title: IL-17+ mast cell/T helper cell axis in the early stages of acne

abstract: Acne is a multifactorial disease driven by physiological changes occurring during puberty in the pilosebaceous unit (PSU) that leads to sebum overproduction and a dysbiosis involving notably Cutibacterium acnes. These changes in the PSU microenvironment lead to a shift from a homeostatic to an inflammatory state. Indeed, immunohistochemical analyses have revealed that inflammation and lymphocyte infiltration can be detected even in the infraclinical acneic stages, highlighting the importance of the early stages of the disease. In this study, we utilized a robust multi-pronged approach that included flow cytometry, confocal microscopy, and bioinformatics to comprehensively characterize the evolution of the infiltrating and resident immune cell populations in acneic lesions, beginning in the early stages of their development. Using a discovery cohort of 15 patients, we demonstrated that the composition of immune cell infiltrate is highly dynamic in nature, with the relative abundance of different cell types changing significantly as a function of clinical lesion stage. Within the stages examined, we identified a large population of CD69+ CD4+ T cells, several populations of activated antigen presenting cells, and activated mast cells producing IL-17. IL-17+ mast cells were preferentially located in CD4+ T cell rich areas and we showed that activated CD4+ T cells license mast cells to produce IL-17. Our study reveals that mast cells are the main IL-17 producers in the early stage of acne, underlying the importance of targeting the IL-17+ mast cell/T helper cell axis in therapeutic approaches.

category: immunology


title: Natural and after colon washing fecal samples: the two sides of the coin for investigating the human gut microbiome

abstract: To date there are several studies focusing on the importance of gut microbiome for human health, however the selection of a universal sampling matrix representative of the microbial biodiversity associated to the gastrointestinal (GI) tract, still represents a challenge. Here we present a study in which, through a deep metabarcoding analysis of the 16S rRNA gene, we compared two sampling matrices, feces (F) and colonic lavage liquid (LL), in order to evaluate their accuracy to represent the complexity of the human gut microbiome. A training set of 37 volunteers was attained and paired F and LL samples were collected from each subject. A preliminary absolute quantification of total 16S rDNA, performed by droplet digital PCR (ddPCR), confirmed that sequencing and taxonomic analysis were performed on same total bacterial abundance obtained from the two sampling methods. The taxonomic analysis of paired samples revealed that, although specific taxa were predominantly or exclusively observed in LL samples, as well as other taxa were detectable only or were predominant in stool, the microbiomes of the paired samples F and LL in the same subject hold overlapping taxonomic composition. Moreover, LL samples revealed a higher biodiversity than stool at all taxonomic ranks, as demonstrated by the Shannon Index and the Inverse Simpsons Index. We also found greater inter-individual variability than intra-individual variability in both sample matrices. Finally, functional differences were unveiled in the gut microbiome detected in the F and LL samples. A significant overrepresentation of 22 and 13 metabolic pathways, mainly occurring in Firmicutes and Proteobacteria, was observed in gut microbiota detected in feces and LL samples, respectively. This suggests that LL samples may allow for the detection of microbes adhering to the intestinal mucosal surface as members of the resident flora that are not easily detectable in stool, most likely representative of a diet-influenced transient microbiota. This first comparative study on feces and LL samples for the study of the human gut microbiome demonstrates that the use of both types of sample matrices may represent a possible choice to obtain a more complete view of the human gut microbiota in response to different biological and clinical questions.

category: molecular biology


title: A dinucleotide tag-based parallel reporter gene assay method

abstract: The causal SNPs leading to increased cancer predisposition mainly function as gene regulatory elements, the evaluation of which largely rely on the parallel reporter gene assay system. However, the common DNA barcode-based parallel reporter gene assay systems are troubled with tag bias, mainly due to the tag nucleotide composition. Here we describe a versatile dinucleotide-tag reporter system (DiR) that enables parallel analysis of regulatory elements with minimized bias based on the next-generation sequencing. The DiR system is also more robust than the classical luciferase assay method, especially in the investigation of moderate-level regulatory elements. We applied DiR-seq assay in the functional evaluation of the prostate cancer risk SNPs in prostate cancer cell lines and disclosed 2, and 6 regulatory SNPs in PC-3 and LNCaP cells. The DiR system has great potential to advance the functional study of risk SNPs that have associations with polygenic diseases.


title: The Consequences of Differential Origin Licensing Dynamics

abstract: MCM complexes are loaded onto chromosomes to license DNA replication origins in G1 phase of the cell cycle, but it is not yet known how mammalian MCM complexes are adequately distributed to both euchromatin and heterochromatin. To address this question, we combined time-lapse live-cell imaging with fixed cell immunofluorescence imaging of single human cells to quantify the relative rates of MCM loading in heterochromatin and euchromatin at different times within G1. We report here that MCM loading in euchromatin is faster than in heterochromatin in very early G1, but surprisingly, heterochromatin loading accelerates relative to euchromatin loading in middle and late G1. These different loading dynamics require ORCA-dependent differences in ORC distribution during G1. A consequence of heterochromatin origin licensing dynamics is that cells experiencing a truncated G1 phase from premature cyclin E expression enter S phase with under-licensed heterochromatin, and DNA damage accumulates preferentially in heterochromatin in the subsequent S/G2 phase. Thus G1 length is critical for sufficient MCM loading, particularly in heterochromatin, to ensure complete genome duplication and to maintain genome stability.


title: The Consequences of Differential Origin Licensing Dynamics in Distinct Chromatin Environments


title: TACSTD2 upregulation is an early reaction to lung infection

abstract: TACSTD2 encodes a transmembrane glycoprotein Trop2 commonly overexpressed in carcinomas. While the Trop2 protein was discovered already in 1981 and first antibody-drug conjugate targeting Trop2 were recently approved cancer therapy, the physiological role of Trop2 is still not fully understood. In this article, we show that TACSTD2/Trop2 expression is evolutionarily conserved in lungs of various vertebrates. By analysis of publicly available transcriptomic data we demonstrate that TACSTD2 level consistently increases in lungs infected with miscellaneous pathogens. Single cell and subpopulation based transcriptomic data revealed that the major source of TACSTD2 transcript are lung epithelial cells and their progenitors and that TACSTD2 is induced directly in lung epithelial cells following infection. This increase may represent a mechanism to maintain/restore epithelial barrier function and contribute to regeneration process in infected/damaged lungs.

category: cell biology


title: Time-dependent toxicity of tire particles on soil nematodes

abstract:
Tire-wear particles (TWPs) are being released into the environment by wearing down during car driving, and are considered an important microplastic pollution source. The chemical additive leaching from these polymer-based materials and its potential effects are likely temporally dynamic, since larger amounts of potentially toxic compounds can gradually increase with contact time of plastic particles with surrounding media. In the present study, we conducted soil toxicity tests using the soil nematode Caenorhabditis elegans with different soil pre-incubation (30 and 75 days) and exposure (short-term exposure, 2 days; lifetime exposure, 10 days) times. Soil pre-incubation increased toxicity of TWPs, and the effective concentrations after the pre-incubation were much lower than environmentally relevant concentrations. The lifetime of C. elegans was reduced faster in the TWP treatment groups, and the effective concentration for lifetime exposure tests were 100- to 1,000-fold lower than those of short-term exposure tests. Water-extractable metal concentrations (Cr, Cu, Ni, Pb, and Zn) in the TWP-soils showed no correlation with nominal TWP concentrations or pre-incubation times, and the incorporated metals in the TWPs may be not the main reason of toxicity in this study. Our results show that toxic effects of TWPs can be time-dependent, both in terms of the microplastic particles themselves and their interactions in the soil matrix, but also because of susceptibility of target organisms depending on developmental stage. It is vital that future work consider these aspects, since otherwise effects of microplastics and TWPs could be underestimated.

Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

category: ecology


title: A quantitative trait variant in Gabra2 underlies increased methamphetamine stimulant sensitivity

abstract: Psychostimulant (methamphetamine, cocaine) use disorders have a genetic component that remains mostly unknown. Here, we conducted genome-wide quantitative trait locus (QTL) analysis of methamphetamine stimulant sensitivity. To facilitate gene identification, we employed a Reduced Complexity Cross between closely related C57BL/6 mouse substrains and examined maximum speed and distance traveled over 30 min following methamphetamine (2 mg/kg, i.p.). For maximum methamphetamine-induced speed following the second and third administration, we identified a single genome-wide significant QTL on chromosome 11 that peaked near the Cyfip2 locus [LOD = 3.5, 4.2; peak = 21 cM (36 Mb)]. For methamphetamine-induced distance traveled, we identified a single genome-wide significant QTL on chromosome 5 that peaked near a functional intronic indel in Gabra2 that codes for the alpha-2 subunit of the GABA-A receptor [LOD = 5.2; peak = 35 cM (67 Mb)]. Striatal cis-expression QTL mapping corroborated Gabra2 as a functional candidate gene underlying methamphetamine-induced distance traveled. CRISPR/Cas9-mediated correction of the mutant intronic deletion on the C57BL/6J background to the wild-type C57BL/6NJ allele was sufficient to reduce methamphetamine-induced locomotor activity toward the wild-type C57BL/6NJ-like level, thus validating the quantitative trait variant (QTV). These studies demonstrate the power and efficiency of Reduced Complexity Crosses in identifying causal genes and variants underlying complex traits. Functionally restoring Gabra2 expression decreased methamphetamine stimulant sensitivity and supports preclinical and human genetic studies implicating the GABA-A receptor in psychostimulant addiction-relevant traits. Importantly, our findings have major implications for investigators studying psychostimulants in the C57BL/6J strain - the gold standard strain in biomedical research.

category: genetics


title: SLOW5: a new file format enables massive acceleration of nanopore sequencing data analysis

abstract: Nanopore sequencing is an emerging genomic technology with great potential. However, the storage and analysis of nanopore sequencing data have become major bottlenecks preventing more widespread adoption in research and clinical genomics. Here, we elucidate an inherent limitation in the file format used to store raw nanopore data - known as FAST5 - that prevents efficient analysis on high-performance computing (HPC) systems. To overcome this, we have developed SLOW5, an alternative file format that permits efficient parallelisation and, thereby, acceleration of nanopore data analysis. For example, we show that using SLOW5 format, instead of FAST5, reduces the time and cost of genome-wide DNA methylation profiling by an order of magnitude on common HPC systems, and delivers consistent improvements on a wide range of different architectures. With a simple, accessible file structure and a ~25% reduction in size compared to FAST5, SLOW5 format will deliver substantial benefits to all areas of the nanopore community.

category: genomics


title: Taming the massive genome of Scots pine with PiSy50k, a new genotyping array for conifer research

abstract:
Scots pine (Pinus sylvestris) is the most widespread coniferous tree in the boreal forests of Eurasia and has major economic and ecological importance. However, its large and repetitive genome presents a challenge for conducting genome-wide analyses such as association studies and genomic selection. We present a new 50K SNP genotyping array for Scots pine research, breeding programs, and other applications. To select the SNP set, we first genotyped 480 Scots pine samples on a 407 540 SNP screening array, and identified 47 712 high-quality SNPs for the final array (called  PiSy50k). Here, we provide details of the design and testing, as well as allele frequency estimates from the discovery panel, functional annotation, tissue-specific expression patterns, and expression level information for the SNPs or corresponding genes, when available. We validated the performance of the PiSy50k array using samples from breeding populations from Finland and Scotland. Overall, 39 678 (83.2%) SNPs showed low error rates (mean = 0.92%). Relatedness estimates based on array genotypes were consistent with the expected pedigrees, and the amount of Mendelian error was negligible. In addition, array genotypes successfully discriminate Scots pine populations from different geographic origins. The PiSy50k array will be a valuable tool for future genetic studies and forestry applications.

Significance statementScots pine is an evolutionary, economically and ecologically impressive coniferous species but its gigantic genome has limited studying e.g. the genetic basis of its functional trait variation. We have developed a genotyping array that facilitates Scots pine genetic research and linking its trait variation to genetic polymorphisms and gene expression levels across the genome.


title: A survey of optimal strategy for signature-based drug repositioning and an application to liver cancer

abstract: Pharmacologic perturbation projects, such as Connectivity Map (CMap) and Library of Integrated Network-based Cellular Signatures (LINCS), have produced many perturbed expression data, providing enormous opportunities for computational therapeutic discovery. However, currently there is no consensus on which methodologies and parameters are the most optimal to conduct such analysis. Aiming to fill this gap, we developed new benchmarking standards for quantitatively estimating drug retrieval performance. Investigations of potential factors influencing drug retrieval were conducted based on these standards. As a result, we determined an optimal strategy for LINCS data-based therapeutic discovery. With this approach, we further identified new therapeutics for liver cancer of which the current treatment modalities remain imperfect. Both computational and experimental results demonstrated homoharringtonine (HHT) could be a promising anti-liver cancer agent. In summary, our findings will not only impact the future applications of LINCS data but also offer new opportunities for therapeutic intervention for liver cancer.

category: bioinformatics


title: Flying in the Face of Adversity: A Drosophila-based Virtual CURE Provides Semester-long Authentic Research Opportunity to the Flipped Classroom.

abstract: A call for the integration of research experiences into all biology curricula has been a major goal for educational reform efforts nationally. Course-Based Undergraduate Research Experiences (CUREs) have been the predominant method of accomplishing this, but their associated costs and complex design can limit their wide adoption. In 2020, the COVID-19 pandemic forced programs to identify unique ways to still provide authentic research experiences while students were virtual. We report here a full guide for the successful implementation of a semester-long virtual CURE that uses Drosophila behavioral assays to explore the connection between pain and addiction with the use of a "lab-in-a-box" sent home to students. Individual components were piloted across three semesters and launched as a 100-level introductory course with 19 students. We found that this course increased science identity and successfully improved key research competencies as per the Undergraduate Research Student Self-Assessment (URSSA) survey. This course is ideal for flipped classrooms ranging from introductory biology to upper-level neuroscience courses and can be integrated directly into the lecture period without the need for building a new course. Given the low cost, recent comfort with virtual learning environments, and the current proliferation of flipped biology classrooms following the 2020 pandemic, this curriculum could serve as an ideal project-based active-learning tool for equitably increasing access to authentic research experiences.

category: scientific communication and education


title: Not all voxels are created equal: reducing estimation bias in regional NODDI metrics using tissue-weighted means

abstract: Neurite orientation dispersion and density imaging (NODDI) estimates microstructural properties of brain tissue relating to the organisation and processing capacity of neurites, which are essential elements for neuronal communication. Descriptive statistics of NODDI tissue metrics are commonly analysed in regions-of-interest (ROI) to identify brain-phenotype associations. Here, the conventional method to calculate the ROI mean weights all voxels equally. However, this produces biased estimates in the presence of CSF partial volume. This study introduces the tissue-weighted mean, which calculates the mean NODDI metric across the tissue within an ROI, utilising the tissue fraction estimate from NODDI to reduce estimation bias. We demonstrate the proposed mean in a study of white matter abnormalities in young onset Alzheimers disease (YOAD). Results show the conventional mean induces significant bias that correlates with CSF partial volume, primarily affecting periventricular regions and more so in YOAD subjects than in healthy controls. Due to the differential extent of bias between healthy controls and YOAD subjects, the conventional mean under- or over-estimated the effect size for group differences in many ROIs. This demonstrates the importance of using the correct estimation procedure when inferring group differences in studies where the extent of CSF partial volume differs between groups. These findings are robust across different acquisition and processing conditions. Bias persists in ROIs at higher image resolution, as demonstrated using data obtained from the third phase of the Alzheimers disease neuroimaging initiative (ADNI); and when performing ROI analysis in template space. This suggests that conventional ROI means of NODDI metrics are biased estimates under most contemporary experimental conditions, the correction of which requires the proposed tissue-weighted mean. The tissue-weighted mean produces accurate estimates of ROI means and group differences when ROIs contain voxels with CSF partial volume. In addition to NODDI, the technique can be applied to other multi-compartment models that account for CSF partial volume, such as the free water elimination method. We expect the technique to help generate new insights into normal and abnormal variation in tissue microstructure of regions typically confounded by CSF partial volume, such as those in individuals with larger ventricles due to atrophy associated with neurodegenerative disease.

category: neuroscience


title: Clonally expanded virus-specific CD8 T cells acquire diverse transcriptional phenotypes during acute, chronic, and latent infections

abstract: CD8+ T cells play a crucial role in the control and resolution of viral infections and can adopt a wide range of phenotypes and effector functions depending on the inflammatory context and the duration and extent of antigen exposure. Similarly, viral infections can exert diverse selective pressures on populations of clonally related T cells. Technical limitations have nevertheless made it challenging to investigate the relationship between clonal selection and transcriptional phenotypes of virus-specific T cells. We therefore performed single-cell T cell receptor (TCR) repertoire and transcriptome sequencing of virus-specific CD8 T cells in murine models of acute, chronic and latent infection. We observed clear infection-specific populations corresponding to memory, effector, exhausted, and inflationary phenotypes. We further uncovered a mouse-specific and polyclonal T cell response, despite all T cells sharing specificity to a single viral epitope, which was accompanied by stereotypic TCR germline gene usage in all three infection types. Persistent antigen exposure during chronic and latent viral infections resulted in a higher proportion of clonally expanded T cells relative to acute infection. We furthermore observed a relationship between transcriptional heterogeneity and clonal expansion for all three infections, with highly expanded clones having distinct transcriptional phenotypes relative to lowly expanded clones. Finally, we developed and utilized a bioinformatic pipeline integrating pseudotime and clonality, termed Clonotyme, to further support a model in which expanded virus-specific CD8+ T cells adopt heterogenic, yet preferentially, effector-like phenotypes. Together our work relates clonal selection to gene expression in the context of viral infection and further provides a dataset and accompanying software for the immunological community.


title: Pathology and immunity after SARS-CoV-2 infection in male ferrets is affected by age and inoculation route

abstract: Improving COVID-19 intervention strategies partly relies on animal models to study SARS-CoV-2 disease and immunity. In our pursuit to establish a model for severe COVID-19, we inoculated young and adult male ferrets intranasally or intratracheally with SARS-CoV-2. Intranasal inoculation established an infection in all ferrets, with viral dissemination into the brain and gut. Upon intratracheal inoculation only adult ferrets became infected. However, neither inoculation route induced observable COVID-19 symptoms. Despite this, a persistent inflammation in the nose was prominent in especially young ferrets and follicular hyperplasia in the bronchi developed 21 days post infection. These effects -if sustained- might resemble long-COVID. Respiratory and systemic cellular responses and antibody responses were induced only in animals with an established infection. We conclude that intranasally-infected ferrets resemble asymptomatic COVID-19 and possibly aspects of long-COVID. Combined with the increasing portfolio to measure adaptive immunity, ferrets are a relevant model for SARS-CoV-2 vaccine research.

category: microbiology


title: Large scale screening discovers clofoctol as an inhibitor of SARS-CoV-2 replication that reduces COVID-19-like pathology

abstract:
Drug repurposing has the advantage of shortening regulatory preclinical development steps. Here, we screened a library of drug compounds, already registered in one or several geographical areas, to identify those exhibiting antiviral activity against SARS-CoV-2 with relevant potency. Of the 1,942 compounds tested, 21 exhibited a substantial antiviral activity in Vero-81 cells. Among them, clofoctol, an antibacterial drug used for the treatment of bacterial respiratory tract infections, was further investigated due to favorable safety profile and pharmacokinetic properties. Notably, the peak concentration of clofoctol that can be achieved in human lungs is more than 20 times higher than its IC50 measured against SARS-CoV-2 in human pulmonary cells. This compound inhibits SARS-CoV-2 at a post-entry step. Lastly, therapeutic treatment of human ACE2 receptor transgenic mice decreased viral load, reduced inflammatory gene expression and lowered pulmonary pathology. Altogether, these data strongly support clofoctol as a therapeutic candidate for the treatment of COVID-19 patients.

SummaryAntivirals targeting SARS-CoV-2 are sorely needed. In this study, we screened a library of drug compounds and identified clofoctol as an antiviral against SARS-CoV-2. We further demonstrated that, in vivo, this compound reduces inflammatory gene expression and lowers pulmonary pathology.


title: Clofoctol inhibits SARS-CoV-2 replication and reduces lung pathology in mice


title: Temperature Evolution Following Joint Loading Promotes Chondrogenesis by Synergistic Cues via Calcium Signaling

abstract: During loading of viscoelastic tissues, part of the mechanical energy is transformed into heat that can locally increase the tissue temperature, a phenomenon known as self-heating. In the framework of mechanobiology, it has been accepted that cells react and adapt to mechanical stimuli. However, the cellular effect of temperature increase as a by-product of loading has been widely neglected. In this work, we focused on cartilage self-heating to present a "thermo-mechanobiological" paradigm, and demonstrate how the synergy of a biomimetic temperature evolution and mechanical loading could influence cell behavior. We thereby developed a customized in vitro system allowing to recapitulate pertinent in vivo physical cues and determined the cells chondrogenic response to thermal and/or mechanical stimuli. Cellular mechanisms of action and potential signaling pathways of thermo-mechanotransduction process were also investigated. We found that co-existence of thermo-mechanical cues had a superior effect on chondrogenic gene expression compared to either signal alone. Specifically, a synergetic effect was observed for upregulation of Sox9 by application of the physiological thermo-mechanical stimulus. Multimodal TRPV4 channels were identified as key mediators of thermo-mechanotransduction process, which becomes ineffective without external calcium sources. We also observed that the isolated temperature evolution, as a by-product of loading, is a contributing factor to the cells response and this could be considered as important as the conventional mechanical loading. Providing an optimal thermo-mechanical environment by synergy of heat and loading portrays new opportunity for development of novel treatments for cartilage regeneration and can furthermore signal key elements for emerging cell-based therapies.

category: bioengineering


title: The Role of Action Potential Waveform in Failure of Excitation Contraction Coupling

abstract: Excitation contraction coupling (ECC) is the process by which electrical excitation of muscle is converted into force generation. Depolarization of skeletal muscle resting potential contributes to failure of ECC in diseases such as periodic paralysis, ICU acquired weakness and possibly fatigue of muscle during vigorous exercise. When extracellular K+ is raised to depolarize the resting potential, failure of ECC occurs suddenly, over a range of several mV of resting potential. While some studies have hypothesized the sudden failure of ECC is due to all-or-none failure of excitation, other studies suggest failure of excitation is graded. Intracellular recordings of action potentials (APs) in individual fibers during depolarization revealed that APs do not fail in an all-or-none manner. Simultaneous imaging of Ca2+ transients during depolarization revealed failure over a narrow range of resting potentials. An AP property that closely correlated with the sudden failure of the Ca2+ transient was the integral of AP voltage with respect to time. We hypothesize the close correlation is due to the combined dependence on time and voltage of Ca2+ release from the sarcoplasmic reticulum. The quantitative relationships established between resting potential, APs and Ca2+ transients provide the foundation for future studies of depolarization-induced failure of ECC in diseases such as periodic paralysis.

category: physiology


title: Using wearable biosensors and ecological momentary assessments for the detection of prolonged stress in real life

abstract:
Emerging efforts toward prevention of stress-related mental disorders have created a need for unobtrusive real-life monitoring of stress-related symptoms. We used ecological momentary assessments (EMA) combined with wearable biosensors to investigate whether these can be used to detect periods of prolonged stress. During stressful high-stake exam (versus control) weeks, participants reported increased negative affect and decreased positive affect. Intriguingly, physiological arousal was decreased on average during the exam week. Time-resolved analyses revealed peaks in physiological arousal associated with both self-reported stress and self-reported positive affect, while the overall decrease in physiological arousal was mediated by lower positive affect during the stress period. We then used machine learning to show that a combination of EMA and physiology yields optimal classification of week types. Our findings highlight the potential of wearable biosensors in stress-related mental-health monitoring, but critically show that psychological context is essential for interpreting physiological arousal detected using these devices.

TeaserSmartwatches combined with daily diaries of mood can detect stress periods using individualized machine learning models.


title: Children are full of optimism, but those rose-tinted glasses are fading: reduced learning from negative outcomes drives hyperoptimism in children.

abstract: Believing that good things will happen in life is essential to maintain motivation and achieve highly ambitious goals. This optimism bias, the overestimation of positive outcomes, may be particularly important during childhood when motivation must be maintained in the face of negative outcomes. In a learning task, we have thus studied the mechanisms underlying the development of optimism bias. Investigating children (8-9 year-olds), early (12-13 year-olds) and late adolescents (16-17 year-olds), we find a consistent optimism bias across age groups. However, children were particularly hyperoptimistic, with the optimism bias decreasing with age. Using computational modelling, we show that this was driven by a reduced learning from worse-than-expected outcomes, and this reduced learning explains why children are hyperoptimistic. Our findings thus show that insensitivity to bad outcomes in childhood helps to prevent taking on an overly realistic perspective and maintain motivation.


title: Varenicline Prevents SARS-CoV-2 Infection In Vitro and in Rhesus Macaques

abstract:
BackgroundSARS-CoV-2 infections have resulted in a global pandemic, but an antiviral therapy for this novel strain of coronavirus does not currently exist. The objective of our study was to investigate the antiviral potential of the nicotinic acetylcholine receptor (nACHR) agonist varenicline tartrate against SARS-CoV-2.

MethodsWe assessed antiviral activity using in vitro human cell assays and we assessed in vivo efficacy in a rhesus macaque model.

ResultsIn vitro studies found that varenicline tartrate, over a range of concentrations, reduced the infectivity of SARS-CoV-2 wildtype, alpha, and beta variants in Calu-3 cells and Caco-2 cells, with maintenance of cell viability. In vivo studies found that varenicline tartrate, administered as a nasal spray to rhesus macaques, reduced SARS-CoV-2 wildtype viral load and inhibited viral replication in the nasal mucosa and upper airway.

ConclusionAlthough the study reported here was exploratory, we have confirmed that the nAChR agonist varenicline has the potential to interact with and inhibit SARS-CoV-2 infection and replication.


title: The SARS-CoV-2 host cell membrane fusion protein TMPRSS2 is a tumor suppressor and its downregulation correlates with increased antitumor immunity and immunotherapy response in lung adenocarcinoma

abstract:
BackgroundTMPRSS2 is a host cell membrane fusion protein for SARS-CoV-2 invading human host cells. It also has an association with cancer, particularly prostate cancer. However, its association with lung cancer remains insufficiently explored. Thus, an in-depth investigation into the association between TMPRSS2 and lung cancer is significant, considering that lung cancer is the leading cause of cancer death and that the lungs are the primary organ SARS-CoV-2 attacks.

MethodsUsing five lung adenocarcinoma (LUAD) genomics datasets, we explored associations between TMPRSS2 expression and immune signatures, cancer-associated pathways, tumor progression phenotypes, and clinical prognosis in LUAD by the bioinformatics approach. Furthermore, we validated the findings from the bioinformatics analysis by performing in vitro experiments with the human LUAD cell line A549 and in vivo experiments with mouse tumor models. We also validated our findings in LUAD patients from Jiangsu Cancer Hospital, China.

ResultsTMPRSS2 expression levels were negatively correlated with the enrichment levels of CD8+ T and NK cells and immune cytolytic activity in LUAD, which represent antitumor immune signatures. Meanwhile, TMPRSS2 expression levels were negatively correlated with the enrichment levels of CD4+ regulatory T cells and myeloid-derived suppressor cells and PD-L1 expression levels in LUAD, which represent antitumor immunosuppressive signatures. However, TMPRSS2 expression levels showed a significant positive correlation with the ratios of immune-stimulatory/immune-inhibitory signatures (CD8+ T cells/PD-L1) in LUAD. It indicated that TMPRSS2 levels had a stronger negative correlation with immune-inhibitory signatures than with immune-stimulatory signatures. TMPRSS2 downregulation correlated with elevated activities of many oncogenic pathways in LUAD, including cell cycle, mismatch repair, p53, and extracellular matrix (ECM) signaling. TMPRSS2 downregulation correlated with increased proliferation, stemness, genomic instability, tumor advancement, and worse survival in LUAD. In vitro and in vivo experiments validated the association of TMPRSS2 deficiency with increased tumor cell proliferation and invasion and antitumor immunity in LUAD. Moreover, in vivo experiments demonstrated that TMPRSS2-knockdown tumors were more sensitive to BMS-1, an inhibitor of PD-1/PD-L1.

ConclusionsTMPRSS2 is a tumor suppressor, while its downregulation is a positive biomarker of immunotherapy in LUAD. Our data provide a connection between lung cancer and pneumonia caused by SARS-CoV-2 infection.


title: Host cellular RNA helicases regulate SARS-CoV-2 infection

abstract:
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has largest RNA genome of approximately 30kb among RNA viruses. The DDX DEAD-box RNA helicase is a multifunctional protein involved in all aspects of RNA metabolism. Therefore, host RNA helicases may regulate and maintain such large viral RNA genome. In this study, I investigated the potential role of several host cellular RNA helicases in SARS-CoV-2 infection. Notably, DDX21 knockdown markedly accumulated intracellular viral RNA and viral production, as well as viral infectivity of SARS-CoV-2, indicating that DDX21 strongly restricts the SARS-CoV-2 infection. As well, MOV10 RNA helicase also suppressed the SARS-CoV-2 infection. In contrast, DDX1, DDX5, and DDX6 RNA helicases were required for SARS-CoV-2 replication. Indeed, SARS-CoV-2 infection dispersed the P-body formation of DDX6 and MOV10 RNA helicases as well as XRN1 exonuclease, while the viral infection did not induce stress granule formation. Accordingly, the SARS-CoV-2 nucleocapsid (N) protein interacted with DDX6, DDX21, and MOV10 and disrupted the P-body formation, suggesting that SARS-CoV-2 N hijacks DDX6 to utilize own viral replication and overcomes their anti-viral effect of DDX21 and MOV10 through as interaction with host cellular RNA helicase. Altogether, host cellular RNA helicases seem to regulate the SARS-CoV-2 infection.

ImportanceSARS-CoV-2 has large RNA genome of approximately 30kb. To regulate and maintain such large viral RNA genome, host RNA helicases may involve in SARS-CoV-2 replication. In this study, I have demonstrated that DDX21 and MOV10 RNA helicases limit viral infection and replication. In contrast, DDX1, DDX5 and DDX6 are required for the SARS-CoV-2 infection. Interestingly, the SARS-CoV-2 infection disrupted P-body formation and attenuated or suppressed stress granule formation. Thus, SARS-CoV-2 seems to hijack host cellular RNA helicases to play a proviral role by facilitating viral infection and replication and, by suppressing host innate immune system.


title: The MpsB protein contributes to both the toxicity and immune evasion capacity of Staphylococcus aureus.

abstract: Understanding the role specific bacterial factors play in the development of severe disease in humans is critical if new approaches to tackle such infections are to be developed. In this study we focus on genes we have found to be associated with patient outcome following bacteraemia caused by the major human pathogen Staphylococcus aureus. By examining the contribution these genes make to the ability of the bacteria to survive exposure to the antibacterial factors found in serum, we identify three novel serum resistance associated genes, mdeA, mpsB and yycH. Detailed analysis of an MpsB mutant supports its previous association with the slow growing SCV phenotype of S. aureus, and we demonstrate that the effect this mutation has on membrane potential prevents the activation of the Agr quorum sensing system, and as a consequence the mutant bacteria do not produce cytolytic toxins. Given the importance of both toxin production and immune evasion to the ability of S. aureus to cause disease, we believe these findings explain the role of the mpsB gene as a mortality-associate locus during human disease.


title: Locating and Quantifying Carbon Steel Corrosion Rates Linked to Fungal B20 Biodiesel Degradation

abstract:
Fungi that degrade B20 biodiesel in storage tanks have also been linked to microbiologically influenced corrosion (MIC). A member of the filamentous fungal genus Byssochlamys, and a yeast from the genus Wickerhamomyces were isolated from heavily contaminated B20 storage tanks from multiple Air Force bases. Although these taxa were linked to microbiologically influenced corrosion in situ, precise measurement of their corrosion rates and pitting severity on carbon steel was not available. In the experiments described here, we directly link fungal growth on B20 biodiesel to higher corrosion rates and pitting corrosion of carbon steel under controlled conditions. When these fungi were growing solely on B20 biodiesel for carbon and energy, consumption of FAME and n-alkanes was observed. The corrosion rates for both fungi were highest at the interface between the B20 biodiesel and the aqueous medium, where they acidified the medium and produced deeper pits than abiotic controls. Byssochlamys produced the most corrosion of carbon steel and produced the greatest pitting damage. This study characterizes and quantifies the corrosion of carbon steel by fungi that are common in fouled B20 biodiesel through their metabolism of the fuel, providing valuable insight for assessing MIC associated with storage and dispensing B20 biodiesel.

IMPORTANCEBiodiesel is widely used across the United States and worldwide, blended with ultralow sulfur diesel in various concentrations. In this study we were able to demonstrate that the filamentous fungi Byssochlamys AF004 and the yeast Wickerhamomyces SE3 were able to degrade fatty acid methyl esters and alkanes in biodiesel causing increases in acidity. Both fungi also accelerated the corrosion of carbon steel, especially at the interface of the fuel and water, where their biofilms were located. This research provides controlled, quantified measurements and the localization of microbiologically influenced corrosion caused by common fungal contaminants in biodiesel fuels.


title: Exploitation of the Secretory Pathway by SARS-CoV-2 Envelope

abstract: The beta-coronavirus SARS-CoV-2 is the causative agent of the current global COVID-19 pandemic. Coronaviruses are enveloped RNA viruses. Assembly and budding of coronavirus particles occur at the Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC), with the structural proteins Nucleocapsid, Spike, Membrane and Envelope facilitating budding and release of virions into the secretory pathway lumen. This allows viral release which can occur through delivery of virus particles to deacidified lysosomes and subsequent lysosomal secretion. Coronaviral Envelope proteins are necessary for coronavirus assembly, play important roles in replication and can form oligomeric cation channels. Whilst synthesised in the ER, the mechanism by which Envelope achieves its steady state localisation to the ERGIC remains unclear. Here, we used fluorescent reporters to illuminate the Envelope protein from SARS-CoV-2. We discovered that internal tagging of this protein is necessary to preserve the functionality of a C-terminal ER-export motif and to allow localisation of Envelope to the ERGIC. Using this non-disruptive form of tagging, we used proximity biotinylation to define the vicinal proteome of wild type and ER-restricted versions of Envelope. We show that both Envelope and the presence of its ER-export motif contribute to the packaging of nucleocapsid into virus like particles. Finally, using our labelled versions of Envelope, we discovered that a minor pool of this protein is delivered to lysosomes. We show that lysosomal Envelope is oligomeric and can contribute to pH neutralisation in these organelles.


title: Interacting effects of cold snaps, rain, and agriculture on the fledging success of a declining aerial insectivore

abstract: Climate change predicts the increased frequency, duration, and intensity of inclement weather periods, such as unseasonably low temperatures (i.e., cold snaps) and prolonged precipitation. Many migratory species have advanced the phenology of important life history stages, and as a result will likely be exposed to these periods of inclement spring weather more often, thus risking reduced fitness and population growth. For declining avian species, including aerial insectivores, anthropogenic landscape changes such as agricultural intensification are another driver of population declines. These landscape changes may affect the foraging ability of food provisioning parents, and reduce the survival of nestlings exposed to inclement weather, through for example pesticide exposure impairing thermoregulation and punctual anorexia. Breeding in agro-intensive landscapes may thus exacerbate the negative effects of inclement weather under climate change. We observed that a significant reduction in the availability of insect prey occurred when daily maximum temperatures fell below 18.3{degrees}C, and thereby defined any day where the maximum temperature fell below this value as a day witnessing a cold snap. We then combined daily information on the occurrence of cold snaps and measures of precipitation to assess their impact on the fledging success of Tree Swallows (Tachycineta bicolor) occupying a nest box system placed across a gradient of agricultural intensification. Estimated fledging success of this declining aerial insectivore was 36.2% lower for broods experiencing four cold snap days during the 12 days post hatching period versus broods experiencing none, and this relationship was worsened when facing more precipitation. We further found that the overall negative effects of a brood experiencing periods of inclement weather was exacerbated in more agro-intensive landscapes. Our results indicate that two of the primary hypothesized drivers of many avian population declines may interact to further increase the rate of declines in certain landscape contexts.


title: Inbreeding and learning affect fitness and colonization of new host plants, a behavioral innovation in the spider mite Tetranychus urticae

abstract: Habitat fragmentation increases the isolation of natural populations resulting in reduced genetic variability and increased species extinction risk. Behavioral innovation through learning, i.e., the expression of a new learned behavior in a novel context, can help animals colonize new suitable and increasingly fragmented habitats. It has remained unclear, however, how reduced genetic variability affects learning for colonizing more or less suitable habitats. Here, we show that inbreeding in a subsocial invertebrate, the spider mite Tetranychus urticae, reduces novel host plant colonization and reproductive fitness. When provided with the possibility to learn from previous experience with a host plant species, outbred mites showed aversive learning ability, but inbred mites did not adapt their behavior. We further found a putative general cost of learning in both inbred and outbred mites. Our results reveal that inbreeding affects the learning component of behavioral innovation for host plant colonization.


title: Detection of potential new SARS-CoV-2 Gamma-related lineage in Tocantins shows the spread and ongoing evolution of P.1 in Brazil

abstract: After more than a year of the pandemic situation of COVID-19, the United Kingdom (UK), South Africa, and Brazil became the epicenter of new lineages of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Variants of Concern (VOCs) were identified through a continuous genomic surveillance global effort, the B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and P.1 (Gamma) harboring a constellation set of mutations. This research aims to: (i) report the predominance of the Gamma (P.1) lineage presenting the epidemiological situation of the SARS-CoV-2 genomic surveillance at the state of Tocantins, and (ii) describe the emergence of possible new mutations and viral variants with the potential new lineage (P1-related) represented by 8 genomes from the Tocantins harboring the mutation L106F in ORF3a. At the moment, 6,687 SARS-CoV-2 genomes from GISAID carry this mutation. The whole-genome sequencing has an important role in understanding the evolution and genomic diversity of SARS-CoV-2, thus, the continuous monitoring will help in the control measures and restrictions imposed by the secretary of health of the state to prevent the spread of variants.


title: Single Cell T Cell Receptor Repertoire Profiling for Dogs

abstract:
BackgroundSpontaneous cancers in companion dogs are increasingly recognized as robust models of human disease. This recognition has led to translational clinical trials in companion dogs with osteosarcoma, lymphoma, melanoma, squamous cell carcinoma, and soft tissue sarcoma. The ability to precisely track tumor-specific immune responses in such clinical trials would benefit from reagents to perform species-specific single cell T cell receptor sequencing (scTCRseq). This technology defines clones of T cells reacting to immune interventions and can help identify the specific epitope of response. Single cell gene expression data give insights into the activity and polarization of the T cell. To date, scTCRseq has not been demonstrated for canine samples.

MethodsSamples from two responding dogs in a trial of an autologous deglycosylated melanoma vaccine were selected to demonstrate applicability of scTCRseq in a cancer immunotherapy setting. A single-cell suspension of cryopreserved peripheral blood mononuclear cells (PBMC) was prepared for 10X single cell sequencing. Full length 10X cDNA was amplified using a custom-designed nested PCR of the alpha/beta V(D)J region. A library made from this enriched product (scTCRseq) and a 10X gene expression (GEX) library (scRNAseq) were sequenced on the NovaSeq 6000.

Results1,850-2,172 estimated V(D)J-expressing cells yielded 87-103.7 million reads with 73.8%-75.8% mapped to a V(D)J gene (beta/alpha chains ratio 1.5:1). 43 TRAJ, 29 TRAV, 12 TRBJ, and 22 TRBV gene segments were observed representing 72.9%, 51.8%, 100%, and 62.9% of all known V and J gene segments respectively. A large diversity of clonotypes was captured with 966-1,253 TRA/TRB clonotypes identified. Both dogs also exhibited a small number of highly abundant T cell clonotypes suggesting the presence of an anti-tumor T cell population. GEX enriched libraries successfully defined large clusters of CD8+ and CD4+ T cells that overlapped with V(D)J-expressing cells.

DiscussionThe developed reagents successfully generated scTCRseq data, for the first time, which allowed the T cell repertoire to be surveyed in dogs responding to anti-tumor immunotherapy. These reagents will allow longitudinal tracking of anti-tumor T cell dynamics in canine cancer immunotherapy trials.


title: Designed architectural proteins that tune DNA looping in bacteria

abstract: Architectural proteins alter the shape of DNA, often by distorting the double helix and introducing sharp kinks that relieve strain in tightly-bent DNA structures. Here we design and test artificial architectural proteins based on a sequence-specific Transcription Activator-like Effector (TALE) protein, either alone or fused to a eukaryotic high mobility group B (HMGB) DNA-bending domain. We hypothesized that TALE protein binding would stiffen DNA to bending and twisting, acting as an architectural protein that antagonizes the formation of small DNA loops. In contrast, fusion to an HMGB domain was hypothesized to generate a targeted DNA-bending architectural protein that facilitates DNA looping. We provide evidence from E. coli Lac repressor gene regulatory loops supporting these hypotheses in living bacteria. Both data fitting to a thermodynamic DNA looping model and sophisticated molecular modeling support the interpretation of these results. We find that TALE protein binding inhibits looping by stiffening DNA to bending and twisting, while the Nhp6A domain enhances looping by bending DNA without introducing twisting flexibility. Our work illustrates artificial approaches to sculpt DNA geometry with functional consequences. Similar approaches may be applicable to tune the stability of small DNA loops in eukaryotes.

category: biochemistry


title: Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization

abstract: Post-translational modifications (PTMs) within splicing factor RNA-binding proteins (RBPs), such as phosphorylation, regulate several critical steps in RNA metabolism including spliceosome assembly, alternative splicing and mRNA export. Notably, the arginine-/serine-rich (RS) domains in SR proteins are densely modified by phosphorylation compared with the remainder of the proteome. Previously, we showed that dephosphorylation of SRSF2 regulated increased interactions with similar arginine-rich RBPs U1-70K and LUC7L3. In this work, we dephosphorylated nuclear extracts using phosphatase in vitro and analyzed equal amounts of detergent-soluble and -insoluble fractions by mass spectrometry-based proteomics. Correlation network analysis resolved 27 distinct modules of differentially soluble nucleoplasm proteins. We found classes of arginine-rich RBPs that decrease in solubility following dephosphorylation and enrich to the insoluble pelleted fraction, including the SR protein family and the SR-like LUC7L RBP family. Importantly, increased insolubility was not observed across broad classes of RBPs. Phosphorylation regulated SRSF2 structure, as dephosphorylated SRSF2 formed high molecular weight oligomeric species in vitro. Reciprocally, phosphorylation of SRSF2 by serine-/arginine protein kinase 2 (SRPK2) in vitro prevented high molecular weight SRSF2 species formation. Furthermore, we pharmacologically inhibited SRPKs in mammalian cells and observed increased cytoplasmic granules as well as the formation of cytoplasmic SRSF2 tubular structures that associate with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation may be a critical modification that prevents arginine-rich RBP insolubility and oligomerization.


title: Structural Insights into the Venus flytrap Mechanosensitive Ion Channel Flycatcher1

abstract: Flycatcher1 (FLYC1), a MscS homolog, has recently been identified as a candidate mechanosensitive (MS) ion channel involved in Venus flytrap prey recognition. FLYC1 is larger and its sequence diverges from previously studied MscS homologs, suggesting it has unique structural features that contribute to its function. Here, we characterized FLYC1 by cryo-electron microscopy, molecular dynamics simulations, and electrophysiology. Akin to bacterial MscS and plant MSL1 channels, we find that FLYC1 central core includes side portals in the cytoplasmic cage that regulate ion conduction, by identifying critical residues that modulate channel conductance. Topologically unique cytoplasmic flanking regions can adopt  up or  down conformations, making the channel asymmetric. Disruption of an up conformation-specific interaction severely delays channel deactivation by 40-fold likely due to stabilization of the channel open state. Our results illustrate novel structural features and likely conformational transitions that regulate mechano-gating of FLYC1.


title: Discovering cancer driver genes and pathways using stochastic block model graph neural networks

abstract:
The identification of genes and pathways responsible for the transformation of normal cells into malignant ones represents a pivotal step to understand the aetiology of cancer, to characterise progression and relapse, and to ultimately design targeted therapies. The advent of high-throughput omic technologies has enabled the discovery of a significant number of cancer driver genes, but recent genomic studies have shown these to be only necessary but not sufficient to trigger tumorigenesis. Since most biological processes are the results of the interaction of multiple genes, it is then conceivable that tumorigenesis is likely the result of the action of networks of cancer driver and non-driver genes.

Here we take advantage of recent advances in graph neural networks, combined with well established statistical models of network structure, to build a new model, called Stochastic Block Model Graph Neural Network (SBM-GNN), which predicts cancer driver genes and cancer mediating pathways directly from high-throughput omic experiments. Experimental analysis of synthetic datasets showed that our model can correctly predict genes associated with cancer and recover relevant pathways, while outperforming other state-of-the-art methods.

Finally, we used SBM-GNN to perform a pan-cancer analysis, where we found genes and pathways directly involved in the hallmarks of cancer controlling genome stability, apoptosis, immune response, and metabolism.


title: PhenoExam: an R package and Web application for the examination of phenotypes linked to genes and gene sets

abstract: Gene set based phenotype enrichment analysis (detecting phenotypic terms that emerge as significant in a set of genes) can improve the rate of genetic diagnoses amongst other research purposes. To facilitate diverse phenotype analysis, we developed PhenoExam, a freely available R package for tool developers and a web interface for users, which performs: (1) phenotype and disease enrichment analysis on a gene set; (2) measures statistically significant phenotype similarities between gene sets and (3) detects significant differential phenotypes or disease terms across different databases. PhenoExam achieves these tasks by integrating databases or resources such as the HPO, MGD, CRISPRbrain, CTD, ClinGen, CGI, OrphaNET, UniProt, PsyGeNET, and Genomics England Panel App. PhenoExam accepts both human and mouse genes as input. We developed PhenoExam to assist a variety of users, including clinicians, computational biologists and geneticists. It can be used to support the validation of new gene-to-disease discoveries, and in the detection of differential phenotypes between two gene sets (a phenotype linked to one of the gene set but no to the other) that are useful for differential diagnosis and to improve genetic panels. We validated PhenoExam performance through simulations and its application to real cases. We demonstrate that PhenoExam is effective in distinguishing gene sets or Mendelian diseases with very similar phenotypes through projecting the disease-causing genes into their annotation-based phenotypic spaces. We also tested the tool with early onset Parkinsons disease and dystonia genes, to show phenotype-level similarities but also potentially interesting differences. More specifically, we used PhenoExam to validate computationally predicted new genes potentially associated with epilepsy. Therefore, PhenoExam effectively discovers links between phenotypic terms across annotation databases through effective integration. The R package is available at https://github.com/alexcis95/PhenoExam and the Web tool is accessible at https://snca.atica.um.es/PhenoExamWeb/.


title: Extent, impact, and mitigation of batch effects in tumor biomarker studies using tissue microarrays

abstract: Tissue microarrays (TMAs) have been used in thousands of cancer biomarker studies. To what extent batch effects, measurement error in biomarker levels between slides, affects TMA-based studies has not been assessed systematically. We evaluated 20 protein biomarkers on 14 TMAs with prospectively collected tumor tissue from 1,448 primary prostate cancers. In half of the biomarkers, more than 10% of biomarker variance was attributable to between-TMA differences (range, 1-48%). We implemented different methods to mitigate batch effects (R package batchtma), tested in plasmode simulation. Biomarker levels were more similar between mitigation approaches compared to uncorrected values. For some biomarkers, associations with clinical features changed substantially after addressing batch effects. Batch effects and resulting bias are not an error of an individual study but an inherent feature of TMA-based protein biomarker studies. They always need to be considered during study design and addressed analytically in studies using more than one TMA.

category: pathology


title: THE LIFESPAN AND OXIDATIVE STRESS BETWEEN FERAL AND MANAGED HONEY BEE COLONIES

abstract: Molecular damage caused by oxidative stress may lead to organismal aging and resulted in acute mortality in organisms. Oxidative stress resistance and longevity are closely linked. Honey bees are the most important managed pollinator in agriculture but the long-term survival of honey bees is seriously threatened. Feral honey bee colonies displayed persistence to Varroa mites. However, it is unknown whether feral honey bees are stress-resistant or survive longer than managed bee populations. More work is needed to determine the impact of oxidative stress on honey bee health and survival. We used the paired colony design to determine the lifespan and levels of oxidative stress on worker bees from either a feral or a managed colony. Each pair of colonies shared similar foraging resources. Results exhibit longer survival time and lifespans of foragers in feral colonies than the managed colonies. The levels of oxidative stress from the lipid damage of feral colonies are higher than the managed colonies, indicating a tolerant mechanism not a repair mechanism to survive. Our study provided new insights into colony difference of physiology and oxidative stress resistance between feral honey bees and commercial stocks.


title: TMEM120A/TACAN inhibits mechanically activated Piezo2 channels

abstract: Mechanically activated Piezo2 channels are key mediators of light touch and proprioception in mice and humans. Relatively little is known about what other proteins regulate Piezo2 activity in a cellular context. TACAN (TMEM120A) was proposed to act as a high threshold mechanically activated ion channel in nociceptive dorsal root ganglion (DRG) neurons. Here we find that TACAN co-expression robustly reduced mechanically activated Piezo2 currents, but did not inhibit mechanically activated Piezo1 and TREK1 currents. TACAN co-expression did not affect cell surface expression of either Piezo1 or Piezo2 and did not have major effects on the cortical actin or tubulin cytoskeleton. TACAN expression alone did not result in the appearance of mechanically activated currents above background. In addition, TACAN and Piezo2 expression in DRG neurons overlapped, and siRNA mediated knockdown of TACAN did not decrease the proportion of slowly adapting mechanically activated currents, but resulted in an increased proportion of rapidly adapting currents. Our data do not support TACAN being a mechanically activated ion channel, and identify it as a negative modulator of Piezo2 channel activity.


title: Single-cell analyses of immune thrombocytopenic patients reveal multiorgan dissemination of high-affinity autoreactive plasma cells

abstract: The major therapeutic goal for immune thrombocytopenia (ITP) is to restore normal platelet counts using drugs to promote platelet production or by interfering with mechanisms responsible for platelet destruction. 80% of patients possess anti-integrin IIb{beta}3 (GPIIbIIIa) IgG autoantibodies causing platelet opsonization and phagocytosis. The spleen is considered the primary site of autoantibody production by autoreactive B cells and platelet destruction. The immediate failure in ~50% of patients to recover a normal platelet count after anti-CD20 Rituximab-mediated B cell depletion and splenectomy suggest that autoreactive, rituximab-resistant, IgG-secreting B cells (IgG-SC) reside in other anatomical compartments. We analyzed >3,300 single IgG-SC from spleen, bone marrow and/or blood of 27 patients with ITP revealing high inter-individual variability in affinity for GPIIbIIIa with variations over 3 logs. IgG-SC dissemination and range of affinities were however similar per patient. Longitudinal analysis of autoreactive IgG-SC upon treatment with anti-CD38 mAb daratumumab demonstrated variable outcomes, from complete remission to failure with persistence of high-affinity anti-GPIIbIIIa IgG-SC in the bone marrow. This study demonstrates the existence and dissemination of high-affinity autoreactive plasma cells in multiple anatomical compartments of patients with ITP that may cause the failure of current therapies.


title: Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformans  enables rapid gene deletion and tagging

abstract: Cryptococcus neoformans, the most common cause of fungal meningitis, is a basidiomycete haploid budding yeast with a complete sexual cycle. Genome modification by homologous recombination is feasible using biolistic transformation and long homology arms, but the method is arduous and unreliable. Recently, multiple groups have reported the use of CRISPR-Cas9 as an alternative to biolistics, but long homology arms are still necessary, limiting the utility of this method. Since the S. pyogenes Cas9 derivatives used in prior studies were not optimized for expression in C. neoformans, we designed, synthesized, and tested a fully C. neoformans-optimized Cas9. We found that a Cas9 harboring only common C. neoformans codons and a consensus C. neoformans intron together with a TEF1 promoter and terminator and a nuclear localization signal (C. neoformans-optimized CAS9 or "CnoCAS9") reliably enabled genome editing in the widely-used KN99 C. neoformans strain. Furthermore, editing was accomplished using donors harboring short (50 bp) homology arms attached to marker DNAs produced with synthetic oligonucleotides and PCR amplification. We also demonstrated that prior stable integration of CnoCAS9 further enhances both transformation and homologous recombination efficiency; importantly, this manipulation does not impact virulence in animals. We also implemented a universal tagging module harboring a codon-optimized fluorescent protein (mNeonGreen) and a tandem Calmodulin Binding Peptide-2X FLAG Tag that allows for both localization and purification studies of proteins for which the corresponding genes are modified by short homology-directed recombination. These tools enable short-homology genome engineering in C. neoformans.


title: Pathogens, Virulence and Resistance Genes Surveillance with Metagenomics Can Pre-empt Dissemination and Escalation of Untreatable Infections: A Systematic Review and Meta-analyses.

abstract:
BackgroundThe dissemination of pathogens carrying genetic elements such as antimicrobial resistance genes (ARGs), mobile-genetic elements (MGEs), virulome and methylome have a negative impact on food and environment safety, water quality and animal and human health. The applications of metagenomics to monitor and identify the prevalence/endemicity and emergence of these pathogenic agents from different sources were examined.

MethodsArticles published in English language up to October 2020 were searched for on PubMed. Qualitative and quantitative data extracted from the included articles were translated into charts and maps. GraphPad Prism 9.0.2 was used to undertake statistical analysis using descriptive and column statistics, Chi-square, ANOVA, Wilcoxons signed-rank, and one-sample t-test.

ResultsIn all, 143 articles from 39 countries from Europe, America, Asia, and Africa were quantitatively analysed. Metagenomes from sewage/wastewater, surface water samples (ocean, sea, river lake, stream and tap water), WWTP, effluents and sludge samples contained pathogenic bacteria (Aeromonas, Acinetobacter, Pseudomonas, Streptococcus, Bacteroides, Escherichia coli, Salmonella enterica, Klebsiella pneumoniae and Acinetobacter baumannii), viruses (Adenovirus, Enterovirus, Hepatovirus, Mamastrovirus and Rotavirus) and parasites (Acanthamoeba, Giardia, Entamoeba, Blastocystis and Naegleria). Integrons, plasmids, transposons, insertion sequences, prophages and integrative and conjugative elements were identified. ARGs mediating resistance to important antibiotics, including {beta}-lactams, aminoglycosides, fluoroquinolones, and tetracycline, and virulence factors such as secretion system, adherence, antiphagocytosis, capsule, invasion, iron uptake, hemolysin, and flagella.

ConclusionClinically important pathogens, ARGs, and MGEs were identified in diverse clinical, environmental, and animal sources through metagenomics, which can be used to determine the prevalence and emergence of known and unknown pathogens and ARGs.

Importance/significanceGlobal metagenomic analyses of drinking water, effluents, influents, un/-treated sewage, WWTPs, sludge, rivers, lakes, soil, sediments, biosolid, air and plants. showed the global distribution of diverse clinically important ARGs on mobile genetic elements, antimicrobial-resistant bacteria (ARB) and pathogens, metal resistance genes, and virulence genes in almost all environments. These depict the importance of shot-gun metagenomics as a surveillance tool for AMR and infectious disease control to safeguard water & food quality as well as public health from water- and food-borne outbreaks of pathogenic and ARB infections. More concerning was the identification of ARGs to last-resort antibiotics i.e., carbapenems, colistin, & tigecycline.


title: The development of ovine gastric and intestinal organoids for studying ruminant host-pathogen interactions

abstract: Gastrointestinal (GI) infections in sheep have significant implications for animal health, welfare and productivity, as well as being a source of zoonotic pathogens. Interactions between pathogens and epithelial cells at the mucosal surface play a key role in determining the outcome of GI infections; however, the inaccessibility of the GI tract in vivo significantly limits the ability to study such interactions in detail. We therefore developed ovine epithelial organoids representing physiologically important gastric and intestinal sites of infection, specifically the abomasum (analogous to the stomach in monogastrics) and ileum. We show that both abomasal and ileal organoids form self-organising three-dimensional structures with a single epithelial layer and a central lumen that are stable in culture over serial passage. We performed RNA-seq analysis on abomasal and ileal tissue from multiple animals and on organoids across multiple passages and show the transcript profile of both abomasal and ileal organoids cultured under identical conditions are reflective of the tissue from which they were derived and that the transcript profile in organoids is stable over at least five serial passages. In addition, we demonstrate that the organoids can be successfully cryopreserved and resuscitated, allowing long-term storage of organoid lines, thereby reducing the number of animals required as a source of tissue. We also report the first published observations of a helminth infecting gastric and intestinal organoids by challenge with the sheep parasitic nematode Teladorsagia circumcincta, demonstrating the utility of these organoids for pathogen co-culture experiments. Finally, the polarity in the abomasal and ileal organoids can be inverted to make the apical surface directly accessible to pathogens or their products, here shown by infection of apical-out organoids with the zoonotic enteric bacterial pathogen Salmonella enterica serovar Typhimurium. In summary, we report a simple and reliable in vitro culture system for generation and maintenance of small ruminant intestinal and gastric organoids. In line with 3Rs principals, use of such organoids will reduce and replace animals in host-pathogen research.


title: Antimicrobial activity of a repurposed harmine-derived compound on extensively drug-resistant Acinetobacter baumannii clinical isolates

abstract:
SynopsisO_ST_ABSObjectivesC_ST_ABSThe spread of antibiotic resistant bacteria is an important threat for human healthcare. Acinetobacter baumannii bacteria impose one of the major issues, as multidrug- to pandrug-resistant strains have been found, rendering some infections untreatable. In addition, A. baumannii is a champion in surviving in harsh environments, being capable of resisting to disinfectants and to persist prolonged periods of desiccation. Due to the high degree of variability found in A. baumannii isolates, the search for new antibacterials is challenging. Here, we screened a compound library to identify compounds active against recent isolates of A. baumannii bacteria.

MethodsA repurposing drug screen was undertaken to identify A. baumannii growth inhibitors. One hit was further characterized by determining its IC50 and testing its activity on 43 recent clinical A. baumannii isolates, amongst which 40 are extensively drug- and carbapenem-resistant strains.

ResultsThe repurposing screen led to the identification of a harmine-derived compound, called HDC1, which proved to have bactericidal activity on the multidrug-resistant AB5075-VUB reference strain with an IC50 of 48.23 {micro}M. In addition, HDC1 impairs growth of all 43 recent clinical A. baumannii isolates.

ConclusionsWe identified a compound with inhibitory activity on all tested, extensively drug-resistant clinical A. baumannii isolates.


title: Bacterial community composition of the sea grape Caulerpa lentillifera: a comparison between healthy and diseased states

abstract: The bacterial communities of the sea grape Caulerpa lentillifera were studied during a disease outbreak in Vietnam. The Rhodobacteraceae and Rhodovulum dominated the composition of healthy C. lentillifera. Clear differences between healthy and diseased cases were observed at order, genus and Operational Taxonomic Unit (OTU) level. Bacterial diversity was lower in healthy C. lentillifera, probably because of antimicrobial compounds from the macroalgae and/or from Clostridium, Cutibacterium or Micrococcus bacteria. The likely beneficial role of Bradyrhizobium, Paracoccus and Brevundimonas strains on nutrient cycling and phytohormone production was discussed. The white coloration of diseased C. lentillifera may not only be associated with pathogens but also with an oxidative response. Aquibacter, Winogradskyella and other OTUs of the family Flavobacteriaceae were hypothesized as detrimental bacteria, this family comprises some well-known seaweed pathogens. Moreover, Thalassobius OTU 2935 and 1635 may represent detrimental Rhodobacteraceae. Phycisphaera together with other Planctomycetes and Woeseia were probably saprophytes of C. lentillifera. This study offers pioneering insights on the co-occurrence of C. lentillifera-attached bacteria, potential detrimental or beneficial microbes, and a baseline for understanding the C. lentillifera holobiont. Further metagenomic and biotechnological approaches are needed to confirm functions of some microbes on this macroalgae to enhance food security in the tropics.


title: Oxidative phosphorylation is required for powering motility and development of the sleeping sickness parasite Trypanosoma brucei within the tsetse fly vector

abstract: The single-celled parasite Trypanosoma brucei causes sleeping sickness in humans and nagana in livestock and is transmitted by hematophagous tsetse flies. Lifecycle progression from mammalian bloodstream form to tsetse midgut form and, subsequently, infective salivary gland form depends on complex developmental steps and migration within different fly tissues. As the parasite colonises the glucose-poor insect midgut, its ATP production is thought to depend on activation of mitochondrial amino acid catabolism via oxidative phosphorylation. This process involves respiratory chain complexes and the F1FO-ATP synthase, and it requires protein subunits of these complexes that are encoded in the parasites mitochondrial DNA (kinetoplast or kDNA). Here we show that a progressive loss of kDNA-encoded functions correlates with an increasingly impaired ability of T. brucei to initiate and complete its development in the tsetse. First, parasites with a mutated F1FO-ATP synthase with a reduced capacity for oxidative phosphorylation can initiate differentiation from bloodstream to insect form, but they are unable to proliferate in vitro. Unexpectedly, these cells can still colonise the tsetse midgut. However, these parasites exhibit a motility defect and are severely impaired in colonising or migrating to subsequent tsetse tissues. Second, parasites with a fully disrupted F1FO-ATP synthase complex that is completely unable to produce ATP by oxidative phosphorylation can still differentiate to the first insect stage in vitro but die within a few days and cannot establish a midgut infection in vivo. Third, mutant parasites lacking kDNA entirely can initiate differentiation but die within 24 h. Together, these three scenarios show that efficient ATP production via oxidative phosphorylation is not essential for initial colonisation of the tsetse vector, but it is required to power trypanosome migration within the fly.


title: CRISPRCas9-mediated gene disruption of endogenous co-receptors confers broad resistance to HIV-1 in human primary cells and humanized mice

abstract: In this project, we investigated the CRISPR/Cas9 system for creating HIV resistance by targeting the human CCR5 and CXCR4 genes, which encode cellular co-receptors required for HIV-1 infection. Using a clinically scalable system for transient ex vivo delivery of Cas9/gRNA ribonucleoprotein (RNP) complexes, we demonstrated that CRISPR-mediated disruption of CCR5 and CXCR4 in T-lymphocytes cells significantly reduced surface expression of the co-receptors, thereby establishing resistance to HIV-1 infection by CCR5 (R5)-tropic, CXCR4 (X4)-tropic, and dual (R5/X4)-tropic strains. CRISPR-mediated disruption of the CCR5 alleles in human CD34+ hematopoietic stem and progenitor cells (HSPCs) led to the differentiation of HIV-resistant macrophages. In human CD4+ T cells transplanted into a humanized mouse model, disruption of CXCR4 inhibited replication of X4-tropic HIV-1, thus leading to the virus-mediated enrichment CXCR4-disrupted cells in the peripheral blood and spleen. However, in human CD4+ T cells with both CCR5 and CXCR4 disruption, we observed poor engraftment in bone marrow, although significant changes were not observed in the lung, spleen, or peripheral blood. This study establishes a clinically scalable strategy for the dual knockout of HIV-1 co-receptors as a therapeutic strategy, while also raising caution of disrupting CXCR4, which may abate engraftment of CD4+ T cells in bone marrow.


title: A Genetic Screen for Suppressors of Cryptic 5' Splicing in C. elegans Reveals Roles for KIN17 and PRCC in Maintaining Both 5' and 3' Splice Site Identity

abstract:
Pre-mRNA splicing is an essential step of eukaryotic gene expression carried out by a series of dynamic macromolecular protein/RNA complexes, known collectively and individually as the spliceosome. This series of spliceosomal complexes define, assemble on, and catalyze the removal of introns. Molecular model snapshots of intermediates in the process have been created from cryo-EM data, however, many aspects of the dynamic changes that occur in the spliceosome are not fully understood. Caenorhabditis elegans follow the GU-AG rule of splicing, with almost all introns beginning with 5 GU and ending with 3 AG. These splice sites are identified early in the splicing cycle, but as the cycle progresses and "custody" of the pre-mRNA splice sites is passed from factor to factor as the catalytic site is built, the mechanism by which splice site identity is maintained or re-established through these dynamic changes is unclear. We performed a genetic screen in C. elegans for factors that are capable of changing 5 splice site choice. We report that KIN17 and PRCC are involved in splice site choice, the first functional splicing role proposed for either of these proteins. Previously identified suppressors of cryptic 5 splicing promote distal cryptic GU splice sites, however, mutations in KIN17 and PRCC instead promote usage of an unusual proximal 5 splice site which defines an intron beginning with UU, separated by 1nt from a GU donor. We performed high-throughput mRNA sequencing analysis and found that mutations in PRCC but not KIN17 changed 5 splice sites genome-wide, promoting usage of nearby non-consensus sites. We further found that mutations in KIN17 and PRCC changed dozens of 3 splice sites, promoting non-consensus sites upstream of canonical splice sites. Our work has uncovered both fine and coarse mechanisms by which the spliceosome maintains splice site identity during the complex assembly process.

Author SummaryPre-mRNA splicing is an essential step of gene regulation, carried out by an unusual molecular machine, the spliceosome. Unlike other molecular machines, such as ribosomes, that simply assemble and catalyze chemical reactions, "the spliceosome" is a highly-dynamic cycle, carried out by 5 specialized small nuclear RNAs and over 100 proteins, which sequentially join, rearrange, and withdraw from the splicing assembly during each splicing cycle. These assemblies initially choose "splice sites" where the pre-mRNA will be cut, and then undergo multiple rearrangements to finally form the active site which catalyzes the splicing reactions which remove an intron from a pre-mRNA. We are currently in the midst of a "resolution revolution", with ever-clearer cryo-EM snapshots of stalled complexes allowing researchers to visualize moments in time in the splicing cycle. These models are illuminating, but do not always elucidate mechanistic functioning, therefore our lab takes a complementary approach, using the power of genetics in a multicellular animal to gain functional insights into the spliceosome. Using a C .elegans genetic screen, we have found novel functional splicing roles for two proteins, KIN17 and PRCC. Our results suggest that the spliceosome does not just rely on its initial identification of the splice site, but in a later step, re-identifies where to cut. We liken this two-stage identification to using a microscope by first using the coarse focus to find the area of interest, and then using the fine focus to adjust as needed. This work moves us closer to full mechanistic understanding of how the spliceosome chooses where to cut a pre-mRNA message.


title: Proper control of R-loop homeostasis is required for maintenance of gene expression and neuronal function during aging

abstract: Age-related loss of cellular function and increased cell death are characteristic hallmarks of aging. While defects in gene expression and RNA metabolism have been linked with age-associated human neuropathies, it is not clear how the changes that occur in aging neurons contribute to loss of gene expression homeostasis. R-loops are RNA-DNA hybrids that typically form co-transcriptionally via annealing of the nascent RNA to the template DNA strand, displacing the non-template DNA strand. Dysregulation of R-loop homeostasis has been associated with both transcriptional impairment and genome instability. Importantly, a growing body of evidence links R-loop accumulation with cellular dysfunction, increased cell death and chronic disease onset. Here, we characterized the R-loop landscape in aging Drosophila melanogaster photoreceptor neurons and showed that bulk R-loop levels increased with age. Further, genome-wide mapping of R-loops revealed that transcribed genes accumulated R-loops over gene bodies during aging, which correlated with decreased expression of long and highly expressed genes. Importantly, while photoreceptor-specific down-regulation of Top3{beta}, a DNA/RNA topoisomerase associated with R-loop resolution, lead to decreased visual function, overexpression of Top3{beta} or nuclear-localized RNase H1, which resolves R-loops, enhanced positive light response during aging. Together, our studies highlight the functional link between dysregulation of R-loop homeostasis, gene expression and visual function during aging.


title: Horizontal Transfer of Histone H3 by Mammalian Cells

abstract: Histones are small, highly basic, nuclear proteins that serve as structural elements to condense DNA into chromatin and regulate its accessibility1. Although histones released from dying cells can act as extracellular signaling molecules, all intracellular histone molecules are assumed to have originated from the cell in which they reside2-6. Here we show that histone H3 is horizontally transferred between viable cells. Using an ER-targeted biotin ligase to detect secreted proteins7,8, we serendipitously discovered that histone H3 is selectively secreted by autophagic cells relative to histones H2A, H2B, and H4. Specific H3 posttranslational modifications are enriched or depleted on secreted H3 relative to intracellular H3, suggesting that specific PTMs dictate H3s ability to be secreted or the information it conveys to neighboring cells. Remarkably, we found that secreted H3 can enter the nuclei of recipient cells in an autophagy-independent and cell contactindependent manner ex vivo and in vivo. These findings have implications for cell-cell communication during nutrient deprivation, hypoxia, and perhaps other forms of cellular stress, and for the ability to deliver macromolecules across cell membranes.


title: STEM CELL TECHNOLOGY PROVIDES NOVEL TOOLS TO UNDERSTAND HUMAN VARIATION IN Plasmodium falciparum MALARIA

abstract: Plasmodium falciparum interacts with several human cell types during their complex life cycle, including erythrocytes and hepatocytes. The enuclated nature of erythrocytes makes them inaccessible to genetic tools, which in turn makes studying erythrocyte proteins involved in malaria invasion and development particularly difficult. Here we overcome this limitation using stem cell technology to develop a universal differentiation protocol for in vitro derivation of erythrocytes from a variety of stem cell lines of diverse origin. This allows manipulation of erythrocytic genes and examination of their impact on the parasite by flow cytometric detection of parasite haemozoin. Deletion of Basigin, the essential receptor for P. falciparum, abrogates invasion, while other less studied proteins such as ATP2B4 have a minor effect. Reprogramming of induced pluripotent stem cells from -thalassemia primary samples shows reduced infection levels, demonstrating this approach is useful for understanding the effect of natural human polymorphisms on the disease.


title: Synaptotagmin 2 is ectopically overexpressed in excitatory presynapses of a widely used CaMK2a-Cre mouse line

abstract: The CaMKII-Cre mouse line, one of the earliest established Cre driver lines, has resulted in over 800 papers to date. Here, we demonstrate that the second most widely used CaMKII-Cre line, Tg(Camk2a-cre)2Gsc (or CamiCre), shows ectopic overexpression of synaptotagmin 2, the most efficient Ca2+ sensor for fast synchronous neurotransmitter release, in excitatory presynapses of Cre+ brains. Moreover, RNA-seq analysis showed aberrant expression in Cre+ hippocampus, including upregulation of immediate early genes, such as Arc and Fos, and genes presumably derived from bacterial artificial chromosome transgene, such as Slc6a7. Most importantly, CamiCre+ mice showed functional phenotypes, such as hyperactivity and enhanced associative learning, suggesting neural activities are affected. These unexpected results suggest difficulties in interpreting results from studies using the CamiCre line and raise awareness of potential pitfalls in the use of Cre driver lines in general.


title: Collateral damage: Identification and characterisation of spontaneous mutations causing deafness from a targeted knockout programme

abstract: Mice carrying targeted mutations are important for investigating gene function and the role of genes in disease, but the process of culturing embryonic stem cells during the making of a targeted allele offers opportunities for spontaneous mutations to arise. Identifying spontaneous mutations relies on the detection of phenotypes segregating independently of targeted alleles, and many phenotypes are easy to miss if not specifically looked for. Here we present data from a large, targeted knockout programme in which mice were analysed through a phenotyping pipeline. Twenty-five lines out of 1311 displayed different deafness phenotypes that did not segregate with the targeted allele. We have identified 8 different mutations causing deafness in 16 of these 25 lines and characterised the resulting phenotypes. Our data show that spontaneous mutations with observable effects on phenotype are a common side effect of intensive breeding programmes, including those underlying targeted mutation programmes.


title: Efficient linear dsDNA tagging using deoxyuridine excision

abstract: Site-specific strategies for exchanging segments of dsDNA are important for DNA library construction and molecular tagging. Deoxyuridine (dU) excision is an approach for generating 3 ssDNA overhangs in gene assembly and molecular cloning procedures. Unlike approaches that use a multi-base pair motif to specify a DNA cut site, dU excision requires only a dT[->]dU substitution. Consequently, excision sites can be embedded in biologically active DNA sequences by placing dU substitutions at non-perturbative positions. In this work, I describe a molecular tagging method that uses dU excision to exchange a segment of a dsDNA strand with a long synthetic oligonucleotide. The core workflow of this method, called deoxyUridine eXcision-tagging (dUX-tagging), is an efficient one-pot reaction: strategically positioned dU nucleotides are excised from dsDNA to generate a 3 overhang so that additional sequence can be appended by annealing and ligating a tagging oligonucleotide. The tagged DNA is then processed by one of two procedures to fill the 5 overhang and remove excess tagging oligo. To facilitate its widespread use, all dUX-tagging procedures exclusively use commercially available reagents. As a result, dUX-tagging is a concise and easily implemented approach for high-efficiency linear dsDNA tagging.


title: Simultaneous in vivo Pharmacokinetics and Pharmacodynamics of TDF Intravaginal Ring during High-dose Vaginal SHIV Challenge in Pigtail Macaques

abstract: The demonstration of complete protection of macaques in a repeated low dose virus challenge by a tenofovir disoproxil fumarate (TDF) intravaginal ring (IVR) and the success of the dapivirine IVR in clinical trials highlighted the potential of IVRs as pre- exposure prophylaxis against HIV. Efficacy of TDF ring was not investigated in sexually active women. Our understanding of the mechanisms of protection is limited. To address this knowledge gap, we performed simultaneous pharmacokinetic and pharmacodynamic analysis of a TDF-IVR at the site of SIV challenge in pigtail macaques at the anatomical and cellular level. Specifically, we challenged TDF-IVR administered pigtail macaques with a single high dose of a non-replicative SIV-based vector containing a dual reporter system that helped us to identify the earliest targets of SIV infection within the mucosa. Two and three days after challenge, the macaques were euthanized and tenofovir (TFV) concentrations were measured in the female reproductive tract (FRT) by HPLC-MS/MS to correlate drug concentrations and SIV-vector transduction efficiency. TFV formed a gradient through the mucosal tissue, with the highest concentrations near the ring, in the upper vagina and endocervix. Despite this, several transduction events were identified with the most common sites being in the ovaries. Moreover, proviral DNA was detected in the cervix and vagina. Thus, our studies demonstrate an uneven distribution of TFV in the FRT of macaques after release from a TDF-IVR that leads to incomplete FRT protection from high viral dose challenge.

category: pharmacology and toxicology


title: Aspirin Protective Effect Against Cyclophosphamide Hematological Toxicity In Experimental Animals

abstract:
Bone marrow toxicity is the most important factor limiting the use of cytotoxic drugs like alkylating agents in cancer treatment. Recently PG synthase enzyme inhibitors have been reported to potentiate the cytotoxic effects of these agents on cancer cells but little is known if they can affect the toxicity of these agents on bone marrow or other tissues. Cyclophosphamide is one of the most commonly used alkylating agent.

In the present work, the effect of these PG synthase enzyme inhibitors, aspirin on cyclophosphamide myelotoxicity was determined employing the peripheral blood count to reflect bone marrow injury. The effect on body weight changes caused by cyclophosphamide was also determined.

O_LICyclophosphamide in doses of 25, 50 and 75 mg/kg i. v. produced as a dose dependent reduction in total WBC count, granulocyte, non granulocyte, and Hb% which was maximum on second day after injection and still present on 5th day post injection. It also produced a dose dependent reduction in body weight on day 5 after injection.
C_LIO_LIAspirin in doges of 75, 150 and 300 mg/kg i. m. protected against the reduction in WBC counts  measured for 5 days after injection of cyclophosphamide (50 mg/kg). This protection was not dose dependent, though it was more optimum with 300 mg/kg and disappeared largely when a dose of 450 mg/kg was used. Aspirin did not prevent the changes in Hb% but retard the reduction in body weight caused by cyclophosphamide.
C_LIO_LIIt is concluded that aspirin can help to reduce injury and enhance recovery from bone marrow toxicity caused by cytotoxic agents such as the alkylating drugs cyclophosphamide for which no specific antidote is available. Aspirin produces this effect possibly by eliminating the harmful inhibitory effect of excess PGs or leukotrienes, released by bone marrow injury on growth factors of haemopoietic progenitor cells.
C_LI

The magnitude of this protection on WBC counts does not seem to differ between either PG synthase enzyme inhibitors or steroids when used alone or in combination although a synergistic effect in protecting erythropoiesis is observed.


title: Establishment of Strigolactone-Producing Bacterium-Yeast Consortium

abstract: Strigolactones (SLs) are a class of phytohormones playing diverse roles in plant growth and development, yet the limited access to SLs is largely impeding SL-based foundational investigations and applications. Here, we developed Escherichia coli-Saccharomyces cerevisiae consortia to establish a microbial biosynthetic platform for the synthesis of various SLs, including carlactone, carlactonic acid, 5-deoxystrigol (5DS, 6.65{+/-}1.71 {micro}g/L), 4-deoxyorobanchol (4DO, 3.46{+/-}0.28 {micro}g/L), and orobanchol (OB, 19.36{+/-}5.20 {micro}g/L). The SL-producing platform enabled us to conduct functional identification of CYP722Cs from various plants as either OB or 5DS synthase. It also allowed us to quantitatively compare known variants of plant SL biosynthetic enzymes in the microbial system. The titer of 5DS was further enhanced through pathway engineering to 47.3 {micro}g/L. This work provides a unique platform for investigating SL biosynthesis and evolution and lays the foundation for developing SL microbial production process.

category: synthetic biology


title: Activity-dependent LTP in the dentate gyrus promotes epileptic seizures

abstract: Epilepsy is a devastating brain disorder whose cellular mechanisms remain poorly understood. Excitatory mossy cells (MCs) in the dentate gyrus of the hippocampus are implicated in temporal lobe epilepsy, the most common form of focal epilepsy in adults. However, the role of MCs during initial seizures, before MC loss occurs, is unclear. Here, we show that initial seizures induced with kainic acid (KA) intraperitoneal injection in adult mice, a well-established model of experimental epilepsy, not only increased MC and granule cell (GC) activity in vivo, but also triggered a BDNF-dependent long-term potentiation at MC-GC synapses (MC-GC LTP). In vivo induction of MC-GC LTP worsened KA-induced seizures, whereas selective MC silencing and Bdnf genetic removal from GCs, which abolishes LTP, were both anti-epileptic. Thus, initial seizures strengthen MC-GC synaptic transmission, thereby promoting epileptic activity. Our findings reveal a potential mechanism of epileptogenesis that may help develop therapeutic strategies for early intervention.


title: TAILORED DC INDUCE PROTECTIVE HIV-1 SPECIFIC POLYFUNCTIONAL CD8+ T CELLS IN THE LYMPHOID TISSUE FROM HUMANIZED BLT MICE

abstract:
Effective function of CD8+ T cells and enhanced innate activation of dendritic cells (DC) in response to HIV-1 is linked to protective antiviral immunity in controllers. Manipulation of DC targeting the master regulator TANK-binding Kinase 1 (TBK1) might be useful to acquire controller-like properties. Here, we evaluated the impact of TBK1-primed DC inducing protective CD8+ T cell responses in lymphoid tissue and peripheral blood and their association with reduced HIV-1 disease progression in vivo in the humanized bone marrow, liver and thymus (hBLT) mouse model. A higher proportion of hBLT-mice vaccinated with TBK1-primed DC exhibited less severe CD4+ T cell depletion following HIV-1 infection compared to control groups. This was associated with infiltration of CD8+ T cells in the white pulp from the spleen, reduced spread of infected p24+ cells to secondary lymphoid organs and with preserved abilities of CD8+ T cells from the spleen and blood of vaccinated animals to induce specific polyfunctional responses upon antigen stimulation. Therefore, TBK1-primed DC might be an useful tool for subsequent vaccine studies.

Author summaryEmulating protective immunological characteristics from individuals capable of spontaneously controlling HIV-1 infection might be useful for the development of a protective vaccine. Enhanced function of dendritic cells (DC) in these HIV-1 controllers depends on the activation of TANK-binding Kinase 1 (TBK1) and might associate with protective T cells. Our study evaluated the ability of DCs trained through TBK1 activation inducing protective adaptive immune responses against HIV-1 and reducing disease progression in vivo, using a humanized mouse model. Our data indicate that mice vaccinated with tailored DC exhibit delayed disease progression, increased induction of protective CD8+ T lymphocyte subsets in the lymphoid tissue and blood upon antigen recognition. Therefore, trained-DC might be an useful tool for future HIV-1 vaccine designs.