title: Evidence Of We-Representation In Monkeys When Acting Together

abstract: A hallmark of successful evolution resides in the ability to adapt our actions to those of others, optimizing collective behaviour, to achieve goals otherwise unattainable by individuals acting alone. We have previously shown that macaques constitute a good model to analyse joint behavior, since they are able to coordinate their actions in a dyadic context. In the present work, we investigated whether monkeys could improve their joint-action performance, under special visuomotor conditions. The behavior of 5 monkeys was analyzed in isometric center-out tasks, requiring hand force application in different directions, either individually or together with a partner. Manipulating the presence or absence of a pre-instruction about the future action condition (SOLO or TOGETHER), allowed us to investigate on the existence of a "we-representation" in macaque monkeys. We found that pre-cueing the future action context increased the chances of dyadic success, also thanks to the emergence of an optimal kinematic setting, that ultimately facilitates inter-individual motor coordination. Our results offer empirical evidence in macaques of a "We-representation" during collective behavior, that once is cued in advance has an overall beneficial effect on joint performance.

category: animal behavior and cognition


title: Fast-spiking interneurons of the premotor cortex contribute to action planning

abstract: Planning and execution of voluntary movement depend on the contribution of distinct classes of neurons in primary motor and premotor areas. However, the specific functional role of GABAergic cells remains only partly understood. Here, electrophysiological and computational analyses are employed to compare directly the response properties of putative pyramidal (PNs) and fast-spiking, GABAergic neurons (FSNs) during licking and forelimb retraction in mice. Recordings from anterolateral motor cortex and rostral forelimb area, reveal that FSNs fire earlier and for a longer duration than PNs, with the exception of a subset of early-modulated PNs in deep layers. Computational analysis reveals that FSNs carry vastly more information than PNs about the onset of movement. While PNs differently modulate their discharge during distinct motor acts, most FSNs respond with a stereotyped increase in firing rate. Accordingly, the informational redundancy was greater among FSNs than PNs. These data suggest that a global rise of inhibition contributes to early action planning.

category: physiology


title: Differential receptor crosstalk in DRD1-DRD2 heterodimer upon phasic and tonic dopamine signals

abstract: In response to phasic and tonic release, dopamine neurotransmission is regulated by its receptor subtypes, mainly dopamine receptor type 1 and 2 (DRD1 and DRD2). These dopamine receptors are known to form a heterodimer, however the receptor crosstalk between DRD1 and DRD2 was only suspected by measuring their downstream signaling products, due to the lack of methodology for selectively detecting individual activity of different dopamine receptors. Here, we develop red DRD1 sensor (R-DRD1) and green DRD2 sensor (G-DRD2) which can specifically monitor the real-time activity of DRD1 and DRD2, and apply these multicolor sensors to directly measure the receptor crosstalk in the DRD1-DRD2 heterodimer. Surprisingly, we discover that DRD1 activation in the heterodimer is inhibited only at micromolar phasic concentration of dopamine, while DRD2 activation is selectively inhibited at nanomolar tonic dopamine level. Differential receptor crosstalk in the DRD1-DRD2 heterodimer further modulates their downstream cAMP level. These data imply a novel function of the DRD1-DRD2 heterodimer at physiological dopamine levels of phasic and tonic release. Our approach utilizing multicolor receptor sensors will be useful to discover novel function of GPCR heterodimers.

category: neuroscience


title: The role of accelerated growth plate fusion in the absence of SOCS2 on osteoarthritis vulnerability

abstract:
Osteoarthritis is the most prevalent systemic musculoskeletal disorder characterised by articular cartilage degeneration and subchondral bone (SCB) sclerosis. Here we sought to examine the contribution of accelerated growth to osteoarthritis development using a murine model of excessive longitudinal growth. Suppressor of cytokine signalling 2 (SOCS2) is a negative regulator of growth hormone (GH) signalling, thus mice deficient in SOCS2 (Socs2-/-) display accelerated bone growth. We examined vulnerability of Socs2-/- mice to osteoarthritis following surgical induction of disease (destabilisation of the medial meniscus (DMM)), and with ageing, by histology and micro-CT. We observed significant increase in number (WT DMM: 532{+/-}56; WT sham: 495{+/-}45; KO DMM: 169{+/-}49; KO sham: 187{+/-}56; P<0.01) and density (WT DMM: 2.2{+/-}0.9; WT sham: 1.2{+/-}0.5; KO DMM: 13.0{+/-}0.5; KO sham: 14.4{+/-}0.7) of growth plate bridges in Socs2-/- in comparison to wild-type (WT). Histological examination of WT and Socs2-/- knees revealed articular cartilage damage with DMM in comparison to sham (WT DMM: 3.4{+/-}0.4; WT sham: 0.3{+/-}0.05 (P<0.05); KO DMM: 3.2{+/-}0.8; KO sham: 0.8{+/-}0.3). Articular cartilage lesion severity scores (mean and maximum) were similar in WT and Socs2-/- mice with either DMM, or with ageing. Micro-CT analysis revealed significant decreases in SCB thickness, epiphyseal trabecular number and thickness in the medial compartment of Socs2-/-, in comparison to WT (P<0.001). DMM had no effect on the SCB thickness in comparison to sham in either genotype. Together these data suggest that enhanced GH signalling through SOCS2 deletion accelerates growth plate fusion, however this has no effect on osteoarthritis vulnerability in this model.

Summary statementDeletion of SOCS2 results in accelerated growth plate fusion, however this has no effect on osteoarthritis vulnerability.

category: pathology


title: A mitochondrial long-chain fatty acid oxidation defect leads to uncharged tRNA accumulation and activation of the integrated stress response in the mouse heart

abstract: The heart relies mainly on mitochondrial fatty acid {beta}-oxidation (FAO) for its high energy requirements. Cardiomyopathy and arrhythmias can be severe complications in patients with inherited defects in mitochondrial long-chain FAO, reinforcing the importance of FAO for cardiac health. However, the pathophysiological mechanisms that underlie the cardiac abnormalities in long-chain FAO disorders remain largely unknown. Here, we investigated the cardiac transcriptional adaptations to the FAO defect in the long-chain acyl-CoA dehydrogenase (LCAD) knockout (KO) mouse. We found a prominent activation of the integrated stress response (ISR) mediated by the eIF2/ATF4 axis in both fed and fasted states, accompanied by a reduction in cardiac protein synthesis during a short period of food withdrawal. Notably, we found an accumulation of uncharged tRNAs in LCAD KO hearts, consistent with a reduced availability of cardiac amino acids, in particular, glutamine. We replicated the activation of the cardiac ISR in hearts of mice with a muscle-specific deletion of carnitine palmitoyltransferase 2 deletion (Cpt2M-/-). Our results show that perturbations in amino acid metabolism caused by long-chain FAO deficiency impact cardiac metabolic signaling, in particular the ISR, and may play a role in the associated cardiac pathology.


title: Pseudomonas arenae sp. nov., Pseudomonas glycinis sp. nov. and Pseudomonas harudinis sp. nov., three novel bacterial species and plant endophytes

abstract: Three novel Pseudomonas species associated with healthy plants are described from the United States. They are Pseudomonas arenae sp. nov. from soybean in Missouri and Phragmites sp. in New Jersey; Pseudomonas glycinis sp. nov. from Vaccinium macrocarpon fruit in Massachusetts, groundwater in Tennessee and soybean in Indiana; and Pseudomonas harudinis sp. nov. from Phragmites sp. in New Jersey. No pathogenic strains are known for any of the novel species based on genome comparisons to assemblies in GenBank.

category: microbiology


title: System characterization of dynamic biological systems through improved data analysis.

abstract: Determining the functional development and dominant competitive strategy in microbial community enrichments is complicated by the extensive measurement campaigns required for off-line system analysis. This study demonstrates that detailed system characterization of aerobic pulse fed enrichments can be established using on-line measurements combined with automated data analysis. By incorporating the physicochemical processes in on-line data processing with a Particle Filter and kinetic process model, an accurate reconstruction of the dominant biological rates can be made. We hereby can differentiate between storage compound production and biomass growth in sequencing batch bioreactors. The method proposed allows for close monitoring of changes in functional behavior of long-running enrichment cultures, without the need for off-line samples, therewith enabling the identification of new insights in process dynamics with a minimal experimental effort. Even though a specific example application of the method proposed is described here, the approach can readily be extended to a wide range of dynamic experimental systems that can be characterized based on on-line measurements.


title: Influenza A virus defective viral genomes are inefficiently packaged into virions relative to wild-type genomic RNAs

abstract:
Deletion-containing viral genomes (DelVGs) are commonly produced during influenza A virus infection and have been implicated in influencing clinical infection outcomes. Despite their ubiquity, the specific molecular mechanisms that govern DelVG formation and their packaging into defective interfering particles (DIPs) remain poorly understood. Here, we utilized next-generation sequencing to analyze DelVGs that form de novo early during infection, prior to packaging. Analysis of these early DelVGs revealed that deletion formation occurs in clearly defined hotspots and is significantly associated with both direct sequence repeats and enrichment of adenosine and uridine bases. By comparing intracellular DelVGs with those packaged into extracellular virions, we discovered that DelVGs face a significant bottleneck during genome packaging relative to wild type genomic RNAs. Surprisingly, packaged DelVGs exhibited no signs of enrichment for specific deletion characteristics suggesting that all DelVGs are equally limited in packaging efficiency. Our data provide the first unbiased, high-resolution portrait of the diversity of DelVGs that are generated by the IAV replication machinery and shed light on the mechanisms that underly DelVG formation.

ImportanceDefective interfering particles (DIPs) are commonly produced by RNA viruses and have been implicated in modulating clinical infection outcomes, hence, there is increasing interest in the potential of DIPs as antiviral therapeutics. For influenza viruses, DIPs are formed by the packaging of genomic RNAs harboring internal deletions. Despite decades of study, the mechanisms that drive the formation of these deletion-containing viral genomes (DelVGs) remain elusive. Here, we used a specialized sequencing pipeline to characterize the first wave of DelVGs that form during influenza virus infection. This dataset provides an unbiased profile of the deletion-forming preferences of the influenza virus replicase. Additionally, by comparing the early intracellular DelVGs with those that get packaged into extracellular virions, we described a significant segment-specific bottleneck that limits DelVG packaging relative to wild type viral RNAs. Altogether, these findings reveal factors that govern the production of both DelVGs and DIPs during influenza virus infection.


title: Insights into the secondary and tertiary structure of the Bovine Viral Diarrhea Virus Internal Ribosome Entry Site

abstract: The Internal Ribosome Entry Site (IRES) RNA of Bovine viral diarrhea virus (BVDV), an economically significant Pestivirus, is required for the cap-independent translation of viral genomic RNA. Thus, it is essential for viral replication and pathogenesis. We applied a combination of high-throughput biochemical RNA structure probing (SHAPE-MaP) and in silico modeling approaches to gain insight into the secondary and tertiary structures of BVDV IRES RNA. Our study demonstrated that BVDV IRES RNA forms in solution a modular architecture composed of three distinct structural domains (I-III). Two regions within domain III are engaged in tertiary interactions to form an H-type pseudoknot. Computational modeling of the pseudoknot motif provided a fine-grained picture of the tertiary structure and local arrangement of helices in the BVDV IRES. Furthermore, comparative genomics and consensus structure predictions revealed that the pseudoknot is evolutionarily conserved among many Pestivirus species. These studies provide detailed insight into the structural arrangement of BVDV IRES RNA H-type pseudoknot and encompassing motifs that likely contribute to the optimal functionality of viral cap-independent translation element.


title: Development of an STR panel for a non-native population of an endangered species

abstract: The establishment of non-native populations of threatened and legally protected species can have many implications for the areas where these species have been introduced. Non-native populations of threatened species have the potential to be exploited and therefore the subject of legal protection, while conversely, if they have become invasive in their introduced range, there is the likelihood that population control will be carried out to reduce abundance and negative impacts associated with introduced species. From both a legal and invasive species monitoring standpoint, it is important to know how many individuals are present. Short tandem repeats (STRs) were developed for the hog deer, an endangered species that was introduced following European settlement to Victoria, Australia using Illumina MiSeq sequencing technology. These markers were combined with previous STRs characterised for hog deer to create a 29-plex identification system. A total of 224 samples were genotyped across the population in Victoria, and further analyses of null allele frequencies, deviation from Hardy-Weinberg equilibrium, and the removal of monomorphic or low amplifying markers resulted in a final marker panel of 17 loci. Despite low values for number of alleles at each locus (2-4), probability of identity showed sufficient discrimination power, with an average probability of identity at 9.46 x 10-7, and a probability of sibling identity of 7.67 x 10-4 across all sites. These findings show that it is feasible to create an informative DNA profiling system that can distinguish between individuals for applications in both wildlife forensic and population control research.

category: molecular biology


title: Hypoxia induces transcription of DOT1L in articular cartilage to protect against osteoarthritis

abstract: Osteoarthritis is the most prevalent joint disease worldwide and a leading source of pain and disability. To date, this disease lacks curative treatment as underlying molecular mechanisms remain largely unknown. The histone methyltransferase DOT1L protects against osteoarthritis, and DOT1L-mediated H3K79 methylation is reduced in human and mouse osteoarthritic joints. Thus, restoring DOT1L function seems to be critical to preserve joint health. However, DOT1L-regulating molecules and networks remain elusive, in the joint and beyond. Here, we identify transcription factors and networks that regulate DOT1L gene expression using a novel bioinformatics pipeline. Thereby, we unravel an undiscovered link between the hypoxia pathway and DOT1L. We provide unprecedented evidence that hypoxia enhances DOT1L expression and H3K79 methylation via hypoxia-inducible factor-1 alpha (HIF-1). Importantly, we demonstrate that DOT1L contributes to the protective effects of hypoxia in articular cartilage and osteoarthritis. Intra-articular treatment with a selective hypoxia mimetic in mice after surgical induction of osteoarthritis restores DOT1L function and stalls disease progression. Collectively, our data unravel a novel molecular mechanism that protects against osteoarthritis with hypoxia inducing DOT1L transcription in cartilage. Local treatment with a selective hypoxia mimetic in the joint restores DOT1L function and could be an attractive therapeutic strategy for osteoarthritis.

category: cell biology


title: Elimination Of Aberrantly Specified Cell Clones Is Independent Of Interfacial Myosin II Accumulation

abstract: Spatial organization of differently fated cells within an organ is essential and needs to be maintained during development. This is largely implemented via compartment boundaries that serve as barriers between distinct cell types. Biased accumulation of junctional non-muscle Myosin II along the interface between differently fated groups of cells contributes to boundary integrity and maintains its shape via increased tension [1-4]. Here we test whether interfacial Myosin driven tension is responsible for the elimination of aberrantly specified cells that would otherwise compromise compartment organization. To this end, we genetically reduce Myosin II levels in three different patterns: in both wild-type and misspecified cells, only in misspecified cells and specifically at the interface between wild-type and aberrantly specified cells. We find that recognition and elimination of aberrantly specified cells do not rely on tensile forces driven by interfacial Myosin cables. Moreover, apical constriction of misspecified cells and their separation from wild type neighbors occurs even when Myosin level is greatly reduced. Thus, we conclude that the forces that drive elimination of aberrantly specified cells are largely independent of Myosin II.

category: developmental biology


title: The effects of host availability and fitness on  Aedes albopictus  blood feeding patterns in New York

abstract: Aedes albopictus is a competent vector of numerous pathogens, representing a range of transmission cycles involving unique hosts. Despite the important status of this vector, variation in its feeding patterns is poorly understood. We examined the feeding patterns of Ae. albopictus utilizing resting collections in Long Island, New York, and contextualized blood meal sources with host availability measured by household interviews and camera traps. We identified 90 blood meals, including 29 human, 22 cat, 16 horse, 12 opossum, 5 dog, 2 goat, and 1 rabbit, rat, squirrel and raccoon. Our study is the first to quantitatively assess Ae. albopictus feeding patterns in the context of host availability of wild animals in addition to humans and domestic animals. Host feeding indices showed that cats and dogs were fed upon disproportionately often compared to humans. Forage ratios suggested a tendency to feed on cats and opossums and to avoid raccoons, squirrels, and birds. This feeding pattern was different from another published study from Baltimore, where Ae. albopictus fed more often on rats than humans. To understand if these differences were due to host availability or mosquito population variation, we compared the fitness of Long Island and Baltimore Ae. albopictus after feeding on rat and human blood. In addition, we examined fitness within the Long Island population after feeding on human, rat, cat, horse, and opossum blood. Together, our results do not show major mosquito fitness differences by blood hosts, suggesting that fitness benefits do not drive Northeastern Ae. albopictus feeding patterns.

category: ecology


title: Lack of food intake during shift work alters the heart transcriptome and leads to cardiac fibrosis and inflammation in rats

abstract: Many epidemiological studies revealed that shift work is associated with increased risk of cardiovascular diseases. However, the underlying mechanisms remain poorly understood. An experimental model of shift work in rats has been shown to recapitulate the metabolic disorders observed in human shift workers, and used to demonstrate that restricting food consumption outside working hours prevents shift work-associated obesity and metabolic disturbance. Here we used this model to characterize the effects of shift work in the heart. We show that experimental shift work reprograms the heart cycling transcriptome independently of food consumption. While phases of rhythmic gene expression are distributed across the 24-hour day in control rats, they are clustered towards discrete times in shift workers. Additionally, preventing food intake during shift work affects the expression level of hundreds of genes in the heart. Many of them are found in transcriptional signatures associated with pressure overload and cardiac hypertrophy, and encode for components of the extracellular matrix and inflammatory markers. Consistent with this, the heart of shift worker rats not eating during work exhibits fibrosis and is colonized by immune cells. While maintaining food access during shift work has less effects on gene expression, genes found in transcriptional signatures of cardiac hypertrophy remain affected, and the heart of shift worker rats exhibits fibrosis without inflammation. Together, our findings provide insights into how shift work affects cardiac function, and suggest that some interventions aiming at mitigating metabolic disorders in shift workers may have adverse effects on cardiovascular diseases.

category: genomics


title: An ultrasensitive method for analysis of viral spike N-glycoforms

abstract: Viruses can evade the host immune system by displaying numerous glycans on their surface "spike-proteins" that cover immune epitopes. We have developed an ultrasensitive "single pot" method to assess glycan occupancy and the extent of glycan processing from high-mannose to complex forms at each N-glycosylation site. Though aimed at characterizing glycosylation of viral spike-proteins as potential vaccines, this method is applicable for analysis of site-specific glycosylation of any glycoprotein.

category: biochemistry


title: DeGlyPHER: an ultrasensitive method for analysis of viral spike N-glycoforms


title: Label-free super-resolution chemical imaging of biomedical specimens

abstract: Raman microscopy provides chemically selective imaging by exploiting intrinsic vibrational properties of specimens. Yet, a fast acquisition, low phototoxicity, and non-specific (to a vibrational/electronic mode) super-resolution method has been elusive for tissue imaging. We demonstrate a single-pixel-based approach, combined with robust structured illumination, that enables fast super-resolution in stimulated Raman scattering microscopy at low power levels. The methodology is straightforward to implement and compatible with thick biological specimens, therefore paving the way for probing complex biological systems when exogenous labelling is challenging.

category: biophysics


title: The EphB2-MYC Axis is a Major Determinant of Barrett's Pathobiology and a Therapeutic Vulnerability in Esophageal Cancer

abstract:
Esophageal adenocarcinoma (EAC), a highly aggressive cancer with limited therapeutic options, often arises in the backdrop of a molecularly-complex esophageal metaplasia disorder, Barretts Esophagus (BE). Using transcriptomics and systems biology analyses of treatment-naive malignant/pre-malignant biopsy tissues, we found Eph receptor B2 (EphB2) tyrosine kinase signaling to be frequently hyperactivated during early stages of EAC development, and across the BE-EAC continuum. Functional studies revealed EphB2 to be an upstream post-translational regulator of c-MYC activity and as a key molecular dependency in BE/EAC. Single-cell transcriptomics in a porcine esophageal 3D spheroid model showed enhanced EphB2 and MYC activity to be significantly associated with BE-like cell fate. shRNA-based knockdown of EphB2 or small molecule inhibitors of MEK, that modulate MYC protein stability, proved effective in suppressing EAC tumor growth in vivo. These findings point to EphB2-MYC axis as an early promoter of EAC and a novel therapeutic vulnerability in this increasingly-prevalent esophageal malignancy.

STATEMENT OF SIGNIFICANCEWe identify EphB2 signaling as a potential master regulator and early promoter of esophageal adenocarcinoma, and the proto-oncogene MYC as a key downstream effector of EphB2 function. Targeting the EphB2-MYC axis could be a promising therapeutic strategy for these often refractory and lethal EAC tumors.

category: cancer biology


title: Neuronal NADPH oxidase in Parkinson disease pathogenesis

abstract:
Mitochondrial dysfunction and oxidative stress are strongly implicated in the pathogenesis of Parkinsons disease (PD) and there is evidence that mitochondrially-generated superoxide can activate NADPH oxidase 2 (NOX2), which is a major enzymatic generator of superoxide. Although NOX2 has been examined in the context of PD, previous studies have focused on microglial function; the role of neuronal NOX2 in PD pathogenesis remains to be defined. Here we devised and validated a proximity ligation assay for NOX2 activity and demonstrated that in human PD and 2 models thereof, neuronal NOX2 is highly active in substantia nigra dopamine neurons. Further, NOX2 activity is responsible for accumulation, post-translational modification and oligomerization of -synuclein as well as activation of leucine-rich repeat kinase 2 (LRRK2). Administration of a brain-penetrant, specific NOX2 inhibitor prevented NOX2 activation and its downstream effects in vivo in a rat model of PD. We conclude that neuronal NOX2 is a major contributor to oxidative stress in PD, to -synuclein pathology and to LRRK2 activation in idiopathic PD. In this context, NOX2 inhibitors hold potential as a disease-modifying therapy in PD.

SummaryIn dopamine neuron, NADPH oxidase isoform 2 amplifies the oxidative stress-related pathogenic cascade in Parkinsons disease


title: A Novel Pharmacogenetic Model for Highly Efficient Ablation of Oligodendrocyte Progenitor Cells in the Adult Mouse CNS

abstract: Approaches to investigate adult oligodendrocyte progenitor cells (OPCs) by targeted cell ablation in the rodent central nervous system have been limited by methodological challenges resulting in only partial and transient OPC depletion. We have developed a novel pharmacogenetic model of conditional OPC ablation, resulting in the elimination of 99.7% of all OPCs throughout the brain. By combining recombinase-based transgenic and viral strategies for targeting of OPCs and ventricular-subventricular zone (V-SVZ)-derived neural precursor cells (NPCs), we found that new PDGFRA-expressing cells born in the V-SVZ repopulated the OPC-deficient brain starting 12 days after OPC ablation. Our data reveal that OPC depletion induces V-SVZ-derived NPCs to generate vast numbers of PDGFRA+/NG2+ cells with the capacity to migrate and proliferate extensively throughout the dorsal anterior forebrain. Further application of this novel approach to ablate OPCs will advance knowledge of the function of both OPCs and oligodendrogenic NPCs in health and disease.


title: Assessing the role of cold-shock protein C: A novel regulator of Acinetobacter baumannii biofilm formation and virulence

abstract: Acinetobacter baumannii is a formidable opportunistic pathogen that is notoriously difficult to eradicate from hospital settings and can spread quickly via healthcare personnel despite preventive measures. This resilience is often attributed to a proclivity for biofilm formation, which grants A. baumannii a higher tolerance towards external stress, desiccation, and antimicrobials. Despite this, little is known regarding the mechanisms orchestrating A. baumannii biofilm formation. Herein, we performed RNA-seq on biofilm and planktonic populations for the multidrug resistant isolate, AB5075, and identified 438 genes with altered expression. To assess the potential role of genes upregulated within biofilms, we tested the biofilm forming capacity of their respective mutants from an A. baumannii transposon library. In so doing, we uncovered 24 genes whose disruption led to reduced biofilm formation. One such element, cold shock protein C (cspC), produced a mucoidal, non-mucoviscous colony phenotype. RNA-sequencing of this mutant revealed the down regulation of pili and fimbriae in the cspC mutant, which would explain the decreased biofilm observed. Using MIC assays, we note that the mutant displayed increased antibiotic tolerance that we hypothesize is mediated by overexpression of multi-drug efflux pumps and altered mRNA stability of their corresponding transcriptional repressor. Finally, we show that CspC is required for survival during oxidative stress and challenge by the human immune system, and plays a pivotal role during systemic infection. Collectively, our work identifies a cadre of new biofilm associated genes within A. baumannii and provides insight into the global regulatory network of this emerging human pathogen.


title: A chemotactic sensor controls Salmonella-host cell interaction

abstract: Intimate cell contact and subsequent type three secretion system-dependent cell invasion are key steps in host colonization of Salmonella. Adhesion to complex glycostructures at the apical membrane of polarized cells is mediated by the giant adhesin SiiE. This protein is secreted by a type 1 secretion system (T1SS) and needs to be retained at the bacterial surface to exert its adhesive function. Here, we show that SiiE surface expression was linked to the presence of L-aspartate sensed by the Salmonella-specific methyl-accepting chemotaxis protein CheM. Bacteria lacking CheM were attenuated for invasion of polarized cells, whereas increased invasion was seen with Salmonella exposed to the non-metabolizable aspartate analog -methyl-D, L-aspartate (MeAsp). While components of the chemotaxis phosphorelay or functional flagella were dispensable for the increased invasion, CheM directly interacted with proteins associated with the SiiE T1SS arguing for a novel non-canonical signaling mechanism. As a result, CheM attractant signaling caused a shift from secreted to surface-retained and adhesion-competent SiiE. Thus, CheM controls the virulence function of SiiE in a precise spatio-temporal fashion depending on the host micro-milieu.


title: The histone chaperone Spt6 is required for normal recruitment of the capping enzyme Abd1 to transcribed regions

abstract: The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating co-transcriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII interacting factors between wild-type cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. The altered association of some of these factors could be attributed to changes in steady-state protein levels. However, Abd1, the mRNA cap methyltransferase, had decreased association with RNAPII after Spt6 depletion despite unchanged Abd1 protein levels, showing a requirement for Spt6 in mediating the Abd1-RNAPII interaction. Genome-wide studies showed that Spt6 is required for maintaining the level of Abd1 over transcribed regions, as well as the level of Spt5, another protein known to recruit Abd1 to chromatin. Abd1 levels were particularly decreased at the 5 ends of genes after Spt6 depletion, suggesting a greater need for Spt6 in Abd1 recruitment over these regions. Together, our results show that Spt6 is important in regulating the composition of the transcription elongation complex and reveal a previously unknown function for Spt6 in the recruitment of Abd1.


title: Mesothelial cells are not a source of adipocytes in mice

abstract: Visceral adipose tissue (VAT) depots are associated with the adverse metabolic consequences of obesity, such as insulin resistance. The developmental origin of VAT depots and the identity and regulation of adipocyte progenitor cells have been active areas of investigation. In recent years, a paradigm of mesothelial cells as a source of VAT adipocyte progenitor cells has emerged based on lineage-tracing studies using the Wilms tumor gene, Wt1, as a marker for cells of mesothelial origin. Here we show that Wt1 expression in adipose tissue is not limited to the mesothelium, but is also expressed by a distinct preadipocyte population in both mice and humans. We identify keratin 19 (Krt19) as a highly-specific marker for the adult mouse mesothelium, and demonstrate that Krt19-expressing mesothelial cells do not differentiate into visceral adipocytes. These results contradict the assertion that the VAT mesothelium can serve as a source of adipocytes.


title: Scale-dependent tipping points of bacterial colonization resistance

abstract: Bacteria are efficient colonizers of a wide range of secluded micro-habitats, such as soil pores, skin follicles, or intestinal crypts. How the structural diversity of these habitats modulates microbial self-organization remains poorly understood, in part because of the challenge to specifically manipulate the physical structure of microbial environments. Using a microfluidic device to grow bacteria in crypt-like incubation chambers of systematically varied lengths, we show that small variations in the physical structure of the micro-habitat can drastically alter bacterial colonization success and resistance against invaders. Small crypts are un-colonizable, intermediately sized crypts can stably support dilute populations, while beyond a second critical lengthscale, populations phase-separate into a dilute and a jammed region. The jammed state is characterized by extreme colonization resistance, even if the resident strain is suppressed by an antibiotic. Combined with a flexible biophysical model, we demonstrate that colonization resistance and associated priority effects can be explained by a crowding-induced phase transition, which results from a competition between proliferation and density-dependent cell leakage. The emerging sensitivity to scale underscores the need to control for scale in microbial ecology experiments. Systematic flow-adjustable lengthscale variations may serve as a promising strategy to elucidate further scale-sensitive tipping points and to rationally modulate the stability and resilience of microbial colonizers.


title: Overdominant mutations restrict adaptive loss of heterozygosity at linked loci

abstract:
Loss of heterozygosity is a common mode of adaptation in asexual diploid populations. Because mitotic recombination frequently extends the full length of a chromosome arm, the selective benefit of loss of heterozygosity may be constrained by linked heterozygous mutations. In a previous laboratory evolution experiment with diploid yeast, we frequently observed homozygous mutations in the WHI2 gene on the right arm of Chromosome XV. However, when heterozygous mutations arose in the STE4 gene, another common target on Chromosome XV, loss of heterozygosity at WHI2 was not observed. Here we show that mutations at WHI2 are partially dominant and that mutations at STE4 are overdominant. We test whether beneficial heterozygous mutations at these two loci interfere with one another by measuring loss of heterozygosity at WHI2 over 1,000 generations for [~]300 populations that differed initially only at STE4 and WHI2. We show that the presence of an overdominant mutation in STE4 reduces, but does not eliminate, loss of heterozygosity at WHI2. By sequencing 40 evolved clones, we show that populations with linked overdominant and partially dominant mutations show less parallelism at the gene level, more varied evolutionary outcomes, and increased rates of aneuploidy. Our results show that the degree of dominance and the phasing of heterozygous beneficial mutations can constrain loss of heterozygosity along a chromosome arm, and that conflicts between partially dominant and overdominant mutations can affect evolutionary outcomes.

SIGNIFICANCE STATEMENTIn diploid populations, it is beneficial for partially dominant beneficial mutations to lose heterozygosity, but it is deleterious for overdominant beneficial mutations to do so. Because loss-of-heterozygosity tracts often encompass entire chromosome arms, a conflict will arise when a partially dominant beneficial mutation and an overdominant beneficial mutation exist in close proximity. We demonstrate that this conflict occurs, and that it restricts loss of heterozygosity, resulting in more variable evolutionary outcomes.

category: evolutionary biology


title: Site-specific epigenetic marks in Trypanosoma brucei transcription termination, antigenic variation, and proliferation

abstract: In Trypanosoma brucei, genes assemble into polycistronic transcription units (PTUs). Transcription termination sites (TTSs) hold deposition sites for three non-essential chromatin factors, histone variants (H3v and H4v) and a DNA modification (base J, a hydroxyl-glucosyl dT). Here, I found that H4v is a major sign for transcription termination at TTSs and readthrough transcription machineries progress until they encounter the next bidirectional transcription start site. While having a secondary function at TTSs, H3v is important for monoallelic transcription of telomeric antigen genes. The simultaneous absence of both histone variants leads to proliferation and replication defects, which are exacerbated by the J deficiency, accompanied by accumulation of sub-G1 population. Base J likely contributes to DNA replication and cell-cycle control. I propose that the coordinated actions of H3v, H4v and J function in concert for cellular fate determination and provide compensatory mechanisms for each other in chromatin organization, transcription, and replication.

category: genetics


title: Concentration matters: different stomatal CO2-responses at sub-ambient and above-ambient CO2 levels

abstract: Stomatal pores, formed of paired guard cells, mediate CO2 uptake for photosynthesis and water loss via transpiration in plants. Globally rising atmospheric CO2 concentration triggers stomatal closure, contributing to increased leaf temperature and reduced nutrient uptake due to lower transpiration rate1. Hence, it is important to understand the signalling pathways that control elevated CO2-induced stomatal closure to identify targets for breeding climate-ready crops. CO2-induced stomatal closure can be studied by increasing CO2 concentration from ambient to above-ambient concentrations2,3, or elevation of CO2 levels from sub-ambient to above-ambient4,5. Previous experiments comparing ferns with angiosperms suggested that stomatal responses to CO2 may be different, when changing CO2 levels in the sub-ambient or above-ambient ranges3,6. Here, we set out to test this by comparing CO2-induced stomatal closure in key guard cell signalling mutants in response to CO2 elevation from 100 to 400 ppm or 400 to 800 ppm. We show that signalling components that contribute to CO2-induced stomatal closure are different in the sub-ambient and above-ambient CO2 levels, with guard cell slow-type anion channel SLAC1 involved mainly in above-ambient CO2-induced stomatal closure.

category: plant biology


title: Dynamic Runx1 chromatin boundaries affect gene expression in hematopoietic development

abstract: The transcription factor RUNX1 is a critical regulator of developmental hematopoiesis and is frequently disrupted in leukemia. Runx1 is a large, complex gene that is expressed from two alternative promoters under the spatiotemporal control of multiple hematopoietic enhancers. To dissect the dynamic regulation of Runx1 in hematopoietic development, we analyzed its three-dimensional chromatin conformation in mouse embryonic stem cell (ESC) differentiation cultures. Runx1 resides in a 1.1 Mb topologically associating domain (TAD) demarcated by convergent CTCF motifs. As ESCs differentiate to mesoderm, chromatin accessibility, Runx1 enhancer-promoter (E-P) interactions, and CTCF-CTCF interactions increased in the TAD, along with initiation of Runx1 expression from the P2 promoter. Differentiation to hematopoietic progenitor cells was associated with the formation of tissue-specific sub-TADs over Runx1, a shift in E-P interactions, P1 promoter demethylation, and robust expression from both Runx1 promoters. Deletions of promoter-proximal CTCF sites at the sub-TAD boundaries had no obvious effects on E-P interactions but led to partial loss of domain structure, mildly affected gene expression, and delayed hematopoietic development. Together, our analyses of gene regulation at a large multi-promoter developmental gene revealed that dynamic sub-TAD chromatin boundaries play a role in establishing TAD structure and coordinated gene expression.


title: New cellular models of undifferentiated pleomorphic sarcoma and malignant peripheral nerve sheath tumor

abstract: Undifferentiated pleomorphic sarcoma (UPS) and malignant peripheral nerve sheath tumor (MPNST) are aggressive soft tissue sarcomas that do not respond well to current treatment modalities. The limited availability of UPS and MPNST cell lines makes it challenging to identify potential therapeutic targets in a laboratory setting. Understanding the urgent need for improved treatments for these tumors and the limited cellular models led us to generate additional cell lines to study these rare cancers further. Patient-derived tumors were used to establish 5 new UPS models, including one radiation-associated UPS--UPS060.1, UPS271.1, UPS511, UPS0103, and RIS620--and 3 new models of MPNST--MPNST007, MPNST3813E, and MPNST4970. This study examined the utility of the new cell lines as sarcoma models by assessing tumorigenic potential and mutation status for known sarcoma-related genes. All the cell lines formed colonies and migrated in vitro. The in vivo tumorigenic potential of each cell line was determined by either subcutaneous injection of cells or implantation of tumor tissue into immunocompromised mice. UPS060.1, UPS271.1, and UPS511 cells formed tumors in mice upon subcutaneous injection. UPS0103 and RIS620 tumor implants formed tumors in vivo, as did MPNST007 and MPNST3813E tumor implants. Mutation analysis of a panel of genes frequently mutated in sarcomas showed that two of the three MPNST cell lines had NF1 mutations. Two of the three MPNST cell lines had mutations in polycomb repressive complex 2 members. These new cellular models provide the scientific community with powerful tools for detailed studies of sarcomagenesis and investigate novel therapies for UPS and MPNST.


title: Human immunocompetent Choroid-on-Chip: a novel tool for studying ocular effects of biological drugs

abstract:
Disorders of the eye leading to visual impairment are a major issue that affects millions of people. On the other side ocular toxicities were described for e.g. molecularly targeted therapies in oncology and may hamper their development. Current ocular model systems feature a number of limitations affecting human-relevance and availability. To find new options for pharmacological treatment and assess mechanisms of toxicity, hence, novel complex model systems that are human-relevant and readily available are urgently required. Here, we report the development of a human immunocompetent Choroid-on-Chip (CoC), a human cell-based in vitro model of the choroid layer of the eye integrating melanocytes and microvascular endothelial cells, covered by a layer of retinal pigmented epithelial cells. Immunocompetence is achieved by perfusion of peripheral immune cells. We demonstrate controlled immune cell recruitment into the stromal compartments through a vascular monolayer and in vivo-like cytokine release profiles. To investigate applicability for both efficacy testing of immunosuppressive compounds as well as safety profiling of immunoactivating antibodies, we exposed the CoCs to cyclosporine and tested CD3 bispecific antibodies.

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category: bioengineering


title: Host transcriptional response to SARS-CoV-2 infection in COVID-19 patients

abstract:
BackgroundOne of the most perplexing aspects of infection with the SARS-CoV-2 virus has been the variable response elicited in its human hosts. Investigating the transcriptional changes in individuals affected by COVID-19 can help understand and predict the degree of illness and guide clinical outcomes in diverse backgrounds.

MethodsAnalysis of host transcriptome variations via RNA sequencing from naso/oropharyngeal swabs of COVID-19 patients.

ResultsWe report strong upregulation of the innate immune response, especially type I interferon pathway, upon SARS-CoV-2 infection. Upregulated genes were subjected to a comparative meta-analysis using global datasets to identify a common network of interferon stimulated and viral response genes that mediate the host response and resolution of infection. A large proportion of mis-regulated genes showed a reduction in expression level, suggesting an overall decrease in host mRNA production. Significantly downregulated genes included those encoding olfactory, taste and neuro-sensory receptors. Many pro-inflammatory markers and cytokines were also downregulated or remained unchanged in the COVID-19 patients. Finally, a large number of non-coding RNAs were identified as down-regulated, with a few of the lncRNAs associated with functional roles in directing the response to viral infection.

ConclusionsSARS-CoV-2 infection results in the robust activation of the bodys innate immunity. Reduction of gene expression is well correlated with the clinical manifestations and symptoms of COVID-19 such as the loss of smell and taste, and myocardial and neurological complications. This study provides a critical dataset of genes that will enhance our understanding of the nature and prognosis of COVID-19.

category: bioinformatics


title: Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate

abstract:
NMR-assisted crystallography - the synergistic combination of solid-state NMR, X-ray crystallography, and first-principles computational chemistry - holds remarkable promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in the enzyme active site - including hydrogen atom locations and tautomeric equilibria - it offers insight into structure, dynamics, and function. Here, we make use of this combined approach to characterize the -aminoacrylate intermediate in tryptophan synthase, a defining species for pyridoxal-5'-phosphate-dependent enzymes on the {beta}-elimination and replacement pathway. By uniquely identifying the protonation states of ionizable sites on the cofactor, substrates, and catalytic side chains, as well as the location and orientation of structural waters in the active site, a remarkably clear picture of structure and reactivity emerges. Most incredibly, this intermediate appears to be mere tenths of angstroms away from the preceding transition state in which the {beta}-hydroxyl of the serine substrate is lost. The position and orientation of the structural water immediately adjacent to the substrate {beta}-carbon suggests not only the fate of the hydroxyl group, but also the pathway back to the transition state and the identity of the active site acid-base catalytic residue. Reaction of this intermediate with benzimidazole (BZI), an isostere of the natural substrate, indole, shows BZI bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the BZI position, indole is positioned with C3 in contact with the -aminoacrylate C{beta} and aligned for nucleophilic attack.

Significance StatementThe determination of active site protonation states is critical to gaining a full mechanistic understanding of enzymatic transformations; yet hydrogen positions are challenging to extract using the standard tools of structural biology. Here we make use of a joint solid-state NMR, X-ray crystallography, and first-principles computational approach that unlocks the investigation of enzyme catalytic mechanism at this fine level of chemical detail. For tryptophan synthase, this allows us to peer along the reaction coordinates into and out of the -aminoacrylate intermediate. Through this process, we are developing a high-resolution probe for structural biology that is keenly sensitive to proton positions - rivaling that of neutron diffraction, yet able to be applied under conditions of active catalysis to microcrystalline and non-crystalline materials.


title: Monocyte-derived transcriptome signature indicates antibody-dependent cellular 1 phagocytosis as the primary mechanism of vaccine-induced protection against HIV-1

abstract: A gene signature previously correlated with mosaic adenovirus 26 vaccine protection in simian immunodeficiency virus (SIV) and SHIV challenge models in non-human primates (NHP). In this report we investigated presence of this signature as a correlate of reduced risk in human clinical trials and potential mechanism for protection. The absence of this gene signature in the DNA/rAd5 human vaccine trial which did not show efficacy, strengthens our hypothesis that this signature is only enriched in studies that demonstrated protection. This gene signature was enriched in the partially effective RV144 human trial that administered the ALVAC/protein vaccine, and we find that the signature associates with both decreased risk of HIV-1 acquisition and increased vaccine efficacy. Total RNA-seq in a clinical trial that used the same vaccine regimen as the RV144 HIV vaccine implicated antibody-dependent cellular phagocytosis (ADCP) as a potential mechanism of vaccine protection. CITE-seq profiling of 53 surface markers and transcriptomes of 53,777 single cells from the same trial, showed that genes in this signature were primarily expressed in cells belonging to the myeloid lineage including monocytes, which are major effector cells for ADCP. The consistent association of this transcriptome signature with vaccine efficacy represents a tool both to identify potential mechanisms, as with ADCP here, and to screen novel approaches to accelerate development of new vaccine candidates.

category: systems biology


title: Reversal of the adipostat control of torpor during migration in hummingbirds

abstract: Many small endotherms use torpor to reduce metabolic rate and manage daily energy balance. However, the physiological "rules" that govern torpor use are unclear. We tracked torpor use and body composition in ruby-throated hummingbirds (Archilochus colubris), a long-distance migrant, throughout the summer using respirometry and quantitative magnetic resonance. During the mid-summer, birds entered torpor at consistently low fat stores ([~]5% of body mass), and torpor duration was negatively related to evening fat load. Remarkably, this energy-emergency strategy was abandoned in the late summer when birds accumulated fat for migration. Migrating birds were more likely to enter torpor on nights when they had higher fat stores, and fat gain was positively correlated with the amount of torpor used. These findings demonstrate the versatility of torpor throughout the annual cycle and suggest a fundamental change in physiological feedback between adiposity and torpor during migration. Moreover, this study highlights the underappreciated importance of facultative heterothermy in migratory ecology.

category: zoology


title: CD147 antibody specifically and effectively inhibits infection and cytokine storm of SARS-CoV-2 variants

abstract: SARS-CoV-2 and its variants are raging worldwide. Unfortunately, the global vaccination is not efficient enough to attain a vaccine-based herd-immunity and yet no special and effective drug is developed to contain the spread of the disease. Previously we have identified CD147 as a novel receptor for SARS-CoV-2 infection. Here, we demonstrated that CD147 antibody effectively inhibits infection and cytokine storm caused by SARS-CoV-2 variants. In CD147KO VeroE6 cells, infections of SARS-CoV-2, its variants (B.1.1.7, B.1.351) and pseudovirus mutants (B.1.1.7, B.1.351, B.1.525, B.1.526 (S477N), B.1.526 (E484K), P.1, P.2, B.1.617.1, B.1.617.2) were decreased. Meanwhile, CD147 antibody effectively blocked the entry of variants and pseudomutants in VeroE6 cells, and inhibited the expression of cytokines. A model of SARS-CoV-2-infected hCD147 transgenic mice was constructed, which recapitulated the features of exudative diffuse alveolar damage and dynamic immune responses of COVID-19. CD147 antibody could effectively clear the virus and alveolar exudation, resolving the pneumonia. We found the elevated level of cyclophilin A (CyPA) in plasma of severe/critical cases, and identified CyPA as the most important proinflammatory intermediate causing cytokine storm. Mechanistically, spike protein of SARS-CoV-2 bound to CD147 and initiated the JAK-STAT pathway, which induced expression of CyPA. CyPA reciprocally bound to CD147, triggered MAPK pathway and consequently mediated the expression of cytokine and chemokine. In conclusion, CD147 is a critical target for SARS-CoV-2 variants and CD147 antibody is a promising drug to control the new wave of COVID-19 epidemic.

category: immunology


title: SAR11 Cells Rely on Enzyme Multifunctionality to Transport and Metabolize a Range of Polyamine Compounds

abstract:
In the ocean surface layer and cell culture, the polyamine transport protein PotD of SAR11 bacteria is often one of the most abundant proteins detected. Polyamines are organic cations produced by all living organisms and are thought to be an important component of dissolved organic matter (DOM) produced in planktonic ecosystems. We hypothesized that SAR11 cells transport and metabolize multiple polyamines and use them as sources of carbon and nitrogen. Metabolic footprinting and fingerprinting were used to measure the uptake of five polyamine compounds (putrescine, cadaverine, agmatine, norspermidine, and spermidine) in two SAR11 strains that represent the majority of SAR11 cells in the surface ocean environment, Ca. Pelagibacter st. HTCC7211 and C. P. ubique st. HTCC1062. Both strains transported all five polyamines and concentrated them to micromolar or millimolar intracellular concentrations. Both strains could use most of the polyamines to meet their nitrogen requirements, but we did not find evidence of use as carbon sources. We propose potABCD transports cadaverine, agmatine, and norspermidine, in addition to its usual substrates of spermidine and putrescine, and that spermidine synthase, speE, is reversible, catalyzing the breakdown of spermidine and norspermidine, in addition to its usual biosynthetic role. These findings provide support for the hypothesis that enzyme multifunctionality enables streamlined cells in planktonic ecosystems to increase the range of DOM compounds they oxidize.

ImportanceGenome streamlining in SAR11 bacterioplankton has resulted in a small repertoire of genes, yet paradoxically they consume a substantial fraction of primary production in the oceans. Enzyme multifunctionality is hypothesized to be an adaptation that increases the range of organic compounds oxidized by cells in environments where selection favors genome minimization. We provide experimental support for this hypothesis by demonstrating that SAR11 cells use multiple polyamine compounds and propose that a small set of multifunctional genes catalyze this metabolism. We also report polyamine uptake rates can exceed metabolism, resulting in high intracellular concentrations of these nitrogen-rich compounds and an increase in cell size. Increases in cytoplasmic solute concentrations during transient episodes of high nutrient exposure has previously been observed in SAR11 cells and may be a feature of their strategy for maximizing the share of labile DOM acquired when in competition with other cell types.

category: microbiology


title: Toxoplasma gondii Toxolysin 4 contributes to efficient parasite egress from host cells

abstract:
Egress from host cells is an essential step in the lytic cycle of T. gondii and other apicomplexan parasites; however, only a few parasite secretory proteins are known to affect this process. The putative metalloproteinase Toxolysin 4 (TLN4) was previously shown to be an extensively processed microneme protein, but further characterization was impeded by the inability to genetically ablate TLN4. Herein we show that TLN4 has the structural properties of an M16 family metalloproteinase, that it possesses proteolytic activity on a model substrate, and that genetic disruption of TLN4 reduces the efficiency of egress from host cells. Complementation of the knockout strain with the TLN4 coding sequence significantly restored egress competency, affirming that the phenotype of the {Delta}tln4 parasite was due to the absence of TLN4. This work identifies TLN4 as the first metalloproteinase and the second microneme protein to function in T. gondii egress. The study also lays a foundation for future mechanistic studies defining the precise role of TLN4 in parasite exit from host cells.

IMPORTANCEAfter replicating within infected host cells, the single celled parasite Toxoplasma gondii must rupture out of such cells in a process termed egress. Although it is known that T. gondii egress is an active event that involves disruption of host-derived membranes surrounding the parasite, very few proteins that are released by the parasite are known to facilitate egress. In this study we identify a parasite secretory protease that is necessary for efficient and timely egress, thus laying the foundation for understanding precisely how this protease facilitates T. gondii exit from host cells.


title: Metagenomic sequencing for rapid identification of Xylella fastidiosa from leaf samples

abstract:
Xylella fastidiosa (Xf) is a globally distributed plant pathogenic bacterium. The primary control strategy for Xf diseases is eradicating infected plants; therefore, timely and accurate detection is necessary to prevent crop losses and further pathogen dispersal. Conventional Xf diagnostics primarily relies on quantitative PCR (qPCR) assays. However, these methods do not consider new or emerging variants due to pathogen genetic recombination and sensitivity limitations. We developed and tested a metagenomics pipeline using in-house short-read sequencing as a complementary approach for affordable, fast, and highly accurate Xf detection. We used metagenomics to identify Xf to strain level in single and mixed infected plant samples at concentrations as low as one picogram of bacterial DNA per gram of tissue. We also tested naturally infected samples from various plant species originating from Europe and the United States. We identified Xf subspecies in samples previously considered inconclusive with real-time PCR (Cq > 35). Overall, we showed the versatility of the pipeline by using different plant hosts and DNA extraction methods. Our pipeline provides taxonomic and functional information for Xf diagnostics without extensive knowledge of the disease. We hope this pipeline can be used for early detection of Xf and incorporated as a tool to inform disease management strategies.

IMPORTANCEXylella fastidiosa (Xf) destructive outbreaks in Europe highlight this pathogens capacity to expand its host range and geographical distribution. The current disease diagnostic approaches are limited by a multiple-step process, biases to known sequences, and detection limits. We developed a low-cost, user-friendly metagenomic sequencing tool for Xf detection. In less than three days, we were able to identify Xf subspecies and strains in field-collected samples. Overall, our pipeline is a diagnostics tool that could be easily extended to other plant-pathogen interactions and implemented for emerging plant threat surveillance.


title: Assessing antibiotic tolerance of Staphylococcus aureus derived directly from patients by the Replica Plating Tolerance Isolation System- REPTIS

abstract:
Antibiotic tolerant Staphylococcus aureus pose a great challenge to clinicians as well as to microbiological laboratories and are one reason for treatment failure. Antibiotic tolerant strains survive transient antibiotic exposure despite being fully susceptible in vitro. Thus, fast and reliable methods to detect tolerance in the routine microbiology laboratory are urgently required.

We therefore evaluated the feasibility of the replica plating tolerance isolation system (REPTIS) to detect antibiotic tolerance in S. aureus isolates derived directly from patients suffering from different types of infections and investigated possible connections to clinical presentations and patient characteristics.

One hundred twenty-five S. aureus isolates were included. Replica plating of the original resistance testing plate was used to assess regrowth in the zones of inhibition, indicating antibiotic tolerance. Bacterial regrowth was assessed after 24 and 48 hours of incubation and an overall regrowth score (ORS) was assigned. Regrowth scores were compared to the clinical presentation.

Bacterial regrowth was high for most antibiotics targeting protein synthesis and relatively low for antibiotics targeting other cellular functions such as DNA-replication, transcription and cell wall synthesis, with the exception of rifampicin. Isolates with a blaZ penicillinase had lower regrowth in penicillin and ampicillin. Low ORSs were more prevalent among isolates recovered from patients with immunosuppression or methicillin-resistant S. aureus (MRSA) isolates.

In conclusion, REPTIS is useful to detect antibiotic tolerance in clinical microbiological routine diagnostics. Rapid detection of antibiotic tolerance offers a new diagnostic readout that might allow more tailored treatments in the future.


title: Analysis of nucleic acids extracted from rapid diagnostic tests reveals a significant proportion of false positive test results associated with recent malaria treatment

abstract: Surveillance programs often use malaria rapid diagnostic tests (RDTs) to determine the proportion of the population carrying parasites in their peripheral blood to assess the malaria transmission intensity. Despite an increasing number of reports on false-negative and false-positive RDT results, there is a lack of systematic quality control activities for RDTs deployed in malaria surveillance programs. Our study provides a larger scale comparative evaluation of RDTs used in the 2018 Malaria Indicator Survey (MIS) conducted on Bioko Island, Equatorial Guinea. We conducted a molecular analysis by extraction of nucleic acids from 1,800 negative and 1,065 positive RDTs followed by qPCR analysis. These results were combined with a dataset collected in a comprehensive questionnaire from each MIS participant. Of the 2,865 RDTs that were collected in 2018 on Bioko Island and analysed in our study, 4.7% had a false-negative result. These false-negative RDT results were associated with low parasite density infections. In a substantial proportion of samples, we identified masked pfhrp2 and pfhrp3 gene deletions in which at least one P. falciparum strain carried a gene deletion. Among all positive RDTs analysed, 28.4% were tested negative by qPCR and therefore considered to be false-positive. Analysing the questionnaire data collected from the participants, this high proportion of false-positive RDT results could be explained by PfHRP2 antigen persistence after recent malaria treatment. We conclude that malaria surveillance depending solely on RDTs needs well-integrated quality control procedures assessing the extend and impact of reduced sensitivity and specificity of RDTs on malaria control programs.


title: The amino acid substitution affects cellular response to mistranslation

abstract: Mistranslation, the mis-incorporation of an amino acid not specified by the "standard" genetic code, occurs in all organisms. tRNA variants that increase mistranslation arise spontaneously and engineered tRNAs can achieve mistranslation frequencies approaching 10% in yeast and bacteria. Interestingly, human genomes contain tRNA variants with the potential to mistranslate. Cells cope with increased mistranslation through multiple mechanisms, though high levels cause proteotoxic stress. The goal of this study was to compare the genetic interactions and the impact on transcriptome and cellular growth of two tRNA variants that mistranslate at a similar frequency but create different amino acid substitutions in Saccharomyces cerevisiae. One tRNA variant inserts alanine at proline codons whereas the other inserts serine for arginine. Both tRNAs decreased growth rate, with the effect being greater for arginine to serine than for proline to alanine. The tRNA that substituted serine for arginine resulted in a heat shock response. In contrast, heat shock response was minimal for proline to alanine substitution. Further demonstrating the significance of the amino acid substitution, transcriptome analysis identified unique up- and downregulated genes in response to each mistranslating tRNA. Number and extent of negative synthetic genetic interactions also differed depending upon type of mistranslation. Based on the unique responses observed for these mistranslating tRNAs, we predict that the potential of mistranslation to exacerbate diseases caused by proteotoxic stress depends on the tRNA variant. Furthermore, based on their unique transcriptomes and genetic interactions, different naturally occurring mistranslating tRNAs have the potential to negatively influence specific diseases.

category: molecular biology


title: Transcriptomic profiling of human orbital fat and differentiating orbital fibroblasts

abstract:
Structured AbstractO_ST_ABSPurposeC_ST_ABSOrbital fat hyperplasia has a central role in the manifestations of thyroid-associated orbitopathy (TAO). To better understand the pathways involved in adipogenesis in TAO, we have used transcriptomic methods to analyze gene expression in control and TAO patients, as well as in differentiating orbital fibroblasts (OFs).

MethodsWe performed bulk RNA sequencing (RNA-Seq) on intraconal orbital fat to compare gene expression in control and TAO patients. We treated cultured OFs derived from TAO patients with media containing dexamethasone, insulin, rosiglitazone, and isobutylmethylxanthine (IBMX) to induce adipogenesis. We used single nuclear RNA-Seq (snRNA-Seq) profiling of treated OFs to compare gene expression over time in order to identify pathways that are involved in orbital adipogenesis in vitro and compared the dynamic patterns of gene expression identify differences in gene expression in control and TAO orbital fat.

ResultsOrbital fat from TAO and control patients segregate with principal component analysis (PCA). Numerous signaling pathways are enriched in orbital fat isolated from TAO patients. SnRNA-Seq of orbital fibroblasts undergoing adipogenesis reveals differential expression of adipocyte-specific genes over the developmental time course. Furthermore, genes that are enriched in TAO orbital fat are also upregulated in orbital adipocytes that differentiate in vitro, while genes that are enriched in control orbital fat are enriched in orbital fibroblasts prior to differentiation.

ConclusionsDifferentiating orbital fibroblasts serve as a model to study orbital fat hyperplasia seen in TAO. We demonstrate that the insulin-like growth factor-1 receptor (IGF-1R) and Wnt signaling pathways are differentially expressed early in orbital adipogenesis.

PrecisTo understand the pathways involved in adipogenesis in TAO, we used transcriptomic methods to analyze gene expression in control and TAO patients, as well as in differentiating OFs. We demonstrate that the IGF-1R and Wnt signaling pathways are differentially expressed during orbital adipogenesis.


title: Human norovirus infection of primary B cells triggers immune activation in vitro

abstract:
Human norovirus (HNoV) is a global health and socio-economic burden, estimated to infect every individual at least five times during their lifetime. The underlying mechanism for the potential lack of long-term immune protection from HNoV infections is not understood and prompted us to investigate HNoV susceptibility of primary human B cells and its functional impact. Primary B cells isolated from whole-blood were infected with HNoV-positive stool samples and harvested 3 days post infection (dpi) to assess viral RNA yield by RT-qPCR. A 3-18 fold increase in HNoV RNA yield was observed in 50-60% donors. Infection was further confirmed in B cells derived from splenic and lymph node biopsies. Next, we characterized infection of whole-blood derived B cells by flow cytometry in specific functional B cell subsets (naive CD27-IgD+, memory switched CD27+IgD-, memory unswitched CD27+IgD+ and double-negative CD27-IgD-). While susceptibility of subsets was similar, we observed changes in B cell subsets distribution upon infection that were recapitulated after treatment with HNoV virus-like particles and mRNA encoding for HNoV NS1-2 protein. Importantly, treatment of immortalized BJAB B cell lines with the predicted recombinant NS1 protein triggered cell proliferation, increased ATP production, and induced metabolic changes, as detected by means of CFSE/Ki67 staining, seahorse analysis and metabolomics, respectively. These data demonstrate the susceptibility of primary B cells to HNoV infection and suggest that the secreted NS1 protein affects B cell function, proliferation and metabolism in vitro, which could have implications for viral pathogenesis and immune response in vivo.

ImportanceHuman norovirus (HNoV) is the most prevalent causative agent of gastroenteritis worldwide. Infection results in a self-limiting disease that can become chronic and severe in the immunocompromised, elderly and infants. There are currently no approved therapeutic and preventative strategies to limit the health and socio-economic burden associated with HNoV infections. Moreover, HNoV does not elicit life-long immunity as repeat infections are common, presenting a challenge for vaccine development. Given the importance of B cells for humoral immunity, we investigated susceptibility and impact of HNoV infection on human B cells. We found that HNoV replicates in human primary B cells derived from blood, spleen and lymph nodes specimens and induces functional changes in B cells, mediated in part by the non-structural protein NS1. Because of the secreted nature of NS1, we put forward the hypothesis that HNoV infection can modulate bystander B cell function with potential implications in systemic immune response.


title: Zika virus infection of pregnant Ifnar1-/- mice triggers strain-specific differences in fetal outcomes

abstract:
Zika virus (ZIKV) is a flavivirus that causes a constellation of adverse fetal outcomes collectively termed Congenital Zika Syndrome (CZS). However, not all pregnancies exposed to ZIKV result in an infant with apparent defects. During the 2015-2016 American outbreak of ZIKV, CZS rates varied by geographic location. The underlying mechanisms responsible for this heterogeneity in outcomes have not been well defined. Therefore, we sought to characterize and compare the pathogenic potential of multiple Asian/American-lineage ZIKV strains in an established Ifnar1-/- pregnant mouse model. Here, we show significant differences in the rate of fetal demise following maternal inoculation with ZIKV strains from Puerto Rico, Panama, Mexico, Brazil, and Cambodia. Rates of fetal demise broadly correlated with maternal viremia but were independent of fetus and placenta virus titer, indicating that additional underlying factors contribute to fetus outcome. Our results, in concert with those from other studies, suggest that subtle differences in ZIKV strains may have important phenotypic impacts. With ZIKV now endemic in the Americas, greater emphasis needs to be placed on elucidating and understanding the underlying mechanisms that contribute to fetal outcome.

IMPORTANCEZika virus (ZIKV) actively circulates in 89 countries and territories around the globe. ZIKV infection during pregnancy is associated with adverse fetal outcomes including birth defects, microcephaly, neurological complications, and even spontaneous abortion. Rates of adverse fetal outcomes vary between regions, and not every pregnancy exposed to ZIKV results in birth defects. Not much is known about how or if the infecting ZIKV strain is linked to fetal outcomes. Our research provides evidence of phenotypic heterogeneity between Asian/American-lineage ZIKV strains and provides insight into the underlying causes of adverse fetal outcomes. Understanding ZIKV strain-dependent pathogenic potential during pregnancy and elucidating underlying causes of diverse clinical sequelae observed during human infections is critical to understanding ZIKV on a global scale.


title: S-Palmitoylation-Dependent Regulation of Cardiomyocyte Rac1 Signaling Activity and Cardiac Hypertrophy

abstract: S-palmitoylation is a reversible lipid modification that regulates trafficking, localization, activity, and/or stability of protein substrates by serving as a fatty acid anchor to cell membranes. However, S-palmitoylation-dependent control of signal transduction in cardiomyocytes and its effects on cardiac physiology are not well understood. We performed an in vivo gain-of-function screen of zinc finger Asp-His-His-Cys (zDHHC) family S-acyl transferases that catalyze S-palmitoylation and identified the Golgi-localized enzyme zDHHC3 as a critical regulator of cardiac maladaptation. The closely-related enzyme, zDHHC7, also induced severe cardiomyopathy but this effect was not observed with overexpression of plasma membrane enzyme zDHHC5, endoplasmic reticulum enzyme zDHHC6, or Golgi enzyme zDHHC13. To identify effectors that may underlie zDHHC3-induced cardiomyopathy we performed quantitative site-specific S-acyl proteomics in zDHHC3-overexpressing cells that revealed the small GTPase Rac1 as a novel substrate. We generated cardiomyocyte-specific transgenic mice overexpressing zDHHC3, which develop severe cardiac disease. Cardiomyopathy and congestive heart failure in zDHHC3 transgenic mice are preceded by enhanced S-palmitoylation of Rac1 and induction of additional Rho family small GTPases including RhoA, Cdc42, and the Rho family-specific chaperone RhoGDI. In contrast, transgenic mice overexpressing an enzymatically-dead mutant of zDHHC3 do not exhibit this profound induction of RhoGTPase signaling or develop cardiac disease. Rac1 S-palmitoylation, plasma membrane localization, activity, and downstream hypertrophic signaling were substantially increased in zDHHC3 overexpressing hearts. Taken together, these data suggest inhibition of zDHHC3/7 S-acyl transferase activity at the cardiomyocyte Golgi or disruption of Rac1 S-palmitoylation as novel therapeutic strategies to treat cardiac disease or other diseases associated with enhanced RhoGTPase signaling.


title: Liquid-Liquid Phase Separation Protects Amyloidogenic and Aggregation-Prone Peptides in Heterologous Expression Systems

abstract: Studying pathogenic effects of amyloids requires homogeneous amyloidogenic peptide samples. Recombinant production of these peptides is challenging due to their susceptibility to aggregation and chemical modifications. Thus, chemical synthesis is primarily used to produce amyloidogenic peptides suitable for high resolution structural studies. Here, we exploited the shielded environment of protein condensates formed via liquid-liquid phase separation (LLPS) as a protective mechanism against premature aggregation. We designed a fusion protein tag undergoing LLPS in E. coli and linked it to highly amyloidogenic peptides, including A{beta} amyloid. We find that the fusion proteins form membraneless organelles during overexpression and remain soluble. We also developed a facile purification method of functional A{beta} peptides free of chromatography steps. The strategy exploiting LLPS can be applied to other amyloidogenic, hydrophobic, and repetitive peptides that are otherwise difficult to produce.


title: Atto 465 derivative is a nuclear stain with unique excitation and emission spectra for multiplex immunofluorescence histochemistry

abstract: The architecture of a biologic response is inextricably linked with the tissue architecture of the target site. Multiplex immunofluorescence (mIF) is an effective technique for the maximal visualization of multiple target proteins in situ. This powerful tool is limited by the spectral overlap separation of the currently available synthetic fluorescent dyes. The fluorescence excitation wavelengths ranging between 405nm and 488nm are rarely used in mIF imaging and serve as a logical additional slot for a fluorescent probe. In the present study, we demonstrate that Atto 465-pentafluoroaniline (Atto 465-p), a derivative of the fluorescent dye Atto 465, can serve as a nuclear stain in the violet-blue region of the visible spectrum. This opens the 405nm channel, traditionally used for nuclear stain, for detection of another experimental target. This increases the flexibility of the mIF panel and, with appropriate staining and microscopy, enables the quantitative analysis of at least six targets in one tissue section.


title: Introduction of Surface Loops as a Tool for Encapsulin Functionalization.

abstract:
Encapsulin based protein cages are nanoparticles with different biomedical applications, such as targeted drug delivery or imaging agents. These particles are biocompatible and can be produced in bacteria, allowing large scale production and protein engineering. In order to use these bacterial nanocages in different applications, it is important to further explore the potential of their surface modification and optimize their production. In this study we design and show new surface modifications of the Thermotoga maritima (Tm) and Brevibacterium linens (Bl) encapsulins. Two new loops on Tm encapsulin with a His-tag insertion after the residue 64 and the residue 127, and the modification of the C-terminal on Bl encapsulin, are reported. The multi-modification of the Tm encapsulin enables up to 240 different functionalities on the cage surface, resulting from 4 potential modifications per protein subunit. We furthermore report an improved protocol giving a better stability and providing a notable increase of the production yield of the cages. Finally, we tested the stability of different encapsulin variants over a year and the results show a difference in stability arising from the tag insertion position. These first insights in the structure-property relationship of encapsulins, with respect to the position of a function loop, allow for further study of the use of these protein nanocages in biomedical applications.

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title: High-Throughput Measurement and Machine Learning-Based Prediction of Collision Cross Sections for Drugs and Drug Metabolites

abstract: Drug metabolite identification is a bottleneck of drug metabolism studies. Ion mobility-mass spectrometry (IM-MS) enables the measurement of collision cross section (CCS), a unique physical property related to an ions gas-phase size and shape, which can be used to increase the confidence in the identification of unknowns. A current limitation to the application of IM-MS to the identification of drug metabolites is the lack of reference CCS values. In this work, we present the production of a large-scale database of drug and drug metabolite CCS values, assembled using high-throughput in vitro drug metabolite generation and a rapid IM-MS analysis with automated data processing. Subsequently, we used this database to train a machine learning-based CCS prediction model, employing a combination of conventional 2D molecular descriptors and novel 3D descriptors. This novel prediction model enables the prediction of different CCS values for different protomers, conformers, and positional isomers for the first time.


title: Histone acetyltransferases and deacetylases are required for virulence, conidiation, DNA damage repair, and multiple stresses resistance of Alternaria alternata

abstract:
Histone acetylation, which is critical for transcriptional regulation and various biological processes in eukaryotes, is a reversible dynamic process regulated by HATs and HDACs. This study determined the function of 6 histone acetyltransferases (HATs) (Gcn5, RTT109, Elp3, Sas3, Sas2, Nat3) and 6 histone deacetylases (HDACs) (Hos2, Rpd3, Hda1, Hos3, Hst2, Sir2) in the phytopathogenic fungus Alternaria alternata by analyzing targeted gene deletion mutants. Our data provide evidence that HATs and HDACs are both required for mycelium growth, cell development and pathogenicity as many gene deletion mutants ({Delta}Gcn5,{Delta} RTT109,{Delta} Elp3,{Delta} Sas3,{Delta} Nat3,{Delta} Hos2, and{Delta} Rpd3) displayed reduced growth, conidiation or virulence at varying degrees. In addition, HATs and HDACs are involved in the resistance to multiple stresses such as oxidative stress (Sas3, Gcn5, Elp3, RTT109, Hos2), osmotic stress (Sas3, Gcn5, RTT109, Hos2), cell wall-targeting agents (Sas3, Gcn5, Hos2), and fungicide (Gcn5, Hos2).{Delta} Gcn5,{Delta} Sas3 and{Delta} Hos2 displayed severe growth defects on sole carbon source medium suggesting a vital role of HATs and HDACs in carbon source utilization. More SNPs were generated in{Delta} Gcn5 in comparison to wild-type when they were exposed to ultraviolet ray. Moreover,{Delta} RTT109,{Delta} Gcn5 and{Delta} Hos2 showed severe defects in resistance to DNA-damaging agents, indicating the critical role of HATs and HDACs in DNA damage repair. These phenotypes correlated well with the differentially expressed genes in{Delta} Gcn5 and{Delta} Hos2 that are essential for carbon sources metabolism, DNA damage repair, ROS detoxification, and asexual development. Furthermore, Gcn5 is required for the acetylation of H3K4. Overall, our study provides genetic evidence to define the central role of HATs and HDACs in the pathological and biological functions of A. alternata.

ImportanceIn filamentous fungi, HATs and HDACs have been found to be involved in growth, development, virulence, synthesis of secondary metabolites and multi-stress resistance. However, the role of all HATs and HDACs in the same fungal pathogen has not been systematically explored. Our study revealed that HATs and HDACs are required for the vegetative growth, conidiation, pathogenicity, multiple stresses resistance, DNA damage repair, and carbon source utilization in A. alternata. Moreover, HATs and HDACs identified in A. alternata are highly conserved in many notorious pathogenic fungi, such as Magnaporthe grisea and Candida albicans. Therefore, this study not only systematically reveals the biological functions of twelve histone acetylation-required enzymes for the first time, but also provides a foundation for future investigations in A. alternata or other pathogenic fungi.


title: Specific bacterial cell wall components influence the stability of Coxsackievirus B3

abstract:
Enteric viruses infect the mammalian gastrointestinal tract and lead to significant morbidity and mortality worldwide. Data indicate that enteric viruses can utilize intestinal bacteria to promote viral replication and pathogenesis. However, the precise interactions between enteric viruses and bacteria are unknown. Here we examined the interaction between bacteria and Coxsackievirus B3, an enteric virus from the picornavirus family. We found that bacteria enhance the infectivity of Coxsackievirus B3 (CVB3) in vitro. Notably, specific bacteria are required as gram-negative Salmonella enterica, but not Escherichia coli, enhanced CVB3 infectivity and stability. Investigating the cell wall components of both S. enterica and E. coli revealed that structures in the O-antigen or core of lipopolysaccharide, a major component of the gram-negative bacterial cell wall, were required for S. enterica to enhance CVB3. To determine if these requirements were necessary for similar enteric viruses, we investigated if S. enterica and E. coli enhanced infectivity of poliovirus, another enteric virus in the picornavirus family. We found that, in contrast to CVB3, these bacteria enhanced the infectivity of poliovirus in vitro. Overall, these data indicate that distinct bacteria enhance CVB3 infectivity and stability, and specific enteric viruses may have differing requirements for their interactions with specific bacterial species.

ImportancePrevious data indicate that several enteric viruses utilize bacteria to promote intestinal infection and viral stability. Here we show that specific bacteria and bacterial cell wall components are required to enhance infectivity and stability of Coxsackievirus B3 in vitro. These requirements are likely enteric virus-specific as the bacteria for CVB3 differs from poliovirus, a closely related virus. Therefore, these data indicate that specific bacteria and their cell wall components dictate the interaction with various enteric viruses in distinct mechanisms.


title: An innovative approach for determining composite wheat quality index to identify quality enriched genotypes - insights and implications

abstract: Ranking test entries or test sites on a quality basis is very difficult in wheat as value addition is perceived by several grain properties and end-products. Here, a novel approach has been developed and tested by deriving wheat quality index based on principal component analysis of 13 physico-chemical grain parameters and 3 end products of 45 wheat varieties. Depending upon the observed index range (0.15 to 0.71), the cultivars were assorted into 3 distinct classes i.e. elite, moderate and poor. The top group ascertained high quality standards of grain suited for bread and chapati whereas bottom group assured better cookies quality. This technique was also tested to differentiate quality enriched test sites within a zone or demarcate the most suited production environments to harness good quality wheat. The index will have an implication on farmers (premium price for varietal segregation), industry (product specific quality cultivars), and consumers (superior quality products).


title: Time-resolved scanning ion conductance microscopy for three-dimensional tracking of nanoscale cell surface dynamics

abstract: Nanocharacterization plays a vital role in understanding the complex nanoscale organization of cells and organelles. Understanding cellular function requires high-resolution information about how the cellular structures evolve over time. A number of techniques exist to resolve static nanoscale structure of cells in great detail (super-resolution optical microscopy1, EM2, AFM3). However, time-resolved imaging techniques tend to either have lower resolution, are limited to small areas, or cause damage to the cells thereby preventing long-term time-lapse studies. Scanning probe microscopy methods such as atomic force microscopy (AFM) combine high-resolution imaging with the ability to image living cells in physiological conditions. The mechanical contact between the tip and the sample, however, deforms the cell surface, disturbs the native state, and prohibits long-term time-lapse imaging. Here, we develop a scanning ion conductance microscope (SICM) for high-speed and long-term nanoscale imaging. By utilizing recent advances in nanopositioning4, nanopore fabrication5, microelectronics6, and controls engineering7 we developed a microscopy method that can resolve spatiotemporally diverse three-dimenional processes on the cell membrane at sub-5nm axial resolution. We tracked dynamic changes in live cell morphology with nanometer details and temporal ranges of sub-second to days, imagining diverse processes ranging from endocytosis, micropinocytosis, and mitosis, to bacterial infection and cell differentiation in cancer cells. This technique enables a detailed look at membrane events and may offer insights into cell-cell interactions for infection, immunology, and cancer research.


title: Gastrin producing syngeneic mesenchymal stem cells protect non-obese diabetic mice from type 1 diabetes

abstract: Progressive destruction of pancreatic islet {beta}-cells by immune cells is a primary feature of type 1 diabetes (T1D) and therapies that can restore the functional {beta}-cell mass are needed to alleviate disease progression. Here, we report the use of mesenchymal stromal/stem cells (MSCs) for the production and delivery of Gastrin, a peptide-hormone which is produced by intestinal cells and fetal islets and can increase {beta}-Cell mass, to promote protection from T1D. A single injection of syngeneic MSCs that were engineered to express Gastrin (Gastrin-MSCs) caused a significant delay in hyperglycemia in non-obese diabetic (NOD) mice compared to engineered control-MSCs. Similar treatment of early-hyperglycemic mice caused the restoration of euglycemia for a considerable duration, and these therapeutic effects were associated with protection of, and/or higher frequencies of, insulin producing islets and less severe insulitis. While the overall immune cell phenotype was not affected profoundly upon treatment using Gastrin-MSCs or upon in vitro culture, pancreatic lymph node cells from Gastrin-MSC treated mice, upon ex vivo challenge with self-antigen, showed a Th2 and Th17 bias, and diminished the diabetogenic property in NOD-Rag1 deficient mice suggesting a disease protective immune modulation under Gastrin-MSC treatment associated protection from hyperglycemia. Overall, this study shows the potential of production and delivery of Gastrin in vivo, by MSCs, in protecting insulin producing {beta}-cells and ameliorating the disease progression in T1D.


title: Lung Epithelial Cells Can Produce Antibodies Participating In Adaptive Humoral Immune Responses

abstract: It is generally believed that the main source of antibodies is B lymphocytes. In this study, our results first revealed that B cell-deficient mice could not produce antibodies specific for TI-Ags; however, mice with B cell deficiency could produce TD-Ag-specific antibodies, although antibody production was delayed compared with that in BALB/c mice after primary TD-Ag challenge. Subsequently, we identified that mouse lung epithelial cells could produce and secrete Ig, including IgM, IgA or IgG, which could display TD-Ag-specific antibody activity. Notably, the production of TD-Ag-specific antibodies by lung epithelial cells was found to be dependent on CD4+ T cells but not CD8+ T cells. Our findings indicate for the first time that B cells are not the only source of TD-Ag-specific antibodies but are essential for rapid TD-Ag-specific antibody production by non-B cells. This discovery may reveal a new mechanism for the production of specific antibodies.


title: Clinical Strains of Pseudomonas aeruginosa secret LasB protease to induce hemorrhagic diffused alveolar damage and acute lung injury in mice

abstract:
BackgroundAcute lung injury and acute respiratory distress syndrome are most often caused by bacterial pneumonia, characterized by severe dyspnea and high mortality. Knowledge about the lung injury effects of current clinical bacteria strains is lacking. The aim of this study was to investigate the ability of representative pathogenic bacteria isolated from patients to cause ALI/ARDS in mice and identify the virulent factors.

Method7 major bacteria species were isolated from clinical sputum and instilled in to mice airway unilaterally. Histology study was used to judge the lung injury effect. Virulence genes were examined by PCR. Sequence type of P. aeruginosa strains were identified by MLST. LC-MS/MS was used to identify the suspicious protein bands. LasB was purified through DEAE-cellulose column and its toxicity was tested both in vitro and in vivo.

ResultsStaphylococcus aureus, Streptococcus pneumoniae, Streptococcus agalactiae, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli were randomly separated and tested 3 times. Among them, gram-negative bacteria are more potential than gram-positive bacteria to cause acute lung injury. However, P. aeruginosa is the only pathogen which induced diffused alveolar damage, hemorrhage and hyaline membrane in the lung of mice. The lung injury effect is associated to the excreted matrix metalloproteinase LasB of P. aeruginosa. Purified LasB recapitulated hemorrhagic acute lung injury identical to P. aeruginosa infection in vivo. We found this was due to the powerful degradation effect of LasB on both lung extracellular matrix and key proteins in coagulation cascade without inducing cellular apoptosis.

ConclusionP. aeruginosa strains are most capable to induce ALI/ARDS among major clinical pathogenic bacteria, this ability is specifically attributed to their LasB production.


title: Evolutionary assembly of cooperating cell types in an animal chemical defense system

abstract: A long-standing challenge in biology is explaining how the functions of multicellular organs emerge from the underlying evolution of cell types. We deconstructed evolution of an organ novelty: a rove beetle gland that secretes a defensive cocktail. We show that gland function was pieced together via assembly of two cell types that manufacture distinct compounds. One cell type forms a chemical reservoir in the beetles abdomen and produces alkane and ester compounds. We demonstrate that this cell type is a hybrid of cuticle cells and ancient pheromone and adipocyte-like cells, and executes its function via a mosaic of enzymes sourced from each parental cell type. The second cell type synthesizes noxious benzoquinones using a chimeric pathway derived from conserved cellular energy and cuticle formation pathways. We present evidence that evolution of each cell type was shaped by coevolution between the two cell types: the benzoquinones produced by the second cell type dissolve in solvents produced by the first, yielding a potent secretion that confers adaptive value onto the gland as a whole. Our findings illustrate how cooperation between cell types can arise, generating new, organ-level behaviors.


title: Optimizing parameters for using the parallel auditory brainstem response (pABR) to quickly estimate hearing thresholds

abstract:
ObjectivesTimely assessments are critical to providing early intervention and better hearing and spoken language outcomes for children with hearing loss. To facilitate faster diagnostic hearing assessments in infants, the authors developed the parallel auditory brainstem response (pABR), which presents randomly timed trains of tone pips at five frequencies to each ear simultaneously. The pABR yields high-quality waveforms that are similar to the standard, single-frequency serial ABR but in a fraction of the recording time. While well-documented for standard ABRs, it is yet unknown how presentation rate and level interact to affect responses collected in parallel. Furthermore, the stimuli are yet to be calibrated to perceptual thresholds. Therefore, this study aimed to determine the optimal range of parameters for the pABR and to establish the normative stimulus level correction values for the ABR stimuli.

DesignTwo experiments were completed, each with a group of 20 adults (18 - 35 years old) with normal hearing thresholds ([≤] 20 dB HL) from 250 to 8000 Hz. First, pABR electroencephalographic (EEG) responses were recorded for six stimulation rates and two intensities. The changes in component wave V amplitude and latency were analyzed, as well as the time required for all responses to reach a criterion signal-to-noise ratio of 0 dB. Second, behavioral thresholds were measured for pure tones and for the pABR stimuli at each rate to determine the correction factors that relate the stimulus level in dB peSPL to perceptual thresholds in dB nHL.

ResultsThe pABR showed some adaptation with increased stimulation rate. A wide range of rates yielded robust responses in under 15 minutes, but 40 Hz was the optimal singular presentation rate. Extending the analysis window to include later components of the response offered further time-saving advantages for the temporally broader responses to low frequency tone pips. The perceptual thresholds to pABR stimuli changed subtly with rate, giving a relatively similar set of correction factors to convert the level of the pABR stimuli from dB peSPL to dB nHL.

ConclusionsThe optimal stimulation rate for the pABR is 40 Hz, but using multiple rates may prove useful. Perceptual thresholds that subtly change across rate allow for a testing paradigm that easily transitions between rates, which may be useful for quickly estimating thresholds for different configurations of hearing loss. These optimized parameters facilitate expediency and effectiveness of the pABR to estimate hearing thresholds in a clinical setting.

HighlightsO_LIThe pABR yields robust responses across stimulus rates and intensities.
C_LIO_LIThe optimal rate is 40 Hz, but using multiple rates may prove useful.
C_LIO_LIThe pABR shows some adaptation with increased stimulation rate.
C_LIO_LIExtended analysis windows improve response detection for low stimulus frequencies.
C_LIO_LIBehavioral thresholds subtly change across pABR rate, giving similar dB nHL values.
C_LI

category: neuroscience