between autistic and typically developing children than the naive approach, supporting this as a promising solution to improve the study of heterogeneous populations in which motion is common.


title: Accounting for motion in resting-state fMRI: What part of the spectrum are we characterizing in autism spectrum disorder?


title: Theory for weakly polydisperse cytoskeleton filaments

abstract: Cytoskeleton filaments have the extraordinary ability to change conformations dynamically in response to alterations of the number density of actins/tubulin, number density and type of binding agents, and electrolyte concentration. This property is crucial for eukaryotic cells to achieve specific biological functions in different cellular compartments. Conventional approaches on biopolymers solution break down for cytoskeleton filaments because they entail several approximations to treat their polyelectrolyte and mechanical properties. In this article, we introduce a novel density functional theory for polydisperse, semiflexible cytoskeleton filaments. The approach accounts for the equilibrium polymerization kinetics, length and orientation filament distributions, as well as the electrostatic interaction between filaments and the electrolyte. This is essential for cytoskeleton polymerization in different cell compartments generating filaments of different lengths, sometimes long enough to become semiflexible. We characterized the thermodynamics properties of actin filaments in electrolyte aqueous solutions. We calculated the free energy, pressure, chemical potential and second virial coefficient for each filament conformation. We also calculated the phase diagram of actin filaments solution and compared with the corresponding results in in-vitro experiments.


title: Scalable in situ single-cell profiling by electrophoretic capture of mRNA

abstract: Methods to spatially profile the transcriptome are dominated by a trade-off between resolution and throughput. Here, we developed a method named EEL FISH that can rapidly process large tissue samples without compromising spatial resolution. By electrophoretically transferring RNA from a tissue section onto a capture surface, EEL speeds up data acquisition by reducing the amount of imaging needed, while ensuring that RNA molecules move straight down towards the surface, preserving single-cell resolution. We applied EEL on eight entire sagittal sections of the mouse brain and measured the expression patterns of up to 440 genes to reveal complex tissue organisation. Moreover, EEL enabled the study of challenging human samples by removing autofluorescent lipofuscin, so that we could study the spatial transcriptome of the human visual cortex. We provide full hardware specification, all protocols and complete software for instrument control, image processing, data analysis and visualization.


title: Bulk and Single-nucleus Transcriptomics Highlight Intra-telencephalic and Somatostatin Neurons in Alzheimer's Disease

abstract:
BackgroundCortical neuron loss is a pathological hallmark of late-onset Alzheimers disease (AD). However, it remains unclear which neuronal subtypes are most vulnerable to degeneration and contribute most to cognitive decline.

MethodsWe analyzed postmortem bulk brain RNA-sequencing (RNAseq) data collected from three studies of aging and AD comprising six neocortical regions (704 individuals; 1037 samples). We estimated relative cell type proportions from each brain sample using neuronal subclass-specific marker genes derived from ultra-high depth single-nucleus RNAseq data (snRNAseq). We associated cell type proportions with AD across all samples using mixed-effects mega-analyses. Bulk tissue analyses were complemented by analyses of three AD snRNAseq datasets using the same cell type definitions and diagnostic criteria (51 individuals). Lastly, we identified cell subtype associations with specific neuropathologies, cognitive decline, and residual cognition.

ResultsIn our mega-analyses, we identified the strongest associations of AD with fewer somatostatin (SST) inhibitory neurons ({beta}=-0.48, pbonf=8.98x10-9) and intra-telencephalic (IT) excitatory neurons ({beta}=-0.45, pbonf =4.32x10-7). snRNAseq-based cell type proportion analyses especially supported the association of SST neurons. Analyses of cell type proportions with specific AD-related phenotypes in ROS/MAP consistently implicated fewer SST neurons with greater brain-wide postmortem tau and beta amyloid ({beta}=-0.155, pFDR=3.1x10-4) deposition, as well as more severe cognitive decline prior to death ({beta}=0.309, pFDR=3.9x10-6). Greater IT neuron proportions were associated strongly with improved cognition ({beta}=0.173, pFDR=8.3x10-5) and residual cognition ({beta}=0.175, pFDR=1.2x10-5), but not canonical AD neuropathology.

ConclusionsProportionally fewer SST and IT neurons were significantly associated with AD diagnosis across multiple studies and cortical regions. These findings support seminal work implicating somatostatin and pyramidal neurons in the pathogenesis of AD and improves our current understanding of neuronal vulnerability in AD.


title: A whole-cell recording database of neuromodulatory action in the adult neocortex

abstract:
BackgroundThe recent release of two large intracellular electrophysiological databases now allows high-dimensional systematic analysis of mechanisms of information processing in the neocortex. Here, to complement these efforts, we introduce a freely and publicly available database that provides a comparative insight into the role of various neuromodulatory transmitters in controlling neural information processing.

FindingsA database of in vitro whole-cell patch-clamp recordings from primary somatosensory and motor cortices (layers 2/3) of the adult mice (2-15 months old) from both sexes is introduced. A total of 464 current-clamp experiments from identified excitatory and inhibitory neurons are provided. Experiments include recordings with (i) Step-and-Hold protocol during which the current was transiently held at 10 steps, gradually increasing in amplitude, (ii) "Frozen Noise" injections that model the amplitude and time-varying nature of synaptic inputs to a neuron in biological networks. All experiments follow a within neuron across drug design which includes a vehicle control and a modulation of one of the following targets in the same neuron: dopamine and its receptors D1R, D2R, serotonin 5HT1f receptor, norepinephrine Alpha1, and acetylcholine M1 receptors.

ConclusionsThis dataset is the first to provide a systematic and comparative insight into the role of the selected neuromodulators in controlling cellular excitability. The data will help to mechanistically address how bottom-up information processing can be modulated, providing a reference for studying neural coding characteristics and revealing the contribution of neuromodulation to information processing.


title: A neural network model of hippocampal contributions to category learning

abstract: In addition to its critical role in encoding individual episodes, the hippocampus is capable of extracting regularities across experiences. This ability is central to category learning, and a growing literature indicates that the hippocampus indeed makes important contributions to this kind of learning. Using a neural network model that mirrors the anatomy of the hippocampus, we investigated the mechanisms by which the hippocampus may support novel category learning. We simulated three category learning paradigms and evaluated the networks ability to categorize and to recognize specific exemplars in each. We found that the trisynaptic pathway within the hippocampus--connecting entorhinal cortex to dentate gyrus, CA3, and CA1--was critical for remembering individual exemplars, reflecting the rapid binding and pattern separation functions of this circuit. The monosynaptic pathway from entorhinal cortex to CA1, in contrast, was responsible for detecting the regularities that define category structure, made possible by the use of distributed representations and a slower learning rate. Together, the simulations provide an account of how the hippocampus and its constituent pathways support novel category learning.


title: Cisplatin neurotoxicity targets specific subpopulations and K+ channels in tyrosine-hydroxylase positive dorsal root ganglia neurons

abstract: Among the features of cisplatin chemotherapy-induced peripheral neuropathy are chronic pain and innocuous mechanical hypersensitivity. The complete etiology of the latter remains unknown. Here, we show that cisplatin targets a heterogeneous population of tyrosine hydroxylase-positive (TH+) primary afferent dorsal root ganglion neurons (DRGNs) within the primary afferent dorsal root ganglia in mice, determined using single-cell transcriptome and electrophysiological analyses. TH+ DRGNs regulate innocuous mechanical sensation through C-low threshold mechanoreceptors. A differential assessment of wild-type and vitamin E deficient TH+ DRGNs revealed heterogeneity and specific functional phenotypes. The TH+ DRGNs comprise; fast-adapting eliciting one action potential (AP; 1-AP), moderately-adapting ([≥] 2-APs), in responses to square-pulse current injection, and spontaneously firing (SF). Cisplatin increased the input resistance and AP frequency but reduced the temporal coding feature of 1-AP and [≥] 2-APs neurons. By contrast, cisplatin has no measurable effect on the SF neurons. Vitamin E reduced the cisplatin-mediated increased excitability, but did not improve the TH+ neuron temporal coding properties. Cisplatin mediates its effect by targeting outward K+ current, likely carried by through K2P18.1 (Kcnk18), discovered through the differential transcriptome studies and heterologous expression. Studies show a potential new cellular target for chemotherapy-induced peripheral neuropathy and implicate the possible neuroprotective effects of vitamin E in cisplatin chemotherapy.


title: Exogenous capture accounts for fundamental differences between prosaccade and antisaccade performance

abstract: Oculomotor circuits generate eye movements based on the physical salience of objects and current behavioral goals, exogenous and endogenous influences, respectively. However, the interactions between exogenous and endogenous mechanisms and their dynamic contributions to target selection have been difficult to resolve because they evolve extremely rapidly. In a recent study (Salinas et al., 2019), we achieved the necessary temporal precision using an urgent variant of the antisaccade task wherein motor plans are initiated early and choice accuracy depends sharply on when exactly the visual cue information becomes available. Empirical and modeling results indicated that the exogenous signal arrives [~]80 ms after cue onset and rapidly accelerates the (incorrect) plan toward the cue, whereas the informed endogenous signal arrives [~]25 ms later to favor the (correct) plan away from the cue. Here, we scrutinize a key mechanistic hypothesis about this dynamic, that the exogenous and endogenous signals act at different times and independently of each other. We test quantitative model predictions by comparing the performance of human participants instructed to look toward a visual cue versus away from it under high urgency. We find that, indeed, the exogenous response is largely impervious to task instructions; it simply flips its sign relative to the correct choice, and this largely explains the drastic differences in psychometric performance between the two tasks. Thus, saccadic choices are strongly dictated by the alignment between salience and behavioral goals.


title: Parent-Child Autonomic Synchrony During Vicarious Extinction Learning in Pediatric PTSD

abstract:
Children learn about threat and safety in their environment in part from their caregivers, a process which may be disrupted in child psychopathology. This transmission may be seen through biological measures like peripheral nervous system outputs such as skin conductance (SCR). Fear learning deficits have been observed in fear-related disorders like PTSD but have received little study in terms of parent-child learning transmission. We used a vicarious fear extinction paradigm to examine whether biological synchrony (SCR and heart rate variability [HRV]) is a potential mechanism by which children learn safety cues from their parents. In this pilot study, 16 dyads (PTSD n=11, typically developing [TD] n=5) underwent a vicarious fear extinction paradigm. We used cross-recurrence quantification analysis (CRQA) to assess SCR and HRV synchrony between parent-child dyads. We then used a linear model to examine group differences between PTSD dyads and TD dyads. For SCR, a significant group difference (p=.037) indicated that TD dyads had higher SCR synchrony compared to PTSD dyads. For HRV, there were no group differences between PTSD and TD dyads (p=.325). In exploratory analyses, increased synchrony was related to an overall decreased autonomic reactivity during recall of fear (p=0.032). These results suggest that SCR synchrony, but not HRV, may be a potential mechanism that allows for fear and safety learning in youth. While these data are preliminary, they provide novel insights on how disruptions in vicarious extinction learning may contribute to fear-related disorders in youth. Furthermore, this study suggests novel approaches to studying dyadic trauma-focused therapies which critically rely on parental coaching to model appropriate fear responses to help their child to recover from trauma.

Significance StatementThis study provides evidence that biological synchrony is a potential mechanism through which youth learn threat and safety cues through their parents. We found that youth with PTSD have lower synchrony with their caregiver, and that synchrony was related to decreased overall autonomic reactivity, suggesting that synchrony may be indicative of overall safety learning. Further, decreased synchrony during fear extinction may potentially underlie the etiology of fear related disorders such as PTSD. These novel approaches may improve our understanding of dyadic trauma-focused therapies which critically rely on parental coaching to model appropriate fear responses to help their child to recover from trauma.


title: Cattle T Cell Phenotyping by an 8-Colour, 10-Parameter Panel

abstract: Purpose and appropriate sample typesThis multiplex staining panel was developed to differentiate cattle T cells into conventional (CD4 and CD8) and unconventional ({gamma}{delta}-TCR) subsets as well as their stage of differentiation and activation. The combination of CD45RO and CD62L allows the identification of naive (TNaive), central memory (TCM), effector memory (TEM) and terminal effector (TTE) T cells. Activated cattle T cells (TAV) can be identified by the cell surface expression of CD25. This panel was developed using cryopreserved cattle peripheral blood mononuclear cells (PBMCs) and tested on fresh as well as stimulated PBMCs. Therefore, this 8-colour, 10-parameter flow cytometry panel simultaneously identifies cattle TNaive, TAV, TCM, TEM, TTE and {gamma}{delta}-TCR cells. This panel will improve our ability to examine T cell response to pathogens and vaccines in cattle including the potential to identify previously undescribed subpopulations. Furthermore, this panel can be readily optimised for other bovid species as many of these reagents are likely to cross react.

category: immunology


title: Optimized Multicolour Immunofluorescence Panel for Cattle T Cell Phenotyping by an 8-Colour, 10-Parameter Panel


title: Comparative transcriptome investigation of Nosema ceranae infecting eastern honeybee workers

abstract: Apis cerana is the original host for Nosema ceranae, a widespread fungal parasite resulting in bee nosemosis, which leads to severe losses for apiculture industry throughout the world. However, knowledge of N. ceranae infecting eastern honeybees is extremely limited. Currently, the mechanism underlying N. ceranae infection is still largely unknown. Based on our previously gained high-quality transcriptome datasets, comparative transcriptomic investigation was conducted in this work, with a focus on virulence factor-associated differentially expressed genes (DEGs). Microscopic observation showed that A. c. cerana workers midguts were effectively infected after inoculation with clean spores of N. ceranae. Totally, 1411, 604, and 38 DEGs were identified from NcCK vs. NcT1, NcCK vs. NcT2 and NcT1 vs. NcT2 comparison groups. Venn analysis showed that ten up-regulated genes and nine down-regulated ones were shared by aforementioned comparison groups. GO category indicated these DEGs were involved in a series of functional terms relevant to biological process, cellular component, and molecular function, such as metabolic process, cell part, and catalytic activity. Additionally, KEGG pathway analysis suggested that the DEGs were engaged in an array of pathways of great importance, such as metabolic pathway, glycolysis, and biosynthesis of secondary metabolites. Further, expression clustering analysis demonstrated that majority of genes encoding virulence factors such as ricin B lectins and polar tube proteins displayed apparent up-regulation, whereas a few virulence factor-associated genes such as hexokinase gene and 6-phosphofructokinase gene presented down-regulation during the fungal infection. Finally, the expression trend of 14 DEGs was confirmed by RT-qPCR, validating the reliability of our transcriptome datasets. These results together demonstrated that an overall alteration of the transcriptome of N. ceranae occurred during the infection of A. c. ceranae workers, and most of virulence factor-related genes were induced to activation to promote the fungal invasion. Our findings not only lay a foundation for clarifying the molecular mechanism underlying N. ceranae infection of eastern honeybee workers, but also shed light on developing novel targets for microsporidiosis control.

category: molecular biology


title: Explainable t-SNE for single-cell RNA-seq data analysis

abstract:
BackgroundSingle-cell RNA (scRNA-seq) sequencing technologies trigger the study of individual cell gene expression and reveal the diversity within cell populations. To measure cell-to-cell similarity based on their transcription and gene expression, many dimension reduction methods are employed to retrieve corresponding low-dimensional embeddings of input scRNA-seq data to conduct clustering. However, the methods lack explainability and may not perform well with scRNA-seq data because they are not customized for high-dimensional sparse scRNA-seq data.

ResultsIn this study, we propose an explainable t-SNE: cell-driven t-SNE (c-TSNE) that fuses cell differences reflected from biologically meaningful distance metrics for input data. Our study shows that the proposed method not only enhances the interpretation of the original t-SNE visualization but also demonstrates favorable single cell segregation performance on benchmark datasets compared to state-of-the-art peers. The robustness analysis shows that the proposed cell-driven t-SNE demonstrates robustness to dropout and noise in clustering. It provides a novel and practical way to investigate the interpretability of t-SNE in scRNA-seq data analysis. Unlike the general assumption that the explainability of a machine learning method may need to compromise with learning efficiency, the proposed explainable t-SNE improves both clustering efficiency and explainability. More importantly, our work suggests that widely used t-SNE can be easily misused in existing scRNA-seq analysis, because its default Euclidean distance can bring biases or meaningless results in cell difference evaluation for high-dimensional sparse scRNA-seq data. To the best of our knowledge, it is the first explainable t-SNE proposed in scRNA-seq analysis and will inspire other explainable machine learning method development in the field.

ConclusionThe proposed explainable t-SNE outperforms classic t-SNE and its peers in meaningful visualization and segregation. The poor performance of the classic t-SNE highlights the importance of developing explainable machine learning methods in scRNA-seq analysis. The explainable t-SNE is a data-centric customized ML enhance efficiency in data analysis through bringing more biological insights and interpretations.

category: bioinformatics


title: Enhanced food motivation in obese mice is controlled by D1R expressing spiny projection neurons in the nucleus accumbens.

abstract: Obesity is a chronic relapsing disorder that is caused by an excess of caloric intake relative to energy expenditure. In addition to homeostatic feeding mechanisms, there is growing recognition of the involvement of food reward and motivation in the development of obesity. However, it remains unclear how brain circuits that control food reward and motivation are altered in obese animals. Here, we tested the hypothesis that signaling through pro-motivational circuits in the core of the nucleus accumbens (NAc) is enhanced in the obese state, leading to invigoration of food seeking. Using a novel behavioral assay that quantifies physical work during food seeking, we confirmed that obese mice work harder than lean mice to obtain food, consistent with an increase in the relative reinforcing value of food in the obese state. To explain this behavioral finding, we recorded neural activity in the NAc core with both in vivo electrophysiology and cell-type specific calcium fiber photometry. Here we observed greater activation of D1-receptor expressing NAc spiny projection neurons (NAc D1SPNs) during food seeking in obese mice relative to lean mice. With ex vivo slice physiology we identified both pre- and post-synaptic mechanisms that contribute to this enhancement in NAc D1SPN activity in obese mice. Finally, blocking synaptic transmission from D1SPNs decreased physical work during food seeking and attenuated high-fat diet-induced weight gain. These experiments demonstrate that obesity is associated with a selective increase in the activity of D1SPNs during food seeking, which enhances the vigor of food seeking. This work also establishes the necessity of D1SPNs in the development of diet-induced obesity, establishing these neurons as a potential therapeutic target for preventing obesity.


title: EZ Clear for simple, rapid, and robust mouse whole organ clearing

abstract: Tissue clearing for whole organ cell profiling has revolutionized biology and imaging for exploration of organs in three-dimensional space without compromising tissue architecture. But complicated, laborious procedures, or expensive equipment, as well as the use of hazardous, organic solvents prevents the widespread adoption of these methods. Here we report a simple and rapid tissue clearing method, EZ Clear, that can clear whole adult mouse organs in 48 hours in just three simple steps. Samples stay at room temperature and remain hydrated throughout the clearing process, preserving endogenous and synthetic fluorescence, without altering sample size. After wholemount clearing and imaging, EZ Cleared samples can be further processed for downstream embedding and cryosectioning followed by standard histology or immunostaining, without loss of endogenous or synthetic fluorescence signal. Overall, the simplicity, speed, and flexibility of EZ Clear make it easy to adopt and apply to diverse approaches in biomedical research.


title: Sequence Coverage Visualizer: A web application for protein sequence coverage 3D visualization

abstract: Protein structure is connected with its function and interaction and plays an extremely important role in protein characterization. As one of the most important analytical methods for protein characterization, Proteomics is widely used to determine protein composition, quantitation, interaction, and even structures. However, due to the gap between identified proteins by proteomics and available 3D structures, it was very challenging, if not impossible, to visualize proteomics results in 3D and further explore the structural aspects of proteomics experiments. Recently, two groups of researchers from DeepMind and Baker lab have independently published protein structure prediction tools that can help us obtain predicted protein structures for the whole human proteome. Although there is still debate on the validity of some of the predicted structures, it is no doubt that these represent the most accurate predictions to date. More importantly, this enabled us to visualize the majority of human proteins for the first time. To help other researchers best utilize these protein structure predictions, we present the Sequence Coverage Visualizer (SCV), http://scv.lab.gy, a web application for protein sequence coverage 3D visualization. Here we showed a few possible usages of the SCV, including the labeling of post-translational modifications and isotope labeling experiments. These results highlight the usefulness of such 3D visualization for proteomics experiments and how SCV can turn a regular result list into structural insights. Furthermore, when used together with limited proteolysis, we demonstrated that SCV can help validate and compare different protein structures, including predicted ones and existing PDB entries. By performing limited proteolysis on native proteins at various time points, SCV can visualize the progress of the digestion. This time-series data further allowed us to compare the predicted structure and existing PDB entries. Although not deterministic, these comparisons could be used to refine current predictions further and represent an important step towards a complete and correct protein structure database. Overall, SCV is a convenient and powerful tool for visualizing proteomics results.


title: An in silico method to assess antibody fragment polyreactivity

abstract: Antibodies are essential biological research tools and important therapeutic agents, but some exhibit non-specific binding to off-target proteins and other biomolecules. Such polyreactive antibodies compromise screening pipelines, lead to incorrect and irreproducible experimental results, and are generally intractable for clinical development. We designed a set of experiments using a diverse naive synthetic camelid antibody fragment ( nanobody) library to enable machine learning models to accurately assess polyreactivity from protein sequence (AUC > 0.8). Moreover, our models provide quantitative scoring metrics that predict the effect of amino acid substitutions on polyreactivity. We experimentally tested our models performance on three independent nanobody scaffolds, where over 90% of predicted substitutions successfully reduced polyreactivity. Importantly, the model allowed us to diminish the polyreactivity of an angiotensin II type I receptor antagonist nanobody, without compromising its pharmacological properties. We provide a companion web-server that offers a straightforward means of predicting polyreactivity and polyreactivity-reducing mutations for any given nanobody sequence.

category: pharmacology and toxicology


title: Semi-field evaluations of three botanically derived repellents against the blacklegged tick, Ixodes scapularis (Acari: Ixodidae)

abstract: Three compounds derived from botanicals sources, ethyl perillyl carbonate, geranyl isovalerate, and citronellyl cyclobutane carboxylate, were tested for repellent activity against Ixodes scapularis Say in a semi-field trial. Tick drags were treated with the compounds or with N, N-diethyl-m-toluamide (DEET) at high (0.25mg/cm2) or low (0.15mg/cm2) concentrations. Negative controls included untreated drags and drags treated with acetone, the carrier for all repellents. Freshly treated drags (within 20 minutes) were used to collect I. scapularis ticks at a county park in Wisconsin. To assess effectiveness, we measured tick encounter rates, detachment rate, and time to detachment. None of the repellent treatments resulted in significantly fewer encounters compared to both control treatments. However, the percentage of ticks that detached within 3 min was significantly higher on drags treated with repellents compared to controls. DEET was the most effective, repelling 69.7 - 87% of ticks by 3 min, but the effectiveness of the three test compounds was still high, ranging from 42% to 87% of ticks detaching by 3 min. For time to detachment, there were no significant differences between DEET and the three test compounds. We conclude that these botanically-derived repellents were effective against I. scapularis in a semi-field trial and could be viable alternatives to DEET.

category: zoology


title: Flexible cue anchoring strategies enable stable head direction coding in both sighted and blind animals

abstract:
Vision plays a crucial role in instructing the brains spatial navigation systems. However, little is known about how vision loss affects the neuronal encoding of spatial information. Here, recording from head direction (HD) cells in the anterior dorsal nucleus of the thalamus in mice, we find stable and robust HD tuning in blind animals. In contrast, placing sighted animals in darkness significantly impairs HD cell tuning. We find that blind mice use olfactory cues to maintain stable HD tuning and that prior visual experience leads to refined HD cell tuning in blind adult mice compared to congenitally blind animals. Finally, in the absence of both visual and olfactory cues, the HD attractor network remains intact but the preferred firing direction of HD cells continuously drifts over time. We thus demonstrate remarkable flexibility in how the brain uses diverse sensory information to generate a stable directional representation of space.

HighlightsO_LIHead direction (HD) cell tuning in ADn is robust in blind animals, but unstable in sighted animals placed in the dark
C_LIO_LIBlind mice use olfaction to stabilize HD cell tuning
C_LIO_LIPrior visual experience leads to refined HD cell tuning in blind adult mice
C_LIO_LIIn the absence of both vision and olfaction, the HD attractor network in ADn remains intact but the preferred firing direction of HD cells continuously drifts
C_LI


title: A Precision Functional Atlas of Network Probabilities and Individual-Specific Network Topography

abstract: The brain is organized into a broad set of functional neural networks. These networks and their various characteristics have been described and scrutinized through in vivo resting state functional magnetic resonance imaging (rs-fMRI). While the basic properties of networks are generally similar between healthy individuals, there is vast variability in the precise topography across the population. These individual differences are often lost in population studies due to population averaging which assumes topographical uniformity. We leveraged precision brain mapping methods to establish a new open-source, method-flexible set of precision functional network atlases: the Masonic Institute for the Developing Brain (MIDB) Precision Brain Atlas. Using participants from the Adolescent Brain Cognitive Development (ABCD) study, single subject precision network maps were generated with two supervised network-matching procedures (template matching and non-negative matrix factorization), an overlapping template matching method for identifying integration zones, as well as an unsupervised community detection algorithm (Infomap). From these individualized maps we also generated probabilistic network maps and integration zones for two demographically-matched groups of n[~]3000 each. We demonstrate high reproducibility between groups (Pearsons r >0.999) and between methods (r=0.96), revealing both regions of high invariance and high variability. Compared to using parcellations based on groups averages, the MIDB Precision Brain Atlas allowed us to derive a set of brain regions that are largely invariant in network topography across populations, which provides more reproducible statistical maps of executive function in brain-wide associations. We also explore an example use case for probabilistic maps, highlighting their potential for use in targeted neuromodulation. The MIDB Precision Brain Atlas is expandable to alternative datasets and methods and is provided open-source with an online web interface to encourage the scientific community to experiment with probabilistic atlases and individual-specific topographies to more precisely relate network phenomenon to functional organization of the human brain.


title: Antisense oligonucleotide-based therapeutic strategy for progranulin-deficient frontotemporal dementia

abstract: GRN mutations cause frontotemporal dementia (FTD) due to haploinsufficiency of progranulin. Several microRNAs, including miR-29b, negatively regulate progranulin protein levels. Antisense oligonucleotides (ASOs) are emerging as a promising therapeutic modality for neurological diseases, but ASO-based strategies for increasing target protein levels are limited. Here, we tested if ASOs can increase progranulin levels by sterically blocking the miR-29b binding site in the 3 UTR of the human GRN mRNA. We found 16 ASOs that increase progranulin protein levels in a dose-dependent manner in neuroglioma cells. A subset of these ASOs also increased progranulin levels in iPSC-derived neurons and in a humanized GRN mouse model. Ribosomal profiling experiments revealed that the ASOs increase the rate of progranulin translation. Consistent with this, ASO treatment increased levels of newly synthesized progranulin protein. In FRET-based assays, the ASOs effectively competed miR-29b from binding to the GRN 3 UTR RNA. Together, our results demonstrate that ASOs can be used to effectively increase target protein levels by partially blocking miR binding sites. This ASO strategy may be therapeutically feasible for progranulin-deficient FTD as well as other conditions of haploinsufficiency.


title: Antisense oligonucleotide strategies to increase progranulin levels for frontotemporal dementia


title: Autophagy is impaired in fetal hypoplastic lungs and rescued by administration of amniotic fluid stem cell extracellular vesicles

abstract:
RationalePulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH) is characterized by reduced branching morphogenesis, which is responsible for poor clinical outcomes. Administration of amniotic fluid stem cell extracellular vesicles (AFSC-EVs) rescues branching morphogenesis in rodent fetal models of pulmonary hypoplasia. Herein, we hypothesized that AFSC-EVs exert their regenerative potential by affecting autophagy, a process required for normal lung development.

ObjectivesTo evaluate autophagy in hypoplastic lungs throughout gestation and establish whether AFSC-EV administration improves branching morphogenesis through autophagy-mediated mechanisms.

MethodsEVs were isolated from c-kit+ AFSC conditioned medium by ultracentrifugation and characterized for size, morphology, and EV markers. Branching morphogenesis was inhibited in rat fetuses by nitrofen administration to dams and in human fetal lung explants by blocking RAC1 activity with NSC23766. Expression of autophagy activators (BECN1 and ATG5) and adaptor (SQSTM1/p62) was analyzed in vitro (rat and human fetal lung explants) and in vivo (rat fetal lungs). Mechanistic studies on rat fetal primary lung epithelial cells were conducted using inhibitors for microRNA-17 and -20a contained in the AFSC-EV cargo and known to regulate autophagy.

Measurements and Main ResultsRat and human models of fetal pulmonary hypoplasia showed reduced autophagy mainly at pseudoglandular and canalicular stages. AFSC-EV administration restored autophagy in both pulmonary hypoplasia models by transferring miR-17[~]92 cluster members contained in the EV cargo.

ConclusionsAFSC-EV treatment rescues branching morphogenesis partly by restoring autophagy through miRNA cargo transfer. This study enhances our understanding of pulmonary hypoplasia pathogenesis and creates new opportunities for fetal therapeutic intervention in CDH babies.


title: Dynamic control systems that mimic natural regulation of catabolic pathways enable rapid production of lignocellulose-derived bioproducts

abstract: Expanding the catabolic repertoire of engineered microbial bioproduction hosts enables more complete use of complex feedstocks such as lignocellulosic hydrolysates and deconstructed mixed plastics, but the deleterious effects of existing expression systems limit the maximum carry capacity for heterologous catabolic pathways. Here, we demonstrate use of a conditionally beneficial oxidative xylose catabolic pathway to improve performance of a Pseudomonas putida strain that has been engineered for growth-coupled bioconversion of glucose into the valuable bioproduct cis,cis-muconic acid. In the presence of xylose, the pathway enhances growth rate, and therefore productivity, by >60%, but the metabolic burden of constitutive pathway expression reduces growth rate by >20% in the absence of xylose. To mitigate this growth defect, we develop a xylose biosensor based on the XylR transcription factor from Caulobacter crescentus NA1000 to autonomously regulate pathway expression. We generate a library of engineered xylose-responsive promoters that cover a three order-of-magnitude range of expression levels to tune pathway expression. Using structural modeling to guide mutations, we engineer XylR with two and three orders-of-magnitude reduced sensitivity to xylose and L-arabinose, respectively. A previously developed heterologous xylose isomerase pathway is placed under control of the biosensor, which improves the growth rate with xylose as a carbon source by 10% over the original constitutively expressed pathway. Finally, the oxidative xylose catabolic pathway is placed under control of the biosensor, enabling the bioproduction strain to maintain the increased growth rate in the presence of xylose, without the growth defect incurred from constitutive pathway expression in the absence of xylose. Utilizing biosensors to autonomously regulate conditionally beneficial catabolic pathways is generalizable approach that will be critical for engineering bioproduction hosts bacteria with the wide range of catabolic pathways required for bioconversion of complex feedstocks.

category: synthetic biology


title: Climate change impacts on the abiotic degradation of acyl-homoserine lactones in the fluctuating conditions of marine biofilms

abstract:
Marine biofilms are functional communities that shape habitats by providing a range of structural and functional services integral to coastal ecosystems. Impacts of climate change on biological aspects of such communities are increasingly studied, but impacts on the chemicals that mediate key interactions of biofilm organisms have largely been overlooked. Acyl-homoserine lactones (AHLs), crucial bacterial signals within biofilms, are known to degrade through pH and temperature-dependent hydrolysis. However, the impact of climate change on AHLs and thus on biofilm form and function is presently unknown. This study investigates the impact of changes in pH and temperature on the hydrolysis rate, half-life time and quantitative abundance of different AHLs on daily and seasonal timescales for current conditions and future climate change scenarios.

We established the mathematical relationships between pH, hydrolysis rates/ half-life times and temperature, which revealed that natural daily pH-driven changes within biofilms cause the greatest fluctuations in AHL concentration (up to 9-fold). Season-dependant temperature enhanced or reduced the observed daily dynamics, leading to higher winter and lower summer concentrations and caused a shift in timing of the highest and lowest AHL concentration by up to two hours. Simulated future conditions based on climate change projections caused an overall reduction of AHL degradation and led to higher AHL concentrations persisting for longer across both the daily and seasonal cycles.

This study provides valuable quantitative insights into the theoretical natural dynamics of AHL concentrations. We highlight critical knowledge gaps on the scale of abiotic daily and seasonal fluctuations affecting estuarine and coastal biofilms and on the biofilms buffering capacity. Detailed experimental studies of daily and seasonal dynamics of AHL concentrations and assessment of the potential implications for a suite of more complex interactions are required. Substantial fluctuations like those we show in this study, particularly with regards to concentration and timing, will likely have far reaching implications for fundamental ecosystem processes and important ecosystem services such as larval settlement and coastal sediment stabilisation.


title: COVID-19 vaccine booster induces a strong CD8+ T cell response against Omicron variant epitopes in HLA-A*02:01+ individuals

abstract:
The >30 mutated residues in the Omicron spike protein have led to its rapid classification as a new SARS-CoV-2 variant of concern. As a result, Omicron may escape from the immune system, decreasing the protection provided by COVID-19 vaccines. Preliminary data shows a weaker neutralizing antibody response to Omicron compared to the ancestral SARS-CoV-2 virus, which can be increased after a booster vaccine. Here, we report that CD8+ T cells can recognize Omicron variant epitopes presented by HLA-A*02:01 in both COVID-19 recovered and vaccinated individuals, even 6 months after infection or vaccination. Additionally, the T cell response was stronger for Omicron variant epitopes after the vaccine booster. Altogether, T cells can recognize Omicron variants, especially in vaccinated individuals after the vaccine booster.

One-Sentence SummaryCD8+ T cells response against Omicron variant epitopes is stronger after the vaccine booster.


title: matOptimize: A parallel tree optimization method enables online phylogenetics for SARS-CoV-2

abstract:
Phylogenetic tree optimization is necessary for precise analysis of evolutionary and transmission dynamics, but existing tools are inadequate for handling the scale and pace of data produced during the COVID-19 pandemic. One transformative approach, online phylogenetics, aims to incrementally add samples to an ever-growing phylogeny, but there are no previously-existing approaches that can efficiently optimize this vast phylogeny under the time constraints of the pandemic. Here, we present matOptimize, a fast and memory-efficient phylogenetic tree optimization tool based on parsimony that can be parallelized across multiple CPU threads and nodes, and provides orders of magnitude improvement in runtime and peak memory usage compared to existing state-of-the-art methods. We have developed this method particularly to address the pressing need during the COVID-19 pandemic for daily maintenance and optimization of a comprehensive SARS-CoV-2 phylogeny. Thus, our approach addresses an important need for daily maintenance and refinement of a comprehensive SARS-CoV-2 phylogeny.

Significance StatementPhylogenetic trees have been central to genomic surveillance, epidemiology, and contact tracing efforts during the COVD-19 pandemic. With over 6 million SARS-CoV-2 genome sequences now available, maintaining an accurate, comprehensive phylogenetic tree of all available SARS-CoV-2 sequences is becoming computationally infeasible with existing software, but is essential for getting a detailed picture of the virus evolution and transmission. Our novel phylogenetic software, matOptimize, is helping refine possibly the largest-ever phylogenetic tree, containing millions of SARS-CoV-2 sequences, thus providing an unprecedented resolution for studying the pathogens evolutionary and transmission dynamics.

category: evolutionary biology


title: An antibody targeting the N-terminal domain of SARS-CoV-2 disrupts the spike trimer

abstract: The protective human antibody response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus focuses on the spike (S) protein which decorates the virion surface and mediates cell binding and entry. Most SARS-CoV-2 protective antibodies target the receptor- binding domain or a single dominant epitope ( supersite) on the N terminal domain (NTD). Here, using the single B cell technology LIBRA-seq, we isolated a large panel of NTD-reactive and SARS-CoV-2 neutralizing antibodies from an individual who had recovered from COVID-19. We found that neutralizing antibodies to the NTD supersite commonly are encoded by the IGHV1-24 gene, forming a genetic cluster that represents a public B cell clonotype. However, we also discovered a rare human antibody, COV2-3434, that recognizes a site of vulnerability on the SARS-CoV-2 S protein in the trimer interface and possesses a distinct class of functional activity. COV2-3434 disrupted the integrity of S protein trimers, inhibited cell-to-cell spread of virus in culture, and conferred protection in human ACE2 transgenic mice against SARS-CoV-2 challenge. This study provides insight about antibody targeting of the S protein trimer interface region, suggesting this region may be a site of virus vulnerability.


title: Immunogenicity of convalescent and vaccinated sera against clinical isolates of ancestral SARS-CoV-2, beta, delta, and omicron variants

abstract: The omicron variant of concern (VOC) of SARS-CoV-2 was first reported in November 2021 in Botswana and South Africa. Omicron has evolved multiple mutations within the spike protein and the receptor binding domain (RBD), raising concerns of increased antibody evasion. Here, we isolated infectious omicron from a clinical specimen obtained in Canada. The neutralizing activity of sera from 65 coronavirus disease (COVID-19) vaccine recipients and convalescent individuals against clinical isolates of ancestral SARS-CoV-2, beta, delta, and omicron VOCs was assessed. Convalescent sera from unvaccinated individuals infected by the ancestral virus during the first wave of COVID-19 in Canada (July, 2020) demonstrated reduced neutralization against beta and omicron VOCs. Convalescent sera from unvaccinated individuals infected by the delta variant (May-June, 2021) neutralized omicron to significantly lower levels compared to the delta variant. Sera from individuals that received three doses of the Pfizer or Moderna vaccines demonstrated reduced neutralization of the omicron variant relative to ancestral SARS-CoV-2. Sera from individuals that were naturally infected with ancestral SARS-CoV-2 and subsequently received two doses of the Pfizer vaccine induced significantly higher neutralizing antibody levels against ancestral virus and all VOCs. Importantly, infection alone, either with ancestral SARS-CoV-2 or the delta variant was not sufficient to induce high neutralizing antibody titers against omicron. This data will inform current booster vaccination strategies, and we highlight the need for additional studies to identify longevity of immunity against SARS-CoV-2 and optimal neutralizing antibody levels that are necessary to prevent infection and/or severe COVID-19.


title: The SARS-CoV-2 Omicron (B.1.1.529) variant exhibits altered pathogenicity, transmissibility, and fitness in the golden Syrian hamster model

abstract:
The newly emerging SARS-CoV-2 Omicron (B.1.1.529) variant first identified in South Africa in November 2021 is characterized by an unusual number of amino acid mutations in its spike that renders existing vaccines and therapeutic monoclonal antibodies dramatically less effective. The in vivo pathogenicity, transmissibility, and fitness of this new Variant of Concerns are unknown. We investigated these virological attributes of the Omicron variant in comparison with those of the currently dominant Delta (B.1.617.2) variant in the golden Syrian hamster COVID-19 model. Omicron-infected hamsters developed significantly less body weight losses, clinical scores, respiratory tract viral burdens, cytokine/chemokine dysregulation, and tissue damages than Delta-infected hamsters. The Omicron and Delta variant were both highly transmissible (100% vs 100%) via contact transmission. Importantly, the Omicron variant consistently demonstrated about 10-20% higher transmissibility than the already-highly transmissible Delta variant in repeated non-contact transmission studies (overall: 30/36 vs 24/36, 83.3% vs 66.7%). The Delta variant displayed higher fitness advantage than the Omicron variant without selection pressure in both in vitro and in vivo competition models. However, this scenario drastically changed once immune selection pressure with neutralizing antibodies active against the Delta variant but poorly active against the Omicron variant were introduced, with the Omicron variant significantly outcompeting the Delta variant. Taken together, our findings demonstrated that while the Omicron variant is less pathogenic than the Delta variant, it is highly transmissible and can outcompete the Delta variant under immune selection pressure. Next-generation vaccines and antivirals effective against this new VOC are urgently needed.

One Sentence SummaryThe novel SARS-CoV-2 Omicron variant, though less pathogenic, is highly transmissible and outcompetes the Delta variant under immune selection pressure in the golden Syrian hamster COVID-19 model.


title: Site-specific adaptation mechanisms of an oral pathobiont in the oral and gut mucosae

abstract:
Periodontal inflammation leads to oral dysbiosis with the expansion of oral pathobionts. Besides the pathogenic role of oral pathobionts during periodontal inflammation, studies have revealed that oral pathobionts contribute to diseases in distant organs beyond the oral mucosa. For example, the oral pathobiont Klebsiella aerogenes, which accumulates in the oral mucosa during periodontitis in mice, can exacerbate colitis when it ectopically colonizes the gastrointestinal tract. However, the precise mechanisms by which oral pathobionts establish their colonization in extra-oral mucosal sites remains incompletely understood. We performed high-throughput in vivo genetic screening to identify fitness genes required for the adaptation of the oral pathobiont K. aerogenes to different mucosal sites - the oral and gut mucosae - at the steady state and during inflammation. In addition, the global transcriptome of K. aerogenes in different environments was analyzed. We determined that K. aerogenes employs genes related to iron acquisition and chaperone usher pili, which are encoded on a newly identified genomic locus named "locus of colonization in the inflamed gut" (LIG), for adaptation in the gut mucosa, particularly during inflammation. In contrast, the LIG virulence factors are not required for K. aerogenes to adapt to the oral mucosa. Thus, oral pathobionts likely exploit distinct adaptation mechanisms in their ectopically colonized intestinal niche compared to their original niche.

Author SummaryThe symbiotic bacterial community evolves uniquely at each body site. However, under certain circumstances, symbionts may disseminate far from their original niche and colonize a site ectopically. It has been reported that oral resident bacteria associated with periodontal disease, namely oral pathobionts, contribute to gastrointestinal diseases, such as inflammatory bowel disease and colorectal cancer, through ectopic colonization of the gut. However, the molecular mechanisms by which oral pathobionts adapt to the gut environment remain largely unexplored. In this study, using a model oral pathobiont Klebsiella aerogenes, we screened genes essential for the colonization of the pathobiont at different mucosal sites. We discovered that the oral pathobiont uses distinct virulence mechanisms at two different mucosal sites - the oral and gut mucosae. Understanding the strategies of bacteria localized in ectopic organs may lead to the development of strategies to prevent disease caused by the dissemination of pathogenic oral bacteria.


title: AAV11 permits efficient retrograde targeting of projection neurons

abstract:
Viral tracers that permit efficient retrograde targeting of projection neurons are powerful vehicles for structural and functional dissections of the neural circuit and for the treatment of brain diseases. Recombinant adeno-associated viruses (rAAVs) are the most potential candidates because they are low-toxic with high-level transgene expression and minimal host immune responses. Currently, some rAAVs based on capsid engineering for retrograde tracing have been widely used in the analysis and manipulation of neural circuits, but suffer from brain area selectivity and inefficient retrograde transduction in certain neural connections. Here, we discovered that the recombinant adeno-associated virus 11 (rAAV11) exhibits potent retrograde labeling of projection neurons with enhanced efficiency to rAAV2-retro in some neural connections. Combined with calcium recording technology, rAAV11 can be used to monitor neuronal activities by expressing Cre recombinase or calcium-sensitive functional probe. In addition, we further showed the suitability of rAAV11 for astrocyte targeting. These properties make rAAV11 a promising tool for the mapping and manipulation of neural circuits and gene therapy of some neurological and neurodegenerative disorders.

HighlightsO_LINaturally occurring AAV serotype capsid exhibits robust retrograde functionality
C_LIO_LIImproved distribution properties and retrograde transport efficiency
C_LIO_LICan express Cre recombinase or calcium-sensitive functional probe for neural circuits monitoring
C_LIO_LICan specifically target astrocytes
C_LI


title: Bifidobacterium longum attenuates ovariectomy-induced bone loss via modulating the Immunoporotic Breg-Treg-Th17 cell axis

abstract: Discoveries in the last few years have emphasized the existence of an enormous breadth of communication between osteo-immune system. These discoveries fuel novel approaches for the treatment of several bone-pathologies including osteoporosis, an inflammatory bone anomaly affecting more than 500 million people globally. Bifidobacterium longum (BL) is preferred probiotic of choice due to its varied immunomodulatory potential in alleviating various inflammatory diseases. Here, we evaluate the effect of BL in ovariectomy (ovx)-induced post-menopausal osteoporotic mice model. Our in vitro findings reveal that BL suppresses the differentiation and functional activity of RANKL-induced osteoclastogenesis in both mouse bone marrow cells and human PBMCs. Our in vivo data clearly establish that BL exhibits osteoprotective potential via modulating the "immunoporotic" Breg-Treg-Th17 cell-axis. Furthermore, {micro}CT and bone mechanical strength data support that BL supplementation significantly enhanced bone mass and strength, and improved microarchitecture in ovx mice. Remarkably, alteration in frequencies of CD19+CD1dhiCD5+ Bregs, CD4+Foxp3+IL-10+ Tregs, and CD4+Ror{gamma}t+IL-17+ Th17 immune cells in distinct lymphoid organs along with serum-cytokine data (enhanced anti-osteoclastogenic cytokines IFN-{gamma} and IL-10 and reduced osteoclastogenic-cytokines IL-6, IL-17, and TNF-) strongly support the immunomodulatory potential of BL. Altogether our findings establish a novel osteo-protective and immunoporotic potential of BL in augmenting bone health under osteoporotic conditions.


title: Standard selection treatments with sulfadiazine limit Plasmodium yoelii host-to-vector transmission

abstract: Some early antimalarial drugs have been repurposed for experimental applications, thus extending their utility well beyond the point when resistance becomes prevalent in circulating parasite populations. One such drug is sulfadiazine, which is an analog of p-aminobenzoic acid (pABA), and acts as a competitive inhibitor of dihydropteroate synthase, which is an essential enzyme in the parasites folate synthesis pathway that is required for DNA synthesis. Sulfadiazine treatment of mice infected with P. yoelii and P. berghei is routinely used to enrich for gametocytes by killing asexual blood stage parasites, but it is not well known if the exposed gametocytes are perturbed or if there is a detrimental effect on transmission. To determine if there was a significant effect of sulfadiazine exposure upon host-to-vector transmission, we transmitted Plasmodium yoelii (17XNL strain) parasites to Anopheles stephensi mosquitoes and evaluated the prevalence of infection (percent of mosquitoes infected) and intensity of infection (number of oocysts per infected mosquito) under different sulfadiazine treatment conditions of the mouse or of the mosquitoes. We observed that parasites exposed to sulfadiazine either in the mouse host or in the mosquito vector had a reduction in both the number of mosquitoes that became infected and in the intensity of infection compared to untreated controls. We also observed that provision of freshly prepared pABA in the mosquito sugar water could only marginally overcome the defects caused by sulfadiazine treatment. In contrast, we determined that gametocytes exposed to sulfadiazine were able to be fertilized and develop into morphologically mature ookinetes in vitro, and thus that sulfadiazine exposure in the host may be reversible if the drug is washed out and the parasites are supplemented with pABA in the culture media. Overall, this indicates that sulfadiazine dampens host-to-vector transmission, and that this inhibition can only be partially overcome by exposure to fresh pABA in vivo and in vitro. Because gametocytes are of great interest for developing transmission blocking interventions, we recommend that less disruptive approaches for gametocyte enrichment be used in order to study minimally perturbed parasites.


title: Transatlantic spread of highly pathogenic avian influenza H5N1 by wild birds from Europe to North America in 2021

abstract:
Highly pathogenic avian influenza (HPAI) viruses of the A/Goose/Guangdong/1/1996 lineage (GsGd), which threaten the health of poultry, wildlife and humans, are spreading across Asia, Europe and Africa, but are currently absent from Oceania and the Americas. In December 2021, H5N1 HPAI viruses were detected in poultry and a free-living gull in St. Johns, Newfoundland and Labrador, Canada. Phylogenetic analysis showed that these viruses were most closely related to HPAI GsGd viruses circulating in northwestern Europe in spring 2021. Analysis of wild bird migration suggested that these viruses may have been carried across the Atlantic via Iceland, Greenland/Arctic or pelagic routes. The here documented incursion of HPAI GsGd viruses into North America raises concern for further virus spread across the Americas by wild bird migration.

One-Sentence SummaryDetection of H5N1 highly pathogenic avian influenza in Canada raises concern for spread in the Americas by migratory birds.


title: A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase

abstract: Pre-mRNA splicing is indispensable for eukaryotic gene expression. Splicing inhibition causes cell cycle arrest and cell death, which are the reasons for the potent antitumor activity of splicing inhibitors. Here, we found that truncated proteins are involved in cell cycle arrest and cell death upon splicing inhibition. We analyzed pre-mRNAs accumulated in the cytoplasm where translation occurs, and found that a truncated form of the p27 CDK inhibitor, named p27*, is translated from pre-mRNA and accumulates in G2-arrested cells. Overexpression of p27* caused G2-phase arrest through inhibiting CDK-cyclin complexes. Conversely, knockout of p27* accelerated resumption of cell proliferation after washout of splicing inhibitor. Interestingly, p27* was resistant to proteasomal degradation. We propose that cells produce truncated proteins differing functionally from the original proteins via pre-mRNA translation only under splicing-deficient conditions in response to such conditions.


title: Overexpression of miR5505 enhanced drought and salt resistance in rice (Orayza sativa)

abstract: Rice is one of the most important crops in the world and half of the world population consumes it as their staple food. The abiotic stresses caused by drought, salt and other stresses have severely impacted rice production. MicroRNAs (miRNAs) are a type of small non-coding RNAs which widely reported as gene regulators, suppressing genes expression by degradation mRNA or translation inhibition. Previously, high-throughput sequencing has found a conserved miRNA miR5505 responding to drought stress in Dongxiang wild rice (DXWR). Several other studies also revealed that miR5505 was involved in rice stress responses. We further studied the effect of miRNA in drought and salt tolerance by overexpression it in rice. 2 in 18 successfully transformed transgenic lines with higher miR5505 expression were selected and then drought and salt resistance ability were evaluated. Both transgenic lines showed stronger drought and salt tolerance than wild-type (WT). Putative targets of miR5505 were identified by psRNATarget and several of them were found stress-related. RNA-seq found 1,980 differentially expressed genes (DEGs) in transgenic lines. Among them, 978 genes were down-regulated. Three genes were predicted by psRNATarget and two of them might be stress-related. We also found various environmental stress cis-acting elements in upstream of miR5505 promoter through Software PlantCARE. In all, we improved rice drought and salt tolerance by overexpressing miR5505, and the generated putative targets and cis-acting elements also suggested miR5505 might play important roles in the regulation of drought and salt responses.


title: Proteasome granular localization is regulated through mitochondrial respiration and kinase signaling

abstract:
In yeast, proteasomes are enriched in cell nuclei where they execute important cellular functions. Nutrient-stress can change this localization indicating proteasomes respond to the cells metabolic state. However, the signals that connect these processes remain poorly understood. Carbon starvation triggers a reversible translocation of proteasomes to cytosolic condensates known as proteasome storage granules (PSGs). Surprisingly, we observed strongly reduced PSG levels when cells had active cellular respiration prior to starvation. This suggests the mitochondrial activity of cells is a determining factor in the response of proteasomes to carbon starvation. Consistent with this, upon inhibition of mitochondrial function we observed proteasomes relocalize to granules. These links between proteasomes and metabolism involve specific signaling pathways, as we identified a MAP kinase cascade that is critical to the formation of proteasome granules after respiratory growth but not following glycolytic growth. Furthermore, the yeast homolog of AMP kinase, Snf1, is important for proteasome granule formation induced by mitochondrial inhibitors, while dispensable for granule formation following carbon starvation. We propose a model where mitochondrial activity promotes proteasome nuclear localization.

SummaryWe determined a role for mitochondrial respiration in regulating proteasome granule formation and identified the cell integrity MAP kinase pathway and Snf1 kinase as regulatory factors.

category: cell biology


title: cGAS recruitment to micronuclei is dictated by pre-existing nuclear chromatin status.

abstract: Micronuclei (MN) are aberrant cytosolic compartments containing broken genomic fragments or whole lagging chromosomes. MN envelopes irreversibly rupture, allowing the viral receptor cGAS to localize to MN and initiate an inflammatory signalling cascade. Here, we demonstrate that MN envelope rupture is not sufficient for cGAS localization. Unlike MN that arise following ionizing radiation (IR), ruptured MN generated from acute transcription stressors DRB or siSRSF1 are refractory to cGAS localization. Recruitment of cGAS to MN is blocked by inhibiting the histone methyltransferase DOT1L prior to IR exposure, demonstrating that cGAS recruitment to MN is dictated by nuclear chromatin organization at the time of DNA damage. Loss of cGAS+ MN, caused either by acute transcription stressors or by preventing DOT1L-deposited histone methylation, corresponded to significantly decreased cGAS-dependent inflammatory signalling. These results implicate nuclear chromatin organization in micronuclear composition and activity, influencing the ability of damage-induced MN to retain cytosolic proteins upon rupture.


title: Optineurin mediates delivery of membrane from transferrin receptor-positive endosomes to autophagosomes

abstract: Autophagy is a conserved quality control mechanism that removes damaged proteins, organelles and invading bacteria through lysosome-mediated degradation. During autophagy several organelles including endoplasmic reticulum, mitochondria, plasma membrane and endosomes contribute membrane for autophagosome formation. However, the mechanisms and proteins involved in membrane delivery to autophagosomes are not clear. Optineurin (OPTN), a cytoplasmic adaptor protein, is involved in promoting maturation of phagophores into autophagosomes; it is also involved in regulating endocytic trafficking and recycling of transferrin receptor (TFRC). Here, we have examined the role of optineurin in the delivery of membrane from TFRC-positive endosomes to autophagosomes. Only a small fraction of autophagosomes was positive for TFRC, indicating that TFRC-positive endosomes could contribute membrane to a subset of autophagosomes. The percentage of TFRC-positive autophagosomes was reduced in Optineurin knockout mouse embryonic fibroblasts (Optn-/- MEFs) in comparison with normal MEFs. Upon over-expression of optineurin, the percentage of TFRC-positive autophagosomes was increased in Optn-/- MEFs. Unlike wild-type optineurin, a disease-associated mutant, E478G, defective in ubiquitin binding, was not able to enhance formation of TFRC-positive autophagosomes in Optn-/- MEFs. TFRC degradation mediated by autophagy was decreased in optineurin deficient cells. Our results suggest that optineurin mediates delivery of TFRC and perhaps associated membrane from TFRC-positive endosomes to autophagosomes, and this may contribute to autophagosome formation.


title: Hypercapnia limits β-catenin mediated alveolar type 2 cell progenitor function by altering Wnt production from adjacent fibroblasts

abstract: Persistent symptoms and radiographic abnormalities suggestive of failed lung repair are among the most common symptoms in patients with COVID-19 after hospital discharge. In mechanically ventilated patients with ARDS secondary to SARS-CoV-2 pneumonia, low tidal volumes to reduce ventilator-induced lung injury necessarily elevate blood CO2 levels, often leading to hypercapnia. The role of hypercapnia on lung repair after injury is not completely understood. Here, we show that hypercapnia limits {beta}-catenin signaling in alveolar type 2 (AT2) cells, leading to reduced proliferative capacity. Hypercapnia alters expression of major Wnts in PDGFRa+-fibroblasts from those maintaining AT2 progenitor activity and towards those that antagonize {beta}-catenin signaling and limit progenitor function. Activation of {beta}-catenin signaling in AT2 cells, rescues the inhibition AT2 proliferation induced by hypercapnia. Inhibition of AT2 proliferation in hypercapnic patients may contribute to impaired lung repair after injury, preventing sealing of the epithelial barrier, increasing lung flooding, ventilator dependency and mortality.


title: Persistence of parental age effect on somatic mutation rates across generations in Arabidopsis.

abstract: In the model plant Arabidopsis thaliana, parental age is known to affect somatic mutation rates in their immediate progeny and here we show that this age dependent effect persists across successive generations. Using a set of detector lines carrying the mutated uidA gene, we examined if a particular parental age maintained across five consecutive generations affected the rates of base substitution (BSR), intrachromosomal recombination (ICR), frameshift mutation (FS), and transposition. The frequency of functional GUS reversions were assessed in seedlings as a function of identical/different parental ages across generations. In the context of a fixed parental age, BCR/ICR rates were unaffected in the first three generations, then dropped significantly in the 4th and increased in most instances in the 5th generation. On the other hand, with advancing parental ages, BSR/ICR rates remained high in the first two/three generations, with a striking resemblance in the pattern of mutation rates. We adopted a novel approach of identifying and tagging flowers pollinated on a particular day, thereby avoiding biases due to potential emasculation induced stress responses. Our results suggest a time component in counting the number of generations a plant has passed through self-fertilization at a particular age in determining the somatic mutation rates.


title: Growth orientations, rather than heterogeneous growth rates, dominate jaw joint morphogenesis in the larval zebrafish

abstract: In early limb embryogenesis, synovial joints acquire specific shapes which determine joint motion and function. The process by which the opposing cartilaginous joint surfaces are moulded into reciprocal and interlocking shapes, called joint morphogenesis, is one of the least understood aspect of joint formation and the cell-level dynamics underlying it are yet to be unravelled. In this research, we quantified key cellular dynamics involved in growth and morphogenesis of the zebrafish jaw joint and synthesised them in a predictive computational simulation of joint development. Cells in larval zebrafish jaw joints labelled with cartilage markers were tracked over a forty-eight hour time window using confocal imaging. Changes in distance and angle between adjacent cell centroids resulting from cell rearrangement, volume expansion and extracellular matrix (ECM) deposition were measured and used to calculate the rate and direction of local tissue deformations. We observed spatially and temporally heterogeneous growth patterns with marked anisotropy over the developmental period assessed. There was notably elevated growth at the level of the retroarticular process of the Meckels cartilage, a feature known to undergo pronounced shape changes during zebrafish development. Analysis of cell dynamics indicated a dominant role for cell volume expansion in growth, with minor influences from ECM volume increases and cell intercalation. Cell proliferation in the joint was minimal over the timeframe of interest. Synthesising the dynamic cell data into a finite element model of jaw joint development resulted in accurate shape predictions. Our biofidelic computational simulation demonstrated that zebrafish jaw joint growth can be reasonably approximated based on cell positional information over time, where cell positional information derives mainly from cell orientation and cell volume expansion. By modifying the input parameters of the simulation, we were able to assess the relative contributions of heterogeneous growth rates and of growth orientation. The use of uniform rather than heterogeneous growth rates only minorly impacted the shape predictions whereas isotropic growth fields resulted in altered shape predictions. The simulation results suggest that growth anisotropy is the dominant influence on joint growth and morphogenesis. This study addresses the gap of the cellular processes underlying joint morphogenesis, with implications for understanding the aetiology of developmental joint disorders such as developmental dysplasia of the hip and arthrogryposis.


title: Enumeration of Citrus endophytic bacterial communities based on illumine metagenomics technique

abstract: Citrus is a valuable crop in Pakistan because it is rich in vitamin C and antioxidants. Huanglongbing (HLB) has an influence on citrus production around the world caused by a bacterium "Candidatus liberibacter asiaticus" (CLas), africanus and americanus. The structure and diversity of bacterial species in various ecosystems can be quickly examined using NGS. This approach is considerably quicker and more precise than outdated methods. Healthy or citrus greening infected leaf samples of Grapefruit, Citrus aurantifolia, and Citrus reticulata Blanco was used for diversity analysis. In this study high throughput, NGS technique was used to access the population of both cultivable and non-cultivable bacterial endophytes from citrus leaves, by using PCR amplicons of 16S rDNA sequences (V5-V7 regions) with Illumina Hi seq. As a result, a total number of 68,722 sequences were produced from the test samples. According to the NGS-based diversity classification, the most common genera of exploited bacterial endophytes were Proteobacteria, Firmicutes, Bacteroides, Cyanobacteria, and Actinobacteria. Citrus aurantifolia and Citrus paradisi showed almost equal diversity, whereas Citrus reticulata Blanco had a higher proportion of Proteobacteria and Cyanobacteria in their leaves. To determine alpha diversity (AD), additional data was analyzed using statistical indices such as Shannon, Chao1, and Simpson. According to the inverse Simpson diversity index, the abundance of the microbial population in six different citrus samples was 0.48, 0.567, and 0.163, respectively. The metagenomics of microbiota in plant tissues was successfully recorded by NGS technology, which can help us learn more about the interactions between plants and microbes. This research is the first step toward a better understanding of 16SrRNA-based metagenomics from citrus in Pakistan using Illumina (Hi seq) Technology.

category: genetics


title: Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons

abstract:
Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here, we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. Using mouse genetics, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase subunit Tir1. Rapid degradation of condensin I and condensin II - two essential regulators of mitotic chromosome structure - revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues.

HighlightsO_LIAuxin-inducible degradation of endogenously tagged proteins in living mice and a range of primary cells.
C_LIO_LIMost but not all cell types are competent for degradation
C_LIO_LIDosage of the tagged protein, E3 ligase substrate receptor and ligand can all determine degradation kinetics
C_LIO_LIRapid degradation of condensin subunits in lymphocytes reveals stage-specific requirements during cell division
C_LI