title: Distinct roles of glutamine metabolism in benign and malignant cartilage tumors with IDH mutations

abstract: Enchondromas and chondrosarcomas are common cartilage neoplasms that are either benign or malignant respectively. The majority of these tumors harbor mutations in either IDH1 or IDH2. Glutamine metabolism has been implicated as a critical regulator of tumors with IDH mutations. Chondrocytes and chondrosarcomas with mutations in the IDH1 or IDH2 genes showed enhanced glutamine utilization in downstream metabolism. Using genetic and pharmacological approaches, we demonstrated that glutaminase-mediated glutamine metabolism played distinct roles in enchondromas and chondrosarcomas with IDH1 or IDH2 mutations. Deletion of glutaminase in chondrocytes with Idh1 mutation increased the number and size of enchondroma-like lesions. Pharmacological inhibition of glutaminase in chondrosarcoma xenografts reduced overall tumor burden. Glutamine affected cell differentiation and viability in these tumors differently through different downstream metabolites. During murine enchondroma-like lesion development, glutamine-derived -ketoglutarate promoted hypertrophic chondrocyte differentiation and regulated chondrocyte proliferation. In human chondrosarcoma, glutamine-derived non-essential amino acids played an important role in preventing cell apoptosis. This study reveals that glutamine metabolism can play distinct roles in benign and malignant cartilage tumors sharing the same genetic mutations. Inhibiting GLS may provide a therapeutic approach to suppress chondrosarcoma tumor growth.

category: cancer biology


title: Elevated METTL9 is associated with peritoneal dissemination in human scirrhous gastric cancers

abstract:
Methylation, the most common chemical modification of cellular components such as DNA, RNA, and proteins, impacts biological processes including transcription, RNA processing, and protein dynamics. Although abnormal expression of methyltransferase can lead to various diseases including cancers, little is known about the relationship between methyltransferase and cancers. Here we aimed to understand the role of methyltransferase in cancer metastasis. We found that elevated methyltransferase-like 9 (METTL9) is closely associated with the acquisition of metastatic activity in human scirrhous gastric cancers. The stable knockdown of METTL9 via an shRNA vector technique in our original metastatic cells from scirrhous gastric cancer patients significantly inhibited migration and invasion. In metastatic cells, METTL9 protein is predominantly localized in mitochondria, and the METTL9 knockdown significantly reduced mitochondrial Complex I activity. METTL9 can be a promising molecular target to inhibit peritoneal dissemination of scirrhous gastric cancers. This report is the first to describe the relationship between METTL9 and cancer metastasis.

HighlightsO_LIElevated METTL9 correlates with metastasis in human scirrhous gastric cancer.
C_LIO_LIThis is the first report on the biological relationship between METTL9 and metastasis.
C_LIO_LIMETTL9 protein localizes mainly in mitochondria in metastatic scirrhous gastric cancer.
C_LIO_LIMETTL9 knockdown reduces mitochondrial Complex I activity to decrease cell migration and invasion in metastatic scirrhous gastric cancer.
C_LIO_LIMETTL9 holds promise against peritoneal dissemination of scirrhous gastric cancer.
C_LI


title: Elevated levels of active Transforming Growth Factor β1 in the subchondral bone relate spatially to cartilage loss and impaired bone quality in human knee osteoarthritis

abstract:
ObjectiveOver-activity of transforming growth factor {beta}1 (TGF{beta}1) in subchondral bone has a direct causal role in rodent models of knee osteoarthritis (OA), which can be blocked by TGF{beta}1 neutralisation. In this study, we investigated whether the spatially distributed level of active TGF{beta}1 in human subchondral bone associates with the characteristic structural, cellular and molecular parameters of human knee OA.

DesignSubchondral bone samples (35 OA arthroplasty patients, aged 69{+/-}9 years) were obtained from regions below either macroscopically present or denuded cartilage. Bone samples were processed to determine the concentration of active TGF{beta}1 (ELISA) and gene-specific mRNA expression (RT-PCR). Synchrotron micro-CT imaging was utilised to assess the bone microstructure, bone mineralization, the osteocyte lacunar network and bone matrix vascularity. Finally, samples were histologically examined for cartilage OARSI grading, quantification of tartrate resistant acid phosphatase positive cells and bone marrow micro-vasculature.

ResultsSubchondral bone below severely degenerated/depleted cartilage, characterised by impaired bone matrix quality due to sclerotic microarchitecture, disorganised collagen, high heterogeneity of the mineral distribution, contained increased concentrations of active TGF{beta}1, compared to adjacent areas with more intact cartilage. In addition, increased levels of active TGF{beta}1 related directly to increased bone volume while increased OARSI grade associated directly with morphometric characteristics (size, shape and orientation) of osteocyte lacunae.

ConclusionThese results indicate that increased active TGF{beta}1 associates spatially with impaired bone quality and the disease severity of human OA. This study therefore suggests that TGF{beta}1 could be a therapeutic target to prevent or reduce human disease progression.

category: pathology


title: Systemic and local immune response to intraocular AAV vector administration in non-human primates

abstract: The positive clinical outcomes in adeno-associated virus (AAV)-mediated retinal gene therapy have often been attributed to the low immunogenicity of AAVs along with the immune-privilege of the eye. However, several recent preclinical studies and clinical trials have shown potential for inflammatory responses to AAV mediated gene therapy. Our current understanding of the factors contributing to intraocular inflammation such as the existence of serum antibodies against AAVs prior to injection and their contribution to increases in antibody levels post-injection is incomplete. The parameters that regulate the generation of new antibodies in response to the AAV capsid or transgene post-injection after intraocular administration are also insufficiently described. In this study we carried out a retrospective analysis of the pre-existing serum antibodies in correlation with changes in antibody levels after intraocular injections of AAV in non-human primates (NHPs). We analyzed NHP serums for the presence of both Binding Antibodies (BABs), as well as a subset of these called Neutralizing Antibodies (NABs) that impede AAV transduction upon binding. We observed significantly higher pre-existing serum BABs against AAV8 compared to other serotypes. We observed a dose-dependent increase in both BABs and NABs in the serums collected post-injection, irrespective of the serotype or the mode of injection. Lastly, we were able to demonstrate a co-relation between the serum BAB levels with clinical grading of inflammation and levels of transgene expression.

category: immunology


title: The consequences of reciprocally exchanging the genomic sites of Integration Host Factor (IHF) subunit production for subunit stoichiometry and bacterial physiology in Salmonella enterica serovar Typhimurium

abstract:
Integration host factor (IHF) is a heterodimeric nucleoid-associated protein that plays roles in bacterial nucleoid architecture and genome-wide gene regulation. The ihfA and ihfB genes encode the subunits and are located 350 kilobase pairs apart, in the Right replichore of the Salmonella chromosome. IHF is composed of one IhfA and one IhfB subunit. Despite this 1:1 stoichiometry, mass spectrometry revealed that IhfB is produced in 2-fold excess over IhfA. We re-engineered Salmonella to exchange reciprocally the protein-coding regions of ihfA and ihfB, such that each relocated protein-encoding region was driven by the expression signals of the others gene. Mass spectrometry showed that in this  rewired strain, IhfA is produced in excess over IhfB, correlating with enhanced stability of the hybrid ihfB-ihfA mRNA that was expressed from the ihfB promoter. Nevertheless, the rewired strain grew at a similar rate to the wild type, had identical cell morphology, and was similar in competitive fitness. However, compared to the wild type, it was less motile, had growth-phase-specific reductions in SPI-1 and SPI-2 gene expression and was engulfed at a higher rate by RAW macrophage. Our data show that while exchanging the physical locations of its ihf genes and the rewiring of their regulatory circuitry are well tolerated in Salmonella, genes involved in the production of type 3 secretion systems exhibit dysregulation accompanied by altered phenotypes.

IMPACT STATEMENTIntegration Host Factor (IHF) is an abundant nucleoid-associated protein that organises DNA architecturally, influencing gene expression globally in Salmonella and other bacteria. IHF is composed of two related, non-identical, subunits, produced by genes that are 350 kilobase pairs apart. Each ihf gene has unique expression controls and is embedded in a complex genetic network that supports mRNA translation. Given that the subunits are thought to be required in a 1:1 ratio to form functional IHF, we were surprised by this physical and regulatory separation. We rewired the Salmonella genome so that each subunit was produced using the others regulatory signals and gene location. This revealed a high degree of tolerance to the effects of this rewiring. However, we discovered that bacterial motility was disrupted, as was the expression of virulence genes that have been acquired by horizontal gene transfer. Proteomic analysis using mass spectroscopy (MS) showed the extent of the alterations to cell composition. Our MS data also demonstrated that the subunits of IHF are not produced in a 1:1 ratio in either the wild type or the rewired strain. We discuss this finding in terms of the ability of each subunit to stabilise its partner.

DATA SUMMARYO_LIWhole genome sequence data for strain OrfSwapihfA-ihfB are available from the European Nucleotide Archive with accession number ERS4653309.
C_LIO_LIData from mass spectrometry analyses are available via ProteomeXchange with identifier PXD027465 (login: reviewer_pxd027465@ebi.ac.uk and password: GVeNIUB2).
C_LIO_LIAll supporting data have been provided in the article or through supplementary data files.
C_LI

RepositoriesWhole genome sequence data for strain OrfSwapihfA-ihfB are available from the European Nucleotide Archive with accession number ERS4653309. Data from mass spectrometry analyses are available via ProteomeXchange with identifier PXD027465 (login: reviewer_pxd027465@ebi.ac.uk and password: GVeNIUB2).

category: microbiology


title: Structural architecture of a dimeric paramyxovirus polymerase complex

abstract: Human parainfluenza virus type 3 (hPIV3), a member of non-segmented, negative-strand RNA viruses (nsNSVs), is the second most common cause of severe respiratory disease in pediatrics. The transcription and replication processes of nsNSVs are catalyzed by a multi-functional RNA-dependent RNA polymerase (RdRp) complex composed of the large protein (L) and the phosphoprotein (P). Previous studies have shown that the polymerase can adopt a dimeric form, however, the structure of the dimer and how it functions are not understood. Here we determined the cryo-EM structure of hPIV3 L-P complex at 2.7 [A] with substantial structural details. A putative catalytic magnesium ion could be built in our structure, and structural comparison revealed atomic features conserved with other RNA viruses. Interactions identified between the two priming and intrusion loops and the connector domain potentially trigger the spatial movement of three C-terminal L domains for different steps of transcription and replication. Structural comparison with other nsNSV RdRps suggests common features of L-P binding. Furthermore, we report for the first time the structural basis of the L-L interaction in the partially modelled dimeric L-P structure, in which the connector domain of one L is positioned at the putative RNA template entry of the other L. Based on these findings, we propose a model by which L dimerization promotes efficient conversion of nascent RNA into a template.

category: molecular biology


title: Targeting mitochondrial double-stranded RNAs ameliorates autoimmune characteristics of Sjogrens syndrome

abstract:
ObjectiveSjogrens syndrome (SS) is a systemic autoimmune disease that targets the exocrine glands, resulting in impaired saliva and tear secretion. To date, type I interferons (I-IFNs) are increasingly recognized as pivotal mediators in SS, but their endogenous drivers have not been elucidated. This study investigates the role of mitochondrial double-stranded RNAs (mt-dsRNAs) in regulating I-IFN response in SS.

MethodsSaliva and tear from SS patients and controls (n=73 for saliva and n=16 for tear), the salivary glands of the SS-prone-non-obese-diabetic mouse, and primary human salivary glandular cells were screened for mt-dsRNAs by RT-qPCR. The human salivary cell line (NS-SV-AC) grown as three-dimensional spheroids were subject to dsRNA stress to measure mt-dsRNA induction and recapitulation of SS glandular inflammatory features. Acetylcholine, SS-IgG, upadacitinib (JAK1 inhibitor), or 2-C'-methyladenosine (mitochondrial transcription inhibitor) were applied to characterize the roles of mt-dsRNAs. To identify endogenous dsRNA-sensor and confirm the mitochondrial origin of cytoplasmic dsRNAs, the immunoprecipitation of dsRNAs was performed.

Resultsmt-dsRNAs were elevated in the SS specimens with salivary ND5 and tear CYTB1 being statistically associated with secretory dysfunction/inflammation and corneal/conjunctival damage, respectively. Stimulation of the spheroids with dsRNA stress of poly I:C induced mt-dsRNAs, p-PKR, and I-IFNS via the JAK1/STAT pathway whereas the inhibition of mt-RNA synthesis or JAK1 attenuated the glandular signature. The inhibitory effect of acetylcholine on mt-dsRNAs and I-IFNS induction was reversed by SS-IgG.

Conclusionmt-dsRNAs amplify the impact of dsRNA stress on SS glandular signatures in vitro, potentially propagating a pseudo-viral signal in the SS target tissue.

SummaryMitochondrial double-stranded RNA levels were elevated in the tear and saliva of SS patients, which was associated with secretory dysfunction and tissue inflammation. These RNAs amplified type I interferon signature as well as glandular phenotypes reported in SS. Inhibitors of mitochondrial RNA transcription or JAK1 in salivary gland acinar cell spheroids attenuated the mitochondrial RNA-mediated changes.


title: Mitochondrial double-stranded RNAs as a pivotal mediator in the pathogenesis of Sjogrens syndrome


title: Cell-based and cell-free firefly luciferase complementation assay to quantify Human Immunodeficiency Virus type 1 Rev-Rev interaction

abstract: Rev is an essential regulatory protein of Human Immunodeficiency Virus type 1 (HIV) that is found in the nucleus of infected cells. Rev multimerizes on the Rev-response element (RRE) of HIV RNA to facilitate the export of intron-containing HIV mRNAs from the nucleus to the cytoplasm, and, as such, HIV cannot replicate in the absence of Rev. We have developed cell-intact and cell-free assays based upon a robust firefly split-luciferase complementation system, both of which quantify Rev-Rev interaction. Using the cell-based system we show that additional Crm1 did not impact the interaction whereas excess Rev reduced it. Furthermore, when a series of mutant Revs were tested, there was a strong correlation between the results of the cell-based assay and the results of a functional Rev trans-complementation infectivity assay. Of interest, a camelid nanobody (NB) that was known to inhibit Rev function enhanced Rev-Rev interaction in the cell-based system. We observed a similar increase in Rev-Rev interaction in a cell-free system, when cell lysates expressing NLUC-Rev or CLUC-Rev were simply mixed. In the cell-free system Rev-Rev interaction occurred within minutes and was inhibited by excess Rev. The levels of interaction between the mutant Revs tested varied by mutant type. Treatment of Rev lysates with RNAse minimally reduced the degree of interaction whereas addition of HIV RRE RNA enhanced the interaction. Purified GST-Rev protein inhibited the interaction. The Z-factor (Z) for the cell-free system was ~0.85 when tested in 96-well format, and anti-Rev NB enhanced the interaction in the cell-free system. Thus, we have developed both cell-intact and cell-free systems that can reliably, rapidly, and reproducibly quantify Rev-Rev interaction. These assays, particularly the cell-free one, may be useful in screening and identifying compounds that inhibit Rev function on a high throughput basis.


title: Variants in ALDH1A2 reveal an anti-inflammatory role for retinoic acid and a new class of disease-modifying drugs in osteoarthritis

abstract:
Over 40% of individuals will develop osteoarthritis (OA) during their lifetime, yet there are currently no licensed disease modifying treatments for this disabling condition. Common polymorphic variants in ALDH1A2, that encodes the key enzyme in the synthesis of all-trans retinoic acid (atRA), have been associated with severe hand OA. In this study, we sought to elucidate the biological significance of this association. We first confirmed that ALDH1A2 risk variants were associated with hand OA in UK Biobank. Articular cartilage was acquired from 33 consenting individuals with hand OA at the time of routine hand OA surgery. They were stratified by genotype and RNA sequencing performed. A reciprocal relationship between ALDH1A2 mRNA and inflammatory genes was observed. Articular cartilage injury up-regulates similar inflammatory genes by a process that we have previously termed mechanoflammation, and which we believe is a primary driver of OA. Cartilage injury was also associated with a concomitant drop in atRA-dependent genes, indicative of cellular atRA levels, and both responses to injury were reversed using talarozole, a retinoic acid metabolism blocking agent (RAMBA). Suppression of mechanoflammation by talarozole was mediated by a peroxisome proliferator activated receptor (PPAR)-{gamma} dependent mechanism. Talarozole, delivered by minipump, was able to suppress mechano-inflammatory genes in articular cartilage in vivo 6h after mouse knee joint destabilization, and reduced cartilage degradation and osteophyte formation after 4 weeks. These data show that boosting atRA suppresses mechanoflammation in the articular cartilage in vitro and in vivo, and identifies RAMBAs as potential disease modifying drugs in OA.

One Sentence SummaryAnalysis of hand OA cartilage stratified by ALDH1A2 polymorphic variants reveals a targetable, anti-inflammatory role for retinoic acid in OA.


title: The key features of SARS-CoV-2 leader and NSP1 required for viral escape of NSP1-mediated repression

abstract: SARS-CoV-2, responsible for the ongoing global pandemic, must overcome a conundrum faced by all viruses. To achieve its own replication and spread, it simultaneously depends on and subverts cellular mechanisms. At the early stage of infection, SARS-CoV-2 expresses the viral nonstructural protein 1 (NSP1), which inhibits host translation by blocking the mRNA entry tunnel on the ribosome; this interferes with the binding of cellular mRNAs to the ribosome. Viral mRNAs, on the other hand, overcome this blockade. We show that NSP1 enhances expression of mRNAs containing the SARS-CoV-2 leader. The first stem-loop (SL1) in viral leader is both necessary and sufficient for this enhancement mechanism. Our analysis pinpoints specific residues within SL1 (three cytosine residues at the positions 15, 19 and 20) and another within NSP1 (R124) which are required for viral evasion, and thus might present promising drug targets. Additionally, we carried out analysis of a functional interactome of NSP1 using BioID and identified components of anti-viral defense pathways. Our analysis therefore suggests a mechanism by which NSP1 inhibits the expression of host genes while enhancing that of viral RNA. This analysis helps reconcile conflicting reports in the literature regarding the mechanisms by which the virus avoids NSP1 silencing.


title: Glucose controls co-translation of structurally related mRNAs via the mTOR and eIF2 pathways in human pancreatic beta cells

abstract:
Pancreatic beta cell response to glucose is critical for the maintenance of normoglycemia. A strong transcriptional response was classically described in rodent models but, interestingly, not in human cells. In this study, we exposed human pancreatic beta cells to an increased concentration of glucose and analysed at a global level the mRNAs steady state levels and their translationalability. Polysome profiling analysis showed an early acute increase in protein synthesis and a specific translation regulation of more than 400 mRNAs, independently of their transcriptional regulation. We clustered the co-regulated mRNAs according to their behaviour in translation in response to glucose and discovered common structural and sequence mRNA features. Among them mTOR- and eIF2-sensitive elements have a predominant role to increase mostly the translation of mRNAs encoding for proteins of the translational machinery. Furthermore, we show that mTOR and eIF2 pathways are independently regulated in response to glucose, participating to a translational reshaping to adapt beta cell metabolism. The early acute increase in the translation machinery components prepare the beta cell for further protein demand due to glucose-mediated metabolism changes.

AUTHOR SUMMARYAdaptation and response to glucose of pancreatic beta cells is critical for the maintenance of normoglycemia. Its deregulation is associated to Diabetic Mellitus (DM), a significant public health concern worldwide with an increased incidence of morbidity and mortality. Despite extensive research in rodent models, gene expression regulation in response to glucose remains largely unexplored in human cells. In our work, we have tackled this question by exposing human EndoC-BH1 cells to high glucose concentration. Using polysome profiling, the gold standard technique to analyse cellular translation activity, we observed a global protein synthesis increase, independent from transcription activity. Among the specific differentially translated mRNAs, we found transcripts coding for ribosomal proteins, allowing the cell machinery to be engaged in a metabolic response to glucose. Therefore, the regulation in response to glucose occurs mainly at the translational level in human cells, and not at the transcriptional level as described in the classically used rodent models.

Furthermore, by comparing the features of the differentially translated mRNAs, and classifying them according to their translational response, we show that the early response to glucose occurs through the coupling of mRNA structure and sequence features impacting translation and regulation of specific signalling pathways. Collectively, our results support a new paradigm of gene expression regulation on the translation level in human beta cells.

category: genomics


title: Stomatal responses at different vegetative stages of selected maize varieties of Bangladesh under water deficit condition

abstract: Drought stress causes stomatal behavior change in most plants. Water deficit condition caused by drought is one of the most significant abiotic factors reducing plant growth, development, reproductive efficiency, and photosynthesis, resulting in yield loss. Maize (Zea mays L.) holds a superior position among all the cereals due to its versatile use in the food, feed, and alcohol industries. A common demonstrative feature of a complex network of signaling pathways led by predominantly abscisic acid under drought conditions is stomatal aperture reduction or stomatal closure, which allows the plant to reduce water loss through the stomatal pore and to sustain a long time on water deficit condition. This study analyses the stomatal density, stomatal closure percentages, and guard cell aperture reduction using a microscopy-based rapid & simple method to compare guard cell response & morphological variations of three hybrid maize varieties viz. BHM (BARI hybrid maize)-7, BHM-9, and BHM-13 developed by Bangladesh Agricultural Research Institute (BARI). A drought treatment was applied to all varieties at two different vegetative stages, vegetative stage 3 (V3) and V5, until they reach V4 and V6, respectively. After drought exposure at the V4 stage, the percentage of closed stomata of BHM-7, BHM-9, and BHM-13 was 21%, 23%, and 33%, respectively. The reduction in the guard cell aperture ratio of BHM-7, BHM-9, and BHM-13 was 14.83%, 10.92%, and 33.85%, respectively. At the V6 stage, for the second set of plants, the closed stomata of BHM-7, BHM-9, and BHM-13 were 18%, 21%, and 34%, respectively. The rate of reduction in guard cell aperture ratio of BHM-7, BHM-9, and BHM-13 was 5.52%, 2.48%, and 38.75%, respectively. Therefore, BHM-13 showed maximum drought adaptation capacity compared to BHM-7 and BHM-9 due to the highest percentage of closed stomata and the highest percentage of reduction in aperture ratio.

category: plant biology


title: Detecting Phytophthora cinnamomi associated with dieback disease on Carya cathayensis using loop-mediated isothermal amplification

abstract: Chinese hickory (Carya cathayensis Sarg.) is an economically and ecologically important nut plant in China. Dieback and basal stem necrosis have been observed in the plants since 2016, and its recent spread has significantly affected plant growth and nut production. Therefore, a survey was conducted to evaluate the disease incidence at five sites in Linan County, China. The highest incidence was recorded at the Tuankou site at up to 11.39% in 2019. The oomycete, Phytophthora cinnamomi, was isolated from symptomatic plant tissue and plantation soil using baiting and selective media-based detection methods and identified. Artificial infection with the representative P. cinnamomi ST402 isolate produced vertically elongated discolorations in the outer xylem and necrotic symptoms in C. cathayensis seedlings in a greenhouse trial. Molecular detections based on loop-mediated isothermal amplification (LAMP) specific to P. cinnamomi ST402 were conducted. Result indicated that LAMP detection showed a high coherence level with the baiting assays for P. cinnamomi detection in the field. This study provides the evidence of existence of high-pathogenic P. cinnamomi in the C. cathayensis plantation soil in China and the insights into a convenient tool developed for conducting field monitoring of this aggressive pathogen.


title: Monitoring photogenic ecological phenomena: Social network site images reveal spatiotemporal phases of Japanese cherry blooms

abstract:
Some ecological phenomena are visually engaging and widely celebrated. Consequently, these have the potential to generate large footprints in the online and social media image records which may be valuable for ecological research. Cherry tree blooms are one such event, especially in Japan where they are a cultural symbol (Sakura, ). For centuries, the Japanese have celebrated Hanami (flower viewing) and the historical data record of the festival allows for phenological studies over this period, one application of which is climate reconstruction. Here we analyse Flickr social network site data in an analogous way to reveal the cherry blossoms seasonal sweep from southern to northern Japan over a twelve-week period.

Our method analyses data filtered using geographical constraints, multi-stage text-tag classification, and machine vision, to assess image content for relevance to our research question and use it to estimate historic cherry bloom times. We validated our estimated bloom times against official data, demonstrating the accuracy of the approach. We also investigated an out of season Autumn blooming that has gained worldwide media attention. Despite the complexity of human photographic and social media activity and the relatively small scale of this event, our method can reveal that this bloom has in fact been occurring over a decade.

The approach we propose in our case study enables quick and effective monitoring of the photogenic spatiotemporal aspects of our rapidly changing world. It has the potential to be applied broadly to many ecological phenomena of widespread interest.

category: ecology


title: Color, activity period, and eye structure in four lineages of ants: pale, nocturnal species have evolved larger eyes and larger facets than their dark, diurnal congeners

abstract: The eyes of insects display an incredible diversity of adaptations to enhance vision across the gamut of light levels that they experience. One commonly studied contrast is the difference in eye structure between nocturnal and diurnal species, with nocturnal species typically having features that enhance eye sensitivity such as larger eyes, larger eye facets, and larger ocelli. In this study, we compared eye structure between workers of closely related nocturnal and diurnal above ground foraging ant species (family Formicidae) in four genera (Myrmecocystus, Aphaenogaster, Temnothorax, Veromessor). In all four genera, nocturnal species tend to have little cuticular pigment (pale), while diurnal species are fully pigmented (dark), hence we could use cuticle coloration as a surrogate for activity pattern. Across three genera (Myrmecocystus, Aphaenogaster, Temnothorax), pale species, as expected for nocturnally active animals had larger eyes, larger facet diameters, and larger visual spans compared to their dark, more day active congeners. This same pattern occurred for one pale species of Veromessor, but not the other. There were no consistent differences between nocturnal and diurnal species in interommatidial angles and eye parameters both within and among genera. Hence, the evolution of eye features that enhance sensitivity in low light levels do not appear to have consistent correlated effects on features related to visual acuity. A survey across several additional ant genera found numerous other pale species with enlarged eyes, suggesting these traits evolved multiple times within and across genera. We also compared size of the anterior ocellus in workers of pale versus dark species of Myrmecocystus. In species with larger workers, the anterior ocellus was smaller in pale than in dark species, but this difference mostly disappeared for species with smaller workers. Presence of the anterior ocellus also was size-dependent in the two largest pale species.

category: evolutionary biology


title: Dinophiliformia early neurogenesis suggests the evolution of conservative neural structures across the Annelida phylogenetic tree

abstract: Despite the increasing data concerning the structure of the adult nervous system in various Lophotrochozoa groups, the early events during the neurogenesis of rare and unique groups need clarification. Annelida are a diverse clade of Lophotrochozoa, and their representatives demonstrate a variety of body plans, lifestyles, and life cycles. Comparative data about the early development are available for Errantia, Sedentaria, Sipuncula and Palaeoannelida; however, our knowledge of Dinophiliformia is currently scarce. Representatives of Dinophiliformia are small interstitial worms combining unique morphological features of different Lophotrochozoan taxa and expressing paedomorphic traits. We describe in detail the early neurogenesis of two related species: Dimorphilus gyrociliatus and Dinophilus vorticoides, from the appearance of first nerve cells until the formation of an adult body plan. In both species, the first cells were detected at the anterior and posterior regions at the early trochophore stage and demonstrated positive reactions with pan-neuronal marker anti-acetylated tubulin only. Long fibers of early cells grow towards each other and form longitudinal bundles along which differentiating neurons later appear and send their processes. We propose that these early cells serve as pioneer neurons, forming a layout of the adult nervous system. The early anterior cell of D. vorticoides is transient and present during the short embryonic period, while early anterior and posterior cells in D. gyrociliatus are maintained throughout the whole lifespan of the species. During development, the growing processes of early cells form compact brain neuropile, paired ventral and lateral longitudinal bundles; unpaired medial longitudinal bundle; and commissures in the ventral hyposphere. Specific 5-HT- and FMRFa-immunopositive neurons differentiate adjacent to the ventral bundles and brain neuropile in the middle trochophore and late trochophore stages, i.e. after the main structures of the nervous system have already been established. Processes of 5-HT- and FMRFa-positive cells constitute a small proportion of the tubulin-immunopositive brain neuropile, ventral cords, and commissures in all developmental stages. No 5-HT- and FMRFa-positive cells similar to apical sensory cells of other Lophotrochozoa were detected. We conclude that: (i) like in Errantia and Sedentaria, Dinophiliformia neurogenesis starts from the peripheral cells, whose processes prefigure the forming adult nervous system, (ii) Dinophiliformia early cells are negative to 5-HT and FMRFa antibodies like Sedentaria pioneer cells.

category: developmental biology


title: USP5 enhances SGTA mediated protein quality control

abstract: Mislocalised membrane proteins (MLPs) present a risk to the cell due to exposed hydrophobic amino acids which cause MLPs to aggregate. Previous studies identified SGTA as a key component of the machinery that regulates the quality control of MLPs. Overexpression of SGTA promotes deubiqutination of MLPs resulting in their accumulation in cytosolic inclusions, suggesting SGTA acts in collaboration with deubiquitinating enzymes (DUBs) to exert these effects. However, the DUBs that play a role in this process have not been identified. In this study we have identified the ubiquitin specific peptidase 5 (USP5) as a DUB important in regulating the quality control of MLPs. We show that USP5 is in complex with SGTA, and this association is increased in the presence of an MLP. Overexpression of SGTA results in an increase in steady-state levels of MLPs suggesting a delay in proteasomal degradation of substrates. However, our results show that this effect is strongly dependent on the presence of USP5. We find that in the absence of USP5, the ability of SGTA to increase the steady state levels of MLPs is compromised. Moreover, knockdown of USP5 results in a reduction in the steady state levels of MLPs, while overexpression of USP5 increases the steady state levels. Our findings suggest that the interaction of SGTA with USP5 enables specific MLPs to escape proteasomal degradation allowing selective modulation of MLP quality control. These findings progress our understanding of aggregate formation, a hallmark in a range of neurodegenerative diseases and type II diabetes, as well as physiological processes of aggregate clearance.

category: biochemistry


title: Glycosaminoglycans Modulate Long-Range Mechanical Communication Between Cells in Collagen Networks

abstract:
Cells can sense and respond to mechanical forces in fibrous extracellular matrices (ECM) over distances much greater than their size. This phenomenon, termed long-range force transmission, is enabled by the realignment (buckling) of collagen fibers along directions where the forces are tensile (compressive). However, whether other key structural components of the ECM, in particular glycosaminoglycans (GAGs), can affect the efficiency of cellular force transmission remains unclear. Here we developed a theoretical model of force transmission in collagen networks with interpenetrating GAGs, capturing the competition between tension-driven collagen-fiber alignment and the swelling pressure induced by GAGs. Using this model, we show that the swelling pressure provided by GAGs increases the stiffness of the collagen network by stretching the fibers in an isotropic manner. We found that the GAG-induced swelling pressure can help collagen fibers resist buckling as the cells exert contractile forces. This mechanism impedes the alignment of collagen fibers and decreases long-range cellular mechanical communication. We experimentally validated the theoretical predictions by comparing collagen fiber alignment between cellular spheroids cultured on collagen gels versus collagen-GAG co-gels. We found significantly less alignment of collagen in collagen-GAG co-gels, consistent with the prediction that GAGs can prevent collagen fiber alignment. The roles of GAGs in modulating force transmission uncovered in this work can be extended to understand pathological processes such as the formation of fibrotic scars and cancer metastasis, where cells communicate in the presence of abnormally high concentrations of GAGs.

Statement of significanceGlycosaminoglycans (GAGs) are carbohydrates that are expressed ubiquitously in the human body and are among the key macromolecules that influence development, homeostasis, and pathology of native tissues. Abnormal accumulation of GAGs has been observed in metabolic disorders, solid tumors, and fibrotic tissues. Here we theoretically and experimentally show that tissue swelling caused by the highly polar nature of GAGs significantly affects the mechanical interactions between resident cells by altering the organization and alignment of the collagenous extracellular matrix. The roles of GAGs in modulating cellular force transmission revealed here can guide the design of biomaterial scaffolds in regenerative medicine and provides insights on the role of cell-cell communication in tumor progression and fibrosis.

category: biophysics


title: Genome of a Giant (Trevally): Caranx ignobilis

abstract: Caranx ignobilis, commonly known as the kingfish or giant trevally, is a large, reef-associated apex predator. It is a prized sportfish, targeted heavily throughout its tropical and subtropical range in the Indian and Pacific Oceans, and it has drawn significant interest in aquaculture due to an unusual tolerance for freshwater. In this study, we present a high-quality nuclear genome assembly of a C. ignobilis individual from Hawaiian waters, which have recently been shown to host a genetically distinct population. The assembly has a contig NG50 of 7.3Mbp and scaffold NG50 of 46.3Mbp. Twenty-five of the 203 scaffolds contain 90% of the genome. We also present the raw Pacific Biosciences continuous long-reads from which the assembly was created. A Hi-C dataset (Dovetail Genomics Omni-C) and Illumina-based RNA-seq from eight tissues are also presented; the latter of which can be particularly useful for annotation and studies of freshwater tolerance. Overall, this genome assembly and supporting data is a valuable tool for ecological and comparative genomics studies of kingfish and other carangoid fishes.


title: Seasonal phenology of Coffee Berry Borer (Hypothenemus hampei Ferrari) in Hawaii and the influence of weather on flight activity

abstract: Coffee berry borer (CBB, Hypothenemus hampei Ferrari) is the most serious insect pest of coffee worldwide, yet little is known about its seasonal flight behavior or the effect that weather variables have on its activity. We sampled flying female CBB adults bi-weekly over a three-year period using red funnel traps baited with an alcohol lure at 14 commercial coffee farms on Hawaii Island to characterize seasonal phenology and the influence of five weather variables on flight activity. We captured almost 5 million Scolytid beetles during the sampling period, with 81-93% of the trap catch comprised of CBB. Of the captured non-target beetles, the majority were tropical nut borer, black twig borer and a species of Cryphalus. Two major flight events were consistent across all three years: an initial emergence from January-April that coincided with early fruit development and a second flight during the harvest season from September-December. A linear regression showed a moderate but significant negative relationship between elevation and total trap catch. A generalized additive mixed model (GAMM) revealed that mean daily air temperature has the most significant (positive) effect on CBB flight, with most flight events occurring between 20-26 {degrees}C. Mean daily solar radiation also had a significant positive effect, while maximum daily relative humidity negatively influenced flight at values above [~]94%. Flight was positively influenced by maximum daily wind speeds up to [~]2.5 m/s and cumulative rainfall up to 100 mm, after which activity declined. Our findings provide important insight into CBB flight patterns across a highly variable landscape and will serve as a starting point for the development of flight prediction models.


title: NeuroML-DB: Sharing and characterizing data-driven neuroscience models described in NeuroML

abstract:
As researchers develop computational models of neural systems with increasing sophistication and scale, it is often the case that fully de novo model development is impractical and inefficient. Thus arises a critical need to quickly find, evaluate, re-use, and build upon models and model components developed by other researchers. We introduce the NeuroML Database (NeuroML-DB.org), which has been developed to address this need and to complement other model sharing resources. NeuroML-DB stores over 1,500 previously published models of ion channels, cells, and networks that have been translated to the modular NeuroML model description language. The database also provides reciprocal links to other neuroscience model databases (ModelDB, Open Source Brain) as well as access to the original model publications (PubMed). These links along with Neuroscience Information Framework (NIF) search functionality provide deep integration with other neuroscience community modeling resources and greatly facilitate the task of finding suitable models for reuse.

Serving as an intermediate language, NeuroML and its tooling ecosystem enable efficient translation of models to other popular simulator formats. The modular nature also enables efficient analysis of a large number of models and inspection of their properties. Search capabilities of the database, together with web-based, programmable online interfaces, allow the community of researchers to rapidly assess stored model electrophysiology, morphology, and computational complexity properties. We use these capabilities to perform a database-scale analysis of neuron and ion channel models and describe a novel tetrahedral structure formed by cell model clusters in the space of model properties and features.

Author SummaryComputational models of neurons and their circuits are increasingly used by neuroscience researchers as a tool to probe fundamental aspects of brain function. Here we describe a database of computational models of neurons and networks that makes it easier to evaluate and reuse these models. The models in the database are available in a standard format, called NeuroML, that makes it easier to extend and reuse the models in simulation studies using a wide range of simulation software platforms. The use of this standard format also makes it easier to characterize models in an automated way and analyze relationships across the features of simulated data from model simulations.

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category: neuroscience


title: Reduced plasticity in coupling strength in the SCN clock in aging as revealed by Kuramoto modelling

abstract:
The mammalian circadian clock is located in the suprachiasmatic nucleus (SCN) and consist of a network of coupled neurons, which are entrained to the environmental light-dark cycle. The phase coherence of the neurons is plastic and driven by the length of the day. With aging the capacity to behaviorally adapt to changes in the light regime reduces. The mechanisms underlying photoperiodic adaptation are largely unknown, but are important to unravel for the development of novel interventions to improve the quality of life of the elderly. We analyzed the neuronal synchronization of PER2::LUC protein expression in the SCN of young and old mice entrained to either long or short photoperiod and used the synchronization levels as input for a two-community noisy Kuramoto model. With the Kuramoto model we estimated the coupling strength between and within neuronal subpopulations. The model revealed that the coupling strength between and within subpopulations contributes to photoperiod induced changes in the phase relationship among neurons. We found that the SCN of young mice adapts in coupling strength over a large range, with low coupling strength in long photoperiod and higher coupling strength in short photoperiod. In aged mice we also found low coupling strength in long photoperiod, but strongly reduced capacity to reach high coupling strength in short photoperiod. The inability to respond with an increase in coupling strength shows that manipulation of photoperiod is not a suitable strategy to enhance clock function with aging. We conclude that the inability of aged mice to reach high coupling strength makes aged mice less capable to seasonal adaptation than young mice.

Author SummaryCircadian clocks drive daily rhythms in physiology and behavior. In mammals the clock resides in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN consist of a network of coupled neurons which are synchronized to produce a coherent rhythm. Due to plasticity of the network, seasonal adaptation to short winter days and long summer days occurs. Disturbances in circadian rhythmicity of the elderly have negative health effects, such as neurodegenerative diseases. With the rise in life expectancy this is becoming a major issue. In our paper, we used a model to compare the neuronal coupling in the SCN between young and old animals. We investigated whether exposure to short photoperiod can strengthen coupling among clock cells, and thereby clock function, in old animals. We observed that this is not possible, indicating that simple environmental manipulations are not an option. We suggest that receptor targeted interventions are required, setting the path for further investigation.


title: Physiological noise optimizes multiplexed coding of vibrotactile-like signals in somatosensory cortex

abstract: Neurons can use different aspects of their spiking to simultaneously represent (multiplex) different features of a stimulus. For example, some pyramidal neurons in primary somatosensory cortex (S1) use the rate and timing of their spikes to respectively encode the intensity and frequency of vibrotactile stimuli. Doing so has several requirements. Because they fire at low rates, pyramidal neurons cannot entrain 1:1 with high-frequency (100-600 Hz) inputs and instead must skip (i.e. not respond to) some stimulus cycles. The proportion of skipped cycles must vary inversely with stimulus intensity for firing rate to encode stimulus intensity. Spikes must phase lock to the stimulus for spike times (intervals) to encode stimulus frequency but, in addition, skipping must occur irregularly to avoid aliasing. Using simulations and in vitro experiments in which S1 pyramidal neurons were stimulated with inputs emulating those induced by vibrotactile stimuli, we show that fewer cycles are skipped as stimulus intensity increases, as required for rate coding, and that physiological noise induces irregular skipping without disrupting phase locking, as required for temporal coding. This occurs because the reliability and precision of spikes evoked by small- amplitude, fast-onset signals are differentially sensitive to noise. Simulations confirmed that differences in stimulus intensity and frequency can be well discriminated based on differences in spike rate or timing, respectively, but only in the presence of noise. Our results show that multiplexed coding by S1 pyramidal neurons is facilitated rather than degraded by physiological levels of noise. In fact, multiplexing is optimal under physiologically noisy conditions.


title: The opposing Chloride Cotransporters KCC and NKCC control locomotor activity inconstant light and during long days.

abstract: Cation Chloride Cotransporters (CCCs) regulate intracellular chloride ion concentration ([Cl-]i) within neurons, which can reverse the direction of the neuronal response to the neurotransmitter GABA. Na+ K+ Cl- (NKCC) and K+ Cl- (KCC) cotransporters transport Cl- into or out of the cell, respectively. When NKCC activity dominates, the resulting high [Cl-]i can lead to an excitatory and depolarizing response of the neuron upon GABAA receptor opening, while KCC dominance has the opposite effect. This inhibitory-to-excitatory GABA switch has been linked to seasonal adaption of circadian clock function to changing day length, and its dysregulation is associated with neurodevelopmental disorders such as epilepsy. Constant light normally disrupts circadian clock function and leads to arrhythmic behavior. Here, we demonstrate a function for KCC in regulating Drosophila locomotor activity and GABA responses in circadian clock neurons because alteration of KCC expression in circadian clock neurons elicits rhythmic behavior in constant light. We observed the same effects after downregulation of the Wnk and Fray kinases, which modulate CCC activity in a [Cl-]i-dependent manner. Patch-clamp recordings from clock neurons show that downregulation of KCC results in a more positive GABA reversal potential, while KCC overexpression has the opposite effect. Finally, KCC downregulation represses morning behavioral activity during long photoperiods, while downregulation of NKCC promotes morning activity. In summary, our results support a model in which the regulation of [Cl-]i by a KCC/NKCC/Wnk/Fray feedback loop determines the response of clock neurons to GABA, which is important for adjusting behavioral activity to constant light and long-day conditions.


title: A caged DAMGO for selective photoactivation of endogenous mu opioid receptors

abstract: Photoactivatable drugs and peptides are valuable tools that can drive quantitative studies into endogenous receptor signaling in relatively intact tissue preparations with high spatiotemporal resolution. We previously developed photoactivatable or "caged" variants of the opioid neuropeptides [Leu5]-enkephalin and dynorphin A (1-8), but these reagents activate multiple opioid receptors simultaneously upon photolysis. To achieve selective engagement of mu opioid receptors (MORs), here we describe a photoactivatable derivative of the MOR-selective peptide agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO). Starting from commercially available DAMGO, we appended a negatively charged carboxynitroveratryl (CNV) caging group to the N-terminal tyrosine side chain (Y) to produce CNV-Y-DAMGO. Dose-response curves in a heterologous functional assay revealed that CNV-Y-DAMGO is ~1,000-fold less potent than DAMGO at MORs. In acute hippocampal brain slices, CNV-Y-DAMGO photoactivation with ultraviolet light resulted in rapid, transient suppression of inhibitory synaptic transmission, and this was blocked by the MOR-selective antagonist CTAP. These results validate CNV-Y-DAMGO as a photopharmacological reagent for manipulating endogenous MOR signaling in brain tissue with light.


title: Microtubule assembly by soluble tau impairs vesicle endocytosis and excitatory neurotransmission via dynamin sequestration in Alzheimer's disease synapse model

abstract:
Elevation of soluble wild-type (WT) tau occurs in synaptic compartments in Alzheimers disease. We addressed whether tau elevation affects synaptic transmission at the calyx of Held in brainstem slices. Whole-cell loading of WT human tau (h-tau) in presynaptic terminals at 10-20 {micro}M caused microtubule (MT) assembly and activity-dependent rundown of excitatory neurotransmission. Capacitance measurements revealed that the primary target of WT h-tau is vesicle endocytosis. Blocking MT assembly using nocodazole prevented tau-induced impairments of endocytosis and neurotransmission. Immunofluorescence imaging analyses revealed that MT assembly by WT h-tau loading was associated with an increased bound fraction of the endocytic protein dynamin. A synthetic dodecapeptide corresponding to dynamin-1-pleckstrin-homology domain inhibited MT-dynamin interaction and rescued tau-induced impairments of endocytosis and neurotransmission. We conclude that elevation of presynaptic WT tau induces de novo assembly of MTs, thereby sequestering free dynamins. As a result, endocytosis and subsequent vesicle replenishment are impaired, causing activity-dependent rundown of neurotransmission.

Significance StatementWild-type human recombinant tau loaded in rodent presynaptic terminals inhibited vesicle endocytosis, thereby causing activity-dependent rundown of excitatory transmission. This endocytic block is caused by a sequestration of dynamin by excess microtubules newly assembled by tau and can be rescued by a peptide inhibiting the microtubules-dynamin interaction, or by the microtubule disassembler nocodazole. Thus, synaptic dysfunction can be induced by pathological increase of endogenous soluble tau in Alzheimer disease slice model.


title: Diurnal modulation of multivesicular release controls the efficiency of information transmission at a sensory synapse

abstract: Neuromodulators adapt sensory circuits to changes in the external world or the animals internal state and synapses are key control sites for such plasticity. Less clear is how neuromodulation alters the amount of information transmitted through the circuit. We investigated this question in the context of the diurnal regulation of visual processing in the retina of zebrafish, focusing on ribbon synapses of bipolar cells. We demonstrate that contrast-sensitivity peaks in the afternoon accompanied by a four-fold increase in the average Shannon information transmitted from an active zone. This increase reflects higher synaptic gain, lower spontaneous "noise" and reduced variability of evoked responses. Simultaneously, an increase in the probability of multivesicular events with larger information content increases the efficiency of transmission (bits per vesicle) by factors of 1.5-2.7. This study demonstrates the multiplicity of mechanisms by which a neuromodulator can adjust the synaptic transfer of sensory information.


title: Decoding the olfactory map: targeted transcriptomics link olfactory sensory neurons to glomeruli

abstract:
Sensory processing in vertebrate olfactory systems is organized across olfactory bulb glomeruli, wherein axons of peripheral sensory neurons expressing the same olfactory receptor co-terminate to transmit receptor-specific activity to central neurons. Understanding how receptors map to glomeruli is therefore critical to understanding olfaction. High-throughput spatial transcriptomics is a rapidly advancing field, but low-abundance olfactory receptor expression within glomeruli has previously precluded high-throughput mapping of receptors to glomeruli. Here we combined spatial sectioning along the anteroposterior, dorsoventral, and mediolateral axes with target capture enrichment sequencing to overcome low-abundance target expression. This strategy allowed us to spatially map 86% of olfactory receptors across the olfactory bulb and uncover a relationship between OR sequence and glomerular position.

ONE SENTENCE SUMMARYTargeted enrichment of olfactory receptor mRNA in olfactory bulb sections determines spatial positions for murine glomeruli.


title: Cross-tissue analysis of allelic X-chromosome inactivation ratios resolves features of human development

abstract: X-chromosome inactivation (XCI) is a random, permanent, and developmentally early epigenetic event that occurs during mammalian embryogenesis. We harness these features of XCI to investigate characteristics of early lineage specification events during human development. We initially assess the consistency of X-inactivation and establish a robust set of XCI-escape genes. By analyzing variance in XCI ratios across tissues and individuals, we find that XCI is completed prior to tissue specification and at a time when 6-16 cells are fated for all tissue lineages. Additionally, we exploit tissue specific variability to characterize the number of cells present at the time of each tissues lineage commitment, ranging from approximately 20 cells in liver and whole blood tissues to 80 cells in brain tissues. By investigating variance of XCI ratios using adult tissue, we resolve key features of human development otherwise difficult to ascertain experimentally and develop scalable methods easily applicable to future data.


title: SARS-CoV-2 Spike Protein Regulation of Angiotensin Converting Enzyme 2 and Tissue Renin-Angiotensin Systems: Influence of Biologic Sex

abstract: Angiotensin converting enzyme 2 (ACE2) is an enzyme that limits activity of the renin-angiotensin system (RAS) and also serves as a receptor for the SARS-CoV-2 Spike (S) protein. Binding of S protein to ACE2 causes internalization which activates local RAS. ACE2 is on the X chromosome and its expression is regulated by sex hormones. In this study, we defined ACE2 mRNA abundance and examined effects of S protein on ACE2 activity and/or angiotensin II (AngII) levels in pivotal tissues (lung, adipose) from male and female mice. In lung, ACE2 mRNA abundance was reduced following gonadectomy (GDX) of male and female mice and was higher in XX than XY mice of the Four Core Genotypes (FCG). Reductions in lung ACE2 mRNA abundance by GDX occurred in XX, but not XY FCG female mice. Lung mRNA abundance of ADAM17 and TMPRSS2, enzymes that shed cell surface ACE2 and facilitate viral cell entry, was reduced by GDX in male but not female mice. For comparison, adipose ACE2 mRNA abundance was higher in female than male mice and higher in XX than XY FCG mice. Adipose ADAM17 mRNA abundance was increased by GDX of male and female mice. S protein reduced ACE2 activity in alveolar type II epithelial cells and 3T3-L1 adipocytes. Administration of S protein to male and female mice increased lung AngII levels and decreased adipose ACE2 activity in male but not female mice. These results demonstrate that sex differences in ACE2 expression levels may impact local RAS following S protein exposures.

category: physiology


title: A histone variant condenses flowering plant sperm via chromatin phase separation

abstract:
Sperm chromatin is typically transformed by protamines into a compact and transcriptionally inactive state. Flowering plant sperm cells lack protamines, yet have small, transcriptionally active nuclei with chromatin condensed by an unknown mechanism. Here we show that a histone variant, H2B.8, mediates sperm chromatin and nuclear condensation in Arabidopsis thaliana. Loss of H2B.8 causes enlarged sperm nuclei with dispersed chromatin, whereas ectopic expression in somatic cells produces smaller nuclei with aggregated chromatin, demonstrating that H2B.8 is sufficient for chromatin condensation. H2B.8 aggregates transcriptionally inactive AT-rich chromatin into phase-separated condensates, thus achieving nuclear compaction without reducing transcription. H2B.8 also intermixes inactive AT-rich chromatin and GC-rich pericentromeric heterochromatin, altering higher-order chromatin architecture. Altogether, our results reveal a novel mechanism of nuclear compaction via global aggregation of unexpressed chromatin. We propose that H2B.8 is a flowering plant evolutionary innovation that achieves nuclear condensation compatible with active transcription.

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title: Molecular basis of differential adventitious rooting competence in poplar genotypes

abstract:
O_LIRecalcitrant adventitious root (AR) development is a major hurdle in propagating commercially important woody plants. Although significant progress has been made to identify genes involved in subsequent steps of AR development, the molecular basis of differences in apparent recalcitrance to form AR between easy-to-root and difficult-to-root genotypes remains unknown.
C_LIO_LITo address this, we generated cambium tissue-specific transcriptomic data from stem cuttings of hybrid aspen, T89 (difficult-to-root) and hybrid poplar OP42 (easy-to-root) and used transgenic approaches to verify the role of several transcription factors (TF) in the control of adventitious rooting.
C_LIO_LIIncreased peroxidase activity is positively correlated with better rooting. We found differentially expressed genes encoding Reactive Oxygen Species (ROS) scavenging proteins to be enriched in OP42 compared to T89. A higher number of differentially expressed TF in OP42 compared to T89 cambium cells was revealed by a more intense transcriptional reprograming in the former. PtMYC2, a potential negative regulator, was less expressed in OP42 compared to T89. Using transgenic approaches, we have demonstrated that PttARF17.1 and PttMYC2.1 negatively regulate adventitious rooting.
C_LIO_LIOur results provide insights into the molecular basis of genotypic differences in AR and implicate differential expression of the master regulator MYC2 as a critical player in this process.
C_LI


title: Bile acids gate dopamine transporter mediated currents

abstract: Bile acids (BAs) are molecules derived from cholesterol that are involved in dietary fat absorption. New evidence supports an additional role for BAs as regulators of brain function. Interestingly, sterols such as cholesterol interact with monoamine transporters (MAT), including the dopamine (DA) transporter (DAT) which plays a key role in DA neurotransmission and reward circuitries in the brain. The present study explores interactions of the BA, obeticholic acid (OCA), with DAT and mechanistically defines the regulation of DAT activity via both electrophysiology and molecular modeling. We express murine DAT (mDAT) in Xenopus laevis oocytes and confirm that DA induces an inward current that reaches a steady-state at a negative membrane voltage. Next, we show that OCA triggers an inward current through DAT that is Na+ dependent and not regulated by intracellular calcium. OCA also inhibits the DAT-mediated Li+ leak current, a feature that parallels DA action and indicates direct binding to the transporter. Interestingly, OCA does not alter DA affinity nor the ability of DA to promote a DAT-mediated inward current, suggesting that the interaction of OCA with the transporter is non-competitive, in regard to DA. The current induced by OCA is transient in nature, returning to baseline in the continued presence of the BA. To understand the molecular mechanism of how OCA affects DAT electrical activity, we performed docking simulations. These simulations revealed two potential binding sites that provide important insights into the potential functional relevance of the OCA-DAT interaction. First, in the absence of DA, OCA binds DAT through interactions with D421, a residue normally involved in coordinating the binding of the Na+ ion to the Na2 binding site (Borre et al., 2014;Cheng and Bahar, 2015). Furthermore, we uncover a separate binding site for OCA on DAT, of equal potential functional impact, that is facilitated through the residues DAT R445 and D436. This binding may stabilize the inward-facing open (IFo) state by preventing the re-formation of the IF gating salt bridges, R60-D436 and R445-E428, that are required for DA transport. This study suggests that BAs may represent novel pharmacological tools to regulate DAT function, and possibly, associated behaviors.


title: BNT162b2-Elicited Neutralization of Delta Plus, Lambda, and Other Variants

abstract:
BNT162b2-elicited human sera are known to neutralize the currently dominant Delta SARS-CoV-2 variant. Here, we report the ability of 20 human sera, drawn 2 or 4 weeks after two doses of BNT162b2, to neutralize USA-WA1/2020 SARS-CoV-2 bearing variant spikes from Delta plus (Delta-AY.1, Delta-AY.2), Delta-{Delta}144 (Delta with the Y144 deletion of the Alpha variant), Lambda, and B. 1.1.519 lineage viruses. Geometric mean plaque reduction neutralization titers against Delta-AY.1, Delta-AY.2, and Delta-{Delta}144 viruses are slightly lower than against USA-WA1/2020, but all sera neutralize the variant viruses to titers of [≥]80.

Neutralization titers against Lambda and B. 1.1.519 variants and against USA-WA1/2020 are equivalent. The susceptibility of Delta plus, Lambda, and other variants to neutralization by the sera indicates that antigenic change has not led to virus escape from vaccine-elicited neutralizing antibodies and supports ongoing mass immunization with BNT162b2 to control the variants and to minimize the emergence of new variants.


title: Protective Efficacy of Gastrointestinal SARS-CoV-2 Delivery Against Intranasal and Intratracheal SARS-CoV-2 Challenge in Rhesus Macaques

abstract:
Live oral vaccines have been explored for their protective efficacy against respiratory viruses, particularly for adenovirus serotypes 4 and 7. The potential of a live oral vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), however, remains unclear. In this study, we assessed the immunogenicity of live SARS-CoV-2 delivered to the gastrointestinal tract in rhesus macaques and its protective efficacy against intranasal and intratracheal SARS-CoV-2 challenge. Post-pyloric administration of SARS-CoV-2 by esophagogastroduodenoscopy resulted in limited virus replication in the gastrointestinal tract and minimal to no induction of mucosal antibody titers in rectal swabs, nasal swabs, and bronchoalveolar lavage. Low levels of serum neutralizing antibodies were induced and correlated with modestly diminished viral loads in nasal swabs and bronchoalveolar lavage following intranasal and intratracheal SARS-CoV-2 challenge. Overall, our data show that post-pyloric inoculation of live SARS-CoV-2 is weakly immunogenic and confers partial protection against respiratory SARS-CoV-2 challenge in rhesus macaques.

ImportanceSARS-CoV-2 remains a global threat, despite the rapid deployment but limited coverage of multiple vaccines. Alternative vaccine strategies that have favorable manufacturing timelines, greater ease of distribution and improved coverage may offer significant public health benefits, especially in resource-limited settings. Live oral vaccines have the potential to address some of these limitations; however no studies have yet been conducted to assess the immunogenicity and protective efficacy of a live oral vaccine against SARS-CoV-2. Here we report that oral administration of live SARS-CoV-2 in non-human primates may offer prophylactic benefits, but that formulation and route of administration will require further optimization.


title: Effect of ORF7 of SARS-CoV-2 on the chemotaxis of monocytes and neutrophils in vitro

abstract: Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the most significant public health threats in worldwide. Patients with severe COVID-19 usually have pneumonia concomitant with local inflammation and sometimes a cytokine storm. Specific components of the SARS-CoV-2 virus trigger lung inflammation, and recruitment of immune cells to the lungs exacerbates this process, although much remains unknown about the pathogenesis of COVID-19. Our study of lung type II pneumocyte cells (A549) demonstrated that ORF7, an open reading frame (ORF) in the genome of SARS-CoV-2, induced the production of CCL2, a chemokine that promotes the chemotaxis of monocytes, and decreased the expression of IL-8, a chemokine that recruits neutrophils. A549 cells also had an increased level of IL-6. The results of our chemotaxis transwell assay suggested that ORF7 augmented monocyte infiltration and reduced the number of neutrophils. We conclude that the ORF7 of SARS-CoV-2 may have specific effects on the immunological changes in tissues after infection. These results suggest that the functions of other ORFs of SARS-CoV-2 should also be comprehensively examined.

category: cell biology


title: Prolific Induction of IL-6 in Human Cells by SARS-CoV-2-derived Peptide is Attenuated by Recombinant Human Anti-inflammatory Cytokines made in planta.

abstract: Development of efficient therapies for COVID-19 is the focus of intense research. The cytokine release syndrome was underlined as a culprit for severe outcomes in COVID-19 patients. Interleukin-6 (IL-6) plays a crucial role in human immune responses and elevated IL-6 plasma levels have been associated with the exacerbated COVID-19 pathology. Since non-structural protein 10 (NSP10) of SARS-CoV-2 has been implicated in the induction of IL-6, we designed Peptide (P)1, containing sequences corresponding to amino acids 68-96 of NSP10, and examined its effect on cultured human cells. Treatment with P1 strongly increased IL-6 secretion by the lung cancer cell line NCI-H1792 and the breast cancer cell line MDA-MB-231 and revealed profound cytotoxic activity on Caco-2 colorectal adenocarcinoma cells. Treatment with P2, harbouring a mutation in the zinc knuckle motif of NSP10, caused no IL-6 induction and no cytotoxicity. Pre-treatment with plant-produced human anti-inflammatory cytokines IL-37b and IL-38 effectively mitigated the induction of IL-6 secretion. Our results suggest a role for the zinc knuckle motif of NSP10 in the onset of increased IL-6 plasma levels of COVID-19 patients and for IL-37b and IL-38 as therapeutics aimed at attenuating the cytokine release syndrome.


title: Mitoxantrone dihydrochloride, an FDA approved drug, binds with SARS-CoV-2 NSP1 C-terminal

abstract: One of the major virulence factors of SARS-CoV-2, NSP1, is a vital drug target due to its role in host immune evasion through multiple pathways. NSP1 protein is associated with inhibiting host mRNA translation by binding to the small subunit of ribosome through its C-terminal region. Previously, we have shown the structural dynamics of NSP1 C-terminal region (NSP1-CTR) in different physiological environments. So, it would be very interesting to investigate the druggable compounds that could bind with NSP1-CTR. Here, in this article, we have performed the different spectroscopic technique-based binding assays of an anticancer drug Mitoxantrone dihydrochloride (MTX) against the NSP1-CTR. We have also performed molecular docking followed by computational simulations with two different forcefields up to one microsecond. Overall, our results have suggested good binding between NSP1-CTR and MTX and may have implications in developing therapeutic strategies targeting NSP1 protein of SARS-CoV-2.


title: A screen for mutants deficient in coronatine-mediated suppression of root immunity identifies Arabidopsis SDA1 as a novel integrator of immunity and phytohormone signaling

abstract: Despite the importance of the root immune system in the interaction with rhizosphere microbes, the majority of genetic screens for immunity regulators have been performed in leaves. A previous screen identified 27 hsm (hormone-mediated suppression of MAMP-triggered immunity) mutants that are impaired in jasmonic acid (JA)-mediated suppression of pattern-triggered immunity (PTI) in roots. Here we characterized 16 of the hsm mutants that retain JA sensitivity and are potential negative regulators of root immunity. We found that the majority of hsm mutants show enhanced resistance to Fusarium, a root fungal pathogen; however, only a subset are more resistant to a foliar pathogen. Surprisingly, 12 of 16 hsm mutants are also impaired in abscisic acid (ABA)-mediated suppression of PTI, suggesting a largely shared pathway between JA- and ABA-mediated immune suppression in roots. Although all hsm mutants are insensitive to JA-mediated suppression of root immunity, hsm4 shows hypersensitivity to JA-mediated root growth inhibition and JA-induced gene expression. Consistently, hsm4 is more resistant to leaf pathogens, suggesting that HSM4 is a negative regulator of both root and leaf immunity. Hsm4 was mapped to a mutation in a conserved ARM-repeat protein homologous to yeast SDA1, which has been reported to regulate 60S ribosome biogenesis. As translational reprogramming is a critical layer of immune regulation, this work suggests that AtSDA1 is a novel negative translational regulator of immunity. Additionally, a comprehensive characterization of all 16 hsm mutants provides a genetic toolkit to identify novel mechanisms that regulate root immunity.


title: Fragment-based ab initio phasing of peptidic nanocrystals by MicroED

abstract: Microcrystal electron diffraction (MicroED) is transforming the visualization of molecules from nanocrystals, rendering their three-dimensional atomic structures from previously unamenable samples. Peptidic structures determined by MicroED include naturally occurring peptides, synthetic protein fragments and peptide-based natural products. However, as a diffraction method, MicroED is beholden to the phase problem, and its de novo determination of structures remains a challenge. ARCIMBOLDO, an automated, fragment-based approach to structure determination. It eliminates the need for atomic resolution, instead enforcing stereochemical constraints through libraries of small model fragments, and discerning congruent motifs in solution space to ensure validation. This approach expands the reach of MicroED to presently inaccessible peptidic structures including segments of human amyloids, and yeast and mammalian prions, and portends a more general phasing solution while limiting model bias for a wider set of chemical structures.


title: Deep learning of Cas13 guide activity from high-throughput gene essentiality screening

abstract: Transcriptome engineering requires flexible RNA-targeting technologies that can perturb mammalian transcripts in a robust and scalable manner. CRISPR systems that natively target RNA molecules, such as Cas13 enzymes, are enabling rapid progress in the investigation of RNA biology and advancement of RNA therapeutics. Here, we sought to develop a Cas13 platform for high-throughput phenotypic screening and elucidate the design principles underpinning its RNA targeting efficiency. We employed the RfxCas13d (CasRx) system in a positive selection screen by tiling 55 known essential genes with single nucleotide resolution. Leveraging this dataset of over 127,000 guide RNAs, we systematically compared a series of linear regression and machine learning algorithms to train a convolutional neural network (CNN) model that is able to robustly predict guide RNA performance based on guide sequence alone. We further incorporated secondary features including secondary structure, free energy, target site position, and target isoform percent. To evaluate model performance, we conducted orthogonal screens via cell surface protein knockdown. The final CNN model is able to predict highly effective guide RNAs (gRNAs) within each transcript with >90% accuracy in this independent test set. To provide user interpretability, we evaluate feature contributions using both integrated gradients and SHapley Additive exPlanations (SHAP). We identify a specific sequence motif at guide position 15-24 along with selected secondary features to be predictive of highly efficient guides. Taken together, we derive Cas13d guide design rules from large-scale screen data, release a guide design tool (http://RNAtargeting.org) to advance the RNA targeting toolbox, and describe a path for systematic development of deep learning models to predict CRISPR activity.

category: bioengineering


title: Development of a novel, pan-variant aerosol intervention for COVID-19

abstract:
To develop a universal strategy to block SARS-CoV-2 cellular entry and infection represents a central aim for effective COVID-19 therapy. The growing impact of emerging variants of concern increases the urgency for development of effective interventions. Since ACE2 is the critical SARS-CoV-2 receptor and all tested variants bind to ACE2, some even at much increased affinity (see accompanying paper), we hypothesized that aerosol administration of clinical grade soluble human recombinant ACE2 (APN01) will neutralize SARS-CoV-2 in the airways, limit spread of infection in the lung and mitigate lung damage caused by deregulated signaling in the renin-angiotensin (RAS) and Kinin pathways. Here we show that intranasal administration of APN01 in a mouse model of SARS-CoV-2 infection dramatically reduced weight loss and prevented animal death. As a prerequisite to a clinical trial, we evaluated both virus binding activity and enzymatic activity for cleavage of Ang II following aerosolization. We report successful aerosolization for APN01, retaining viral binding as well as catalytic RAS activity. Dose range-finding and IND-enabling repeat-dose aerosol toxicology testing were conducted in dogs. Twice daily aerosol administration for two weeks at the maximum feasible concentration revealed no notable toxicities. Based on these results, a Phase I clinical trial in healthy volunteers can now be initiated, with subsequent Phase II testing in individuals with SARS-CoV-2 infection. This strategy could be used to develop a viable and rapidly actionable therapy to prevent and treat COVID-19, against all current and future SARS-CoV-2 variants.

One Sentence SummaryPreclinical development and evaluation of aerosolized soluble recombinant human ACE2 (APN01) administered as a COVID-19 intervention is reported.

category: pharmacology and toxicology


title: Network visualisation of synthetic biology designs

abstract: As genetic circuits become more sophisticated, the size and complexity of data about their designs increases. This data captured goes beyond monolithic genetic sequences and towards circuit modularity and functional details, which are beneficial for analyzing circuit performance and establishing design automation techniques. However, the accessibility, visualisation and usability of design data (and metadata) have received relatively little attention to date. Here, we present a method to turn circuit designs into networks and showcase its potential to enhance the utility of design data. Since networks are dynamic structures, initial graphs can be interactively shaped into sub-networks of relevant information based on requirements such as abstraction, hierarchy and protein interactions. Additionally, several visual changes can be applied, such as colouring or clustering nodes based on types (e.g., genes or promoters), resulting in easier comprehension from a user perspective. This approach allows circuit designs to be coupled to other networks, such as metabolic pathways or implementation protocols captured in graph-like formats. Therefore, we advocate using networks to structure, access and improve synthetic biology information.

category: synthetic biology


title: A network approach to genetic circuit designs


title: Direct capsid labeling of infectious HIV-1 by genetic code expansion allows detection of largely complete nuclear capsids and suggests nuclear entry of HIV-1 complexes via common routes

abstract: The cone-shaped mature HIV-1 capsid is the main orchestrator of early viral replication. After cytosolic entry, it transports the viral replication complex along microtubules towards the nucleus. Capsid uncoating from the viral genome apparently occurs beyond the nuclear pore. Observation of post-entry events via microscopic detection of HIV-1 capsid protein (CA) is challenging, since epitope shielding limits immunodetection, and the genetic fragility of CA hampers other labeling approaches. Here, we present a minimally invasive strategy based on genetic code expansion and click chemistry that allows for site-directed fluorescent labeling of HIV-1 CA, while retaining virus morphology and infectivity. Thereby, we could directly visualize virions and subviral complexes using advanced microscopy, including nanoscopy and correlative imaging. Quantification of signal intensities of subviral complexes showed that the amount of CA associated with nuclear complexes in HeLa-derived cells and primary T cells is consistent with a complete capsid and revealed that treatment with the small molecule inhibitor PF74 did not result in capsid dissociation from nuclear complexes. Cone-shaped objects detected in the nucleus by electron tomography were clearly identified as capsid-derived structures by correlative microscopy. High-resolution imaging revealed dose-dependent clustering of nuclear capsids, suggesting that incoming particles may follow common entry routes.


title: Isolation, identification and molecular characterization of Aeromonas spp. from farmed loach (Misgurnus anguillicaudatus)

abstract:
Gram-negative Aeromonas bacteria is known to contaminate aquaculture products and retains the ability to infect wide range of host including fishes, shrimps and humans. This study is designed to evaluate the presence of Aeromonas in healthy loaches (Misgurnus anguillicaudatus) collected from the markets in Hainan and Guangdong provinces of China. Based on the molecular sequencing and phylogenetic analysis with single 16S rRNA and concatenated genes (gyrA and ropD), 104 isolates were identified as Aeromonas species followed by further classification to A. veronii (83.65%), A. jandaei (9.62%), A. hydrophila (3.85%) and A. allosaccharophila (2.88%). More than half of the isolates displayed hemolytic activity of 87.5% and were able to form moderate biofilm (78.26%). Fourteen antibiotics from ten representative classes were screened that demonstrated complete resistance to ampicillin, 89.4% and 68.3% to lincomycin and nalidixic acid. Moreover, a notable detection and prevalence was found in screening of ten virulence-related and nine resistance genes. To our knowledge, it is the first report of its kind demonstrating potential threat of the commercial loaches carrying Aeromonas to human health. These findings will assist professionals working in clinical settings to optimize their prescription accordingly and researchers to access the impacts of Aeromonas resistance on human health.

ImportanceThis study demonstrates high infection rate of loaches by Aeromonas spp. in Hainan and Guangdong provinces of China.

The most prevalent species was Aeromonas veronii.

Most of the isolates displayed hemolytic activity and formed moderate biofilm.

Multiple virulence genes, resistance genes and multi-antibiotic resistance were detected.

Aeromonads risks should be considered in loach industry.


title: Controls on the isotopic composition of microbial methane

abstract: Microbial methane production (methanogenesis) is responsible for more than half of the annual emission of this major greenhouse gas to the atmosphere. Though the stable isotopic composition of methane is often used to characterize its sources and sinks, strictly empirical descriptions of the isotopic signature of methanogenesis currently limit such attempts. We developed a biochemical-isotopic model of methanogenesis by CO2 reduction, which predicts carbon and hydrogen isotopic fractionations, and clumped isotopologue distributions, as functions of the cells environment. We mechanistically explain multiple-isotopic patterns in laboratory and natural settings and show that such patterns constrain the in-situ energetics of methanogenesis. Combining our model with environmental data, we infer that in almost all marine environments and gas deposits, energy-limited methanogenesis operates close to chemical and isotopic equilibrium.


title: Secretin channel-interactors prevent antibiotic influx during type IV pili assembly in P. aeruginosa

abstract: Type IV pili (T4P) are important virulence factors involved in host attachment and other aspects of bacterial pathogenesis. In Gram-negative bacteria, the T4P filament is polymerized from pilin subunits at the platform complex in the inner membrane (IM) and exits the outer membrane (OM) through the OM secretin channel. Although essential for T4P assembly and function, the OM secretin complexes can potentially impair the permeability barrier function of the OM and allow the entry of antibiotics and other toxic molecules. The mechanism by which Gram-negative bacteria prevent secretin-mediated OM leakage is currently not well understood. Here, we report a discovery of SlkA and SlkB (PA5122 and PA5123) that prevent permeation of several classes of antibiotics through the secretin channel of Pseudomonas aeruginosa type IV pili. We found these periplasmic proteins interact with the OM secretin complex and prevent toxic molecules from entering through the channel when there is a problem in the assembly of the T4P IM subcomplexes or when docking between the OM and IM complexes is defective. Thus, our results indicate that the secretin channel-interacting proteins play an important role in maintaining the OM permeability barrier, suggesting they may be attractive targets for potentiators that sensitize Gram-negative pathogens to antibiotics that are normally ineffective at penetrating the OM.


title: Emerging chikungunya virus variants at the E1-E1 inter-glycoprotein spike interface impact virus attachment and Inflammation

abstract:
Chikungunya virus (CHIKV) is a re-emerging arthropod-borne alphavirus and a serious threat to human health. Therefore, efforts toward elucidating how this virus causes disease and the molecular mechanisms underlying steps of the viral replication cycle are crucial. Using an in vivo transmission system that allows intra-host evolution, we identified an emerging CHIKV variant carrying a mutation in the E1 glycoprotein (V156A) in the serum of mice and saliva of mosquitoes. E1 V156A has since emerged in humans during an outbreak in Brazil, co-occurring with a second mutation, E1 K211T, suggesting an important role for these residues in CHIKV biology. Given the emergence of these variants, we hypothesized that they function to promote CHIKV infectivity and subsequent disease. Here, we show that E1 V156A and E1 K211T modulate virus attachment and fusion and impact binding to heparin, a homolog of heparan sulfate, a key entry factor on host cells. These variants also exhibit differential neutralization by anti-glycoprotein monoclonal antibodies, suggesting structural impacts on the particle that may be responsible for altered interactions at the host membrane. Finally, E1 V156A and E1 K211T exhibit increased titers in an adult arthritic mouse model and induce increased foot-swelling at the site of injection. Taken together, this work has revealed new roles for E1 where discrete regions of the glycoprotein are able to modulate cell attachment and swelling within the host.

IMPORTANCEAlphaviruses represent a growing threat to human health worldwide. Chikungunya virus (CHIKV) has rapidly spread to new geographic regions in the last several decades, causing overwhelming outbreaks of disease, yet there are no approved therapeutics. The CHIKV glycoproteins are key determinants of CHIKV adaptation and virulence. In this study, we characterize the naturally emerging E1 glycoprotein variants, V156A and K211T. We demonstrate that E1 V156A and K211T function in virus attachment to cells, a role that until now has been only attributed to the CHIKV E2 glycoprotein. We also demonstrate E1 V156A and K211T to increase foot-swelling in mice. Observing that these variants and other pathogenic variants occur at the E1-E1 inter-spike interface, we highlight this structurally important region as critical for multiple steps during CHIKV infection. Together, these studies further defines the function of E1 in CHIKV infection and can inform the development of therapeutic or preventative strategies.


title: Associations and recovery dynamics of the nasopharyngeal microbiota during influenza-like illness in the aging population

abstract:
BackgroundOlder adults are more susceptible to respiratory pathogens, several of which have been associated with an altered respiratory microbiota. Influenza-like illness (ILI), a disease caused by respiratory pathogens including but not exclusively by influenza virus, is a major health concern in this population. However, there is little information on changes in the nasopharyngeal (NP) microbiota of older adults associated with respiratory infections identified by/ reported as ILI, as well as its dynamics during recovery. Here, we compared the NP microbiota in older adults who presented with ILI (n= 240) to the NP microbiota in older adults not reporting an ILI event (n= 157) during the 2014-2015 influenza season. To investigate the dynamics of the microbiota from the acute phase to the recovery phase of the infection, participants reporting an ILI event were sampled at onset of infection (<72 hours), at 14 days and at 7-9 weeks after infection (recovery sample).

ResultsCross-sectional analysis of the microbiota at the different time-points showed no differences in alpha diversity between the groups. A small but significant effect of the ILI was observed on the microbiota community and structure when compared to controls and recovery samples. Furthermore, the NP microbiota exhibited inter-individual differences in dynamics from onset of ILI to recovery. Corynebacterium, one of the keystone species in the upper respiratory tract, was negatively associated with ILI and its abundance increased after recovery. Potential pathobionts such as Haemophilus, Porphyromonas and Gemella had higher abundances during acute-ILI. Stability and changes in the NP microbial community showed individual dynamics. Key core genera, Corynebacterium, Moraxella and Dolosigranulum exhibited higher inter-individual variability in acute-ILI, but showed comparable variability to controls after recovery. Participants in the ILI group with higher core microbiota abundances at the acute phase showed higher microbiota stability after recovery.

ConclusionsOur findings demonstrate that acute-ILI is associated with small but significant alterations in the phylogenetic structure of the NP microbiota in older adults. The observed variation in the core microbiota suggests potential imbalances in the ecosystem, which could play a role in the recovery of the NP microbiota after an ILI event.


title: MIMOSA2: A metabolic network-based tool for inferring mechanism-supported relationships in microbiome-metabolome data

abstract:
MotivationRecent technological developments have facilitated an expansion of microbiome-metabolome studies, in which a set of microbiome samples are assayed using both genomic and metabolomic technologies to characterize the composition of microbial taxa and the concentrations of various metabolites. A common goal of many of these studies is to identify microbial features (species or genes) that contribute to differences in metabolite levels across samples. Previous work indicated that integrating these datasets with reference knowledge on microbial metabolic capacities may enable more precise and confident inference of such microbe-metabolite links.

ResultsWe present MIMOSA2, an R package and web application for model-based integrative analysis of microbiome-metabolome datasets. MIMOSA2 uses reference databases to construct a community metabolic model based on microbiome data and uses this model to predict differences in metabolite levels across samples. These predictions are compared with metabolomics data to identify putative microbiome-governed metabolites and specific taxonomic contributors to metabolite variation. MIMOSA2 supports various input data types and can be customized to incorporate user-defined metabolic pathways. We demonstrate MIMOSA2s ability to identify ground truth microbial mechanisms in simulation datasets, and compare its results with experimentally inferred mechanisms in a dataset describing honeybee gut microbiota. Overall, MIMOSA2 combines reference databases, a validated statistical framework, and a user-friendly interface to facilitate modeling and evaluating relationships between members of the microbiota and their metabolic products.

Availability and ImplementationMIMOSA2 is implemented in R under the GNU General Public License v3.0 and is freely available as a web server and R package from www.borensteinlab.com/software_MIMOSA2.html.


title: The RNA-binding protein DRBD18 regulates processing and export of the mRNA encoding Trypanosoma brucei RNA-binding protein 10

abstract: The parasite Trypanosoma brucei grows as bloodstream forms in mammalian hosts, and as procyclic forms in tsetse flies. Trypanosome protein coding genes are arranged in polycistronic transcription units, so gene expression regulation depends heavily on post-transcriptional mechanisms. The essential RNA-binding protein RBP10 is expressed only in mammalian-infective forms, where it targets procyclic-specific mRNAs for destruction. We show that developmental regulation of RBP10 expression is mediated by the exceptionally long 7.3 Kb 3-UTR of its mRNA. Different regulatory sequences that can independently enhance mRNA stability and translation in bloodstream forms, or destabilize and repress translation in procyclic forms, are scattered throughout the 3-UTR. The RNA-binding protein DRBD18 is implicated in the export of a subset of mRNAs from the nucleus in procyclic forms. We confirmed that in bloodstream forms, DRBD18 copurifies the outer ring of the nuclear pore, mRNA export proteins and exon junction complex proteins. Loss of DRBD18 in bloodstream forms caused accumulation of several shortened RBP10 mRNA isoforms, with loss of longer species, but RNAi targeting the essential export factor MEX67 did not cause such changes, demonstrating specificity. Long RBP10 mRNAs accumulated in the nucleus, while shorter ones reached the cytoplasm. We suggest that DRBD18 binds to processing signals in the RBP10 3-UTR, simultaneously preventing their use and recruiting mRNA export factors. DRBD18 depletion caused truncation of the 3-UTRs of more than 100 other mRNAs, suggesting that it has an important role in regulating use of alternative processing sites.


title: Selective Modulator of Nuclear Receptor PPARy with Reduced Adipogenic Potential Ameliorates Experimental Nephrotic Syndrome

abstract:
BackgroundGlomerular disease, often manifesting as nephrotic syndrome (NS) with high proteinuria, can be refractory to standard treatment and is typically associated with hypoalbuminemia, hypercholesterolemia and hypercoagulopathy. We hypothesized that the nuclear receptor PPAR{gamma} can be selectively modulated using a novel partial agonist, GQ-16, to gain therapeutic advantage over traditional PPAR{gamma} agonists (e.g. thiazolidinediones) for the treatment of glomerular disease.

MethodsNephropathy was induced with puromycin amino-nucleoside (PAN) in Wistar rats and treated with Pioglitazone (Pio) or GQ-16. Plasma, serum, and urine chemistries were performed, and kidneys, glomeruli, liver, and white adipose tissue (WAT) were harvested. Lipid accumulation and adipogenic gene expression were measured in adipocytes.

ResultsPAN-induced proteinuria was significantly reduced with Pio to 64% of PAN-value. It was reduced robustly with GQ-16 to 81% of PAN, which was comparable to controls. While both GQ-16 and Pio restored glomerular Nphs1 and hepatic Pcsk9 expression and reduced hypercholesterolemia, GQ-16 also restored glomerular Nrf2, and reduced hypoalbuminemia and hypercoagulopathy. Furthermore, RNA-seq analysis identified both common and distinct restored glomerular genes downstream of Pio and GQ-16. Pio but not GQ-16 significantly induced aP2 (fatty acid binding protein) in adipocytes and in WAT. Pio induced more lipid accumulation than GQ-16 in differentiated adipocytes. Both, Pio and GQ-16 induced insulin sensitizing adipokines in WAT with varying degrees.

ConclusionsSelective modulation of PPAR{gamma} by a partial agonist, GQ-16, is more advantageous than pioglitazone in reducing proteinuria and NS associated co-morbidities, while reducing the adipogenic side-effects conferred by traditional PPAR{gamma} full agonists.

Translational StatementThe authors have previously reported that type-II diabetes drugs, thiazolidinediones (PPAR{gamma} agonists), also provide beneficial effects in reducing podocyte and glomerular injury. However, these drugs are associated with adverse effects such as weight gain, and their effects on glomerular disease-associated features are largely unexplored. Their current findings demonstrate that PPAR{gamma} can be selectively modulated by its partial agonist, GQ-16, which reduces proteinuria and improves nephrotic syndrome (NS) with reduced side-effects typically conferred by thiazolidinediones. These findings not only deepen our molecular understanding of the role of PPAR{gamma} in glomerular disease and underscore the potential for partial agonists of PPAR{gamma}, such as GQ-16 as a treatment modality for NS, but also lend the possibility of its potential benefits in diabetic nephropathy.