title: Estimating the time structure of descending activation that generates movements at different speeds

abstract:
In targeted movements of the hand, descending activation patterns must not only generate muscle activation, but must also adjust spinal reflexes from stabilizing the initial to stabilizing the final postural state. We estimate descending activation patterns that change minimally while generating a targeted movement within a given movement time based on a model of the biomechanics, muscle dynamics, and the stretch reflex. The estimated descending activation patterns predict human movement trajectories quite well. Their temporal structure varies across workspace and with movement speed, from monotonic profiles for slow movements to non-monotonic profiles for fast movements. Descending activation patterns at different speeds thus do not result from a not mere rescaling of invariant templates, but reflect varying needs to compensate for interaction torques and muscle dynamics. The virtual attractor trajectories, on which active muscle torques are zero, lie within reachable workspace and are largely invariant movements when represented in end-effector coordinates. Their temporal structure along movement direction changes from linear ramps to "N-shaped" profiles with increasing movement speed.

Author summaryWhat descending patterns of activation drive targeted movements? Based on a model that includes biomechanics, muscles dynamics and the stretch reflex we estimate the descending activation patterns by minimizing their change using optimal control, given a movement target and movement time. The resulting activation patterns predict experimentally observed human movements reasonably well. The temporal structure of the estimated descending activation patterns depends on the speed of the movement, varying from monotonic for slow to non-monotonic for fast movements. This structure reflects the need to compensate for interaction torques and muscle properties. From the model we are able to estimate the virtual attractor trajectories on which all active muscle torques are zero. These lie within reachable workspace, and are relatively uniform across workspace. Their time structure varies from linear ramps for slow movements to N-shaped temporal profiles for fast movements.


title: The little skate genome and the evolutionary emergence of wing-like fin appendages

abstract: Skates are cartilaginous fish whose novel body plan features remarkably enlarged wing-like pectoral fins that allow them to thrive in benthic environments. The molecular underpinnings of this unique trait, however, remain elusive. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins - gene expression, chromatin occupancy and three-dimensional (3D) conformation - we find skate-specific genomic rearrangements that alter the 3D regulatory landscape of genes involved in the planar cell polarity (PCP) pathway. Functional inhibition of PCP signaling resulted in marked reduction of anterior fin size, confirming this pathway as a major contributor of batoid fin morphology. We also identified a fin-specific enhancer that interacts with 3 HOX genes, consistent with the redeployment of Hox gene expression in anterior pectoral fins, and confirmed the potential of this element to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganizations and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.

category: evolutionary biology


title: Quantitation, base identity and distribution of stably incorporated ribonucleotides in nuclear and mitochondrial DNA from murine tissues

abstract: Ribonucleotides are estimated to be the most common non-canonical nucleotides transiently incorporated in DNA. Their presence or failure of their removal can affect genome stability and mutations in factors involved in dNTP pool maintenance or ribonucleotide removal can cause Aicardi-Goutieres syndrome or promote certain human cancers. Here, we have mapped and quantitated ribonucleotides genome-wide, in nine tissues of wild-type mice. We observed tissue-specific variation in number and base identity of incorporated ribonucleotides and present evidence that a number of genomic features, such as tRNA genes, transcription start sites and G-quadruplexes, can increase the frequency of stably incorporated ribonucleotides in their proximity. Moreover, we present the non-random distribution of incorporated ribonucleotides in mtDNA and identified ribonucleotide hotspots. The study presents a framework to understand the physiological role of ribonucleotides in mammalian DNA.

category: genomics


title: Deletion of CEP164 in mouse photoreceptors post-ciliogenesis interrupts ciliary intraflagellar transport (IFT)

abstract:
Centrosomal protein 164 (CEP164) is located at the edge of distal appendages in primary cilia and is necessary for basal body (BB) docking to the apical membrane. To investigate the function of CEP164 before and after BB docking in photoreceptors, we deleted CEP164 during retina embryonic development (Six3Cre), in postnatal rod photoreceptors (iCre75) and in mature retina using tamoxifen induction. BBs dock to the cell cortex during postnatal day 6 (P6) and extend a connecting cilium (CC) and an axoneme. P6 retina-specific knockouts (retCep164-/-) are unable to dock BBs, thereby preventing formation of a CC or outer segments (OS). In rod-specific knockouts (rodCep164-/-), Cre expression starts after P9 and CC/OS form. P16 rodCep164-/- rods have nearly normal OS lengths, and maintain OS attachment through P21 despite loss of CEP164. Intraflagellar transport components (IFT88, IFT57 and IFT140) were reduced at P16 rodCep164-/- BBs and CC tips and nearly absent at P21, indicating impaired intraflagellar transport. Nascent OS discs, labeled with a fluorescent dye on P14 and P18 and harvested on P19, showed continued rodCep164-/- disc morphogenesis but absence of P14 discs mid-distally, indicating OS instability. Tamoxifen induction with PROM1ETCre;Cep164F/F (tamCep164-/-) adult mice affected maintenance of both rod and cone OS. The results suggest that CEP164 is key towards recruitment and stabilization of IFT-B particles at BB/CC. Impairment of IFT may be the main driver of ciliary malfunction observed with hypomorphic CEP164 mutations.

Author summaryCentrosomal protein of 164 kDa (CEP164) is indispensable for docking of basal bodies to apical membranes and formation of primary cilia. Homozygous truncations of CEP164 are associated with severe ciliopathy whereas missense mutations generate milder forms of NPHP. Recessive mutations of CEP164 are associated with nephronophthisis (NPHP), Meckel-Gruber (MKS) and Bardet-Biedl syndromes (BBS) in which primary cilia fail to form, or do not function correctly. We found that deletion of CEP164 in mouse photoreceptors before docking of the basal body (BB) to the apical inner segment membrane prevents elaboration of connecting cilia (CC, equivalent to transition zones) and outer segments (OSs, modified primary cilia). We also generated mouse models in which deletion of CEP164 occurs after OS are established and observed that the BB/CC and axoneme are initially stable in the absence of CEP164. Loss of CEP164, however, diminished recruitment of IFT-B proteins to the BB/CC affecting intraflagellar transport (IFT), which is required for maintenance of cilia and OSs. We propose that, in ciliopathies caused by missense mutations (e.g., X1460WextX57 associated with LCA) impairment of IFT provides a mechanism for ciliary dysfunction and disease.

category: neuroscience


title: Fetal loss in pregnant rhesus macaques infected with high-dose African-lineage Zika virus

abstract:
Countermeasures against Zika virus (ZIKV), including vaccines, are frequently tested in nonhuman primates (NHP). Macaque models are important for understanding how ZIKV infections impact human pregnancy due to similarities in placental development. The lack of consistent adverse pregnancy outcomes in ZIKV-affected pregnancies poses a challenge in macaque studies where group sizes are often small (4-8 animals). Studies in small animal models suggest that African-lineage Zika viruses can cause more frequent and severe fetal outcomes. No adverse outcomes were observed in macaques inoculated with a low dose of African-lineage ZIKV at gestational day (GD) 45. Here, we inoculate eight pregnant rhesus macaques with a higher dose of African-lineage ZIKV at GD 45 to test the hypothesis that adverse pregnancy outcomes are dose-dependent. Three of eight pregnancies ended prematurely with fetal death. ZIKV was detected in both fetal and placental tissues from all cases of early fetal loss. Further refinements of this challenge system (e.g., varying the dose and timing of infection) could lead to an even more consistent, unambiguous fetal loss phenotype for assessing ZIKV countermeasures in pregnancy. These data demonstrate that high-dose inoculation with African-lineage ZIKV causes pregnancy loss in macaques and also suggest that ZIKV-induced first trimester pregnancy could be strain-specific.

Author summaryAlthough pregnant rhesus macaques are susceptible to infection with Zika virus (ZIKV), fetal phenotypes can be subtle and variable. Most macaque studies of ZIKV have involved infection with Asian-lineage viruses because these viruses caused Western Hemisphere outbreaks beginning in 2015. African-lineage ZIKV yields more severe adverse fetal outcomes in small animal models. Here, we provide evidence that pregnant macaques infected late in the first trimester using a high dose of African-lineage ZIKV have frequent ZIKV-associated pregnancy loss. This severe phenotype establishes a new model for evaluating countermeasures and reinforces the idea that African-lineage ZIKV infection may be a frequent cause of pregnancy loss in areas where it is endemic depending on the amount of transmitted virus during infection.

category: pathology


title: Quantifying formations of Neutravidin clusters on biotinylated mixed silane Self-Assembled Monolayers.

abstract: Towards the goal of developing bio-chip / lab-on-a-chip substrates capable of performing highly specific bio-chemical reactions, Neutravidin binding to mixed Biotinylated Silane Self-Assembled Monolayers were studied using Confocal Fluorescence Light Microscopy. Non-specific bindings, specifically the formations of Neutravidin clusters, were quantified. Several experiments were conducted to determine the concentrations of Neutravidin necessary to not saturate surface binding to Biotinylated Self-Assembled Monolayers, determine the effectiveness of using FBS blocking buffers to reduce non-specific binding, optimize the repeatability of Neutravidin binding to Biotinlyated mixed Self-Assembled Monolayers with Silane-PEG-Biotin compositions ranging from 0 to 15%, and quantify background Neutravidin bindings and the corresponding formations of Neutravidin clusters to Self-Assembled Monolayers as Silane-PEG-Biotin percent compositions increase from 0 to 15%. The Neutravidin, Silane-PEG-Biotin, and Silane mPEG concentrations and ratios needed to develop homogeneous Neutravidin films, without the formations of clusters, on the Self-Assembled Monolayers have been determined.

category: bioengineering


title: Optimizing sampling for surface localization in 3D-scanning microscopy

abstract: 3D-scanning fluorescence imaging of living tissue is in demand for less phototoxic acquisition process. For the imaging of biological surfaces, adaptive and sparse scanning schemes have been proven to efficiently reduce the light dose by concentrating acquisitions around the surface. In this paper, we focus on optimizing the scanning scheme at constant photon budget, when the problem is to estimate the position of a biological surface whose intensity profile is modeled as a Gaussian shape. We propose an approach based on the Cramer-Rao Bound to optimize the positions and number of scanning points, assuming signal-dependant Gaussian noise. We show that the optimization problem can be reduced to a few parameters, allowing to define quasi-optimal acquisition strategies, first when no prior knowledge on the surface location is available, and then when the user has a prior on this location.

category: biophysics


title: The generation of HepG2 transmitochondrial cybrids to reveal the role of mitochondrial genotype in idiosyncratic drug-induced liver injury: a translational in vitro study

abstract:
BackgroundEvidence supports an important link between mitochondrial DNA (mtDNA) variation and adverse drug reactions such as idiosyncratic drug-induced liver injury (iDILI). Here we describe the generation of HepG2-derived transmitochondrial cybrids in order to investigate the impact of mtDNA variation upon mitochondrial (dys)function and susceptibility to iDILI against a constant nuclear background. In this study, cybrids were created to contain mitochondrial genotypes of haplogroup H and haplogroup J for comparison.

MethodsBriefly, HepG2 cells were depleted of mtDNA to make rho zero cells before the introduction of known mitochondrial genotypes using platelets from healthy volunteers (n=10), thus generating 10 distinct transmitochondrial cybrid cell lines. The mitochondrial function of each was assessed at basal state and following treatment with compounds associated with iDILI; flutamide, 2-hydroxyflutamide and tolcapone, by ATP assays and extracellular flux analysis.

FindingsOverall, baseline mitochondrial function was similar between haplogroups H and J. However, haplogroup specific responses to mitotoxic drugs were observed; haplogroup J was more susceptible to the inhibition of respiratory complexes I and II, and also to the effects of tolcapone, an uncoupler of mitochondrial respiration.

ConclusionsThis study demonstrates that HepG2 transmitochondrial cybrids can be created to contain the mitochondrial genotype of any individual of interest, thus providing a practical and reproducible system to investigate the cellular consequences of variation in mitochondrial genome against a constant nuclear background. Additionally the results support that that inter-individual variation in mitochondrial genotype and haplogroup may be a factor in determining sensitivity to mitochondrial toxicants.

FundingThis work was supported by the Centre for Drug Safety Science supported by the Medical Research Council, United Kingdom (Grant Number G0700654); and GlaxoSmithKline as part of an MRC-CASE studentship (grant number MR/L006758/1).

category: pharmacology and toxicology


title: Adipose mTORC2 is essential for arborization of sensory neurons in white adipose tissue and whole-body energy homeostasis

abstract: Adipose tissue, via sympathetic and sensory neurons, communicates with the central nervous system (CNS) to mediate energy homeostasis. In contrast to the sympathetic nervous system, the morphology, role and regulation of the sensory nervous system in adipose tissue is poorly characterized. Taking advantage of recent progress in whole-mount three-dimensional imaging of adipose tissue, we identified a neuronal network of calcitonin gene-related protein (CGRP)-positive sensory neurons in white adipose tissue (WAT). Furthermore, we show that adipose mammalian target of rapamycin complex 2 (mTORC2), a major component of the insulin signaling pathway, mediates sensory innervation in WAT. Based on visualization of neuronal networks, mTORC2-deficient WAT displayed reduced arborization of (CGRP)-positive sensory neurons, while sympathetic neurons were unaffected. This selective loss of sensory innervation followed reduced expression of growth-associated protein 43 (GAP43) in CGRP-positive sensory neurons. Finally, we found that loss of sensory innervation in WAT correlated with systemic insulin resistance. Our findings suggest that adipose mTORC2 is necessary for sensory innervation in WAT which likely contributes to WAT-to-CNS communication.

category: physiology


title: Non-cell autonomous small RNA silencing in female gametes and early embryo of Arabidopsis

abstract: In recent years, small RNA movement has been both hypothesized and shown to be an integral part of the epigenetic DNA methylation reprogramming occurring during plant reproduction. It was suggested that the release of epigenetic silencing in accessory cell types or tissues is necessary to reinforce epigenetic silencing in the gametes (egg cell and sperm cells) which would in turn ensure the genomic stability of the next generation plant. Small RNA movement was indeed shown to occur during male gametogenesis. However, the situation within the female gametophyte and in early seed development is mostly unknown. Here, we show that small RNA can induce non-cell autonomous silencing from the central cell towards the egg cell but also from the synergids to the egg cell and central cell. In addition, we also observe a non-cell-autonomous silencing from the central cell or endosperm towards the early embryo. Our data shows, that in addition to the movement of sRNAs during pollen development, sRNA movement also occurs in the female gametes.

category: plant biology


title: The Phytophthora capsici RxLR effector CRISIS2 has roles in suppression of PTI and triggering cell death in host plant

abstract: Pathogen effectors can suppress various plant immune responses, suggesting that they have multiple targets in the host. To understand the mechanisms underlying plasma membrane-associated and effector-mediated immunity, we performed Phytophthora capsici RxLR cell death Inducer Suppressing Immune System (CRISIS) screening. In Nicotiana benthamiana, the cell death induced by the RxLR effector CRISIS2 is inhibited by the irreversible plasma membrane H+-ATPase (PMA) activator fusicoccin. Biochemical and gene silencing analyses revealed that CRISIS2 physically and functionally associates with PMAs and induces host cell death independent of immune receptors. CRISIS2 induces apoplastic alkalization by suppressing PMA activity via its association with the C-terminal regulatory domain of PMA. In planta expression of CRISIS2 significantly enhanced the virulence of P. capsici, whereas host-induced gene silencing of CRISIS2 compromised disease symptom and biomass of P. capsici. Furthermore, co-immunoprecipitation assays revealed that CRISIS2 constitutively associates with BAK1, the co-receptor of pattern recognition receptors (PRRs). CRISIS2 interferes with the FLS2-BAK1 complex induced by flagellin perception and impairs downstream signaling from the PRR complex. Proteomics and gene silencing assays identified putative PRRs that negatively regulate the virulence of P. capsici in N. benthamiana as interactors of CRISIS2 and BAK1. Our study identified a novel RxLR effector playing multiple roles in the suppression of plant defense and induction of cell death to support the pathogen hemibiotrophic life cycle in the host plant.


title: Multicellular PI control for gene regulation in microbial consortia

abstract: We describe two multicellular implementations of the classical P and PI feedback controllers for the regulation of gene expression in a target cell population. Specifically, we propose to distribute the proportional and integral actions over two different cellular populations in a microbial consortium comprising a third target population whose output needs to be regulated. By engineering communication among the different cellular populations via appropriate orthogonal quorum sensing molecules, we are able to close the feedback loop across the consortium. We derive analytical conditions on the biological parameters guaranteeing the regulation of the output of the target population and we validate the robustness and modularity of proposed control schemes via in silico experiments in BSim, a realistic agent-based simulator of bacterial populations.

category: synthetic biology


title: Individual differences in eye drift predict visual acuity

abstract:
Visual acuity is essential for daily activities. It is believed that optics and anatomy alone determine the limits of visual resolution. However, in addition to these factors, eye movements greatly contribute to input dynamics, as the eyes jitter incessantly during fixation, displacing stimuli over many photoreceptors. Here we examined the relation between this motion (ocular drift) and differences in visual acuity in emmetropic observers. We show that: (a) large individual variability exists in the characteristics of ocular drifts; (b) these differences profoundly affect the structure of spatiotemporal signals to the retina; and (c) the spectral distribution of the resulting input signals strongly correlates with visual acuity. Building on these findings, we further show that individual acuity differences can be predicted from the motor behavior elicited by a simple fixation task, without directly measuring acuity. These results shed new light on how oculomotor behavior contributes to individual variability in acuity.

SignificanceHigh visual acuity is essential for humans and other species. Healthy emmetropic individuals differ considerably in their actual acuity limits. Here we show that the pattern of ocular drift during fixation also varies greatly across these observers, and that these large differences in ocular drift lead to sizable changes in the frequency content of the retinal input. We find that individual variability in acuity is accounted for by the way ocular drift reshapes visual input signals, particularly at high spatial frequencies. As a result, differences in high acuity can be predicted by differences in the characteristics of fixational drift. These findings show that besides optical and anatomical factors, ocular drift plays a critical role in modulating the limits of acuity.


title: Reactivity to conditioned threat cues is distinct from exploratory drive in the elevated plus-maze.

abstract: Fear and anxiety are adaptive states that allow humans and animals alike to respond appropriately to threatening cues in their environment. Commonly used tasks for studying behaviour akin to fear and anxiety in rodent models are pavlovian threat conditioning and the elevated plus maze (EPM) respectively. In threat conditioning the rodents learn to associate an aversive event with a specific stimulus or context. The learnt association between the two stimuli (the  memory) can then be recalled by re-exposing the subject to the conditioned stimulus. The elevated plus maze is argued to measure the agoraphobic avoidance of the brightly lit open maze arms in crepuscular rodents. These two tasks have been used extensively, yet research into whether they interact is scarce. We investigated whether recall of an aversive memory, across contextual, odour or auditory modalities, would potentiate anxiety-like behaviour in the elevated plus maze. The data did not support that memory recall, even over a series of timepoints, could influence EPM behaviour. Furthermore, there was no correlation between EPM behaviour and conditioned freezing in independent cohorts tested in the EPM before or after auditory threat conditioning. Further analysis found the production of 22kHz ultrasonic vocalisations revealed the strongest responders to a conditioned threat cue. These results are of particular importance for consideration when using the EPM and threat conditioning to identify individual differences, and the possibility to use the tasks in batteries of tests without cross-task interference.


title: Molecular Characterisation Of Urine And Vaginal Samples And Their Antibiotic Susceptibility Pattern Associated With β-Lactamases Resistance Gene

abstract:
Infection or inflammation of the vagina is called vaginitis. The two most common causes of vaginitis are bacterial vaginitis and Candida vaginitis. The causative organisms may be sexually transmitted or endogenous, usually by Gardnerella vaginalis and Mycoplasma hominis in combination with other anaerobes. Urinary tract infections (UTI) refer to the presence of microbial pathogens within the urinary tract and are caused by a range of pathogens but most commonly by Escherichia coli, Klebsiella pneumoniae etc. Highly increase in antibiotic-resistant among uropathogens, including ESBL producing E.coli, AmpC {beta}-lactamase or carbapenemase-producing Enterobacteriaceae (e.g., New Delhi metallo-{beta}-lactamase [NDM]) are being reported among health care unit. Carbapenem-resistant Enterobacteriaceae (CRE) is a growing concern worldwide and a major challenge by the WHO 2014.

The aim of this study was to determine the occurrence of CTX-M gene and TEM gene and the possible role of ESBL in resistance to antibiotic. A study was conducted on a total of 100 urine and vaginal samples from Dr. B. Lal clinical laboratory and was cultured on Nutrient Agar and MacConkey agar for the detection of etiologic agents. Bacteria isolation, identification & biochemical characterization was done. Kirby-Bauer Disk Diffusion (KBDS) method was used for detection of ESBL-producing strains. A disc of Cefotaxime (30 {micro}g) and Clavulanic acid (30 {micro}g) were placed at a distance of 20 mm on Muller Hilton agar plate along with Imipenem(30{micro}g), Piperacillin/Tazobactam(10{micro}g) and Ceftazidime(30{micro}g). The zone of inhibition generally greater than 20mm was considered as ESBL producer. Total 14 isolates were identified as ESBL producer. In order to find out the plasmid profile of these isolates, plasmid DNA isolation was done. Isolates was found to have one or more than one plasmid gene responsible for the ESBL resistance. This finding concludes that CTX-M gene is most common gene in ESBL producing isolates. It is highly recommended that antibiotic prescription should be monitored according to guideline. The prevalence of ESBL is increasing day by day and necessary steps should be taken to prevent the spread and emergence of resistance.

category: microbiology


title: Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

abstract: The formation of a clathrin-coated vesicle is a major membrane remodeling process that is crucial for membrane traffic in cells. Besides clathrin, these vesicles contain at least 100 different proteins although it is unclear how many are essential for the formation of the vesicle. Here, we show that intracellular clathrin-coated formation can be induced in living cells using minimal machinery and that it can be achieved on various membranes, including the mitochondrial outer membrane. Chemical heterodimerization was used to inducibly attach a clathrin-binding fragment "hook" to an "anchor" protein targeted to a specific membrane. Endogenous clathrin assembled to form coated pits on the mitochondria, termed MitoPits, within seconds of induction. MitoPits are double-membraned invaginations that form preferentially on high curvature regions of the mitochondrion. Upon induction, all stages of CCV formation - initiation, invagination, and even fission - were faithfully reconstituted. We found no evidence for the functional involvement of accessory proteins in this process. In addition, fission of MitoPit-derived vesicles was independent of known scission factors including dynamins and dynamin-related protein 1 (Drp1), suggesting that the clathrin cage generates sufficient force to bud intracellular vesicles. Our results suggest that, following its recruitment, clathrin alone is sufficient for intracellular clathrin-coated vesicle formation.

category: cell biology


title: Niche hypervolume analysis revealed that water pollution reduces reproductive success of endangered landlocked salmon but does not directly impact their population density

abstract: The critical information for conserving endangered species is to identify how different niche dimensions affect key life history stages of a population. However, it is often difficult to quantify how each ecological niche dimension affects different life history stages because environmental factors may affect each fitness component of organisms to various degrees. Here, we applied the recently developed hypervolume method that follows the idea of Hutchinsons n-dimensional hypervolume. We analyzed the niche space of different life history stages of the endangered landlocked salmon Oncorhynchus masou formosanus, the most southerly distributed of all salmonoids. We found no direct effect of water pollution on salmon population density but a significant negative effect on salmon reproductive success. Surprisingly, the niche hypervolume analysis showed that the size of hatching success niche hypervolume was only 17.9% and 18.4% of the natural redd site density or population density, respectively. This result demonstrates a much higher environmental requirement for salmon during the egg stage than that affecting population density or nesting environment. Our results suggest that understanding the behavioral and physiological mechanisms that influence crucial life history stages in the wild is critical to developing effective conservation programs, and the niche hypervolume is a valuable method to achieve this.

category: ecology


title: Dynamic coastal pelagic habitat drives rapid changes in growth and condition of juvenile sockeye salmon (Oncorhynchus nerka) during early marine migration

abstract: Migrating marine taxa encounter diverse habitats that differ environmentally and in foraging conditions over a range of spatial scales. We examined body (RNA/DNA, length-weight residuals) and nutritional condition (fatty acid composition) of juvenile sockeye salmon (Oncorhynchus nerka) in British Columbia, as they migrated through coastal waters that varied oceanographically over tens of kilometers. Fish were sampled in the stratified, productive northern Strait of Georgia (NSoG); the highly mixed, unproductive Johnstone Strait (JS); and the transitional zone of Queen Charlotte Strait (QCS). Body and nutritional condition responded rapidly to changes in prey availability and were lowest in JS with low prey availability, supporting the Tropic Gauntlet Hypothesis, additionally we saw signs of compensatory growth in QCS. Juvenile salmon leaving the SoG in 2016 had significantly lower condition than in 2015, despite higher zooplankton biomass in 2016. We propose that this was due to the higher abundance of low food quality southern zooplankton species in 2016. This study highlights the importance of including food quality as a parameter to understand changes in fish condition and survival between years. Furthermore, small scale variation in oceanographic dynamics impact foraging conditions and need to be considered when assessing early marine survival of juvenile salmon.


title: Croatian Genetic Heritage: Renewed Y Chromosome Story a Decade Later

abstract:
AimTo analyze an additional set of Y-Chromosome genetic markers to acquire a more detailed insight into the diversity of the Croatian population.

MethodsThe total number of 518 Yfiler Plus profiles were genotyped. Allele, haplotype frequencies, and haplotype diversity were calculated using the STRAF software package v2.0.4. Genetic distances were quantified by Rst using AMOVA online tool from the YHRD. The evolutionary history was inferred using the neighbor-joining method of phylogenetic tree construction in MEGAX software. Whit Atheys Haplogroup Predictor v5 was used for additional comparison with available regional and other European populations.

ResultsThe total of 507 haplotypes were used for genetic STR analysis. The interpopulation study on 17 Y-STR markers shows the lowest genetic diversity between the Croatian and Bosnian-Herzegovinian populations and the highest between the Croatian and Irish populations. Additional interpopulation comparison with the original 27 Y-STR markers (for the population with available data) was also performed. A total of 518 haplotypes were used in the determination of haplogroup diversity. Haplogroup I with its sublineage I2a expressed the highest prevalence. Haplogroup R, with its major sublineage R1a, is the second most abundant in the studied Croatian population, except for the subpopulation of Hvar, where E1b1b is the second most abundant haplogroup. Rare haplogroups also confirmed in this study are L, T, and Q. G1 is detected for the very first time in the Croatian population.

ConclusionNew insight into differences between examined subpopulations of Croatia and their possible (dis)similarities with neighboring abroad populations was notified.


title: A Machine Learning Framework to Predict Subcellular Morphology of Endothelial Cells for Digital Twin Generation

abstract: Gaining insight into different cell behaviors is key to better understanding different pathologies. These behaviors may be explained in part through close observation of 3D cell morphology. Therefore, the objective of this research was to develop a machine learning (ML) framework that can predict 3D subcellular morphological variation of endothelial cells (ECs) to generate digital twins. ECs were cultured and their membrane, nucleus, and focal adhesion (FA) sites were stained and imaged with confocal microscopy. The multicellular confocal z stacks were segmented resulting in a total of 60 single-cell stacks. Fifty randomly picked cells were augmented 20-fold to train the ML framework, and the remaining 10 were used for an independent test of prediction accuracy. The ML framework was based on an open-source conditional generative adversarial network (cGAN), which was expanded to make 3D predictions using membrane only as input to predict nucleus and FA morphology. After training the framework, the results on the independent test showed an average prediction accuracy of [~]87% for nucleus and [~]70% for FA sites. The predictions were used to build a digital twin of each EC and compared to their respective ground truth, showing an average [~]79% global accuracy and [~]84% accuracy in FA-Nucleus distribution. The results presented show the effectiveness of the developed ML framework to generate digital twins of Ecs using limited amount of data. These digital twins can be used to couple EC morphology with different behaviors. The ML framework can be potentially expanded to predict morphology of other subcellular structures as well as to study other types of cells.


title: Three-dimensional multilayer concentric bipolar electrodes enhance the selectivity of optic nerve stimulation

abstract:
ObjectiveIntraneural nerve interfaces often operate in a monopolar configuration with a common and distant ground electrode. This configuration leads to a wide spreading of the electric field. Therefore, this approach is suboptimal for intraneural nerve interfaces when selective stimulation is required.

ApproachWe designed a multilayer electrode array embedding three-dimensional concentric bipolar electrodes. First, we validated the higher stimulation selectivity of this new electrode array compared to classical monopolar stimulation using simulations. Next, we compared them in-vivo by intraneural stimulation of the rabbit optic nerve and recording evoked potentials in the primary visual cortex.

Main resultsSimulations showed that three-dimensional concentric bipolar electrodes provide a high localisation of the electric field in the tissue so that electrodes are electrically independent even for high electrode density. Experiments in-vivo highlighted that this configuration leads to evoked responses with lower amplitude and more localised cortical patterns due to the fewer fibres activated by the electric stimulus in the nerve.

SignificanceHighly focused electric stimulation is crucial to achieving high selectivity in fibre activation. The multilayer array embedding three-dimensional concentric bipolar electrodes improves selectivity in optic nerve stimulation. This approach is suitable for other neural applications, including bioelectronic medicine.


title: Nanomechanical and microrheological properties of bladder cancer cells at cellular and spheroid levels

abstract: Cancer progression is associated with changes in cell mechanical and rheological properties that could be probed by atomic force microscopy (AFM). In this study, we applied AFM to measure elastic (by compressing the cells) and viscoelastic (by applying shear stress) properties of bladder cancers in relation to their culture morphology (in single cells, cell monolayers, and spheroids). Three different cell lines, HCV29 (non-malignant cell cancer of ureter), HT1376 (grade III bladder carcinoma), T24 (grade IV transitional cell carcinoma), were investigated. Nanoindentation measurements only differentiate between non-malignant and cancer cells, but it is difficult to distinguish between specific bladder cancers. By applying microrheological measurements, we confirm that non-malignant cells are more rigid than cancer cells but more importantly, it was possible to differentiate between two cancerous cell lines, regardless of the culture conditions. As each of them is characterized by a distinct actin filament network inside the cell, we showed that actin filaments are a key element in defining the rheological properties of spheroids originating from cells having thick actin bundles. Our results showed that HCV29 cells are more rigid than the studied cancer cells, indicating that normal cells are resistant to compressive and shear forces. Therefore, we conclude that cell mechanical and rheological properties may serve as a biophysical marker to distinguish normal and cancer cells of different malignancies.


title: Pinning transition in biofilm structure driven by active layer dynamics

abstract: Surface-attached communities of microbes, known as biofilms, are diverse in their morphologies. Characterising distinct types of biofilm spatial structure, and understanding how they emerge, can shed light on the fundamental biological and biophysical mechanisms involved, and can improve our understanding of evolution in biofilms. Here, we perform long-time individual-based simulations of growing biofilms. We observe distinct types of biofilm spatial structure depending on the parameters, and we classify these into three  phases according to the behaviour of the active layer of growing cells at the biofilm interface. In the unpinned phase, the biofilm is smooth and the active layer is unbroken with no gaps. In the transiently pinned phase, short-lived gaps in the active layer arise, which can cause local parts of the biofilm interface to pin, or become stationary relative to the moving front. In the pinned phase these  pinning sites persist, leading to fingering of the biofilm interface. We show that pinning arises due to the dynamical behaviour of active layer gaps, and observe that the relative magnitudes of the active layer thickness and the active layer fluctuations are important in this process. We demonstrate a direct connection between biofilm pinning and interface roughness, and we show that the pinning phase transition is well described by a control parameter that combines the average and standard deviation of the active layer thickness. Taken together, our work suggests a role for active layer dynamics in controlling pinning of the biofilm interface and hence biofilm morphology.


title: Usefulness of the pancreas as a prime target for histopathological diagnosis of Tilapia parvovirus (TiPV) infection in Nile tilapia, Oreochromis niloticus

abstract: Tilapia parvovirus (TiPV) is an emerging virus reportedly associated with disease and mortality in farmed tilapia. Although previous descriptions of histopathological changes are available, the lesions reported in these are not pathognomonic. Here, we report Cowdry type A inclusion bodies (CAIB) in the pancreas as a diagnostic histopathological feature found in adult Nile tilapia naturally infected with TiPV. This type of inclusion body has been well-known as a histopathological landmark for the diagnosis of other parvoviral infections in shrimp and terrestrial species. Interestingly, this lesion could be exclusively observed in pancreatic acinar cells, both in the hepatopancreas and pancreatic tissue along the intestine. In situ hybridization (ISH) using a TiPV-specific probe revealed the intranuclear presence of TiPV DNA in multiple tissues, including the liver, pancreas, kidney, spleen, gills, and the membrane of oocytes in the ovary. These findings suggest that although TiPV can replicate in several tissue types, CAIB manifest exclusively in pancreatic tissues. In addition to TiPV, most diseased fish were co-infected with Streptococcus agalactiae, and presented with multifocal granulomas secondary to this bacterial infection. Partial genome amplification of TiPV was successful and revealed high nucleotide identity (> 99%) to previously reported isolates. In summary, this study highlights the usefulness of pancreatic tissue as a prime target for histopathological diagnosis of TiPV in diseased Nile tilapia. This pattern may be critical when determining the presence of TiPV infection in new geographic areas, where ancillary testing may not be available. TiPV pathogenesis in this landmark organ warrants further investigation.


title: Arginine-dependent hypusination of the eukaryotic translation initiation factor (eIF)5A drives erythroid lineage differentiation

abstract: Metabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. We discovered that SLC7A1/CAT1-dependent arginine uptake and its catabolism to spermidine control the erythroid specification of HSPCs via activation of eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on the metabolism of spermidine to hypusine and inhibiting hypusine synthesis abrogates erythropoiesis and diverts EPO-stimulated HSPCs to a myeloid fate. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A and induction of mitochondrial function partially rescues erythropoiesis in the absence of hypusine. Within the hypusine network, ribosomal proteins are highly enriched and we identify defective eIF5A hypusination in erythroid pathologies caused by abnormal ribosome biogenesis. Thus, eIF5A-dependent protein synthesis is critical in the branching of erythro-myeloid differentiation and attenuated eIF5A activity characterizes ribosomal protein-linked disorders of ineffective erythropoiesis.


title: Dietary fibre deprivation and bacterial curli exposure shift gut microbiome and exacerbate Parkinson's disease-like pathologies in an alpha-synuclein-overexpressing mouse

abstract: The microbiome-gut-brain axis has been proposed as a pathogenic path in Parkinsons disease (PD). Dietary driven dysbiosis and reduced gut barrier function could facilitate the interaction of toxic external or internal factors with the enteric nervous system, where PD could start. Amyloid bacterial protein such as curli can act as seed to corrupt enteric -synuclein and lead to its aggregation. Misfolded -synuclein can propagate to and throughout the brain. Here, we aimed at understanding if fibre deprivation and amyloidogenic protein curli could, individually or together, exacerbate the phenotype in both enteric and central nervous systems of a transgenic mouse overexpressing wild-type human -synuclein. We analysed the gut microbiome, motor behaviour, gastrointestinal and brain pathologies in these mice. Our findings show that external interventions, akin to unhealthy life habits in humans, can exacerbate PD-like pathologies in mice. We believe that our results shed light on how lifestyle affects PD progression.


title: Whole-brain modeling explains the context-dependent effects of cholinergic neuromodulation

abstract: Integration and segregation are accepted as two fundamental principles of brain organization. The brain manages the transitions between different functional states, more segregated or integrated, through neuromodulatory systems. Recently computational and experimental works suggest a pro-segregation effect of cholinergic neuromodulation. Here, we studied the effects of the cholinergic system on brain functional connectivity using both empirical fMRI data and computational modeling. First, we analyzed the effects of nicotine on functional connectivity and network topology of healthy subjects during resting-state conditions and during an attentional task. Then, to find causal mechanisms about these changes, we employed a wholebrain neural mass model embedded into a human connectome for simulating the effects of nicotine. We found that nicotine incremented functional segregation in both empirical and simulated data, and the effects are context-dependent: observed during task, but not in resting-state. In-task performance correlates with functional segregation, discovering a link between functional network topology and behavior. In the model, the drug was modeled decreasing both the global coupling and local feedback inhibition parameters of the model, consistent with the known cellular effects of acetylcholine. Also, we found that the regional density of the nicotinic acetylcholine 4{beta}2 receptor explains better the effects of nicotine at the whole-brain level. Our results confirm that cholinergic neuromodulation promotes functional segregation in a context-dependent fashion, and suggest that this segregation is suited for simple visual-attentional tasks. In addition, we propose biophysical mechanisms to simulate cholinergic neuromodulation in whole-brain models.


title: Decoding trans-saccadic prediction error

abstract:
We are constantly sampling our environment by moving our eyes, but our subjective experience of the world is stable and constant. One undesirable consequence of eye movements is that stimulus displacement during or shortly after a saccade often goes unnoticed, a phenomenon called the saccadic suppression of displacement. Although we fail to notice such displacements, our oculomotor system computes the post-saccadic prediction errors and adequately adjusts the gaze and future saccadic execution, a phenomenon known as saccadic adaptation. In the present study, we aimed to find a brain signature of the trans-saccadic prediction error that informs the motor system but not explicit perception. We asked human participants to report whether a visual target was displaced during a saccade towards it while recording electroencephalography (EEG). Using multivariate pattern analysis, we were able to differentiate displacements from no displacements, even when participants failed to report the displacement. In other words, we found that trans-saccadic prediction error is represented in the EEG signal 100 ms after the displacement presentation, mainly in occipital and parieto-occipital regions, even in the absence of explicit perception of the displacement.

Significance StatementStability in vision occurs even while performing saccades. One suggested mechanism for this counterintuitive visual phenomenon is that external displacement is suppressed during the retinal remapping caused by a saccade. Here, we shed light on the mechanisms of trans-saccadic stability by showing that displacement information is not entirely suppressed and specifically present in the early stages of visual processing. Such a signal is relevant and computed for oculomotor adjustment despite being neglected for error perception.


title: Taxonomic and functional diversity of aquatic heterotrophs is sustained by dissolved organic matter chemodiversity

abstract: Dissolved organic matter (DOM) is ubiquitous in aquatic ecosystems and fundamental for planetary processes and ecosystem functioning. While the link between microbial community composition and heterotrophic utilization of DOM has been recognized, the full diversity of organic compounds, their bioavailability, degradability and specific influences on the diversity and function of heterotrophs are still not clear. Here we experimentally investigate heterotrophic bacteria in thirty-three freshwater model communities. We identified 34 different heterotrophs growing in ambient lake DOM with taxonomic affiliations matching abundant freshwater bacterioplankton. We further describe 25 different heterotrophs growing in the phycosphere of M. Aeruginosa and 6 heterotrophs growing on the DOM produced by M. Aeruginosa with taxonomic affiliation in accord to phycosphere heterotrophs. In our experiment we observed that heterotrophs that live in the phycosphere remove more dissolved organic carbon than abundant freshwater heterotrophs. Moreover, phycosphere heterotrophs have bigger genomes than abundant lake bacteria. Altogether of the 4224 chemical features that were resolved by LC-MS, only 1229 were seen in all three treatments. None of the common/shared compounds were removed across all the model communities, suggesting contrasting niches of the studied taxa. Altogether our study highlights how each model community, with its unique taxonomic assemblages and organotroph functioning is upkept by the chemodiversity of DOM.


title: Environmental Conditions and Vigilance Behaviour in the Smooth-coated Otter, Lutrogale perspicillata

abstract:
Vigilance is an ecologically important behaviour that acts as a defence mechanism against threats such as predators, competitors, and environmental pressures. Being vigilant can place energetic constraints on an individual, as conducting other behaviours such as grooming can be limited. The smooth-coated otter is a vulnerable species, and understanding baseline behaviour, such as vigilance, and stressor effects can be a crucial step in implementing any conservation strategies. However, literature on this charismatic species is relatively sparse, with the majority of research covering prey items, habitat use and extent of occurrence.

Using camera trap footage along with historical climate datasets, the current study assessed various aspects of vigilance behaviour of the smooth-coated otter. These aspects include how long individuals dedicated to being vigilant, what they prioritised being vigilant over, and how intense their vigilance efforts were

Variables that are expected to change with global climate change such as temperature, and to an extent, air pressure, were associated with increases in vigilance duration. Wind speeds, which are also expected to be affected by climate change, along with increasing numbers of conspecifics, were linked to increased intensity possibly highlighting that increasing ambient movement results in more frequent head rotations. When vigilance is ineffectual however, such during periods of higher rainfall, cloud cover or at night, intensity decreases, and gazing towards conspecifics increases - possibly to reaffirm where safety lies when threats cannot be adequately monitored.

category: animal behavior and cognition


title: Rapid genotyping of viral samples using Illumina short-read sequencing data

abstract: The most important information about microorganisms might be their accurate genome sequence. Using current Next Generation Sequencing methods, sequencing data can be generated at an unprecedented pace. However, we still lack tools for the automated and accurate reference-based genotyping of viral sequencing reads. This paper presents our pipeline designed to reconstruct the dominant consensus genome of viral samples and analyze their within-host variability. We benchmarked our approach on numerous datasets and showed that the consensus genome of samples could be obtained reliably without further manual data curation. Our pipeline can be a valuable tool for fast identifying viral samples. The pipeline is publicly available on the projects github page (https://github.com/laczkol/QVG).

category: bioinformatics


title: Maximum likelihood pandemic-scale phylogenetics

abstract: 1Genomic data plays an essential role in the study of transmissible disease, as exemplified by its current use in identifying and tracking the spread of novel SARS-CoV-2 variants. However, with the increase in size of genomic epidemiological datasets, their phylogenetic analyses become increasingly impractical due to high computational demand. In particular, while maximum likelihood methods are go-to tools for phylogenetic inference, the scale of datasets from the ongoing pandemic has made apparent the urgent need for more computationally efficient approaches. Here we propose a new likelihood-based phylogenetic framework that greatly reduces both the memory and time demand of popular maximum likelihood approaches when analysing many closely related genomes, as in the scenario of SARS-CoV-2 genome data and more generally throughout genomic epidemiology. To achieve this, we rewrite the classical Felsenstein pruning algorithm so that we can infer phylogenetic trees on at least 10 times larger datasets with higher accuracy than existing maximum likelihood methods. Our algorithms provide a powerful framework for maximum-likelihood genomic epidemiology and could facilitate similarly groundbreaking applications in Bayesian phylogenomic analyses as well.


title: Methylene Blue Is a Nonspecific Protein-Protein Interaction Inhibitor with Potential for Repurposing as an Antiviral for COVID-19

abstract: We have previously identified methylene blue, a tricyclic phenothiazine dye approved for clinical use for the treatment of methemoglobinemia and used for other medical applications, as a small-molecule inhibitor of the protein-protein interaction (PPI) between the spike protein of the SARS-CoV-2 coronavirus and ACE2, the first critical step of the attachment and entry of this coronavirus responsible for the COVID-19 pandemic. Here, we show that methylene blue concentration-dependently inhibits this PPI for the spike protein of the original strain as well as for those of variants of concerns such as the D614G mutant and delta (B.1.617.2) with IC50 in the low micromolar range (1-5 M). Methylene blue also showed promiscuous activity and inhibited several other PPIs of viral proteins (e.g., HCoV-NL63 - ACE2, hepatitis C virus E - CD81) as well as others (e.g., IL-2 - IL-2R) with similar potency. This non-specificity notwithstanding, methylene blue inhibited the entry of pseudoviruses bearing the spike protein of SARS-CoV-2 in hACE2-expressing host cells both for the original strain and the delta variant. It also blocked SARS-CoV-2 (B.1.5) virus replication in Vero E6 cells with an IC50 in the low micromolar range (1.7 M) when assayed using quantitative PCR of the viral RNA. Thus, while it seems to be a promiscuous PPI inhibitor with low micromolar activity and it has a relatively narrow therapeutic index, methylene blue inhibits entry and replication of SARS-CoV-2, including several of its mutant variants, and has potential as a possible inexpensive, broad-spectrum, orally bioactive small-molecule antiviral for the prevention and treatment of COVID-19.


title: Detection and measurement of butterfly eyespot and spot patterns using convolutional neural networks

abstract:
Butterflies are increasingly becoming model insects where basic questions surrounding the diversity of their color patterns are being investigated. Some of these color patterns consist of simple spots and eyespots. To accelerate the pace of research surrounding these discrete and circular pattern elements we developed two distinct convolutional neural networks (CNNs) for detection and measurement of butterfly spots and eyespots on digital images of butterfly wings. We tested the accuracy of the detection and of the area measurements using manual identifications and measurements. These methods were able to identify and distinguish marginal eyespots from spots, as well as distinguish these patterns from less symmetrical patches of color. In addition, the measurements of an eyespots central area and surrounding rings were highly accurate. These CNNs offer improvements of eyespot/spot detection and measurements relative to previous methods because it is not necessary to mathematically define the feature of interest. All that is needed is to point out the images that have those features to train the CNN.

Author summaryWe developed two distinct convolutional neural networks (CNNs) for detection and measurement of butterfly spots and eyespots on digital images of butterfly wings. We tested the accuracy of the detection and of the area measurements using manual identifications and measurements. Our methods were able to identify and distinguish marginal eyespots from spots, as well as distinguish these patterns from less symmetrical patches of color. In addition, the measurements of an eyespots central area and surrounding rings were highly accurate.

category: systems biology


abstract:
Macrophages are a major source of pro-inflammatory cytokines in COVID-19. How macrophages sense the causative virus, SARS-CoV-2, to drive cytokine release is, however, unclear. Here, we show that human macrophages do not directly sense and respond to infectious SARS-CoV-2 virions because they lack sufficient ACE2 expression to support virus entry and replication. Over-expression of ACE2 in human macrophages permits SARS-CoV-2 entry and early-stage replication and facilitates macrophage pro-inflammatory and anti-viral responses. ACE2 over-expression does not, however, permit the release of newly synthesised virions from SARS-CoV-2-infected macrophages, consistent with abortive replication. Release of new, infectious SARS-CoV-2 virions from ACE2 over-expressing macrophages only occurred if anti-viral mediator induction was also blocked, indicating that macrophages restrict SARS-CoV-2 infection at two stages of the viral life cycle. These findings resolve the current controversy over macrophage-SARS-CoV-2 interactions and identify a signalling circuit that directly links macrophage recognition of SARS-CoV-2 to restriction of viral replication.

One sentence summaryACE2 is necessary for SARS-CoV-2 infection and sensing by macrophages but not sufficient for productive viral replication.

category: immunology


title: A mRNA vaccine encoding for a RBD 60-mer nanoparticle elicits neutralizing antibodies and protective immunity against the SARS-CoV-2 delta variant in transgenic K18-hACE2 mice

abstract: Two years into the COVID-19 pandemic there is still a need for vaccines to effectively control the spread of novel SARS-CoV-2 variants and associated cases of severe disease. Here we report a messenger RNA vaccine directly encoding for a nanoparticle displaying 60 receptor binding domains (RBDs) of SARS-CoV-2 that acts as a highly effective antigen. A construct encoding the RBD of the delta variant elicits robust neutralizing antibody response with neutralizing titers an order of magnitude above currently approved mRNA vaccines. The construct also provides protective immunity against the delta variant in a widely used transgenic mouse model. We ultimately find that the proposed mRNA RBD nanoparticle-based vaccine provides a flexible platform for rapid development and will likely be of great value in combatting current and future SARS-CoV-2 variants of concern.


title: Humanized mice for sustained Plasmodium vivax blood-stage infection and transmission

abstract: Plasmodium vivax is the most widespread human malaria parasite 1. The presence of extravascular reservoirs 2, the early circulation of infective stages (gametocytes), and relapsing infections arising from dormant liver stages 3 render this parasite particularly difficult to control and eliminate 4. Experimental research is limited by the lack of a continuous culture in vitro system that fulfills the parasites needs,5 namely its tropism for immature CD71+ red blood cells (RBCs). 5 Here, we report a humanized mice model, which upon engraftment of human hematopoietic progenitor and stem cells (HPSCs), exhibits efficient human erythropoiesis. Humanized HIS-HEry mice inoculated with cryopreserved P. vivax samples sustain long-lasting asexual parasite multiplication within CD71+ human RBCs and differentiation into mature gametocytes that can be efficiently transmitted to Anopheles mosquitoes, leading to formation salivary-gland sporozoites. Blood stages can be sequentially transferred to uninfected humanized mice by injection of fresh or frozen infected bone marrow cells, providing a unique murine model for the long-term maintenance of P. vivax isolates. This work offers a novel experimental platform to investigate the biology of RBC invasion and intraerythrocytic P. vivax development in vivo and evaluate new interventions against this elusive human parasite.


title: A potent SARS-CoV-2 antibody neutralizes Omicron variant by disassembling the spike trimer

abstract:
The continuous emergence of novel SARS-CoV-2 variants poses new challenges to the fight against the COVID-19 pandemic. The newly emerging Omicron strain caused serious immune escape and raised unprecedented concern all over the world. The development of antibody targeting conserved and universal epitope is urgently needed. A subset neutralizing antibody(nAbs) against COVID-19 from convalescent patients were isolated in our previous study. Here in this study, we investigated the accommodation of these nAbs to SARS-CoV-2 variants of concerns (VOCs), revealing that IgG 553-49 neutralizes pseudovirus of SARS-CoV-2 Omicron variant. In addition, we determined the cryo-EM structure of SARS-CoV-2 spike complexed with three antibodies targeting different epitopes, including 553-49, 553-15 and 553-60. Notably, 553-49 targets a novel conserved epitope and neutralizes virus by disassembling spike trimers. 553-15, an antibody that neutralizes all the other VOCs except omicron, cross-links two spike trimers to form trimer dimer, demonstrating that 553-15 neutralizes virus by steric hindrance and virion aggregation. These findings suggest the potential to develop 49 and other antibody targeting this highly conserved epitope as promising cocktail therapeutics reagent for COVID-19.

ImportanceThe newly emergence of Omicron strain caused higher immune escape, raising unprecedented concerns about the effectiveness of antibody therapies and vaccines. In this study, we identified a SARS-CoV-2 Omicron neutralizing antibody 553-49, which neutralizes Omicron variant by targeting a completely conserved novel epitope. Besides, we revealed that IgG 553-15 neutralizes SARS-CoV-2 by crosslinking virions and 553-60 functions by blocking receptor binding. Comparison of different RBD epitopes revealed that the epitope of 553-49 is hidden in the S trimer and keeps high conservation during SARS-CoV-2 evolution, making 553-49 a promising therapeutics reagent to fight against the emerging Omicron and future variant of SARS-CoV-2.


title: Genome-resolved metagenomics reveals cohesive metabolic dynamics, metal resistance genes (MRGs) and biogeochemical cycling in a hot spring system

abstract: The hot spring microbiome is a complex assemblage of micro- and geochemistry of this spring have been and macro- organisms, however, the understanding and projection of enzymatic repertoire that access earths integral ecosystem processes remains limited. The composite microbial communities drive global carbon, sulfur, oxygen, iron and nitrogen cycles and other metabolic mechanism involved in heavy metal tolerance and degradation. Interestingly, the Khirganga hot spring microbiome displayed an astounding taxonomical diversity revealed by examination of 41 high and medium qualified metagenome-assembled genomes (MAGs) from at least 12 bacterial and 2 archaeal phyla. Over 1749 genes putatively involved in crucial metabolism of elements viz. nitrogen, phosphorous, sulfur and 598 genes encoding enzymes for metals resistance from cadmium, zinc, chromium, arsenic and copper. The MAGs also possess 229 biosynthetic gene clusters dominated by bacteriocins and terpenes could be exploited in medicinal industries. Their metabolic roles found to be altering linkages in biogeochemical cycles and explored a discerned rate of carbon fixation exclusively in archaeal member Methanospirillum hungatei. Higher Pfam entropy scores in Proteobacteria members highlighting their major contribution in sequestration of ammonia, nitrate and sulfate components as electron acceptors. Through these results, we could postulate that few novel organisms within the community can conduct multiple sequential redox conversions and also considered in reducing emergent difficulties of waste water treatment plants and biotechnological applications.


title: Genetic Diversity of Candidatus Liberibacter asiaticus Based on Four Hypervariable Genomic Regions in China

abstract:
Huanglongbing (HLB; greening disease), caused by Candidatus Liberibacter asiaticus (CLas), is the most damaging citrus disease worldwide. The disease has spread throughout the citrus-producing regions of Guangxi, Guangdong, Fujian, and others in China. A total of 1,789 HLB-like symptomatic or asymptomatic samples were collected from the Guangxi and Fujian provinces of China to decipher the genetic diversity of CLas and its correlation with pathogenicity and host range. The disease was the most severe in orange and the least in pomelo. CLas bacteria associated with the specific geographical and citrus variety infected more than 50% of the HLB-like symptomatic samples. We identified a total of 6,286 minor variations by comparing 35 published CLas genomes and observed a highly heterogeneous variation distribution across the genome. Highly diverse regions, including two prophages, were generally more unstable and prone to lose. In the collected CLas strains, four highly diverse non-prophage segments and three prophage segments in the prophage region were chosen for PCR amplification and genotyping. Four hypervariable regions were used to decipher CLas diversity. A total of 100 strains were divided into four groups, of which 90 strains were clustered into two clades with 15 reported reference genomes, while 10 were grouped in two clades separately from the reported genomes. This study provides insight into the molecular characteristics and genetic variations of different CLas strains in China and might help develop HLB prevention and control strategies.

ImportanceThe objective of the present study is to characterize the hypervariable genomic region sequence, decipher the genetic diversity of CLas strains from China, understand how various strains differ, and lay the foundation for developing HLB prevention and control strategies.


title: Multi-omics analyses of MEN1 missense mutations identify disruption of menin-MLL and menin-JunD interactions as critical requirements for molecular pathogenicity

abstract: Loss-of-function mutations of the multiple endocrine neoplasia type 1 (MEN1) gene are causal to the MEN1 tumor syndrome, but they are also commonly found in sporadic pancreatic neuroendocrine tumors and other types of cancers. The MEN1 gene product, menin, is involved in transcriptional and chromatin regulation, most prominently as an integral component of KMT2A/MLL1 and KMT2B/MLL2 containing COMPASS-like histone H3K4 methyltransferase complexes. In a mutually exclusive fashion, menin also interacts with the JunD subunit of the AP-1 and ATF/CREB transcription factors. After in silico screening of 253 disease-related MEN1 missense mutations, we selected a set of nine menin mutations in surface-exposed residues. The protein interactomes of these mutants were assessed by quantitative mass spectrometry, which indicated that seven of the nine mutants disrupt interactions with both MLL1/2 and JunD complexes. Interestingly, we identified three missense mutations, R52G, E255K and E359K, which predominantly reduce the interaction with MLL1 compared to JunD. This observation was supported by a pronounced loss of binding of the R52G, E255K and E359K mutant proteins at unique MLL1 genomic binding sites with less effect on unique JunD sites. These findings support the general importance of the menin-MLL1 and menin-JunD interactions in MEN1 gene-associated pathogenic conditions.

category: molecular biology


title: Persistent acetylation of newly synthesized histones inhibits DNA replication origin activity

abstract: Histone acetylation is a known regulator of DNA replication dynamics. In the yeast Saccharomyces cerevisiae, newly synthesized histone H3 deposited behind DNA replication forks are acetylated on lysine 56 (H3 K56ac) by the Rtt109 histone acetyltransferase. Two enzymes of the sirtuin family of deacetylases, Hst3 and Hst4, act redundantly to deacetylate this residue throughout chromosomes following S phase. Cells lacking Hst3 and Hst4 present constitutively acetylated H3 K56, which causes sensitivity to replication-blocking genotoxins and slow growth via mechanisms that remain poorly understood. Here, we present the results of a genome-wide screen aimed at identifying haploinsufficient genes that promote cell fitness in response to inhibition of sirtuins by nicotinamide (NAM). We find that heterozygous deletion alleles of genes involved in the regulation of DNA replication origins sensitize cells to NAM. Consistently, haploid cells harboring the hypomorphic temperature sensitive allele cdc7-4 present striking NAM-induced S phase progression defects at their semi-permissive temperature. We further show i) that Rap1-interacting factor 1 (Rif1), an inhibitor of Cdc7-dependent activation of replication origins, causes DNA damage and replication defects in cells exposed to NAM and in hst3{Delta} hst4{Delta} mutants, and ii) that cdc7-4 hst3{Delta} hst4{Delta} triple mutant cells present strong synthetic temperature sensitivity associated with defective activation of replication origin and delayed S phase progression. Our data further demonstrate that such replication defects are not due to intra-S phase checkpoint activation leading to inhibition of origin activity. We finally show that Rtt109-dependent acetylation of histone H3 lysine 56 and its associated Rtt101-Mms1 ubiquitin ligase complex cause the DNA replication defects observed in cdc7-4 hst3{Delta} hst4{Delta} cells. Overall, these results argue that abnormal regulation of nascent chromatin structure negatively influences DNA replication initiation in cells presenting reduced Dbf4-dependent kinase activity including those experiencing replicative stress.


title: Comparison of microbial communities from diverse biological matrices using mock community as an in situ positive control

abstract:
Metataxonomy has become the standard for characterizing the diversity and composition of microbial communities associated with multicellular organisms and their environment. Understanding the interactions between the microbiotas within the same ecosystem is essential for fully understanding the role of microorganisms in evolutionary and ecological processes; however, such comparative studies across diverse biological samples are rare. In particular, currently available protocols assume a uniform DNA extraction, amplification and sequencing efficiency for all sample types and taxa. The addition of a mock community (MC) to biological samples before the DNA extraction step could aid identification of technical biases, but the impact of MC on diversity estimates is unknown. Here, standardized aliquots of bovine faecal samples with high or low biomass were extracted with high or low doses of MC, characterized using standard Illumina technology for metataxonomics, and analysed with custom bioinformatic pipelines. We showed that a MC was an informative in situ positive control provided an estimate of 16S rRNA sample gene copies (which allowed a more direct measure of community size), and detected sample outliers. However, we also demonstrated that if the recommended dose of MC is added to a sample with low biomass, diversity estimates were distorted. Using our results, we recommend MC doses for a range of sample types, including rhizosphere soil, whole invertebrates, and vertebrate faecal samples.

ImportanceThe simultaneous processing of the sample microbiota with a known number of readily identifiable MC cells (co-extracted with the sample cells) or SNA (co-amplified with the sample DNAs) can be a valuable in situ positive control. However, guidelines regarding their application are very limited and do not consider the effect of these controls on sample diversity estimates. We demonstrate that a MC co-extracted with the study sample provides several advantages, such as highlighting bias in DNA extraction of gram positive, identifying potential sample outliers and inferring the number of 16S rRNA gene copies in the sample. However, the incorporation of a MC requires prior knowledge of sample biomass, as high ratios of MC to sample microbiota lead to biased sample diversity estimates. Practical advice on determining the appropriate MC dose for a wide range of sample types, including rhizosphere soil, whole invertebrates and mammalian faecal samples are provided.


title: Structure-forming CAG/CTG repeats interfere with gap repair to cause repeat expansions and chromosome breaks

abstract: Expanded CAG/CTG repeats are sites of DNA damage, leading to changes in repeat length. To determine how ssDNA gap filling affects repeat instability, we inserted (CAG)70 or (CTG)70 repeats into a single-strand annealing (SSA) assay system such that resection and filling in the ssDNA gap would occur across the repeat tract. After resection, when the CTG sequence was the single-stranded template for fill-in synthesis, repeat contractions were elevated and the ssDNA created a fragile site that led to large deletions involving flanking homologous sequences. In contrast, resection was inhibited when CTG was on the resected strand, resulting in repeat expansions. Deleting Rad9, the ortholog of 53BP1, rescued repeat instability and lost viability by increasing resection and fill-in speed. Deletion of Rad51 increased CTG contractions and decreased survival, implicating Rad51 in protecting ssDNA during gap filling. Taken together, DNA sequence within a single-stranded gap determines repair kinetics, fragility, and repeat instability.

category: genetics


title: Live-cell RNA imaging with metabolically incorporated fluorescent nucleosides

abstract: Fluorescence imaging is a powerful method for probing macromolecular dynamics in biological systems, however approaches for cellular RNA imaging are limited to the investigation of individual RNA constructs or bulk RNA labeling methods compatible primarily with fixed samples. Here, we develop a platform for fluorescence imaging of bulk RNA dynamics in living cells. We show that fluorescent bicyclic and tricyclic cytidine analogues can be metabolically incorporated into cellular RNA by overexpression of uridine-cytidine kinase 2 (UCK2). In particular, metabolic feeding with the tricyclic cytidine-derived nucleoside tC combined with quantitative confocal imaging enables the investigation of RNA synthesis, degradation, and trafficking at single-cell resolution. We apply our imaging modality to study RNA metabolism and localization during the oxidative stress response and find that bulk RNA turnover is greatly accelerated upon NaAsO2 treatment. Further, we identify cytoplasmic RNA granules containing RNA transcripts generated during oxidative stress that are distinct from canonical stress granules and P-bodies and co-localize with the RNA helicase DDX6. Taken together, our work provides a powerful approach for live-cell RNA imaging and reveals how cells reshape RNA transcriptome dynamics in response to oxidative stress.

category: biochemistry


title: Regulation of multiple dimeric states of E-cadherin by adhesion activating antibodies revealed through Cryo-EM and X-ray crystallography

abstract: E-cadherin adhesion is regulated at the cell surface, a process that can be replicated by activating antibodies. We use cryo-EM and X-ray crystallography to examine functional states of the cadherin adhesive dimer. This dimer is mediated by N-terminal beta strand-swapping involving Trp2, and forms via a different transient X-dimer intermediate. X-dimers are observed in cryo-EM along with monomers and strand-swap dimers, indicating that X-dimers form stable interactions. A novel EC4-mediated dimer was also observed. Activating Fab binding caused no gross structural changes in E- cadherin monomers but can facilitate strand swapping. Moreover, activating Fab binding is incompatible with the formation of the X-dimer. Both cryo-EM and X-ray crystallography reveal a distinctive twisted strand-swap dimer conformation caused by an outward shift in the N-terminal beta strand that may represent a strengthened state. Thus, regulation of adhesion involves changes in cadherin dimer configurations.


title: Bending stiffness of Candida albicans hyphae reflects adaptive behavior of the fungal cell wall

abstract: The cell wall is a key component of fungi. It constitutes a stiff shell which counteracts internal cell turgor pressure. Its mechanical properties thus contribute to define cell morphology. Measurements of the elastic moduli of the fungal cell wall have been carried out in many species including Candida albicans, a major human opportunistic pathogen. They mainly relied on atomic force microscopy, and mostly considered the yeast form. We developed a parallelized pressure-actuated microfluidic device to measure the bending stiffness of hyphae. We found that the cell wall stiffness lies in the MPa range. We the used three different ways to disrupt cell wall physiology: inhibition of beta-glucan synthesis, a key component of the inner cell wall; application of an hyperosmotic shock triggering a sudden decrease of the hyphal diameter; deletion of two genes encoding GPI-modified cell wall proteins resulting in reduced cell wall thickness. The bending stiffness values were affected to different extents by these environmental stresses or genetic modifications. Overall, our results support the elastic nature of the cell wall and its ability to remodel at the scale of the entire hypha over minutes.


title: Light-sheet mesoscopy with the Mesolens provides fast sub-cellular resolution imaging throughout large tissue volumes.

abstract: Rapid imaging of large biological tissue specimens such as ultrathick sections of mouse brain cannot easily be performed with a standard microscope. Optical mesoscopy offers a solution, but thus far imaging has been too slow to be useful for routine use. We have developed two different illuminators for light-sheet mesoscopy with the Mesolens and we demonstrate their use in high-speed optical mesoscale imaging of large tissue specimens. The first light-sheet approach uses Gaussian optics and is straightforward to implement. It provides excellent lateral resolution and high-speed imaging, but the axial resolution is poor. The second light-sheet is a more complex Airy light-sheet that provides sub-cellular resolution in three dimensions that is comparable in quality to point-scanning confocal mesoscopy, but the light-sheet method of illuminating the specimen reduces the imaging time by a factor of 14. This creates new possibilities for high-content, higher-throughput optical bioimaging at the mesoscale.


title: Hypoxic conditions promote a proliferative, poorly differentiated, and pro-secretory phenotype in COPD lung tissue progenitor cells in vitro

abstract:
Chronic obstructive pulmonary disease patients experience variable symptoms dependent on the presence of an emphysematous versus a chronic bronchitis phenotype. Both presentations can be associated with lung tissue and systemic hypoxia, at its most severe leading to Cor pulmonale. Despite this, minimal attention has been given to the effects of hypoxia at the cellular disease level.

We isolated and cultured progenitor cells from the distal lung tissue of a 64 year-old, male, emphysematous donor in ambient (21%) and hypoxic (2%) oxygen conditions. Proliferative capacity was determined on collagen coated culture plastic and growth-inactivated 3T3-J2 co-cultures. Epithelial (E-cadherin and pan-cytokeratin) and progenitor (TP63, cytokeratin 5) marker expression were examined. Expanded cells were differentiated at air-liquid interface and ciliated, mucous producing, and club cell populations identified.

Isolated cells were positive for the epithelial, pan-cytokeratin and E-cadherin, and progenitor, TP63 and cytokeratin 5, cell markers at isolation and again at passage 5. Passage 5 expanded cells in hypoxia had increased the proportion of TP63 expressing cells by 10% from 51.6 {+/-} 1.2% to 62.6 {+/-} 2.3% (p [≤] 0.01). Proliferative capacity was greater in 3T3J2 co-cultured cells overall and in 2% oxygen this supported the emergence of a proliferation unrestricted population with a limited differentiation capacity. Cells expanded on collagen I in either oxygen underwent differentiation having been expanded with the production of ciliated cells positive for {beta}IV tubulin, and mucin 5ac, mucin 5b and CC10 positive secretory cells. Epithelial barrier formation was reduced significantly (p [≤] 0.0001) in hypoxia-expanded cells compared to normoxia. qRT-PCR showed higher expression of mucins in 2% expanded cells, significantly so with MUC5B (P [≤] 0.05) although mucin protein secretion was greater in 21% expanded cells.

Concomitantly these results demonstrate that hypoxia promotes a proliferative phenotype while reducing the overall differentiation capacity of the cells. Further, the retained differentiation potential becomes skewed to a more secretory phenotype demonstrating that hypoxia may be contributing to disease symptom and severity in COPD patients.


title: The Arabidopsis transcription factor NLP2 regulates early nitrate responses and integrates nitrate assimilation with energy and carbon skeleton supply

abstract:
Nitrate signaling improves plant growth under limited nitrate availability and, hence, optimal resource use for crop production. Ongoing work has identified several transcriptional regulators of nitrate signaling, including the Arabidopsis thaliana transcription factor NIN-LIKE PROTEIN 7 (NLP7), but additional regulators likely remain to be identified. Here, we characterized Arabidopsis NLP2 as a master upstream transcriptional regulator of early nitrate responses that interacts with NLP7 in vivo and shares key molecular features such as nitrate-dependent nuclear localization, a DNA binding motif, and some target genes with NLP7. Additional genetic, genomic and metabolic approaches revealed a specific role for NLP2 in the nitrate-dependent regulation of carbon and energy-related processes that likely influence plant growth under distinct nitrogen environments. Our findings highlight the complementarity and specificity of NLP2 and NLP7 in orchestrating a multi-tiered nitrate regulatory network that links nitrate assimilation with carbon and energy metabolism for efficient nitrogen use and biomass production.

One-sentence summaryNLP2 and NLP7 orchestrate plant responses to nitrate supply and control nitrate- dependent regulation of carbon and energy metabolism.


title: The wheat multi-pathogen resistance gene, Lr67res, confers a novel gain-of-function phenotype involving anion fluxes

abstract:
Partial resistance to multiple biotrophic fungal pathogens in wheat (Triticum aestivum L.) is conferred by the Lr67 gene, which encodes a Sugar Transport Protein 13 (STP13) family hexose-proton symporter variant. Two mutations (G144R, V387L) differentiate the resistant and susceptible protein variants (Lr67res and Lr67sus). The molecular function of the Lr67res protein is not well understood. We functionally characterized the wheat Lr67res protein variant using two heterologous expression systems - Xenopus laevis oocytes and Saccharomyces cerevisiae yeast. Wheat and barley (Hordeum vulgare) were used to verify disease resistance capability of Lr67/STP13 variants. The Lr67res allele, but not Lr67sus, induced large sugar-independent, anion-dominated currents in oocytes and an increased sensitivity to ions in yeast, implicating a novel gain-of-function. We demonstrate that the single mutant variant, Lr67susG144R, confers disease resistance in wheat and that transgenic barley (Hordeum vulgare L.) plants expressing the orthologous HvSTP13 gene carrying the equivalent mutations present in Lr67res exhibited increased resistance to Puccinia hordei. NaCl treatment was found to induce leaf tip necrosis in Lr67res wheat. An Lr67res-like gain-of-function can be introduced into orthologous plant hexose transporters via single amino acid mutation, highlighting the possibility of generating disease resistance in other crop species, especially with gene editing.

One sentence summaryThe wheat Lr67res protein responsible for multipathogen resistance has a gain-of-function over the Lr67sus protein, that is associated with anion fluxes and likely contributes to disease resistance.


title: On the Design of a PID Bio-controller with Set Point Weighting and Filtered Derivative Action

abstract: Effective and robust regulation of biomolecular processes is crucial for designing reliable synthetic bio-devices functioning in uncertain and constantly changing biological environments. Proportional-Integral-Derivative (PID) controllers are undeniably the most common way of implementing feed-back control in modern technological applications. Here, we introduce a highly tunable PID bio-controller with set point weighting and filtered derivative action presented as a chemical reaction network with mass action kinetics. To demonstrate its effectiveness, we apply our PID scheme on a simple biological process of two mutually activated species, one of which is assumed to be the output of interest. To highlight its performance advantages we compare it to PI regulation using numerical simulations in both the deterministic and stochastic setting.


title: Using protein-per-mRNA differences among human tissues in codon optimization

abstract: Codon usage and nucleotide composition of coding sequences have profound effects on protein expression. However, while it is recognized that different tissues have distinct tRNA profiles and codon usages in their transcriptomes, the effect of tissue-specific codon optimality on protein synthesis remains elusive. Here, we leverage existing state-of-the-art transcriptomics and proteomics datasets from the GTEx project and the Human Protein Atlas to compute the protein-to-mRNA ratios of 36 human tissues. Using this as a proxy of translational efficiency, we build a machine learning model that identifies codons enriched or depleted in specific tissues. In particular, we detect two clusters of tissues with an opposite pattern of codon preferences. We then use the identified patterns for the development of CUSTOM, a codon optimizer algorithm which suggests a synonymous codon design in order to optimize protein production in a tissue-specific manner. In a human cell model, we provide evidence that codon optimization should indeed take into account particularities of the translational machinery of the tissues in which the target proteins are expressed and that our approach can design genes with tissue-optimized expression profiles. Altogether, CUSTOM could benefit biological and biotechnological research, such as the design of tissue-targeted therapies and vaccines.


title: Distinct gene programs underpinning 'disease tolerance' and 'resistance' in influenza virus infection

abstract: When challenged with an invading pathogen, the host defense response is engaged to eliminate the pathogen (resistance) and to maintain health in the presence of the pathogen (disease tolerance). However, the identification of distinct molecular programs underpinning disease tolerance and resistance remained obscure. We exploited transcriptional and physiological monitoring across 33 mouse strains, during in vivo influenza virus infection, to identify two host-defense gene programs - one is associated with hallmarks of disease tolerance and the other with hallmarks of resistance. Both programs constitute generic responses in multiple mouse and human cell types. Our study describes the organizational principles of these programs and validates Arhgdia as a regulator of disease-tolerance states in epithelial cells. We further reveal that the baseline disease-tolerance state in macrophages is associated with the pathophysiological response to injury and infection. Our framework provides a paradigm for the understanding of disease tolerance and resistance at the molecular level.


title: The NLRP1 and CARD8 inflammasomes detect reductive stress

abstract: The danger signals that activate the NLRP1 and CARD8 inflammasomes have not been fully established. We recently discovered that cytosolic peptide accumulation activates these inflammasomes. In addition, we found that the oxidized form of TRX1 binds to and represses NLRP1, suggesting that NLRP1 also detects a lack of reactive oxygen species, or reductive stress. However, no agents that induce reductive stress were known to test this premise. Here, we identify and characterize several radical-trapping antioxidants, including JSH-23, that induce reductive stress. We show that these compounds accelerate the proteasome-mediated degradation of the repressive N-terminal fragments of NLRP1 and CARD8, releasing the inflammasome-forming C-terminal fragments from autoinhibition. Moreover, we found that reductive stress and peptide accumulation together trigger much more intense inflammasome activation than either signal alone. Overall, this work validates chemical probes that induce reductive stress, and establishes reductive stress alongside peptide accumulation as the key inflammasome-activating danger signals.


title: Identification and validation of reference genes for RT-qPCR single-gene relative expression analysis in Taenia solium pre-adult stages

abstract:
The larvae-to-adult development on the life cycle of zoonotic parasitic tapeworm Taenia solium involves striking -but clinically unappreciated- events with pivotal importance in cestode biology. Unlike the ones related to the intermediate host, the early-adult stages can be addressed in vitro offering a useful model to study evagination, strobilation and worm development. In the absence of a stage-specific transcriptome, postgenomic data exploration followed by single-gene relative expression analysis by RT-qPCR (reverse transcription-quantitative PCR) are useful strategies to gather information on the regulation of genes of interest during parasite development. However, this approach requires the validation of an endogenous reference gene (RG) to achieve accurate comparisons.

Therefore, we analyzed the expression stability of 17 candidate RGs on the context of the early-adult stages of T. solium classified as non-evaginated and evaginated larvae (cysts). The comprehensive tool RefFinder ranked malate dehydrogenase as the most stable gene within these conditions, and its suitability for relative quantification was validated by normalizing the expression of the transporter TGTP1 gene, known for being upregulated upon evagination. This study is the first attempt in finding reliable normalization standards for transcript exploration in genus Taenia.


title: Stage-specific gene and transcript dynamics in human male germ cells

abstract: Cell differentiation processes are highly dependent on cell stage-specific gene expression, including timely production of alternatively spliced transcripts. One of the most transcriptionally rich tissues is the testis, where the process of spermatogenesis, or generation of male gametes, takes place. To date, germ cell-specific transcriptome dynamics remain understudied due to limited transcript information emerging from short-read sequencing technologies. To fully characterize the transcriptional profiles of human male germ cells and to understand how the human spermatogenic transcriptome is regulated, we compared whole transcriptomes of men with different types of germ cells missing from their testis. Specifically, we compared the transcriptomes of testis lacking germ cells (Sertoli cell-only phenotype; SCO; n=3), with an arrest at the stage of spermatogonia (SPG; n=4), spermatocytes (SPC; n=3), and round spermatids (SPD; n=3), with the transcriptomes of testis with normal and complete spermatogenesis (Normal; n=3). We found between 839 and 4,138 differentially expressed genes (DEGs, log2 fold change [≥] 1) per group comparison, with the most prevalent changes observed between SPG and SPC arrest samples, corresponding to the entry into meiosis. We detected highly germ cell-type specific marker genes among the topmost DEGs of each group comparison. Moreover, applying state-of-the-art bioinformatic analysis we were able to evaluate differential transcript usage (DTU) during human spermatogenesis and observed between 1,062 and 2,153 genes with alternatively spliced transcripts per group comparison. Intriguingly, DEGs and DTU genes showed minimal overlap (< 8%), suggesting that stage-specific splicing is an additional layer of gene regulation in the germline. By generating the most complete human testicular germ cell transcriptome to date, we unravel extensive dynamics in gene expression and alternative splicing during human spermatogenesis.