bluuebunny/crossref_metadata_2025 huggingface dataset:
Single Cell T Cell Receptor Repertoire Profiling for Dogs
Authors: Skidmore Zachary L. , Rindt Hans , Chu Shirley , Fisk Bryan , Fronick Catrina , Fulton Robert , Zhou Mingyi , Bivens Nathan J. , Reinero Carol N. , Griffith Malachi , Bryan Jeffrey N. , Griffith Obi L.
Published: July 2021

Background

Spontaneous 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.

Methods

Samples 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.

Results

1,850-2,172 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.

Discussion

The 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.
Epitope order Matters in multi-epitope-based peptide (MEBP) vaccine design: Anin silicostudy
Authors: Salaikumaran Muthu Raj , Kasamuthu Prasanna Sudharson , Burra V L S Prasad
Published: July 2021

With different countries facing multiple waves, with some SARS-CoV-2 variants more deadly and virulent, the COVID-19 pandemic is becoming more dangerous by the day and the world is facing an even more dreadful extended pandemic with exponential positive cases and increasing death rates. There is an urgent need for more efficient and faster methods of vaccine development against SARS-CoV-2. Compared to experimental protocols, the opportunities to innovate are very high in immunoinformatics/in silicoapproaches especially with the recent adoption of structural bioinformatics in peptide vaccine design. In recent times, multi-epitope-based peptide vaccine candidates (MEBPVCs) have shown extraordinarily high humoral and cellular responses to immunization. Most of the publications claim that respective reported MEBPVC(s) assembled using a set ofin silicopredicted epitopes, to be the computationally validated potent vaccine candidate(s) ready for experimental validation. However, in this article, for a given set of predicted epitopes, it is shown that the published MEBPVC is one among the many possible variants and there is high likelihood of finding more potent MEBPVCs than the published candidate. To test the same, a methodology is developed where novel MEBP variants are derived by changing the epitope order of the published MEBPVC. Further, to overcome the limitations of current qualitative methods of assessment of MEBPVC, to enable quantitative comparison, ranking, and the discovery of more potent MEBPVCs, novel predictors, Percent Epitope Accessibility (PEA), Receptor specific MEBP vaccine potency(RMVP), MEBP vaccine potency(MVP) are introduced. The MEBP variants indeed showed varied MVP scores indicating varied immunogenicity. When the MEBP variants were ranked in descending order of their MVP scores, the published MEBPVC had the least MVP score. Further, the MEBP variants with IDs, SPVC_387 and SPVC_206, had the highest MVP scores indicating these variants to be more potent MEBPVCs than the published MEBPVC and hence should be prioritized for experimental testing and validation. Through this method, more vaccine candidates will be available for experimental validation and testing. This study also opens the opportunity to develop new software tools for designing more potent MEBPVCs in less time. The computationally validated top-ranked MEBPVCs must be experimentally tested, validated, and verified. The differences and deviations between experimental results and computational predictions provide an opportunity for improving and developing more efficient algorithms and reliable scoring schemes and software.
Comparative computational modeling of the bat and human immune response to viral infection with the Comparative Biology Immune Agent Based Model
Authors: Cockrell Chase , An Gary
Published: July 2021

Given the impact of pandemics due to viruses of bat origin there is increasing interest in comparative investigation into the differences between bat and human immune responses. The practice of comparative biology can be enhanced by computational methods used for dynamic knowledge representation to visualize and interrogate the putative differences between the two systems. We present an agent-based model that encompasses and bridges the differences between bat and human responses to viral infection: the Comparative Biology Immune Agent-based Model, or CBIABM. The CBIABM examines differences in innate immune mechanisms between bats and humans, specifically regarding inflammasome activity and Type 1 Interferon dynamics, in terms of tolerance to viral infection. Simulation experiments with the CBIABM demonstrate the efficacy of bat-related features in conferring viral tolerance and also suggest a crucial role for endothelial inflammasome activity as a mechanism for bat systemic viral tolerance and affecting the severity of disease in human viral infections. We hope that this initial study will inspire additional comparative modeling projects to link, compare, and contrast immunological functions shared across different species, and in so doing, provide insight and aid in the preparation for future viral pandemics of zoonotic origin.
Benchmarking supervised signature-scoring methods for single-cell RNA sequencing data in cancer
Authors: Noureen Nighat , Ye Zhenqing , Chen Yidong , Wang Xiaojing , Zheng Siyuan
Published: July 2021

Quantifying the activity of gene expression signatures is common in analyses of single-cell RNA sequencing data. Methods originally developed for bulk samples are often used for this purpose without accounting for contextual differences between bulk and single-cell data. More broadly, these methods have not been benchmarked. Here we benchmark four such supervised methods, including single sample gene set enrichment analysis (ssGSEA), AUCell, Single Cell Signature Explorer (SCSE), and a new method we developed, Jointly Assessing Signature Mean and Inferring Enrichment (JASMINE). Using cancer as an example, we show cancer cells consistently express more genes than normal cells. This imbalance leads to bias in performance by bulk-sample-based ssGSEA in gold standard tests and down sampling experiments. In contrast, single-cell-based methods are less susceptible. Our results suggest caution should be exercised when using bulk-sample-based methods in single-cell data analyses, and cellular contexts should be taken into consideration when designing benchmarking strategies.
Metabolic and transcriptomic profiles of glioblastoma invasion revealed by comparisons between patients and corresponding orthotopic xenografts in mice
Authors: Cudalbu Cristina , Bady Pierre , Lai Marta , Xin Lijing , Gusyatiner Olga , Hamou Marie-France , Lepore Mario , Brouland Jean Philippe , Daniel Roy T. , Hottinger Andreas F. , Hegi Monika E.
Published: July 2021

The invasive behavior of glioblastoma, the most aggressive primary brain tumor, is considered highly relevant for tumor recurrence. However, the invasion zone is difficult to visualize by Magnetic Resonance Imaging (MRI) and is protected by the blood brain barrier, posing a particular challenge for treatment. We report biological features of invasive growth accompanying tumor progression and invasion based on associated metabolic and transcriptomic changes observed in patient derived orthotopic xenografts (PDOX) in the mouse and the corresponding patients’ tumors. The evolution of metabolic changes, followedin vivolongitudinally by1H-Magnetic Resonance Spectroscopy (MRS) at ultra-high field, reflected growth and the invasive properties of the human glioblastoma transplanted into the brains of mice (PDOX). Comparison of MRS derived metabolite signatures, reflecting temporal changes of tumor development and invasion in PDOX, revealed high similarity to spatial metabolite signatures of combined multi-voxel MRS analyses sampled across different areas of the patients’ tumors. Pathway analyses of the transcriptome associated with the metabolite profiles of the PDOX, identified molecular signatures of invasion, comprising extracellular matrix degradation and reorganization, growth factor binding, and vascular remodeling. Specific analysis of expression signatures from the invaded mouse brain, revealed extent of invasion dependent induction of immune response, recapitulating respective signatures observed in glioblastoma. Integrating metabolic profiles and gene expression of highly invasive PDOX provided insights into progression and invasion associated mechanisms of extracellular matrix remodeling that is essential for cell-cell communication and regulation of cellular processes. Structural changes and biochemical properties of the extracellular matrix are of importance for the biological behavior of tumors and may be druggable. Ultra-high field MRS reveals to be suitable forin vivomonitoring of progression in the non-enhancing infiltration zone of glioblastoma.
Promoting long-term forest landscape resilience in the Lake Tahoe basin
Authors: Abelson Eric S. , Reynolds Keith M. , White Angela M. , Long Jonathan W. , Maxwell Charles , Manley Patricia N.
Published: July 2021

Rapid environmental changes expected in the 21stcentury challenge the resilience of wildlands around the world. The western portion of the Lake Tahoe basin (LTW) in California is an important ecological and cultural hotspot that is at risk of degradation from current and future environmental pressures. Historical uses, fire suppression, and a changing climate have created forest landscape conditions at risk of drought stress, destructive fire, and loss of habitat diversity. We prospectively modeled forest landscape conditions for a period of 100 years to evaluate the efficacy of five unique management scenarios in achieving desired landscape conditions across the 23,600 hectares of LTW. Management scenarios ranged from no management other than fire suppression to applying treatments consistent with historical fire frequencies and extent (i.e., regular and broadscale biomass reduction). We developed a decision support tool to evaluate environmental and social outcomes within a single framework to provide a transparent set of costs and benefits; results illuminated underlying mechanisms of forest resilience and provided actionable guidance to decision makers. Sixteen attributes were assessed in the model after assigning weights to each, derived through a survey of stakeholder priorities, so that the contribution of each attribute to evaluations of scenario performance was influenced by the combined priorities of stakeholders. We found that removing forest biomass across the landscape, particularly when accomplished using extensive fire-based removal techniques, led to highly favorable conditions for environmental quality and promoting overall landscape resilience. Environmental conditions resulting from extensive fire-based biomass removal also had nominal variation over time, in contrast with strategies that had less extensive and/or used physical removal techniques, namely thinning. Our analysis provided a transparent approach to data assessment, considering the priorities of stakeholders, to provide insights into the complexities of maintaining optimal conditions and managing landscapes to promote ecosystem resilience in a changing world.
The Lifespan and Levels of Oxidative Stress between Feral and Managed Honey Bee Colonies
Authors: Ward Kilea , Li-Byarlay Hongmei
Published: July 2021

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.
Aspirin Protective Effect Against Cyclophosphamide Hematological Toxicity In Experimental Animals
Authors: Hashim Imad , Al-Attar Zaid , Hamdan Saba Jasim
Published: July 2021

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.

Cyclophosphamide 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.

Aspirin 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.

It 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.

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.
Host cellular RNA helicases regulate SARS-CoV-2 infection
Authors: Ariumi Yasuo
Published: July 2021

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.

Importance

SARS-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.
Highly-Neutralizing COVID-19-Convalescent-Plasmas Potently Block SARS-CoV-2 Replication and Pneumonia in Syrian Hamsters
Authors: Takamatsu Yuki , Imai Masaki , Maeda Kenji , Nakajima Noriko , Higashi-Kuwata Nobuyo , Iwatsuki-Horimoto Kiyoko , Ito Mutsumi , Kiso Maki , Maemura Tadashi , Takeda Yuichiro , Omata Kazumi , Suzuki Tadaki , Kawaoka Yoshihiro , Mitsuya Hiroaki
Published: July 2021

Despite various attempts to treat SARS-CoV-2-infected patients with COVID-19-convalescent plasmas, neither appropriate approach nor clinical utility has been established. We examined the efficacy of administration of highly-neutralizing COVID-19-convalescent plasma (hn-plasmas) and such plasma-derived IgG administration using the Syrian hamster COVID-19 model. Two hn-plasmas, which were in the best 1% of 340 neutralizing-activity-determined convalescent plasma samples, were intraperitoneally administered to SARS-CoV-2-infected hamsters, resulting in significant reduction of viral titers in lungs by up to 32-fold as compared to the viral titers in hamsters receiving control non-neutralizing plasma, while with two moderately neutralizing plasmas (mn-plasmas) administered, viral titer reduction was by up to 6-fold. IgG fractions purified from the two hn-plasmas also reduced viral titers in lungs than those from the two mn-plasmas. The severity of lung lesions seen in hamsters receiving hn-plasmas was minimal to moderate as assessed using micro-computerized tomography, which histological examination confirmed. Western blotting revealed that all four COVID-19-convalescent-plasmas variably contained antibodies against SARS-CoV-2 components including the receptor-binding domain and S1 domain. The present data strongly suggest that administering potent-neutralizing-activity-confirmed COVID-19-convalescent plasmas would be efficacious in treating patients with COVID-19.

Importance

Convalescent plasmas obtained from patients, who recovered from a specific infection, have been used as agents to treat other patients infected with the very pathogen. To treat using convalescent plasmas, despite that more than 10 randomized-controlled-clinical-trials have been conducted and more than 100 studies are currently ongoing, the effects of convalescent plasma against COVID-19 remained uncertain. On the other hand, certain COVID-19 vaccines have been shown to reduce the clinical COVID-19 onset by 94-95%, for which the elicited SARS-CoV-2-neutralizing antibodies are apparently directly responsible. Here, we demonstrate that highly-neutralizing-effect-confirmed convalescent plasmas significantly reduce the viral titers in the lung of SARS-CoV-2-infected Syrian hamsters and block the development of virally-induced lung lesions. The present data provide a proof-of-concept that the presence of highly-neutralizing antibody in COVID-19-convalescent plasmas is directly responsible for the reduction of viral replication and support the use of highly-neutralizing antibody-containing plasmas in COVID-19 therapy with convalescent plasmas.
Locating and Quantifying Carbon Steel Corrosion Rates Linked to Fungal B20 Biodiesel Degradation
Authors: Floyd James G. , Stamps Blake W. , Goodson Wendy J. , Stevenson Bradley S.
Published: July 2021

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.

IMPORTANCE

Biodiesel 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.
Glutathione synthetase overexpression in Acidithiobacillus ferrooxidans improves halotolerance of iron oxidation
Authors: Inaba Yuta , West Alan C. , Banta Scott
Published: July 2021

Acidithiobacillus ferrooxidans are well-studied iron- and sulfur-oxidizing acidophilic chemolithoautotrophs that are exploited for their ability to participate in the bioleaching of metal sulfides. Here, we overexpressed the endogenous glutamate--cysteine ligase and glutathione synthetase genes in separate strains and found that glutathione synthetase overexpression increased intracellular glutathione levels. We explored the impact of pH on the halotolerance of iron oxidation in wild type and engineered cultures. The increase in glutathione allowed the modified cells to grow under salt concentrations and pH conditions that are fully inhibitory to wild type cells. These results indicate that glutathione overexpression can be used to increase halotolerance in A. ferrooxidans and would likely be a useful strategy on other acidophilic bacteria.

Importance

The use of acidophilic bacteria in the hydrometallurgical processing of sulfide ores can enable many benefits including the potential reduction of environmental impacts. The cells involved in bioleaching tend to have limited halotolerance, and increased halotolerance could enable several benefits, including a reduction in the need for fresh water resources. We show that the genetic modification of A. ferrooxidans for the overproduction of glutathione is a promising strategy to enable cells to resist the oxidative stress that can occur during growth in the presence of salt.
Simultaneousin vivoPharmacokinetics and Pharmacodynamics of TDF Intravaginal Ring during High-dose Vaginal SHIV Challenge in Pigtail Macaques
Authors: Halavaty Katarina Kotnik , Ott Adina K. , Maric Danijela , Su Jonathan T. , Matias Edgar , Allen Edward J. , Martin Amy , Pereira Lara , Deyounks Frank , Smith James M. , Kiser Patrick F. , Hope Thomas J.
Published: July 2021

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.
Co-localization of different neurotransmitter transporters on synaptic vesicles is sparse except of VGLUT1 and ZnT3
Authors: Upmanyu Neha , Jin Jialin , von der Emde Henrik , Ganzella Marcelo , Bösche Leon , Malviya Viveka Nand , Zhuleku Evi , Politi Antonio , Ninov Momchil , Silbern Ivan , Leutenegger Marcel , Urlaub Henning , Riedel Dietmar , Preobraschenski Julia , Milosevic Ira , Hell Stefan W , Jahn Reinhard , Sambandan Sivakumar
Published: July 2021

Vesicular transporters (VTs) define the type of neurotransmitter that synaptic vesicles (SVs) store and release. While certain neurons in mammalian brain release multiple transmitters, the prevalence, physiology of such pluralism and if the release occurs from same or distinct vesicle pools is not clear. Using quantitative single vesicle imaging, we show that a small population of neuronal SVs indeed contain different VTs to accomplish corelease. Surprisingly, this population is highly diverse (27 types), expressing distinct dual transporters suggesting corelease of various combinations of neurotransmitters. Using glutamatergic vesicles as an example, we demonstrate that transporter colocalization not only determine the transmitter type but also influences the SV content and synaptic quantal size. Thus, presence of diverse transporters on the same vesicle is bona-fide and, depending on the VT types, this may act as one mechanism to regulate neurotransmitter type, content and release in space and time.
Structural Insights into the Venus flytrap Mechanosensitive Ion Channel Flycatcher1
Authors: Jojoa-Cruz Sebastian , Saotome Kei , Tsui Che Chun (Alex) , Lee Wen-Hsin , Sansom Mark S. P. , Murthy Swetha E. , Patapoutian Ardem , Ward Andrew B.
Published: July 2021

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.
Reliability of Decision-Making and Reinforcement Learning Computational Parameters
Authors: Mkrtchian Anahit , Valton Vincent , Roiser Jonathan P.
Published: July 2021

Computational models can offer mechanistic insight into cognition and therefore have the potential to transform our understanding of psychiatric disorders and their treatment. For translational efforts to be successful, it is imperative that computational measures capture individual characteristics reliably. Here we examine the reliability of reinforcement learning and economic models derived from two commonly used tasks. Healthy individuals (N=50) completed a restless four-armed bandit and a calibrated gambling task twice, two weeks apart. Reward and punishment learning rates from the reinforcement learning model showed good reliability and reward and punishment sensitivity from the same model had fair reliability; while risk aversion and loss aversion parameters from a prospect theory model exhibited good and excellent reliability, respectively. Both models were further able to predict future behaviour above chance within individuals. This prediction was better when based on participants’ own model parameters than other participants’ parameter estimates. These results suggest that reinforcement learning, and particularly prospect theory parameters, as derived from a restless four-armed bandit and a calibrated gambling task, can be measured reliably to assess learning and decision-making mechanisms. Overall, these findings indicate the translational potential of clinically-relevant computational parameters for precision psychiatry.
Designed architectural proteins that tune DNA looping in bacteria
Authors: Tse David , Becker Nicole A. , Young Robert T. , Olson Wilma K. , Peters Justin P. , Schwab Tanya L. , Clark Karl J. , Maher L. James
Published: July 2021

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 fromE. coliLac 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.
Pathology and immunity after SARS-CoV-2 infection in male ferrets is affected by age and inoculation route
Authors: van de Ven Koen , van Dijken Harry , Wijsman Lisa , Gomersbach Angéla , Schouten Tanja , Kool Jolanda , Lenz Stefanie , Roholl Paul , Meijer Adam , van Kasteren Puck , de Jonge Jørgen
Published: July 2021

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.
A 3D gene expression atlas of the floral meristem based on spatial reconstruction of single nucleus RNA sequencing data
Authors: Neumann Manuel , Xu Xiaocai , Smaczniak Cezary , Schumacher Julia , Yan Wenhao , Blüthgen Nils , Greb Thomas , Jönsson Henrik , Traas Jan , Kaufmann Kerstin , Muino Jose M
Published: July 2021

Identity and functions of plant cells are influenced by their precise cellular location within the plant body. Cellular heterogeneity in growth and differentiation trajectories results in organ patterning. Therefore, assessing this heterogeneity at molecular scale is a major question in developmental biology. Single-cell transcriptomics (scRNA-seq) allows to characterize and quantify gene expression heterogeneity in developing organs at unprecedented resolution. However, the original physical location of the cell is lost during the scRNA-seq procedure. To recover the original location of cells is essential to link gene activity with cellular function and morphology. Here, we reconstruct genome-wide gene expression patterns of individual cells in a floral meristem by combining single-nuclei RNA-seq with 3D spatial reconstruction. By this, gene expression differences among meristematic domains giving rise to different tissue and organ types can be determined. As a proof of principle, the data are used to trace the initiation of vascular identity within the floral meristem. Our work demonstrates the power of spatially reconstructed single cell transcriptome atlases to understand plant morphogenesis. The floral meristem 3D gene expression atlas can be accessed at http://threed-flower-meristem.herokuapp.com
A novel post-translational proteomics platform identifies neurite outgrowth impairments in Parkinson’s diseaseGBA-N370Sdopamine neurons
Authors: Bogetofte Helle , Ryan Brent J. , Jensen Pia , Vergoossen Dana L.E. , Barnkob Mike B. , Kiani Lisa , Chughtai Uroosa , Brandes Janine , Vowles Jane , Bunn Fiona , Kilfeather Peter , Fernandes Hugo J.R. , Caffrey Tara , Meyer Morten , Cowley Sally A. , Larsen Martin R. , Wade-Martins Richard
Published: July 2021

The causes of Parkinson’s disease (PD) likely involve complex interactions between environmental factors and susceptibility genes with variants at theGBAlocus encoding the glucocerebrosidase (GCase) enzyme being the strongest common genetic risk factor for PD. To understandGBA-related disease mechanisms, we used a novel multipart-enrichment proteomics and post-translational modification workflow to simultaneously identify peptides with phosphorylation, reversible cysteine-modifications or sialylated N-linked glycosylation, alongside unmodified proteins.

We identified large numbers of dysregulated proteins and post-translational modifications (PTMs) in heterozygousGBA-N370SPD patient induced pluripotent stem cells (iPSC)-derived dopamine neurons. Alterations in glycosylation status of lysosomal proteins identified disturbances in the autophagy-lysosomal pathway, concurrent with upstream perturbations in mTOR phosphorylation and activity inGBA-N370SiPSC-dopamine neurons. In addition, the strategy revealed several native and modified proteins encoded by PD-associated genes to be dysregulated inGBA-N370Sneurons, enhancing our understanding of the wider role ofGBAmutations on the neuronal proteome. Integrated pathway analysis of all datasets revealed impaired neuritogenesis inGBA-N370SPD iPSC-dopamine neurons and identified tau (MAPT) as a key mediator of this process. Using a functional assay, we confirmed neurite outgrowth deficits inGBA-N370SPD neurons and a central role for tau in this process. Furthermore, pharmacological restoration of GCase activity inGBA-N370SPD patient neurons rescued the neurite outgrowth deficit. Overall, this study demonstrates the potential of PTMomics to elucidate novel neurodegeneration-associated pathways and identify phenotypes and potential drug targets in complex disease models.
In Situ Volumetric Imaging and Analysis of FRESH 3D Bioprinted Constructs Using Optical Coherence Tomography
Authors: Tashman Joshua W. , Shiwarski Daniel J. , Ruesch Alexander , Lanni Frederick , Kainerstorfer Jana M. , Feinberg Adam W.
Published: July 2021

As 3D bioprinting has grown as a fabrication technology, so too has the need for improved analytical methods to characterize these engineered constructs. This is especially challenging for soft tissues composed of hydrogels and cells as these materials readily deform, posing a barrier when trying to assess print fidelity and other properties non- destructively. Indeed, given the importance of structure-function relationships in most tissue types, establishing that the 3D architecture of the bioprinted construct matches its intended anatomic design is critical. Here we report development of a multimaterial bioprinting platform with integrated optical coherence tomography (OCT) for in situ volumetric imaging, error detection, and 3D reconstruction. While generally applicable to extrusion-based 3D bioprinting, we also report improvements to the Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting process through new collagen bioink compositions, support bath optical clearing, and machine pathing. This enables high-fidelity 3D volumetric imaging with micron scale resolution over centimeter length scales, the ability to detect a range of print defect types within a 3D volume, and real-time imaging of the printing process at each print layer. These advances provide FRESH and other extrusion-based 3D bioprinting approaches with a comprehensive methodology for quality assessment that has been absent in the field to date, paving the way for translation of these engineered tissues to the clinic and ultimately to achieving regulatory approval.

Teaser

Transparent FRESH support bath enables in situ 3D volumetric imaging and validation of patient-derived tissue constructs.
CellProfiler 4: Improvements in Speed, Utility and Usability
Authors: Stirling David R. , Swain-Bowden Madison J. , Lucas Alice M. , Carpenter Anne E. , Cimini Beth A. , Goodman Allen
Published: July 2021

Background

Imaging data contains a substantial amount of information which can be difficult to evaluate by eye. With the expansion of high throughput microscopy methodologies producing increasingly large datasets, automated and objective analysis of the resulting images is essential to effectively extract biological information from this data. CellProfiler is a free, open source image analysis program which enables researchers to generate modular pipelines with which to process microscopy images into interpretable measurements.

Results

Herein we describe CellProfiler 4, a new version of this software with expanded functionality. Based on user feedback, we have made several user interface refinements to improve the usability of the software. We introduced new modules to expand the capabilities of the software. We also evaluated performance and made targeted optimizations to reduce the time and cost associated with running common large-scale analysis pipelines.

Conclusions

CellProfiler 4 provides significantly improved performance in complex workflows compared to previous versions. This release will ensure that researchers will have continued access to CellProfiler’s powerful computational tools in the coming years.
Trends and Gaps on Philippine Scombrid Research: A bibliometric analysis
Authors: Gadut Jay-Ar L. , Deocaris Custer C. , Alinsug Malona V.
Published: July 2021

Philippine scombrids have been among the top priorities in fisheries research due primarily to their economic value worldwide. Assessment of the number of studies under general themes (diversity, ecology, taxonomy and systematics, diseases and parasites, and conservation) provides essential information to evaluate trends and gaps of research. This study presents a bibliometric evaluation of the temporal trends from 2000-2019 on Philippine scombrid research. Seven out of nine tuna species have SREA values equal to or higher than 1 with Thunnus albacares being the most researched tuna species. Twelve out of thirteen non-tuna species have SREA values less than one, thus indicating low research effort. It was apparent that there were significant differences in the number of studies in each thematic area except in ‘chemical analysis’ and ‘diseases and parasites’ between scombrid groups where there was low research effort observed. The research points at the uneven research distributions between scombrid groups in each thematic area. As locally published research are significantly behind foreign publications in terms of citation index, international collaborations by Filipino researchers have shown an increase research impact. Our study hopes to influence the local and international R&D agenda on Philippine scombrids and promote solidarity among nations towards its conservation and management.
miRNAFinder: A Comprehensive Web Resource for Plant Pre-microRNA Classification
Authors: Lokuge Sandali , Jayasundara Shyaman , Ihalagedara Puwasuru , Kahanda Indika , Herath Damayanthi
Published: July 2021

microRNAs (miRNAs) are known as one of the small non-coding RNA molecules that control the expression of genes at the RNA level, while some operate at the DNA level. They typically range from 20–24 nucleotides in length and can be found in the plant and animal kingdoms as well as in some viruses. Computational approaches have overcome the limitations of the experimental methods and have performed well in identifying miRNAs. Compared to mature miRNAs, precursor miRNAs (pre-miRNAs) are long and have a hairpin loop structure with structural features. Therefore, most in-silico tools are implemented for pre-miRNA identification. This study presents a multilayer perceptron (MLP) based classifier implemented using 180 features under sequential, structural, and thermodynamic feature categories for plant pre-miRNA identification. This classifier has a 92% accuracy, a 94% specificity, and a 90% sensitivity. We have further tested this model with other small non-coding RNA types and obtained 78% accuracy. Furthermore, we introduce a novel dataset to train and test machine learning models, addressing the overlapping data issue in the positive training and testing datasets presented in PlantMiRNAPred for the classification of real and pseudo-plant pre-miRNAs. The new dataset and the classifier that can be used with any plant species are deployed on a web server freely accessible at http://mirnafinder.shyaman.me/.
Bacterial community composition of the sea grape Caulerpa lentillifera: a comparison between healthy and diseased states
Authors: Kopprio Germán A. , Luyen Nguyen Dinh , Cuong Le Huu , Fricke Anna , Kunzmann Andreas , Huong Le Mai , Gärdes Astrid
Published: July 2021

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.
Accurate estimation of cell-type resolution transcriptome in bulk tissue through matrix completion
Authors: Wang Weixu , Zhou Xiaolan , Yao Jun , Wen Haimei , Wang Yi , Sun Mingwan , Zhang Chao , Tao Wei , Zou Jiahua , Ni Ting
Published: July 2021

Single cell RNA-seq (scRNA-seq) has been widely used to uncover cellular heterogeneity, however, the constraints of cost make it impractical as a routine on large patient cohorts. Here we present ENIGMA, a method that accurately deconvolute bulk tissue RNA-seq into single cell-type resolution given the knowledge gained from scRNA-seq. ENIGMA applies a matrix completion strategy to minimize the distance between mixture transcriptome and weighted combination of cell type-specific expression, allowing quantification of cell type proportions and reconstruction of cell type-specific transcriptome. The superior performance of ENIGMA was validated in simulated and realistic datasets, including disease-related tissues, demonstrating its ability in novel biological findings.