title: Optimization of anastomotic technique and gastric conduit perfusion with hyperspectral imaging in an experimental model for minimally invasive esophagectomy

abstract:
ObjectiveTo optimize anastomotic technique and gastric conduit perfusion with hyperspectral imaging (HSI) for total minimally invasive esophagectomy (MIE) with linear stapled anastomosis.

Summary Background DataEsophagectomy is the mainstay of esophageal cancer treatment but anastomotic insufficiency related morbidity and mortality remain challenging for patient outcome.

MethodsA live porcine model (n=50) for MIE was used with gastric conduit formation and linear stapled side-to-side esophagogastrostomy. Four main experimental groups differed in stapling length (3 vs. 6 cm) and anastomotic position on the conduit (cranial vs. caudal). Tissue oxygenation around the anastomotic site was evaluated using HSI and was validated with histopathology.

ResultsThe tissue oxygenation ({Delta}StO2) after the anastomosis remained constant only for the short stapler in caudal position (-0.4{+/-} 4.4%, n.s.) while it dropped markedly in the other groups (short-cranial: -15.6{+/-} 11.5%, p=0.0002; long-cranial: -20.4{+/-} 7.6%, p=0.0126; long-caudal: -16.1{+/-} 9.4%, p<0.0001) Tissue samples from deoxygenated stomach as measured by HSI showed correspondent eosinophilic pre-necrotic changes in 35.7{+/-} 9.7% of the surface area.

ConclusionsTissue oxygenation at the anastomotic site of the gastric conduit during MIE is influenced by stapling technique. Optimal oxygenation was achieved with a short stapler (3 cm) and sufficient distance of the anastomosis to the cranial end of the gastric conduit. HSI tissue deoxygenation corresponded to histopathologic necrotic tissue changes. These findings allow for optimization of gastric conduit perfusion and anastomotic technique in MIE.

Level of EvidenceNot applicable. Translational animal science. Original article.

category: bioengineering


title: Mesophyll conductance response to short-term changes in pCO2 is related to leaf anatomy and biochemistry in diverse C4 grasses

abstract:
O_LIMesophyll CO2 conductance (gm) in C3 species responds to short-term (minutes) changes in environment potentially due to changes in some leaf anatomical and biochemical properties and due to measurement artifacts. Compared to C3 species, there is less information about gm responses to short-term changes in environment conditions like pCO2 across diverse C4 species and the potential determinants of these responses.
C_LIO_LIUsing 16 diverse C4 grasses we investigated the response of gm to short-term changes in CO2 and how this response related to the leaf anatomical and biochemical traits.
C_LIO_LIFor all the measured C4-grasses gm increased as CO2 decreased; however, the percent change in gm varied (+13% to +250%) and significantly related to percent changes in leaf transpiration efficiency (TEi). The percent increase in gm was highest in grasses with thinner mesophyll cell walls and greater leaf nitrogen, activities of phosphoenolpyruvate carboxylase (PEPC), Rubisco and carbonic anhydrase, and a higher affinity of PEPC for bicarbonate.
C_LIO_LIOur study demonstrates that CO2 response of gm varies greatly across diverse C4 grasses and identifies the key leaf anatomical and biochemical traits related to this variation. These findings have implications for improving C4 photosynthetic models, and in attempts to improve TEi through manipulation of gm.
C_LI

category: plant biology


title: An exploratory analysis of 4844 withdrawn articles and their retraction notes.

abstract:
The objective of our study was to obtain an updated image of the dynamic of retractions and retraction notes, retraction reasons for questionable research and publication practices, countries producing retracted articles, and the scientific impact of retractions by studying 4844 PubMed indexed retracted articles published between 2009 and 2020 and their retraction notes.

RESULTSMistakes/inconsistent data account for 32% of total retractions, followed by images(22,5%), plagiarism(13,7%) and overlap(11,5%).

Thirty countries account for 94,79% of 4844 retractions. Top five are: China(32,78%), United States(18,84%), India(7,25%), Japan(4,37%) and Italy(3,75%).

The total citations number for all articles is 140810(Google Scholar), 96000(Dimensions).

Average exposure time(ET) is 28,89 months. Largest ET is for image retractions(49,3 months), lowest ET is for editorial errors(11,2 months).

The impact of retracted research is higher for Spain, Sweden, United Kingdom, United States, and other nine countries and lower for Pakistan, Turkey, Malaysia, and other six countries, including China.

CONCLUSIONSMistakes and data inconsistencies represent the main retraction reason; images and ethical issues show a growing trend, while plagiarism and overlap still represent a significant problem. There is a steady increase in QRP and QPP article withdrawals. Retraction of articles seems to be a technology-dependent process.

The number of citations of retracted articles shows a high impact of papers published by authors from certain countries. The number of retracted articles per country does not always accurately reflect the scientific impact of QRP/QPP articles.

The country distribution of retraction reasons shows structural problems in the organization and quality control of scientific research, which have different images depending on geographical location, economic development, and cultural model.

category: scientific communication and education


title: Signatures of vibration frequency tuning in human neocortex

abstract: The spectral content of vibrations produced in the skin conveys essential information about textures and underlies sensing through hand-held tools. Humans can perceive and discriminate vibration frequency, yet the central representation of this fundamental feature is unknown. Using fMRI, we discovered that cortical responses are tuned for vibration frequency. Voxel tuning was biased in a manner that reflects perceptual sensitivity and the response profile of the Pacinian afferent system. These results imply the existence of tuned populations that may encode naturalistic vibrations according to their constituent spectra.

category: neuroscience


title: Neuropeptidergic regulation of Compulsive Ethanol Seeking in C. elegans

abstract: An improved understanding of the molecular basis of alcohol seeking despite the catastrophic consequences of alcohol abuse is likely to enrich our treatments for Alcohol Use Disorders (AUD) and comorbidities. The compulsive seeking is characterized by an imbalance between the superior drive to substance and disruption in control of substance use. To model the development of compulsive engagement of alcohol seeking, we exploit two distinct behavioral programs of C. elegans in conflict, ethanol preference and avoidance of aversive stimulus, simultaneously. We demonstrate that C. elegans exhibited the recapitulation of the pivotal features of compulsive alcohol seeking in mammals, which are repeated attempts, endurance, and finally aversion-resistant ethanol seeking. We find that the neuropeptide signaling via SEB-3, CRF receptor-like GPCR, facilitates the development of ethanol preference and compels animals to seek ethanol compulsively. Furthermore, our functional genomic approach and behavioral elucidation suggest the interaction between neuropeptidergic signaling, SEB-3 and TKR-1, Neurokinin receptor orthologue, to progress compulsive ethanol seeking behavior.


title: Enhancement of hippocampal-thalamocortical temporal coordination during slow-frequency long-duration anterior thalamic spindles

abstract: Temporal nesting of cortical slow oscillations (SO), thalamic spindles and hippocampal ripples indicates the succession of regional neuronal interactions required for memory consolidation. However how the thalamic activity during spindles organizes hippocampal dynamics remains largely undetermined. We analyzed simultaneous recordings of anterodorsal thalamus and CA1 in mice to determine the contribution of thalamic spindles in cross-regional synchronization. Our results indicated that temporal hippocampo-thalamocortical coupling were more enhanced during slower and longer thalamic spindles. Additionally, spindles occurring closer to SO trough were more strongly coupled to ripples. We found that the temporal association between CA1 spiking/ripples and thalamic spindles was stronger following spatial exploration compared to baseline sleep. We further developed a hippocampal-thalamocortical model to explain the mechanism underlying the duration and frequency-dependent coupling of thalamic spindles to hippocampal activity. Our findings shed light on our understanding of the functional role of thalamic activity during spindles on multi-regional information transfer.


title: Alterations in connectome dynamics in autism spectrum disorder: A harmonized mega- and meta-analysis study using the ABIDE dataset

abstract:
BACKGROUNDNeuroimaging studies have reported functional connectome aberrancies in autism spectrum disorder (ASD). However, the time-varying patterns of connectome topology in ASD individuals and the connection between these patterns and gene expression profiles remain unknown.

METHODSTo investigate case-control differences in dynamic connectome topology, we conducted mega- and meta-analyses of resting-state functional magnetic resonance imaging data of 939 participants (440 ASD patients and 499 healthy controls, all males) from 18 independent sites, selected from the ABIDE (Autism Brain Imaging Data Exchange) dataset. Functional data was preprocessed and analyzed using harmonized protocols, and brain module dynamics was assessed using a multilayer network model. We further leveraged postmortem brain-wide gene expression data to identify transcriptomic signatures associated with ASD-related alterations in brain dynamics.

RESULTSCompared to healthy controls, ASD individuals exhibited a higher global mean and lower standard deviation of whole-brain module dynamics, indicating an unstable and less regionally differentiated pattern. More specifically, ASD individuals showed higher module switching, primarily in the medial prefrontal cortex, posterior cingulate gyrus, and angular gyrus, and lower switching in the visual regions. These alterations in brain dynamics were predictive of social impairments in ASD individuals and were linked with expression profiles of genes primarily involved in the regulation of neurotransmitter transport and secretion, as well as with previously identified autism-related genes.

CONCLUSIONSThis study is the first to identify consistent alterations in brain network dynamics in ASD and the transcriptomic signatures related to those alterations, furthering insights into the biological basis behind this disorder.


title: Severe distortion in the representation of foveal visual image locations in short term memory

abstract: The foveal visual image region provides the human visual system with the highest acuity. However, it is unclear whether such a high fidelity representational advantage is maintained when foveal image locations are committed to short term memory. Here we describe a paradoxically large distortion in foveal target location recall by humans. We briefly presented small, but high contrast, points of light at eccentricities ranging from 0.1 to 12 deg, while subjects maintained their line of sight on a stable target. After a brief memory period, the subjects indicated the remembered target locations via computer controlled cursors. The biggest localization errors, in terms of both directional deviations and amplitude percentage overshoots or undershoots, occurred for the most foveal targets, and such distortions were still present, albeit with qualitatively different patterns, when subjects shifted their gaze to indicate the remembered target locations. Foveal visual images are severely distorted in short term memory.


title: Neural language processing across time, space, frequency and age: MEG-MVPA classification of intertrial phase coherence

abstract: Language is a key part of human cognition. Whereas many neurocognitive abilities decline with age, for language the picture is much less clear and how exactly language processing changes with aging is still unknown. To investigate this, we employed magnetoencephalography (MEG) and recorded neuromagnetic brain responses to auditory linguistic stimuli in healthy participants of younger and older age using a passive task-free paradigm and a range of different linguistic stimulus contrasts, which enabled us to assess neural language processes at multiple levels (lexical, semantic, morphosyntactic). By using machine learning-based classification algorithms to scrutinise intertrial phase coherence of MEG responses in source space, we found significant differences between younger and older participants across several frequency bands and for all tested processing types, which shows multiple changes in the brains neurolinguistic circuits which may be due to both healthy aging in general and compensatory processes in particular.


title: Microglia mediate synaptic plasticity induced by 10 Hz repetitive magnetic stimulation

abstract:
IntroductionRepetitive transcranial magnetic stimulation (rTMS) is a non-invasive brain stimulation technique that is widely used in clinical practice for therapeutic purposes. Nevertheless, the mechanisms that mediate its therapeutic effects remain poorly understood. Recent work implicates that microglia, the resident immune cells of the central nervous system, have a defined role in the regulation of physiological brain function, e.g. the expression of synaptic plasticity. Despite this observation, no evidence exists for a role of microglia in excitatory synaptic plasticity induced by rTMS.

MethodsHere, we used repetitive magnetic stimulation of organotypic entorhino-hippocampal tissue cultures to test for the role of microglia in synaptic plasticity induced by 10 Hz repetitive magnetic stimulation (rMS). For this purpose, we performed PLX3397 (Pexidartinib) treatment to deplete microglia from tissue culture preparations. Using whole-cell patch-clamp recordings, live-cell microscopy, immunohistochemistry and transcriptome analysis, we assessed structural and functional properties of both CA1 pyramidal neurons and microglia to correlate the microglia phenotype to synaptic plasticity.

ResultsPLX3397 treatment over 18 days reliably depletes microglia in tissue cultures, without affecting structural and functional properties of CA1 pyramidal neurons. Microglia-depleted cultures display defects in the ability of CA1 pyramidal neurons to express plasticity of excitatory synapses upon rMS. Notably, rMS induces a moderate release of proinflammatory and plasticity-promoting factors, while microglial morphology stays unaltered.

ConclusionWe conclude that microglia play a crucial role in rMS-induced excitatory synaptic plasticity.


title: Neural basis for adaptive motor behavior during car driving

abstract: Car driving is supported by motor skills trained through continuous daily practice. One of the skills unique to expert drivers is the ability to detect abrupt changes in the driving environment and then quickly adapt their operation mode to the changes. Previous functional neuroimaging studies on motor control investigated the mechanisms underlying behaviors adaptive to changes in control properties of simple experimental devices such as a computer mouse or a joystick. The switching of multiple internal models mainly engages adaptive behaviors and underlies the interplay between the cerebellum and frontoparietal network (FPN) regions as the neural process. However, it remains unclear whether the neural mechanisms identified through an experimental paradigm using such simple devices also underlie practical driving behaviors. In the current study, we measure functional magnetic resonance imaging (fMRI) activities while participants control a realistic driving simulator inside the MRI scanner. Here, the accelerator sensitivity of a virtual car is abruptly changed, requiring participants to respond to this change as quickly as possible. We first compare brain activities before and after the sensitivity change. As a result, sensorimotor areas, including the left cerebellum, increase their activities after the sensitivity change. Moreover, after the change, activity significantly increases in the inferior parietal lobe and dorsolateral prefrontal cortex, parts of the FPN regions. By contrast, the posterior cingulate cortex, a part of the default mode network, deactivates after the sensitivity change. Our results suggest that the neural bases found in previous experiments using the simpler devices can serve as the foundation of adaptive car driving. At the same time, this study also highlights the unique contribution of non-motor-related regions to addressing the high cognitive demands of driving.


title: Le Petit Prince: A multilingual fMRI corpus using ecological stimuli

abstract: Neuroimaging using more ecologically valid stimuli such as audiobooks has advanced our understanding of natural language comprehension in the brain. However, prior naturalistic stimuli have typically been restricted to a single language, which limited generalizability beyond small typological domains. Here we present the Le Petit Prince fMRI Corpus (LPPC-fMRI), a multilingual resource for research in the cognitive neuroscience of speech and language during naturalistic listening (Open-Neuro: ds003643). 49 English speakers, 35 Chinese speakers and 28 French speakers listened to the same audiobook The Little Prince in their native language while multi-echo functional magnetic resonance imaging was acquired. We also provide time-aligned speech annotation and word-by-word predictors obtained using natural language processing tools. The resulting timeseries data are shown to be of high quality with good temporal signal-to-noise ratio and high inter-subject correlation. Data-driven functional analyses provide further evidence of data quality. This annotated, multilingual fMRI dataset facilitates future re-analysis that addresses cross-linguistic commonalities and differences in the neural substrate of language processing on multiple perceptual and linguistic levels.


title: Natural influent bioaugmentation of activated sludge water resource recovery systems: implications for low temperature nitrification and heterotrophic population structures

abstract:
This work aimed at demonstrating the natural bioaugmentation of biological activated sludge systems with nitrifying biomass from influent wastewater in lab-scale reactors. Three sequencing batch reactors (SBR) were fed with sterile synthetic wastewater. While nitrification was complete at a temperature of 8 {degrees}C and a SRT of 20 days, it failed when the temperature was lowered to 5 {degrees}C, and the SRT decreased to 7 days. In the test period, the sterile synthetic wastewater fed to the Test Reactor was supplemented by influent solids harvested at a full-scale treatment facility at a total suspended solids concentration of 100 mg/L, which corresponded to approximately 5 mg-COD/L of nitrifying biomass. Upon this addition, nitrification was restores. Subsequent halting the supply of influent solids to the Test Reactor led a rapid failure of nitrification and washout of nitrifiers from the SBR. Reproducibility was demonstrated by switching the feed composition between the Test and Negative control reactors. PCR-based amplicon sequencing analyses targeting the amoA, and nxrB genes of the Nitrospira genus have shown that the influent wastewater governed the structure and composition of the activated sludge nitrifying populations. The most abundant ammonia-oxidizing bacteria (AOB) and Nitrospira-related nitrite-oxidizing bacteria (NOB) in the influent seeds occurred as the most dominant ones in the activated sludge. This pattern was observed even when the influent seeds varied over time. The heterotrophic populations were less affected by the influent seeds with the activated sludge and raw sewage showing distinct microbial populations based on principal coordinate analysis (PCoA). However, the immigrant populations appeared to modulate the structure of the activated sludge heterotrophic communities to some extent. These findings provide concrete evidence of the presence of active nitrifiers in raw wastewater capable of supporting nitrification in an otherwise non-conducive environment. This may have important implications on process design, operation and optimization of wastewater treatment systems.

HighlightsO_LILab-scale reactors fed sterile synthetic wastewater at low temperatures and SRTs.
C_LIO_LINitrification failed when conditions were adjusted to 5 {degrees}C and a SRT of 7 days.
C_LIO_LINitrification restored by addition of real wastewater influent solids.
C_LIO_LINitrifiers in solids from sewers naturally bioaugment activated sludge systems.
C_LIO_LIActivated sludge models should consider the immigration of nitrifiers with influent.
C_LI

Graphical abstract

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category: microbiology


title: Stepwise progression of β-selection during T cell development as revealed by histone deacetylation inhibition

abstract: During T cell development, the first step in creating a unique T Cell Receptor (TCR) is the genetic recombination of the TCR{beta} chain. The quality of the newly recombined TCR{beta} is assessed at the {beta}-selection checkpoint. Most cells fail this checkpoint and die, but the coordination of the complex events that control fate at the {beta}-selection checkpoint is not yet understood. We shed new light on fate determination during {beta}-selection using a selective inhibitor of histone deacetylase 6, ACY1215. ACY1215 disrupted the {beta}-selection checkpoint. Characterising the basis for this disruption revealed a new, pivotal stage in {beta}-selection, bookended by upregulation of the TCR co-receptors, CD28 and CD2 respectively. Within this  DN3bPre stage, CD5 and Lef1 are upregulated to reflect pre-TCR signalling and their expression correlates with proliferation. During this phase, ACY1215-mediated disruption of the organisation of the {beta}-selection immunological synapse is associated with a breakdown in connectivity of expression of pre-TCR, CD5 and Lef1. Subsequent deregulation of pre-TCR-induced proliferation leads to bypass of the {beta}-selection checkpoint and subsequent failure to progress. We propose that the progressive expression of CD28, CD5 and Lef1, then CD2 reports and modulates the pre-TCR signal to orchestrate passage through the {beta}-selection checkpoint. These findings suggest a refined model of {beta}-selection in which a coordinated increase in expression of pre-TCR, CD28, CD5 and Lef1 provides for escalating TCR signalling strength, and culminates in the expression of CD2 to enable exit from the {beta}-selection checkpoint.

category: developmental biology


title: Stepwise progression of beta-selection during T cell development as revealed by histone deacetylation inhibition


title: A critical assessment of the detailed Aedes aegypti simulation model Skeeter Buster 2 using field experiments of indoor insecticidal control in Iquitos, Peru

abstract:
The importance of mosquitoes in human pathogen transmission has motivated major research efforts into mosquito biology in pursuit of more effective vector control measures. Aedes aegypti is a particular concern in tropical urban areas, where it is the primary vector of numerous flaviviruses, including the yellow fever, Zika, and dengue viruses. With an anthropophilic habit, Ae. aegypti prefers houses, human blood meals, and ovipositioning in water-filled containers. We hypothesized that this relatively simple ecological niche should allow us to predict the impacts of insecticidal control measures on mosquito populations. To do this, we use Skeeter Buster 2 (SB2), a stochastic, spatially explicit, mechanistic model of Ae. aegypti population biology. SB2 builds on Skeeter Buster, which reproduced equilibrium dynamics of Ae. aegypti in Iquitos, Peru. Our goal was to predict the response of mosquito populations to perturbations by indoor insecticidal spraying and widespread destructive insect surveys.

To evaluate SB2, we conducted two field experiments in Iquitos, Peru: a smaller pilot study in 2013 (S-2013) followed by a larger experiment in 2014 (L-2014). Here, we compare model predictions with (pre-viously reported) empirical results from these experiments. In both simulated and empirical populations, repeated spraying yielded substantial yet temporary reductions in adult densities. The proportional effects of spraying were broadly comparable between simulated and empirical results, but we found noteworthy differences. In particular, SB2 consistently over-estimated the proportion of nulliparous females and the proportion of containers holding immature mosquitoes. We also observed less temporal variation in simulated surveys of adult abundance relative to corresponding empirical observations. Our results indicate the presence of ecological heterogeneities or sampling processes not effectively represented by SB2. Although additional empirical research could further improve the accuracy and precision of SB2, our results under-score the importance of non-linear dynamics in the response of Ae. aegypti populations to perturbations, and suggest general limits to the fine-grained predictability of its population dynamics over space and time.

category: ecology


title: iLoci: Robust evaluation of genome content and organization for provisional and mature genome assemblies

abstract:
BackgroundThe rate at which new draft genome assemblies and corresponding annotation versions are being produced has long outpaced the scientific communitys capacity to refine these drafts into "finished," reference-quality data resources to a standard typically expected from dedicated efforts of model organism research communities. Nonetheless, scientists must be able to evaluate newly sequenced genomes in the context of previously published data, requiring summaries of genome content and organization that can be quickly computed, updated, and meaningfully compared. As annotation quality will necessarily vary within and across data sets, the ability to select subsets of only those data that are well supported is critical for distinguishing technical artifacts from biological effects in genome-wide analyses.

ResultsWe introduce a new framework for genome analyses based on parsing an annotated genome assembly into distinct interval loci (iLoci), available as open source software as part of the AEGeAn Toolkit (https://github.com/BrendelGroup/AEGeAn). We demonstrate that iLoci provide an alternative coordinate system that is robust to changes in assembly and annotation versions and facilitates granular quality control of genome data. We discuss how statistics computed on iLoci reflect various characteristics of genome content and organization and illustrate how these statistics can be used to establish a baseline for assessment of the completeness and accuracy of the data. We also introduce a well-defined measure of relative genome compactness and compute other iLocus statistics that reveal genome-wide characteristics of gene arrangements in the whole genome context.

ConclusionsWe present a coherent computational framework that calculates informative statistics from genome assembly/annotation data input. Given the fast pace of assembly/annotation updates, our AEGeAn Toolkit fills a niche in computational genomics based on deriving persistent and species-specific genome statistics. Gene structure model centric iLoci provide a precisely defined coordinate system that can be used to store assembly/annotation updates that reflect either stable or changed assessments. Large-scale application of the approach revealed species and clade specific genome organization in precisely defined computational terms, promising intriguing forays into the forces of shaping genome structure as more and more genome assemblies are being deposited.

category: genomics


title: Monkey Plays Pac-Man with Compositional Strategies and Hierarchical Decision-making

abstract:
Humans can often handle daunting tasks with ease by developing a set of strategies to reduce decision making into simpler problems. The ability to use heuristic strategies demands an advanced level of intelligence and has not been demonstrated in animals. Here, we trained macaque monkeys to play the classic video game Pac-Man. The monkeys decision-making may be described with a strategy-based hierarchical decision-making model with over 90% accuracy. The model reveals that the monkeys adopted the take-the-best heuristic by using one dominating strategy for their decision-making at a time and formed compound strategies by assembling the basis strategies to handle particular game situations. With the model, the computationally complex but fully quantifiable Pac-Man behavior paradigm provides a new approach to understanding animals advanced cognition.

One-Sentence SummaryMacaque monkeys play Pac-Man with strategy-based hierarchical decision making, a cognitive capacity hitherto unknown in them.

category: animal behavior and cognition


title: Color is necessary for specialized face learning in the Northern paper wasp, Polistes fuscatus

abstract: Visual individual recognition requires animals to distinguish among conspecifics based on appearance. Though visual individual recognition has been reported in a range of taxa, the features that animals rely on to discriminate between individuals are often not well understood. Northern paper wasp females, Polistes fuscatus, possess individually distinctive color patterns on their faces, which mediate individual recognition. It is currently unclear what facial features P. fuscatus use to distinguish individuals. The anterior optic tubercle, a chromatic processing brain region, is especially sensitive to social experience during development, suggesting that color may be important for recognition in this species. We sought to test the roles of color in wasp facial recognition. Color may be important simply because it creates a pattern. If this is the case, then wasps should perform similarly when discriminating color or grayscale images of the same faces. Alternatively, color itself may be important for recognition, which would predict poorer performance on grayscale image discrimination relative to color images. We found wasps trained on grayscale faces, unlike those trained on color images, did not perform better than chance. Suggesting that color is necessary for the recognition of an image as a face by the wasp visual system.


title: Discovery of Aminoglycosides as first in class, nanomolar inhibitors of Heptosyltransferase I

abstract: A clinically relevant inhibitor for Heptosyltransferase I (HepI) has been sought after for many years and while many have designed novel small-molecule inhibitors, these compounds lack the bioavailability and potency necessary for therapeutic use. Extensive characterization of the HepI protein has provided valuable insight into the dynamic motions necessary for catalysis that could be targeted for inhibition. With the help of molecular dynamic simulations, aminoglycoside antibiotics were shown to be putative inhibitors for HepI and in this study, they were experimentally determined to be the first in-class nanomolar inhibitors of HepI with the best inhibitor demonstrating a Ki of 600 +/- 90 nM. Detailed kinetic analyses were performed to determine the mechanism of inhibition while circular dichroism spectroscopy, intrinsic tryptophan fluorescence, docking, and MD simulations were used to corroborate kinetic experimental findings. Kinetic analysis methods include Lineweaver-Burk, Dixon, Cornish-Bowden and Mixed-Model of Inhibition which allowed for unambiguous assignment of inhibition mechanism for each inhibitor. In this study, we show that neomycin and kanamycin b are competitive inhibitors against the sugar acceptor substrate while tobramycin exhibits a mixed inhibitory effect and streptomycin is non-competitive. MD simulations also allowed us to suggest that the inhibitors bind tightly and inhibit catalytic dynamics due to a major desolvation penalty of the enzyme active site. While aminoglycosides have long been known as a class of potent antibiotics, they also have been scientifically shown to impact cell membrane stability, and we propose that inhibition of HepI contributes to this effect by disrupting lipopolysaccharide biosynthesis.

category: biochemistry


title: Discovery of Aminoglycosides as First in Class, Nanomolar Inhibitors of Heptosyltransferase I


title: Lamina cribrosa vessel and collagen beam networks are distinct

abstract:
Our goal was to analyze the spatial interrelation between vascular and collagen networks in the lamina cribrosa (LC). Specifically, we quantified the percentages of collagen beams with/without vessels and of vessels inside/outside of collagen beams. To do this, the vasculature of six normal monkey eyes was labelled by perfusion post-mortem. After enucleation, coronal cryosections through the LC were imaged using fluorescence and polarized light microscopy to visualize the blood vessels and collagen beams, respectively. The images were registered to form 3D volumes. Beams and vessels were segmented, and their spatial interrelationship was quantified in 3D. We found that 22% of the beams contained a vessel (range 14% to 32%), and 21% of vessels were outside beams (13% to 36%). Stated differently, 78% of beams did not contain a vessel (68% to 86%), and 79% of vessels were inside a beam (64% to 87%). Individual monkeys differed significantly in the fraction of vessels outside beams (p<0.01 by linear mixed effect analysis), but not in the fraction of beams with vessels (p>0.05). There were no significant differences between contralateral eyes in the percent of beams with vessels and of vessels outside beams (p>0.05). Our results show that the vascular and collagenous networks of the LC in monkey are clearly distinct, and the historical notions that each LC beam contains a vessel and all vessels are within beams are inaccurate. We postulate that vessels outside beams may be relatively more vulnerable to mechanical compression by elevated IOP than are vessels shielded inside of beams.

Research highlights- We combined fluorescence and polarized light microscopy to map in 3D the lamina cribrosa vessels and collagen beams of three pairs of monkey eyes
- Collagen beam and vessel networks of the lamina cribrosa have distinct topologies
- Over half of lamina cribrosa collagen beams did not contain a blood vessel
- One fifth of blood vessels in the lamina cribrosa were outside collagen beams
- Beams with/without vessels and vessels inside/outside beams may respond differently to IOP


title: A novel method for producing functionalized vesicles that efficiently deliver oligonucleotides in vitro and in vivo in mice

abstract: Nano-based delivery systems have enhanced our ability to administer and target drugs and macromolecules to their targets. Oligonucleotide drugs have great therapeutic potential but often have off-target effects and stability issues. Therefore, they are often encapsulated in vesicles with targeting ligands such as antibodies (Ab) to deliver their cargo. Herein, we describe a novel, scalable and straightforward approach to producing functionalized vesicles called the "Functionalized Lipid Insertion Method," which differs from the older "Detergent-Dialysis Method." The latter method required excess detergent and extensive dialysis over many hours to produce the functionalized vesicles. With our method, only the functionalized lipid is detergent-solubilized during the engineering of the vesicle. The approach reduces the dialysis time, keeps the vesicles intact while orienting the targeting moieties of the functionalized lipid toward the outside of the vesicle. Pilot in vitro and in vivo experiments was performed to show the feasibility of our method. Dynamic light scattering (DLS) experiments suggested that the original vesicular structure was relatively unperturbed, and the functionalized lipid was inserted externally. Our approach efficiently delivered oligonucleotides and affected the function of liver cancer HepG2 cells. Furthermore, functionalized vesicles achieved targeted delivery of oligonucleotides in mice without inducing a significant innate immune response. The industrial and therapeutic significance and implications of functionalized vesicles produced by our method are also discussed. Additional experiments and analyses are recommended to bring out the full potential of this molecular delivery technology.


title: A novel method for producing functionalized vesicles that efficiently deliver oligonucleotides in vitro in cancer cells and in vivo in mice


title: Real-time imaging of optic nerve head collagen microstructure and biomechanics using instant polarized light microscopy

abstract:
Current tools lack the temporal or spatial resolution necessary to image many important aspects of the architecture and dynamics of the optic nerve head (ONH). We evaluated the potential of instant polarized light microscopy (IPOL) to overcome these limitations by leveraging the ability to capture collagen fiber orientation and density in a single image. Coronal sections through the ONH of fresh normal sheep eyes were imaged using IPOL while they were stretched using custom uniaxial or biaxial micro-stretch devices. IPOL allows identifying ONH collagen architectural details, such as fiber interweaving and crimp, and has high temporal resolution, limited only by the frame rate of the camera. Local collagen fiber orientations and deformations were quantified using color analysis and image tracking techniques. We quantified stretch-induced collagen uncrimping of lamina cribrosa (LC) and peripapillary sclera (PPS), and changes in LC pore size (area) and shape (convexity and aspect ratio). The simultaneous high spatial and temporal resolutions of IPOL revealed complex ONH biomechanics: i) stretch-induced local deformation of the PPS was nonlinear and nonaffine. ii) under load the crimped collagen fibers in the PPS and LC straightened, without torsion and with only small rotations. iii) stretch-induced LC pore deformation was anisotropic and heterogeneous among pores. Overall, with stretch the pores were became larger, more convex, and more circular. We have demonstrated that IPOL reveals details of collagen morphology and mechanics under dynamic loading previously out of reach. IPOL can detect stretch-induced collagen uncrimping and other elements of the tissue nonlinear mechanical behavior. IPOL showed changes in pore morphology and collagen architecture that will help improve understanding of how LC tissue responds to load.

HighlightsO_LIWe demonstrate that instant polarized light microscopy allows visualization and quantification of changes in optic nerve head collagen morphology and architecture under dynamic loading
C_LIO_LIWe show crimped collagen fibers in the peripapillary sclera and lamina cribrosa straightening under load, without torsion and with only small rotations.
C_LIO_LIWe show that stretch-induced local deformation of the peripapillary sclera was nonlinear and nonaffine.
C_LIO_LIWe show that stretch-induced lamina cribrosa pore deformation was anisotropic and heterogeneous among pores.
C_LIO_LIOur results show this novel imaging technique could help understand the role of collagen microstructure in eye physiology, aging, and in biomechanics-related diseases, such as glaucoma and myopia.
C_LI


title: High-speed live cell interferometry for screening bioprinted organoids

abstract: High-throughput drug screening is an established approach to investigate tumor biology and identify therapeutic leads. Traditional platforms for high-throughput screening use two-dimensional cultures of immortalized cell lines which do not accurately reflect the biology of human tumors. More clinically relevant model systems, such as three-dimensional tumor organoids, can be difficult to screen and scale. For example, manually seeded organoids coupled to destructive endpoint assays allow for the characterization of response to treatment, but do not capture the transitory changes and intra-sample heterogeneity underlying clinically observed resistance to therapy. We therefore developed a pipeline to generate bioprinted tumor organoids linked to label-free, real-time imaging via high-speed live cell interferometry (HSLCI) and machine learning-based quantitation of individual organoids. Bioprinting cells gives rise to 3D organoid structures that preserve tumor histology and gene expression. HSLCI imaging in tandem with machine learning-based image segmentation and organoid classification tools enables accurate, label-free parallel mass measurements for thousands of bioprinted organoids. We demonstrate that our method quantitatively identifies individual organoids as insensitive, transiently sensitive, or persistently sensitive to specific treatments. This opens new avenues for rapid, actionable therapeutic selection using automated tumor organoid screening.


title: Drug screening at single-organoid resolution via bioprinting and interferometry


title: LOCOM: A logistic regression model for testing differential abundance in compositional microbiome data with false discovery rate control

abstract:
MotivationCompositional analysis is based on the premise that a relatively small proportion of taxa are "differentially abundant", while the ratios of the relative abundances of the remaining taxa remain unchanged. Most existing methods of compositional analysis such as ANCOM or ANCOM-BC use log-transformed data, but log-transformation of data with pervasive zero counts is problematic, and these methods cannot always control the false discovery rate (FDR). Further, high-throughput microbiome data such as 16S amplicon or metagenomic sequencing are subject to experimental biases that are introduced in every step of the experimental workflow. McLaren, Willis and Callahan [1] have recently proposed a model for how these biases affect relative abundance data.

MethodsMotivated by [1], we show that the (log) odds ratios in a logistic regression comparing counts in two taxa are invariant to experimental biases. With this motivation, we propose LOCOM, a robust logistic regression approach to compositional analysis, that does not require pseudocounts. We use a Firth bias-corrected estimating function to account for sparse data. Inference is based on permutation to account for overdispersion and small sample sizes. Traits can be either binary or continuous, and adjustment for continuous and/or discrete confounding covariates is supported.

ResultsOur simulations indicate that LOCOM always preserved FDR and had much improved sensitivity over existing methods. In contrast, ANCOM often had inflated FDR; ANCOM-BC largely controlled FDR but still had modest inflation occasionally; ALDEx2 generally had low sensitivity. LOCOM and ANCOM were robust to experimental biases in every situation, while ANCOM-BC and ALDEx2 had elevated FDR when biases at causal and non-causal taxa were differentially distributed. The flexibility of our method for a variety of microbiome studies is illustrated by the analysis of data from two microbiome studies.

Availability and implementationOur R package LOCOM is available on GitHub at https://github.com/yijuanhu/LOCOM in formats appropriate for Macintosh or Windows.

category: bioinformatics


title: Quantifying Cellular Pluripotency and Pathway Robustness through Forman-Ricci Curvature

abstract: In stem cell biology, cellular pluripotency describes the capacity of a given cell to differentiate into multiple cell types. From a statistical physics perspective, entropy provides a statistical measure of randomness and has been demonstrated as a way to quantitate pluripotency when considering biological gene networks. Furthermore, recent theoretical work has established a relationship between Ricci curvature (a geometric measure of "flatness") and entropy (also related to robustness), which one can exploit to link the geometric quantity of curvature to the statistical quantity of entropy. Therefore, this study seeks to explore Ricci curvature in biological gene networks as a descriptor of pluripotency and robustness among gene pathways. Here, we investigate Forman-Ricci curvature, a combinatorial discretization of Ricci curvature, along with network entropy, to explore the relationship of the two quantities as they occur in gene networks. First, we demonstrate our approach on an experiment of stem cell gene expression data. As expected, we find Ricci curvature directly correlates with network entropy, suggesting Ricci curvature could serve as an indicator for cellular pluripotency much like entropy. Second, we measure Forman-Ricci curvature in a dataset of cancer and non-cancer cells from melanoma patients. We again find Ricci curvature is increased in the cancer state, reflecting increased pluripotency or "stemness". Further, we locally examine curvature on the gene level to identify several genes and gene pathways with known relevance to melanoma. In turn, we conclude Forman-Ricci curvature provides valuable biological information related to pluripotency and pathway functionality. In particular, the advantages of this geometric approach are promising for extension to higher-order topological structures in order to represent more complex features of biological systems.

category: systems biology


title: Slow oscillations provide the spatio-temporal framework for long-range neural communication during sleep

abstract: Slow oscillations (SOs, <1Hz) during non-rapid eye movement sleep are thought to reflect sleep homeostasis and support memory consolidation. Yet, the fundamental properties of SOs and their impact on neural network communication are not understood. We used effective connectivity to estimate causal information flow across the electrode manifold during SOs and found two peak of information flow in specific phases of the SO. We show causal communication during non-rapid eye movement sleep peaks during specific phases of the SO, but only across long distances. We confirmed this prediction by cluster analysis demonstrating greater flow in global, compared with local, SOs. Finally, we tested the functional significance of these results by examining which SO properties supported overnight episodic memory improvement, with the underlying assumption that memory consolidation would engage global, long-range communication. Indeed, episodic memory improvement was predicted only by the SO properties with greatest causal information flow, i.e., longest distances between sinks and sources and global, but not local, SOs. These findings explain how NREM sleep (characterized as a state of low brain connectivity) leverages SO-induced selective information flow to coordinate a wide network of brain regions during memory formation.


title: PAX3-FOXO1 coordinates enhancer architecture, eRNA transcription, andRNA polymerase pause release at select gene targets

abstract: Transcriptional control is a highly dynamic process that changes rapidly in response to various cellular and extracellular cues1. Thus, it is difficult to achieve a mechanistic understanding of transcription factor function using traditional genetic deletion or RNAi methods, because these slow approaches make it challenging to distinguish direct from indirect transcriptional effects. Here, we used a chemical-genetic approach to rapidly degrade a canonical transcriptional activator, PAX3-FOXO12-6 to define how the t(2;13)(q35;q14) disrupts normal gene expression programs to trigger cancer. By coupling rapid protein degradation with the analysis of nascent transcription over short time courses, we identified a core transcriptional network that rapidly collapsed upon PAX3-FOXO1 degradation. Moreover, loss of PAX3-FOXO1 impaired RNA polymerase pause release and transcription elongation at regulated gene targets. The activity of PAX3-FOXO1 at enhancers controlling this core network was surprisingly selective and often only a single element within a complex super-enhancer was affected. In addition, fusion of the endogenous PAX3-FOXO1 with APEX2 identified proteins in close proximity with PAX3-FOXO1, including ARID1A and MYOD1. We found that continued expression of PAX3-FOXO1 was required to maintain chromatin accessibility and allow neighboring DNA binding proteins and chromatin remodeling complexes to associate with this small number of regulated enhancers. Overall, this work provides a detailed mechanism by which PAX3-FOXO1 maintains an oncogenic transcriptional regulatory network.

category: molecular biology


title: Allometric scaling of RNA abundance from genes to communities

abstract: The metabolic theory of ecology (MTE) and growth rate hypothesis (GRH) help explain the mechanistic basis of size (allometry) and temperature dependence on growth rate and whole-body-RNA content in organisms. However, testing RNA allometric scaling with next-generation sequencing is yet to be done. Here, we validated the assumptions of GRH and MTE on messenger RNA and ribosome abundance using mock community metatranscriptome analysis. Our findings highlight that fast-growing smaller species harbor greater RNA abundance per mass of tissue compared with species having larger body sizes and slower growth rates, where allometric slopes for genomic and gene-level RNA abundance range from -[1/3] to -1. We found that genome size and body size impose significant constraints in interspecific RNA abundance scaling, while the assumed temperature dependence appeared to be weak. Lastly, allometric scaling integration in community-level models may extend the use of metatranscriptomics as a reliable tool for estimating ecosystem processes.


title: Extracellular release of mitochondrial DNA is triggered by cigarette smoke and is detected in COPD

abstract: Chronic obstructive pulmonary disease (COPD) is characterized by continuous and irreversible inflammation frequently caused by persistent exposure to toxic inhalants such as cigarette smoke (CS). CS may trigger mitochondrial DNA (mtDNA) extrusion into the cytosol, extracellular space, or foster its transfer by extracellular vesicles (EVs). The present study aimed to elucidate whether mtDNA is released upon CS exposure and in COPD. We measured cell-free mtDNA (cf-mtDNA) in the plasma of former smokers affected by COPD, in the serum of mice that developed CS-induced emphysema, and in the extracellular milieu of human bronchial epithelial cells exposed to cigarette smoke extract (CSE). Further, we characterized cells exposed to sublethal and lethal doses of CSE by measuring mitochondrial membrane potential and dynamics, superoxide production and oxidative stress, cell cycle progression, and cytokine expression. Patients with COPD and mice that developed emphysema showed increased levels of cf-mtDNA. In cell culture, exposure to a sublethal dose of CSE decreased mitochondrial membrane potential, increased superoxide production and oxidative damage, dysregulated mitochondrial dynamics, and triggered mtDNA release in extracellular vesicles. The release of mtDNA into the extracellular milieu occurred concomitantly with increased expression of DNase III, DNA-sensing receptors (cGAS, NLRP3), proinflammatory cytokines (IL-1{beta}, IL-6, IL-8, IL-18, CXCL2), and markers of senescence (p16, p21). Exposure to a lethal dose of CSE preferentially induced mtDNA and nuclear DNA release in cell debris. Our findings demonstrate that CS-induced stress triggers mtDNA release and is associated with COPD, supporting cf-mtDNA as a novel signaling response to CS exposure.

category: cell biology


title: Characterization and Structural Prediction of ORF10, ORF7b, ORF7a, ORF6, Membrane Glycoprotein, and Envelope Protein in SARS-CoV-2 Bangladeshi Variant through Bioinformatics Approach

abstract: The acute respiratory disease induced by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has become a global epidemic in just less than a year by the first half of 2020. The subsequent efficient human-to-human transmission of this virus eventually affected millions of people worldwide. The most devastating thing is that the infection rate is continuously uprising and resulting in significant mortality especially among the older age population and those with health co-morbidities. This enveloped, positive-sense RNA virus is chiefly responsible for the infection of the upper respiratory system. The virulence of the SARS-CoV-2 is mostly regulated by its proteins like entry to the host cell through fusion mechanism, fusion of infected cells with neighboring uninfected cells to spread the virus, inhibition of host gene expression, cellular differentiation, apoptosis, mitochondrial biogenesis, etc. But very little is known about the protein structures and functionalities. Therefore, the main purpose of this study is to learn more about these proteins through bioinformatics approaches. In this study, ORF10, ORF7b, ORF7a, ORF6, membrane glycoprotein, and envelope protein have been selected from a Bangladeshi Corona-virus strain G039392 and a number of bioinformatics tools (MEGA-X-V10.1.7, PONDR(R), ProtScale, ProtParam, SCRIBER, NetSurfP v2.0, IntFOLD, UCSF Chimera, and PyMol) and strategies were implemented for multiple sequence alignment and phylogeny analysis with 9 different variants, predicting hydropathicity, amino acid compositions, protein-binding propensity, protein disorders, 2D and 3D protein modeling. Selected proteins were characterized as highly flexible, structurally and electrostatically extremely stable, ordered, biologically active, hydrophobic, and closely related to the proteins of different variants. This detailed information regarding the characterization and structure of proteins of SARS-CoV-2 Bangladeshi variant was performed for the first time ever to unveil the deep mechanism behind the virulence features and also, this robust appraisal paves the future way for molecular docking, vaccine development targeting these characterized proteins.


title: Hierarchical Gaussian Processes and Mixtures of Experts to Model COVID-19 Patient Trajectories

abstract: Gaussian processes (GPs) are a versatile nonparametric model for nonlinear regression and have been widely used to study spatiotemporal phenomena. However, standard GPs offer limited interpretability and generalizability for datasets with naturally occurring hierarchies. With large-scale, rapidly-updating electronic health record (EHR) data, we want to study patient trajectories across diverse patient cohorts while preserving patient subgroup structure. In this work, we partition our cohort of over 2000 COVID-19 patients by sex and ethnicity. We develop and apply a hierarchical Gaussian process and a mixture of experts (MOE) hierarchical GP model to fit patient trajectories on clinical markers of disease progression. A case study for albumin, an effective predictor of COVID-19 patient outcomes, highlights the predictive performance of these models. These hierarchical spatiotemporal models of EHR data bring us a step closer toward our goal of building flexible approaches to capture patient data that can be used in real-time systems*.


title: Dynamic Allostery Highlights the Evolutionary Differences between the CoV-1 and CoV-2 Main Proteases

abstract:
The SARS-CoV-2 coronavirus has become one of the most immediate and widely-studied systems since its identification and subsequent global outbreak from 2019-2020. In an effort to understand the biophysical changes as a result of mutations, the mechanics of multiple different proteins within the SARS-CoV-2 virus have been studied and compared with SARS-CoV-1. Focusing on the main protease (mPro), we first explored the long range dynamic-relationship, particularly in cross-chain dynamics, using the Dynamic Coupling Index (DCI) to investigate the dynamic coupling between the catalytic site residues and the rest of the protein, both inter and intra chain for the CoV-1 and CoV-2 mPro. We found that there is significant cross-chain coupling between these active sites and distal residues in the CoV-2 mPro but it was missing in CoV-1. The enhanced long distance interactions, particularly between the two chains, suggest subsequently enhanced cooperativity for CoV-2. A further comparative analysis of the dynamic flexibility using the Dynamic Flexibility Index (DFI) between the CoV-1 and CoV-2 mPros shows that the inhibitor binding near active sites induces change in flexibility to a distal region of the protein, opposite in behavior between the two systems; this region becomes more flexible upon inhibitor binding in CoV-1 while it becomes less flexible in the CoV-2 mPro. Upon inspection, we show that, on average, the dynamic flexibility of the sites substituted from CoV-1 to CoV-2 changes significantly less than the average calculated across all residues within the structure, indicating that the differences in behaviors between the two systems is likely the result of allosteric influence, where the new substitutions in COV-2 induce flexibility and dynamical changes elsewhere in the structure.

SIGNIFICANCEHere we have conducted a comparative analysis between the SARS-CoV-1 and SARS-CoV-2 mPro systems to shed mechanistic insight on the biophysical changes associated with the mutations between these two enzymes. Our work shows that the CoV-2 mPro system exhibits enhanced cross-chain communication between catalytic site residues and the rest of the structure. Further, both dynamic coupling and dynamic flexibility analyses indicates that, largely, the dynamic changes as evaluated by DCI and DFI occur at sites other than the mutation sites themselves, indicating that the functional differences between these two proteins are a result of dynamic allostery

category: biophysics


title: Olfactory-driven beta band entrainment of limbic circuitry during neonatal development

abstract: Cognitive processing relies on the functional refinement of the limbic circuitry during the first two weeks of life. During this developmental period, when most sensory systems are still immature, the sense of olfaction acts as "door to the world", providing the main source of environmental inputs. However, it is unknown whether early olfactory processing shapes the activity in the limbic circuitry during neonatal development. Here, we address this question by combining simultaneous in vivo recordings from the olfactory bulb (OB), lateral entorhinal cortex (LEC), hippocampus (HP), and prefrontal cortex (PFC) with opto- and chemogenetic manipulations of mitral/tufted cells (M/TCs) in the OB of non-anesthetized neonatal mice. We show that the neonatal OB synchronizes the limbic circuity in beta frequency range. Moreover, it drives neuronal and network activity in LEC, as well as subsequently, HP and PFC via long-range projections from mitral cells (MCs) to HP-projecting LEC neurons. Thus, OB activity shapes the communications within limbic circuits during neonatal development.


title: TREM2-H157Y Increases Soluble TREM2 Production and Reduces Amyloid Pathology

abstract: The p.H157Y variant of TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) has been reported to increase Alzheimers disease (AD) risk. This mutation in the extracellular domain of TREM2 localizes at the cleavage site, leading to enhanced shedding. Here, we generated a novel Trem2 H157Y knock-in mouse model to investigate how this H157Y mutation impacts TREM2 proteolytic processing, synaptic function, and AD-related amyloid pathology. Consistent with previous in vitro findings, TREM2-H157Y increases the amount of soluble TREM2 (sTREM2) in the cortex and serum of mutant mice compared to the wild type controls. Interestingly, the Trem2 H157Y variant enhances synaptic plasticity without affecting microglial density and morphology. In the presence of amyloid pathology, TREM2-H157Y surprisingly accelerates A{beta} clearance and reduces amyloid burden and microgliosis. Taken together, our findings support a beneficial effect of the Trem2 H157Y mutation in synaptic function and in mitigating amyloid pathology. Considering the genetic association of TREM2 p.H157Y with AD, we speculate TREM2-H157Y might increase AD risk through an amyloid-independent pathway, as such its effects on tauopathy and neurodegeneration merit further investigation.


title: Structure selected RBM immunogens prime polyclonal memory responses that neutralize SARS-CoV-2 variants of concern

abstract: Successful control of the COVID-19 pandemic depends on vaccines that prevent transmission. The full-length Spike protein is highly immunogenic but the majority of antibodies do not target the virus: ACE2 interface. In an effort to concentrate the antibody response to the receptor-binding motif (RBM) we generated a series of conformationally-constrained immunogens by inserting solvent-exposed RBM amino acid residues into hypervariable loops of an immunoglobulin molecule. Priming C57BL/6 mice with plasmid (p)DNA encoding these constructs yielded a rapid memory response to booster immunization with recombinant Spike protein. Immune sera antibodies bound strongly to the purified receptor-binding domain (RBD) and Spike proteins. pDNA primed for a consistent response with antibodies efficient at neutralizing authentic WA1 virus and two variants of concern (VOC), B.1.351 and B.1.617.2. These findings demonstrate that immunogens built on structure selection can focus the response to conserved sites of vulnerability shared between wildtype virus and VOCs and induce neutralizing antibodies across variants.

category: immunology


title: In Vitro Activity of Cysteamine Against SARS-CoV-2 Variants Alpha, Beta, Gamma and Delta

abstract: Global COVID-19 pandemic is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Continuous emergence of new variants and their rapid spread are jeopardizing vaccine countermeasures to a significant extent. While currently available vaccines are effective at preventing illness associated with SARS-CoV-2 infection, these have been shown to be less effective at preventing breakthrough infection and transmission from a vaccinated individual to others. Here we demonstrate broad antiviral activity of cysteamine HCl in vitro against major emergent infectious variants of SARS-CoV-2 in a highly permissible Vero cell line. Cysteamine HCl inhibited infection of wild type, alpha, beta, gamma, delta, lambda, and omicron variants effectively. Cysteamine is a very well-tolerated US FDA-approved drug used chronically as a topical ophthalmic solution to treat ocular cystinosis in patients who receive it hourly or QID lifelong at concentrations 6 to 10 times higher than that required to completely inhibit SARS CoV-2 in tissue culture. Application of cysteamine as a topical nasal treatment can potentially1) mitigate existing infection 2) prevent infection in exposed individuals, and 3) limit the contagion in vulnerable populations.


title: In Vitro Activity of Cysteamine Against SARS-CoV-2 Variants


title: Dalpiciclib and Pyrotinib Exert Synergistic Antitumor Effects in Triple Positive Breast Cancer

abstract:
BackgroundThe therapeutic benefit of the standard combination of anti-HER2 and chemotherapy in triple-positive breast cancer (TPBC) is limited even after the addition of endocrine therapy to the regimen. Therefore, treatment optimization is required urgently.

MethodsThrough the drug sensitivity test, the drug combination efficacy of anti-HER2 drug, endocrine drug and CDK4/6 inhibitor to BT474 cells were tested. The underlying molecular mechanisms were investigated using immunofluorescence, western blot analysis, immunohistochemical staining and cell cycle analysis. Potential biomarker which may indicate the responsiveness to drug treatment in triple positive breast cancer was selected out using RNA-sequence and tested using immunohistochemical staining.

ResultsWe found that pyrotinib combined with dalpiciclib showed better efficacy than pyrotinib combined with tamoxifen in BT474 cells. Degradation of HER2 could enhance ER nuclear transportation, whereas cell cycle blockers could reverse this process. This may be the underlying mechanism by which the addition of dalpiciclib was more beneficial than the addition of pyrotinib plus tamoxifen. Furthermore, CALML5 was revealed to be a potential indicator of responsiveness to anti-HER2 therapy plus CDK4/6 inhibition in triple positive breast cancer.

ConclusionOur study provided evidence for the introduction of CDK4/6 inhibitor in the treatment of TPBC and indicated that the combination of anti-HER2 therapy and cell cycle blockers may be a better strategy for TPBC treatment.

FundingThis study was supported by the National Natural Science Foundation of China (#U20A20381, #81872159)


title: Protein complex prediction with AlphaFold-Multimer

abstract: While the vast majority of well-structured single protein chains can now be predicted to high accuracy due to the recent AlphaFold [1] model, the prediction of multi-chain protein complexes remains a challenge in many cases. In this work, we demonstrate that an AlphaFold model trained specifically for multimeric inputs of known stoichiometry, which we call AlphaFold-Multimer, significantly increases accuracy of predicted multimeric interfaces over input-adapted single-chain AlphaFold while maintaining high intra-chain accuracy. On a benchmark dataset of 17 heterodimer proteins without templates (introduced in [2]) we achieve at least medium accuracy (DockQ [3] [≥] 0.49) on 13 targets and high accuracy (DockQ [≥] 0.8) on 7 targets, compared to 9 targets of at least medium accuracy and 4 of high accuracy for the previous state of the art system (an AlphaFold-based system from [2]). We also predict structures for a large dataset of 4,446 recent protein complexes, from which we score all non-redundant interfaces with low template identity. For heteromeric interfaces we successfully predict the interface (DockQ [≥] 0.23) in 70% of cases, and produce high accuracy predictions (DockQ [≥] 0.8) in 26% of cases, an improvement of +27 and +14 percentage points over the flexible linker modification of AlphaFold [4] respectively. For homomeric inter-faces we successfully predict the interface in 72% of cases, and produce high accuracy predictions in 36% of cases, an improvement of +8 and +7 percentage points respectively.


title: A particle-based computational model to analyse remodelling of the red blood cell cytoskeleton during malaria infections

abstract:
Red blood cells can withstand the harsh mechanical conditions in the vasculature only because the bending rigidity of their plasma membrane is complemented by the shear elasticity of the underlying spectrin-actin network. During an infection by the malaria parasite Plasmodium falciparum, the parasite mines host actin from the junctional complexes and establishes a system of adhesive knobs, whose main structural component is the knob-associated histidine rich protein (KAHRP) secreted by the parasite. Here we aim at a mechanistic understanding of this dramatic transformation process. We have developed a particle-based computational model for the cytoskeleton of red blood cells and simulated it with Brownian dynamics to predict the mechanical changes resulting from actin mining and KAHRP-clustering. Our simulations include the three-dimensional conformations of the semi-flexible spectrin chains, the capping of the actin protofilaments and several established binding sites for KAHRP. For the healthy red blood cell, we find that incorporation of actin protofilaments leads to two regimes in the shear response. Actin mining decreases the shear modulus, but knob formation increases it. We show that dynamical changes in KAHRP binding affinities can explain the experimentally observed relocalization of KAHRP from ankyrin to actin complexes and demonstrate good qualitative agreement with experiments by measuring pair cross-correlations both in the computer simulations and in super-resolution imaging experiments.

Author summaryMalaria is one of the deadliest infectious diseases and its symptoms are related to the blood stage, when the parasite multiplies within red blood cells. In order to avoid clearance by the spleen, the parasite produces specific factors like the adhesion receptor PfEMP1 and the multifunctional protein KAHRP that lead to the formation of adhesive knobs on the surface of the red blood cells and thus increase residence time in the vasculature. We have developed a computational model for the parasite-induced remodelling of the actin-spectrin network to quantitatively predict the dynamical changes in the mechanical properties of the infected red blood cells and the spatial distribution of the different protein components of the membrane skeleton. Our simulations show that KAHRP can relocate to actin junctions due to dynamical changes in binding affinities, in good qualitative agreement with super-resolution imaging experiments. In the future, our simulation framework can be used to gain further mechanistic insight into the way malaria parasites attack the red blood cell cytoskeleton.


title: Slow TCA flux implies low ATP production in tumors

abstract: The tricarboxylic acid (TCA) cycle oxidizes carbon substrates to carbon dioxide, with the resulting high energy electrons fed into the electron transport chain to produce ATP by oxidative phosphorylation. Healthy tissues derive most of their ATP from oxidative metabolism, and the remainder from glycolysis. The corresponding balance in tumors remains unclear. Tumors upregulate aerobic glycolysis (the Warburg effect), yet they also typically require an intact TCA cycle and electron transport chain1-6. Recent studies have measured which nutrients contribute carbon to the tumor TCA metabolites7,8, but not tumor TCA flux: how fast the cycle turns. Here, we develop and validate an in vivo dynamic isotope tracing-mass spectrometry strategy for TCA flux quantitation, which we apply to all major mouse organs and to five tumor models. We show that, compared to the tissue of origin, tumor TCA flux is markedly suppressed. Complementary glycolytic flux measurements confirm tumor glycolysis acceleration, but the majority of tumor ATP is nevertheless made aerobically, and total tumor ATP production is suppressed compared to healthy tissues. In murine pancreatic cancer, this is accommodated by downregulation of the major energy-using pathway in the healthy exocrine pancreas, protein synthesis. Thus, instead of being hypermetabolic as commonly assumed, tumors apparently make ATP at a lower than normal rate. We propose that, as cells de-differentiate into cancer, they eschew ATP-intensive processes characteristic of the host tissue, and that the resulting suppressed ATP demand contributes to the Warburg effect and facilitates cancer growth in the nutrient-poor tumor microenvironment.

category: cancer biology


title: Unique Peptide Signatures Of SARS-CoV-2 Against Human Proteome Reveal Variants' Immune Escape And Infectiveness

abstract: SARS-CoV-2 pandemic has emerged the necessity of the identification of sequences sites in viral proteome appropriate as antigenic sites and treatment targets. In the present study, we apply a novel approach for deciphering the virus-host organism interaction, by analyzing the Unique Peptides of the virus with a minimum amino acid sequence length defined as Core Unique Peptides (CrUPs) not of the virus per se, but against the entire proteome of the host organism. The result of this approach is the identification of the CrUPs of the virus itself, which do not appear in the host organism proteome. Thereby, we analyzed the SARS-CoV-2 proteome for identification of CrUPs against the Human Proteome and they are defined as C/H-CrUPs. We found that SARS-CoV-2 include 7.503 C/H-CrUPs, with the SPIKE_SARS2 being the protein with the highest density of C/H-CrUPs. Extensive analysis indicated that the P681R mutation produces new C/H-CrUPs around the R685 cleavage site, while the L452R mutation induces the loss of antigenicity of the NF9 peptide and the strong(er) binding of the virus to ACE2 receptor protein. The simultaneous existence of these mutations in variants like Delta results in the immune escape of the virus, its massive entrance into the host cell, a notable increase in virus formation, and its massive release and thus elevated infectivity.


title: REV7/FANCV Binds to CHAMP1 and Promotes Homologous Recombination Repair

abstract: A critical determinant of DNA repair pathway choice is the HORMA protein REV7, a small abundant adaptor which binds to various DNA repair proteins through its C-terminal seatbelt domain. The REV7 seatbelt binds to the REV3 polymerase to form the Polymerase {zeta} complex, a positive regulator of translesion synthesis (TLS) repair. Alternatively, the REV7 seatbelt binds to SHLD3 in the Shieldin complex, a positive regulator of NHEJ repair. Recent studies have identified another novel REV7 seatbelt-binding protein, CHAMP1 (Chromosome Alignment-Maintaining Phosphoprotein, though its role in DNA repair is unknown. Here, we show that the REV7-CHAMP1 complex promotes homologous recombination (HR) repair by sequestering REV7 from the Shieldin complex. CHAMP1 competes directly with the SHLD3 subunit of the Shieldin complex for a limited pool of C-REV7, thereby inhibiting the REV7-mediated recruitment of the SHLD2 and SHLD1 effector subunits to DNA double strand breaks. CHAMP1 thereby channels DNA repair away from error-prone NHEJ and towards the competing error-free HR pathway. Similarly, CHAMP1 competes with the REV3 component of the POL{zeta} complex, thereby reducing the level of mutagenic TLS repair. CHAMP1 interacts with POGZ in a heterochromatin complex further promoting HR repair. Importantly, in human tumors, CHAMP1 overexpression promotes HR, confers PARP inhibitor resistance, and correlates with poor prognosis. Thus, by binding to either REV3, SHLD3, or CHAMP1 through its seatbelt, the REV7 protein can promote either TLS repair, NHEJ repair, or HR repair respectively.


title: Development of coarse-grained model for a minimal stratum corneum lipid mixture

abstract: Molecular dynamics simulations of mixtures of the ceramide N-(tetracosanoyl)-sphingosine (NS), cholesterol, and a free fatty acid are performed to gain a molecular-level understanding of the structure of the lipids found in the stratum corneum layer of skin. A new coarse-grained model for cholesterol, developed using the multistate iterative Boltzmann inversion method, is compatible with previously developed coarse-grained forcefields for ceramide NS, free fatty acid, and water, and validated against atomistic simulations of these lipids using the CHARMM force field. Self-assembly simulations of multilayer structures using these coarse-grained force fields are performed, revealing that a large fraction of the ceramides adopt extended conformations, which cannot occur in the bilayer structures typically studied using simulation. Cholesterol fluidizes the membrane by promoting packing defects and it is observed that an increase in cholesterol content reduces the bilayer height, due to an increase in interdigitation of the C24 lipid tails, consistent with experimental observations. Through the use of a simple reverse-mapping procedure, a self-assembled coarse-grained multilayer system is used to construct an equivalent structure with atomistic resolution. Simulations of this atomistic structure are found to closely agree with experimentally derived neutron scattering length density profiles. Significant interlayer hydrogen bonding is observed in the inner layers of the atomistic multilayer structure that are not found in the outer layers in contact with water or in equivalent bilayer structures. These results identify several significant differences in the structure and hydrogen bonding of multilayer structures as compared to the more commonly studied bilayer systems, and, as such, highlight the importance of simulating multilayer structures for more accurate comparisons with experiment. These results also provide validation of the efficacy of the coarse-grained forcefields and the framework for multiscale simulation.


title: The zinc-binding motif in tankyrases is required for the structural integrity and proper function of the catalytic domain

abstract: Tankyrases are ADP-ribosylating enzymes that regulate many physiological processes in the cell and they are therefore possible drug targets for cancer and fibrotic diseases. The catalytic ADP-ribosyl-transferase domain of tankyrases contains a unique zinc-binding motif of unknown function. Recently, this motif was suggested to be involved in the catalytic activity of tankyrases. In this work, we set out to study the effect of the zinc-binding motif on activity, stability and structure of human tankyrases. We generated mutants of human TNKS1 and TNKS2 abolishing the zinc-binding capabilities and characterized the proteins biochemically and biophysically in vitro. We further generated a crystal structure of TNKS2, in which the zinc ion was oxidatively removed. Our work shows that the zinc-binding motif in tankyrases is a crucial structural element which is particularly important for the structural integrity of the acceptor site. While mutation of the motif rendered TNKS1 inactive likely due to introduction of major structural defects, the TNKS2 mutant remained active and displayed a different activity profile compared to the wild type.


title: Directional proton conductance in bacteriorhodopsin is driven by concentration gradient, not affinity gradient

abstract: Many microorganisms can harvest energy from sun light to establish electrochemical potential across cell membrane by pumping protons outward. Light driven proton pumping against a transmembrane gradient entails exquisite electronic and conformational reconfigurations at fs to ms time scales. However, transient molecular events along the photocycle of bacteriorhodopsin are difficult to comprehend from noisy electron density maps obtained from multiple experiments. A major challenge arises from the coexisting intermediate populations as a heterogenous conformational mixture continuously evolves over 13 decades in time. This study reports a meta-analysis of the recent time-resolved datasets collected by several consortia. By resolving structural heterogeneity, this in-depth analysis substantially improves the quality of the electron density maps, and provides a clear visualization of the isolated intermediates from I to M. The earliest photoproducts revealed by the deconvoluted maps suggest that a proton transfer uphill against 15 pH units is accomplished by the same physics governing the tablecloth trick. While the Schiff base is displaced at the beginning of the photoisomerization within [~]30 fs, the proton stays due to its inertia. This affinity-independent early deprotonation builds up a steep proton concentration gradient that subsequently drives the directional proton conductance toward the extracellular medium. This mechanism fundamentally deviates from the widely adopted notion on multiple steps of chemical equilibrium driven by light-induced changes of proton affinities. The method of a numerical resolution of concurrent events from mixed observations is also generally applicable.


title: Ferroptosis assassinates tumor (FAST)

abstract: In 2020, nearly 20 million peoples got cancer and nearly 10 million peoples died of cancer, indicating the current therapies do not meet the cancer treatment and cancer remains a great threat to human health and life. New therapies are still in urgent demand. In a recent study, we developed a new effective cancer therapy, gene-interfered ferroptosis therapy (GIFT), by combining cancer cell-specific knockdown of two iron efflux genes (FPN and LCN2) with iron nanoparticles (FeNPs). GIFT shows wide antitumor activity, high cancer specificity, certain cancer eradication potential, and biosafety. To further improve the therapy, we here develop an updated GIFT named as Ferroptosis ASsassinates Tumor (FAST) by knocking down five additional ferroptosis-resistance genes (FSP1, FTH1, GPX4, SLC7A11, NRF2). As a result, we found that FAST showed more significant antitumor activity than GIFT. Especially, FAST eradicated three different types of tumors (leukemia, colon cancer and lung metastatic melanoma) from over 50 percent of cancer mice, making the mice to survive up to 250 days without tumor relapse. FAST also significantly inhibited and prevented growth of spontaneous breast cancer and improved survival in mice. Additionally, FAST showed high pan-antitumor efficacy, high cancer specificity, and in vivo safety.


title: Highly Reversible Tunable Thermal-repressible Split-T7 RNA polymerases (Thermal-T7RNAPs) for dynamic gene regulation

abstract: Temperature is a physical cue that is easy to apply, allowing cellular behaviors to be controlled in a contactless and dynamic manner via heat-inducible/repressible systems. However, existing heat-repressible systems are limited and rely on thermal sensitive mRNA or transcription factors which function at low temperatures, lack tunability, suffer delays or overly-complex. To provide an alternative mode of thermal regulation, we developed a library of compact, reversible and tunable thermal-repressible split-T7 RNA polymerase systems (Thermal-T7RNAPs) which fuses temperature-sensitive domains of Tlpa protein with split-T7RNAP to enable direct thermal control of the T7RNAP activity between 30 - 42 {degrees}C. We generated a large mutant library with varying thermal performances via automated screening framework to extend temperature tunability. Lastly, using the mutants, novel thermal logic circuitry was implemented to regulate cell growth and achieve active thermal control of the cell proportions within co-cultures. Overall, this technology expands avenues for thermal control in biotechnology applications.

category: synthetic biology


title: Functional interaction between Drosophila olfactory sensory neurons and their support cells

abstract: Insects detect volatile chemicals using antennae, which house a vast variety of olfactory sensory neurons (OSNs) that innervate hair-like structures called sensilla where odor detection takes place. In addition to OSNs, the antenna also hosts various support cell types. These include the triad of trichogen, tormogen and thecogen support cells that lie adjacent to their respective OSNs. The arrangement of OSN supporting cells occurs stereotypically for all sensilla and is widely conserved in evolution. While insect chemosensory neurons have received considerable attention, little is known about the functional significance of the cells that support them. For instance, it remains unknown whether support cells play an active role in odor detection, or only passively contribute to homeostasis, e.g. by maintaining sensillum lymph composition. To investigate the functional interaction between OSNs and support cells, we used optical and electrophysiological approaches in Drosophila. First, we characterized the distribution of various supporting cells using genetic markers. By means of an ex vivo antennal preparation and genetically-encoded Ca2+ and K+ indicators, we then studied the activation of these auxiliary cells during odor presentation in adult flies. We observed acute responses and distinct differences in Ca2+ and K+ fluxes between support cell types. Finally, we observed alterations in OSN responses upon thecogen cell ablation in mature adults. Upon inducible ablation of thecogen cells, we notice a gain in mechanical responsiveness to mechanical stimulations during single-sensillum recording, but a lack of change to neuronal resting activity. Taken together, these results demonstrate that support cells play a more active and responsive role during odor processing than previously thought. Our observations thus reveal that support cells functionally interact with OSNs and may be important for the extraordinary ability of insect olfactory systems to dynamically and sensitively discriminate between odors in the turbulent sensory landscape of insect flight.


title: Targeting ON-bipolar cells by AAV gene therapy stably reverses LRIT3-congenital stationary night blindness

abstract:
AAV gene therapies aimed at curing inherited retinal diseases to date have typically focused on photoreceptors and retinal pigmented epithelia within the relatively accessible outer retina. However, therapeutic targeting in diseases such as congenital stationary night blindness (CSNB) that involve defects in ON-bipolar cells (ON-BCs) within the mid-retina has been challenged by the relative inaccessibility of the target cell in intact retinas, the limited transduction efficiency of these cells by existing AAV serotypes, poor availability of established ON-BC-specific promoters, and absence of appropriate patient-relevant large animal models. Here, we demonstrate safe and effective ON-BC targeting by AAV gene therapy in a recently characterized naturally-occurring canine model of CSNB, LRIT3-CSNB. To effectively target ON-BCs, new AAV capsid variants with ON-BC tropism and ON-BC specific modified GRM6 promoters were adopted to ensure cell-specific transgene expression. Notably, subretinal injection of one vector, AAVK9#4-shGRM6-cLRIT3-WPRE, significantly recovered rod-derived b-wave in all treated eyes (6/6) of adult dogs injected at 1-3 years of age. The robust therapeutic effect was evident 7 weeks post-injection and was sustained for at least 1 year in all treated eyes. Scotopic vision was significantly improved in treated eyes based on visually-guided obstacle course navigation. Restoration of LRIT3 signals was confirmed by immunohistochemistry. Thus, we report on the first ON-BC functional rescue in a large animal model using a novel AAV capsid variant and modified promoter construct optimized for ON-BC specificity, thereby establishing both proof-of-concept and a novel translational platform for treatment of CSNB in patients with defects in photoreceptor-to-bipolar signaling.

SignificanceCanine models of inherited retinal diseases have informed and advanced AAV gene therapies targeting specific cells, including photoreceptors in the outer retina, for treatment of blinding diseases in human patients. However, therapeutic targeting of diseases such as congenital stationary night blindness (CSNB) that exhibit defects in ON-bipolar cells (ON-BCs) of the mid-retina remains under-developed. Using a new LRIT3 mutant canine model of CSNB exhibiting ON-BC dysfunction, we tested the ability of a cell-specific AAV capsid and promotor to specifically target ON-BCs for gene delivery. Notably, subretinal injection of AAV-LRIT3 vector demonstrated safety and efficacy with robust and stable rescue of ERG signals and night vision up to 1 year, paving the way for clinical trials in CNSB patients.


title: Nanoengineered DNA origami with repurposed TOP1 inhibitors targeting myeloid cells for the mitigation of neuroinflammation

abstract: Targeting myeloid cells, especially microglia, for the treatment of neuroinflammatory diseases such as multiple sclerosis (MS), is underappreciated. Here, we screened a library of compounds and identified the topoisomerase 1 (TOP1) inhibitor camptothecin (CPT) as a promising drug candidate for microglial modulation. CPT and its FDA-approved analog topotecan (TPT) inhibited inflammatory responses in microglia and macrophages, and ameliorated neuroinflammation in mice. Transcriptomic analysis of sorted microglia revealed an altered transcriptional phenotype following TPT treatment, with Ikzf1 identified as a potential target. Importantly, TOP1 expression was found elevated in several neuroinflammatory conditions, including human MS brains. To achieve targeted delivery to myeloid cells we designed a nanosystem using DNA origami and loaded TPT into it (TopoGami). TopoGami also significantly suppressed the inflammatory response in microglia and mitigated disease progression in MS-like mice. Our findings suggest that TOP1 inhibition represents a therapeutic strategy for neuroinflammatory diseases, and the proposed nanosystem may foster future research and drug development with a demand to target myeloid cells.


title: Gut Microbiome Signatures Linked to HIV-1 Reservoir Size and Viremia Control

abstract:
BackgroundThe potential role of the gut microbiome as a predictor of immune-mediated HIV-1 control in the absence of antiretroviral therapy (ART) is still unknown. In the BCN02 clinical trial, which combined the MVA.HIVconsv immunogen with the latency-reversing agent romidepsin in early-ART treated HIV-1 infected individuals, 23% (3/13) of participants showed sustained low-levels of plasma viremia during 32 weeks of a monitored ART pause (MAP). Here, we present a multi-omics analysis to identify compositional and functional gut microbiome patterns associated with HIV-1 control in the BCN02 trial.

ResultsViremic controllers during the MAP (controllers) exhibited higher Bacteroidales/Clostridiales ratio and lower microbial gene richness before vaccination and throughout the study intervention when compared to non-controllers. Longitudinal assessment indicated that the gut microbiome of controllers was enriched in pro-inflammatory bacteria and depleted in butyrate-producing bacteria and methanogenic archaea. Functional profiling also showed that metabolic pathways, including methanogenesis and carbohydrate biosynthesis, were significantly decreased in controllers. Fecal metaproteome analyses confirmed that baseline functional differences were mainly driven by Clostridiales. Participants with high baseline Bacteroidales/Clostridiales ratio had increased pre-existing immune activation-related transcripts. The Bacteroidales/Clostridiales ratio as well as host immune-activation signatures inversely correlated with HIV-1 reservoir size.

ConclusionsThis proof-of-concept study suggests the Bacteroidales/Clostridiales ratio as a novel gut microbiome signature associated with HIV-1 reservoir size and immune-mediated viral control after ART interruption.


title: Differential timing for glucose assimilation in Prochlorococcus and coexistent microbial populations at the North Pacific Subtropical Gyre

abstract:
The marine cyanobacterium Prochlorococcus can utilize glucose as a source of carbon. However, the relative importance of inorganic and organic carbon assimilation and the timing of glucose assimilation are still poorly understood in these numerically dominant cyanobacteria. Here we investigated whole microbial community and group-specific primary production and glucose assimilation, using incubations with radioisotopes combined with flow cytometry cell sorting. We also studied changes in the microbial community structure in response to glucose enrichments and analyzed the transcription of Prochlorocccus genes involved in carbon metabolism and photosynthesis.

Our results showed a circadian rhythm for glucose assimilation in Prochlorococcus, with maximum assimilation during the midday and minimum at midnight, which was different compared with that of the total microbial community. This suggests that rhythms in glucose assimilation have been adapted in Prochlorococcus to couple the active transport to photosynthetic light reactions producing energy, and possibly to avoid competition from the rest of the microbial community. High-light Prochlorococcus strains showed most transcriptional changes upon glucose enrichment. Pathways involved in glucose metabolism as the pentose phosphate, the Entner-Dudoroff, glycolysis, respiration and glucose transport showed an increase in the transcript level. A few genes of the low-light strains showed opposite changes, suggesting that glucose assimilation has been subjected to diversification along the Prochlorococcus evolution.


title: Emergence of rare carbapenemases (FRI, GES-5, IMI, SFC, and SFH-1) in Enterobacterales isolated from surface waters in Japan

abstract:
ObjectivesCarbapenemase-producing Enterobacterales (CPE) pose serious threats to public health. Compared with clinical CPE, the genetic characteristics of environmental CPE are not well understood. This study aimed to characterize the genetic determinants of carbapenem resistance in CPE isolated from environmental waters in Japan.

MethodsEighty-five water samples were collected from rivers and a lake in Japan. CPE were identified using selective media, and genome sequencing was performed for the obtained isolates (n = 21).

ResultsVarious rare/novel carbapenemases were identified: GES-5 in Raoultella planticola (n = 1), FRI-8 and FRI-11 in Enterobacter spp. (n = 8), IMI-22 and IMI-23 in Serratia ureilytica (n = 3), and SFC-1, SFC-2 and SFH-1 in Serratia fonticola (n = 9). Genomes of 11 isolates could be closed, allowing the elucidation of the genetic contexts of the carbapenemase genes. The blaGES-5 gene was located within a class 1 integron, In2071 (cassette array, blaGES-5-aacA3-aadA16), on a 33 kb IncP6 plasmid. The blaFRI-8 genes were carried on IncFII(Yp) plasmids ranging in size from 191 kb to 244 kb, and the blaFRI-11 genes were carried on 70 kb and 74 kb IncFII(pECLA)/IncR plasmids. The blaIMI-22 and blaIMI-23 genes were colocated on a 107 kb plasmid. The blaSFC and blaSFH-1 genes were found on putative genomic islands inserted at tRNA-Phe genes in chromosomes.

ConclusionsThis study revealed the presence of rare/novel carbapenemases among CPE in aquatic environments, suggesting that the environment may act as a potential reservoir of these minor carbapenemases.


title: Pantothenate biosynthesis is critical for chronic infection by the neurotropic parasite Toxoplasma gondii

abstract: Coenzyme A (CoA) is an essential molecule acting in metabolism, post-translational modification, and regulation of gene expression. While all organisms synthesize CoA, many, including humans, are unable to produce its precursor, pantothenate. Intriguingly, like most plants, fungi and bacteria, parasites of the coccidian subgroup of Apicomplexa, including the human and animal pathogen Toxoplasma gondii, possess all the enzymes required for de novo synthesis of pantothenate. Here, the importance of CoA and pantothenate biosynthesis for the acute and chronic stages of T. gondii infection was dissected through genetic, biochemical and metabolomic approaches, revealing that CoA synthesis is essential for T. gondii tachyzoites, due to the parasites inability to salvage CoA or intermediates of the pathway. In contrast, de novo pantothenate synthesis was only partially active in T. gondii tachyzoites, making the parasite reliant on Pan uptake. However, Pan synthesis proved to be crucial for the establishment of chronic infection, offering a promising target for intervention against the persistent stage of T. gondii.


title: Robust T cell activation requires an eIF3-driven burst in T cell receptor translation

abstract: Activation of T cells requires a rapid surge in cellular protein synthesis. However, the role of translation initiation in the early induction of specific genes remains unclear. Here we show human translation initiation factor eIF3 interacts with select immune system related mRNAs including those encoding the T cell receptor (TCR) subunits TCRA and TCRB. Binding of eIF3 to the TCRA and TCRB mRNA 3-untranslated regions (3-UTRs) depends on CD28 coreceptor signaling and regulates a burst in TCR translation required for robust T cell activation. Use of the TCRA or TCRB 3-UTRs to control expression of an anti-CD19 chimeric antigen receptor (CAR) improves the ability of CAR-T cells to kill tumor cells in vitro. These results identify a new mechanism of eIF3-mediated translation control that can aid T cell engineering for immunotherapy applications.


title: Non-coding function for mRNAs in Focal Adhesion Architecture and Mechanotransduction

abstract: Messenger RNA (mRNA) compartmentalization within the cytosol is well-recognized as a key mechanism of local translation-mediated regulation of protein levels, but whether such localization could be a means of exercising non-coding mRNA function is unknown. Here, we explore non-coding functions for mRNAs associated with focal adhesions (FAs), cellular structures responsible for mediating cell adhesion and response to changes in the extracellular matrix (ECM). Using high-throughput single molecule imaging and genomic profiling approaches, we find that mRNAs with distinct sequence characteristics localize to FAs in different human cell types. Notably, [~]85% of FA-mRNAs are not translationally active at steady state or under conditions of FA dissolution or activation. Untranslated mRNA sequences are anchored to FA based on their functional states by the RNA binding protein, G3BP1, forming biomolecular granules. Removing RNA or G3BP1, but not blocking new polypeptide synthesis, dramatically changes FA protein composition and organization, resulting in loss of cell contractility and cellular ability to adapt to changing ECM. We have therefor uncovered a novel, non-coding role for mRNAs as scaffolds to maintain FA structure and function, broadening our understating of noncanonical mRNA functions.


title: Phenotypic diversity created by a transposable element increases productivity and resistance to competitors in plant populations

abstract: An accumulating body of evidence indicates that natural plant populations harbour a large diversity of transposable elements (TEs). TEs provide genetic and epigenetic variation that can substantially translate into changes in plant phenotypes. Despite the wealth of data on the ecological and evolutionary effects of TEs on plant individuals, we have virtually no information on the role of TEs on populations and ecosystem functioning. On the example of Arabidopsis thaliana, we demonstrate that TE-generated variation creates differentiation in ecologically important functional traits. In particular, we show that Arabidopsis populations with increasing diversity of individuals differing in copy numbers of the ONSEN retrotransposon had higher phenotypic and functional diversity. Moreover, increased diversity enhanced population productivity and reduced performance of interspecific competitors. We conclude that TE-generated diversity can have similar effects on ecosystem as usually documented for other biological diversity effects.