bluuebunny/crossref_metadata_2025 huggingface dataset:
A case of Capnocytophaga canimorsus endocarditis in a non-immunosuppressed host: the value of 16S PCR for diagnosis
Authors: McNicol Mark , Yew Peter , Beattie Gwyn , Loughlin Laura
Published: May 2021

Capnocytophaga canimorsus

is a rare cause of endocarditis and is particularly unusual in non-immunosuppressed hosts. It is associated with animal bites, particularly those from dogs. This case describes a healthy 59-year-old woman, with no identifiable risk factors or dog bite history, who presented with fever of unknown origin. Echocardiography demonstrated an aortic valve mass and root abscess, in keeping with endocarditis, requiring urgent valve replacement surgery. Eight sets of blood cultures were drawn in total; after prolonged incubation, one set grew C. canimorusus. There was initial uncertainty over this being the causative organism, given the lack of immunosuppression or dog bite history, but 16S PCR of the valve identified the same organism, permitting targeted treatment. This case highlights the value of valve 16S PCR as a diagnostic tool in endocarditis.
Whole genome sequence analysis of Shigella from Malawi identifies fluoroquinolone resistance
Authors: Stenhouse George E. , Jere Khuzwayo C. , Peno Chikondi , Bengtsson Rebecca J. , Chinyama End , Mandolo Jonathan , Cain Amy , Iturriza-Gómara Miren , Bar-Zeev Naor , Cunliffe Nigel A. , Cornick Jennifer , Baker Kate S.
Published: May 2021

Increasing antimicrobial resistance and limited alternative treatments have led to fluoroquinolone-resistant

Shigella

strain inclusion on the WHO global priority pathogens list. In this study we characterized multiple

Shigella

isolates from Malawi with whole genome sequence analysis, identifying the acquirable fluoroquinolone resistance determinant qnrS1.
Experimental evolution in morbidostat reveals converging genomic trajectories on the path to triclosan resistance
Authors: Leyn Semen A. , Zlamal Jaime E. , Kurnasov Oleg V. , Li Xiaoqing , Elane Marinela , Myjak Lourdes , Godzik Mikolaj , de Crecy Alban , Garcia-Alcalde Fernando , Ebeling Martin , Osterman Andrei L.
Published: May 2021

Understanding the dynamics and mechanisms of acquired drug resistance across major classes of antibiotics and bacterial pathogens is of critical importance for the optimization of current anti-infective therapies and the development of novel ones. To systematically address this challenge, we developed a workflow combining experimental evolution in a morbidostat continuous culturing device with deep genomic sequencing of population samples collected in time series. This approach was applied to the experimental evolution of six populations of

Escherichia coli

BW25113 towards acquiring resistance to triclosan (TCS), an antibacterial agent in various consumer products. This study revealed the rapid emergence and expansion (up to 100% in each culture within 4 days) of missense mutations in the fabI gene, encoding enoyl-acyl carrier protein reductase, the known TCS molecular target. A follow-up analysis of isolated clones showed that distinct amino acid substitutions increased the drug IC90 in a 3–16-fold range, reflecting their proximity to the TCS-binding site. In contrast to other antibiotics, efflux-upregulating mutations occurred only rarely and with low abundance. Mutations in several other genes were detected at an earlier stage of evolution. Most notably, three distinct amino acid substitutions were mapped in the C-terminal periplasmic domain of CadC protein, an acid stress-responsive transcriptional regulator. While these mutations do not confer robust TCS resistance, they appear to play a certain, yet unknown, role in adaptation to relatively low drug pressure. Overall, the observed evolutionary trajectories suggest that the FabI enzyme is the sole target of TCS (at least up to the ~50 µm level), and amino acid substitutions in the TCS-binding site represent the main mechanism of robust TCS resistance in

E. coli

. This model study illustrates the potential utility of the established morbidostat-based approach for uncovering resistance mechanisms and target identification for novel drug candidates with yet unknown mechanisms of action.
The ARC2 response in Streptomcyes coelicolor requires the global regulatory genes afsR and afsS
Authors: Calvelo Vanessa Yoon , Crisante David , Elliot Marie , Nodwell Justin Rea
Published: May 2021

ARC2 is a synthetic compound, related in structure and mechanism to the antibiotic triclosan, that activates the production of many specialized metabolites in the

Streptomyces

genus of bacteria. In this work, we demonstrate that the addition of ARC2 to

Streptomyces coelicolor

cultures results in considerable alterations in overall gene expression including most notably the specialized metabolic genes. Using actinorhodin production as a model system, we show that the effect of ARC2 depends on the pleiotropic regulators afsR and afsS but not afsK. We find that the constitutive expression of afsS can bypass the need for afsR but not the reverse, while the constitutive expression of afsK had no effect on actinorhodin production. These data are consistent with a model in which ARC2 activates a cell stress response that depends on AfsR activating the expression of the afsS gene such that AfsS then triggers the production of actinorhodin.
Investigation of the Relationship between Delta Inflammatory Markers and Prognosis in Head and Neck Squamous Cell Carcinoma
Authors: Khaymovich Julian , Ko Andrew , Wong Amanda , Zhu Daniel , Gigante Christian , Garikapati Srilakshmi , Jeng Ginnie , Van Der Elst Sarah , Rong Charles , Tham Tristan
Published: May 2021

Purpose

Inflammatory markers, such as Lymphocyte-to-Monocyte Ratio (LMR), Neutrophil-to-Lymphocyte Ratio (NLR), and Platelet-to-Lymphocyte Ratio (PLR), have been shown to hold significant prognostic value in the context of head and neck cancer (HNC). Recently, delta inflammatory markers, the difference between pre and post- treatment inflammatory marker ratios, have been suggested as potentially significant values in predicting cancer prognosis. Our objective was to evaluate the prognostic utility of delta LMR, NLR, and PLR in head and neck squamous cell carcinoma (HNSCC).

Methods

Retrospective cohort study in a tertiary academic hospital setting. Patients diagnosed with HNSCC in the oral cavity, larynx, and oropharynx treated with curative intent treatment were included. The variables collected were age, sex, BMI, alcohol/tobacco exposure, performance scores, ACE-27, tumor characteristics, adjuvant treatment, ECOG score, and lab values. Overall Survival (OS) and Event-Free Survival (EFS) were chosen as endpoints. OS was defined as time from date of treatment to date of last follow-up or death from any cause, and EFS was defined as the start of treatment to any progression, recurrence, or death from any cause. Univariate and multivariate analyses were performed on the primary endpoints.

Results

A total of 89 patients were included from 2010 to 2017. In multivariate analysis, EFS was found to be significantly associated with an N stage of 3 (p=0.0005) and delta LMR > -1.48 (p=0.0241). No significant relationships were uncovered with OS in multivariate analysis.

Conclusion

A higher delta LMR value (>-1.48) was associated with poorer EFS, but was not associated with OS.
Retrospective Analysis of the Utility of Genetic Testing in Predicting Drug Response in Chronic Pain
Authors: Gupta Gaurav , Cpl Emilie Paquet-Proulx , Lalonde Sasha , Burton Kira , LCol Markus Besemann , Amir Minerbi
Published: May 2021

Introduction

Chronic pain is often unrecognized and/or undertreated, and as a result has significant impact on functional abilities, quality of life, societal participation and health care utilization. Medications remain a mainstay of treatment, but selection for any given patient remains a challenge when trying to predict efficacy and/or side effects. There is interest to see whether genetic analysis of how a given drug is processed for a patient can help with rational drug choices. This appears to have some early support in cardiac, psychiatric and acute pain studies. We studied whether genetic analysis of drug processing using the Pillcheck program could have helped in choosing the appropriate medications in a cohort of patients suffering from chronic pain. To our knowledge this type of study has not been completed in this environment and/or patient population.

Methods

We retrospectively studied a 31 patient cohort seen in the Canadian Forces Health Services Unit (Ottawa) Physiatry clinic. All patients suffered from a diagnosed chronic pain condition, completed the Pillcheck genetic drug processing analysis and filled-in questionnaires looking at efficacy and side effects of the drugs. We analyzed the correlation between the Pillcheck predictions and participants’ self-reported treatment efficacy and tolerability. The goal was to explore the clinical utility of Pillcheck results in guiding prescriptions for chronic pain patients.

Results

31 patients returned completed questionnaires and had samples taken. Forty eight percent of the participants were actively treated with one of the study pain medications, and 84% had been taking at least one of these medications and discontinued. Pillcheck scores did not correlate with self-reported efficacy of any of the medications, nor did it correlate with self-reported side effects. Furthermore, active medications were more likely to receive a score indicating caution should be exerted, than were medications which had been discontinued.

Discussion

In a small cohort of pain patients with comorbid psychiatric disorders, genetic profiling using Pillcheck did not seem to correlate with reported benefit or side effect profile of commonly prescribed pain medications. Furthermore, discontinued medications were no more likely to be marked as warranting caution than did actively used medications.

Conclusion

Retrospectively using pharmacogenetics to guide medication selection in Canadian Forces members with chronic pain did not correlate with response or side effects. A larger prospective controlled study, measuring numerous clinical and non- clinical outcomes would be worthwhile in the future before widespread adoption for patients living with chronic pain.
COVID-19 and acute kidney injury in German hospitals 2020
Authors: Walendy Victor , Girndt Matthias , Greinert Daniel
Published: May 2021

Introduction

The SARS-CoV-2 pandemic is a major challenge for patients, healthcare professionals, and populations worldwide. While initial reporting focused mainly on lung involvement, the ongoing pandemic showed that multiple organs can be involved, and prognosis is largely influenced by multi-organ involvement. Our aim was to obtain nationwide retrospective population-based data on hospitalizations with COVID-19 and AKI.

Materials & Methods

We performed a query of G-DRG data for the year 2020 via the Institute for the hospital remuneration system (Institut für das Entgeltsystem im Krankenhaus GmbH, InEK) data portal and therefore included hospitalizations with a secondary diagnosis of RT-PCR proven COVID-19 infection, aged over 15 years. We included hospitalizations with acute kidney injury (AKI) stages 1 to 3. Age-specific and age-standardized hospitalization and in-hospital mortality rates (ASR) per 100.000 person years were calculated, with the German population of 2011 as the standard.

Results

In 2020, there were 16.776.845 hospitalizations in German hospitals. We detected 154.170 hospitalizations with RT-PCR proven COVID-19 diagnosis. The age-standardized hospitalization rate for COVID-19 in Germany was 232,8 per 100.000 person years (95% CI 231,6-233,9). The highest proportion of hospitalizations associated with COVID-19 were in the age group over 80 years. AKI was diagnosed in 16.773 (10,9%) of the hospitalizations with COVID-19. The relative risk of AKI for males was 1,49 (95%CI 1,44-1,53) compared to females. Renal replacement therapy (RRT) was performed in 3.443 hospitalizations, 20,5% of the hospitalizations with AKI. For all hospitalizations with COVID-19, the in-hospital mortality amounted to 19,7% (n= 30.300). The relative risk for in-hospital mortality was 3,87 (95%CI 3,80-3,94) when AKI occurred. The age-standardized hospitalization rates for COVID-19 took a bimodal course during the observation period. The first peak occurred in April (ASR 23,95 per 100.000 person years (95%CI 23,58-24,33)), hospitalizations peaked again in November 2020 (72,82 per 100.000 person years (95%CI 72,17-73,48)). The standardized rate ratios (SRR) for AKI and AKI-related mortality with the overall ASR for COVID-19 hospitalizations in the denominator, decreased throughout the observation period and remained lower in autumn than they were in spring. In contrast to all COVID-19 hospitalizations, the SRR for overall mortality in COVID-19 hospitalizations diverged from hospitalizations with AKI in autumn 2020.

Discussion

Our study for the first time provides nationwide data on COVID-19 related hospitalizations and acute kidney injury in Germany in 2020. AKI was a relevant complication and associated with high mortality. We observed a less pronounced increase in the ASR for AKI-related mortality during autumn 2020. The proportion of AKI-related mortality in comparison to the overall mortality decreased throughout the course of the pandemic.
Motor Function and White Matter Connectivity in Children Treated with Therapeutic Hypothermia for Neonatal Encephalopathy
Authors: Spencer Arthur P.C. , Brooks Jonathan C.W. , Masuda Naoki , Byrne Hollie , Lee-Kelland Richard , Jary Sally , Thoresen Marianne , Goodfellow Marc , Cowan Frances M. , Chakkarapani Ela
Published: May 2021

Therapeutic hypothermia reduces the incidence of severe motor disability, such as cerebral palsy, following neonatal hypoxic-ischemic encephalopathy. However, cooled children without cerebral palsy at school-age demonstrate motor deficits and altered white matter connectivity. In this study, we used diffusion-weighted imaging to investigate the relationship between white matter connectivity and motor performance, measured using the Movement Assessment Battery for Children-2, in school-age children treated with therapeutic hypothermia for neonatal hypoxic ischaemic encephalopathy at birth, who did not develop cerebral palsy (cases), and matched controls. Analysis of tract-level microstructure (33 cases, 36 controls) revealed correlations between total motor scores and fractional anisotropy, in cases but not controls, in the anterior thalamic radiation bilaterally, the inferior fronto-occipital fasciculus bilaterally and both the hippocampal and cingulate gyrus parts of the left cingulum. Analysis of structural brain networks (22 cases, 32 controls), in which edges were determined by probabilistic tractography and weighted by fractional anisotropy, revealed correlations between total motor scores and several whole-brain network metrics in cases but not controls. We then investigated edge-level association with motor function using the network-based statistic. This revealed subnetworks which exhibited group differences in the association between motor outcome and edge weights, for total motor scores as well as for balance and manual dexterity domain scores. All three of these subnetworks comprised numerous frontal lobe regions known to be associated with motor function, including the superior frontal gyrus and middle frontal gyrus. These findings demonstrate an association between impaired motor function and brain organisation in case children.
Development of in-house, indirect ELISAs for the detection of SARS-CoV-2 spike protein-associated serology in COVID-19 patients in Panama
Authors: de la Guardia Carolina , Rangel Giselle , Villarreal Alcibiades , Goodridge Amador , Fernández Patricia L , Lleonart Ricardo
Published: May 2021

COVID-19 is the name of the acute respiratory disease caused by the new coronavirus SARS-CoV-2, a close relative of those that caused the severe outbreaks of SARS and MERS several years ago. Since first appearance on December of 2019, the COVID-19 pandemic has cause extremely high levels of mortality, morbidity, global economic breakdown and the consequent human suffering. While vaccination efforts are not extensive and rapid enough, the main tools to keep the virus under control are still keeping physical distancing, reinforce personal hygiene measures, using masks and early diagnosis of virus infected persons, either symptomatic or not. The main diagnostic test for the confirmation of symptomatic individuals is the detection of viral RNA by reverse transcriptase – quantitative real time PCR (RT-PCR). Additionally, serology techniques, such as ELISA are extremely useful to measure the antibodies generated in humans after virus contact, as well as the direct presence of viral antigens. In this study we aim to assemble and evaluate four ELISAs assays to measure the presence of IgG or IgM specific for the viral Spike protein in COVID-19 patients, using either the full recombinant SARS-CoV-2 Spike protein or the fragment corresponding to the receptor binding domain. As a control, we also analyzed a group of prepandemic serum samples obtained before 2017.

Strong reactivity was observed against both antigens. A few prepandemic samples displayed high OD values, suggesting the possibility of some crossreactivity. All four assays show very good repeatability, both intra- and inter-assay; however, no clear relationship could be detected between positivity and time of sample collection for serology. Receiver operating characteristic analysis allowed the definition of cutoffs and evaluation of performance for each ELISA by estimation of the area under the curve. This performance parameter was high for all tests (AUC range: 0.98-0.995). Multiple comparisons between tests revealed no significant difference between each other (P values: 0.24-0.95). Our results show that both antigens are very effective to reveal both specific IgG and IgM antibodies, with high sensitivity (range 0.929-0.99) and specificity (range 0.933-0.977). The estimated congruence with the RT-PCR test, as estimated by Cohen’s Kappa, indicates a high agreement in all cases (range 0.874-0.937). This test will allow health authorities to have a new tool to estimate seroprevalence, and to manage and improve the serious sanitary situation created by this virus.
Possible role of accessory proteins in the viral replication for the 20I/501Y.V1 (B.1.1.7) SARS-CoV-2 variant
Authors: Nyayanit Dimpal A. , Sarkale Prasad , Shete-Aich Anita , Kumar Abhinendra , Patil Savita , Majumdar Triparna , Baradkar Shreekant , Gawande Pranita , Mohandas Sreelekshmy , Yadav Pragya D
Published: May 2021

The study investigates the replication cycle and transcriptional pattern of the B.1.1.7 variant. It was observed that the B.1.1.7 variant required a longer maturation time. The transcriptional response demonstrated higher expression of ORF6 and ORF8 compared to nucleocapsid transcript till the eclipse period which might influence higher viral replication. The number of infectious viruses titer is higher in the B.1.1.7, despite a lesser copy number than B.1, indicating higher infectivity.
Point of care testing for detection of coronaviruses including SARS-CoV-2 from saliva without treating RNA in advance
Authors: Muraoka Masaaki , Kawaguchi Osamu , Mizukoshi Mikio , Sejima Shunsuke
Published: May 2021

Coronavirus disease 2019 (COVID-19) outbreak was reported to the WHO (World Health Organization) as an outbreak on end of 2019, afterwards pandemic on the worldwide in 2020. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has be reported it to cause COVID-19, and highly transmissible. Therefore, it is important that it is rapidly and continuously detected and monitored on site so that the infection is prevented. Namely, POCT (point of care testing) may be important to control cross infection of SARS-CoV-2. At present, the routine confirmation of SARS-CoV-2 is based on detection of sequence unique in the virus RNA by nucleic acid amplification tests (NAATs) such as rRT-PCR. Taking POCT into account, it is clear that it takes time and labour very much or much. Thus, it was our purpose this time to contribute to develop POCT for microbes such as SARS-CoV-2, and thus needed to improve NAATs method.

First, combining the mobile real-time RT-PCR (rRT-PCR) device PCR1100 with the appropriate rRT-PCR reagent, we found that it is possible to detect RNA of SARS-CoV-2 for less 14 minutes with equivalent accuracy to conventional devices. Next, we found that the above method made it possible for us to detect coronaviruses by direct rRT-PCR without pre-treatments. Furthermore, it also made clear that coronaviruses in saliva could be detected by the similar direct rRT-PCR method.

Hence, it was concluded that this method made it possible to detect virus in saliva without treating in advance (extraction, purification, concentration, etc.), and moreover, samples would be able to be collected with non-invasive. For this reason, we suggest that this method is useful for POCT of coronaviruses including SARS-CoV-2.
Forecasting COVID-19 Spreading in Canada using Deep Learning
Authors: Khennou Fadoua , Akhloufi Moulay A.
Published: May 2021

Abstract

The novel coronavirus disease 2019 (COVID-19) is disrupting all aspects of our lives as the global spread of the virus continues. In this difficult period, various research projects are taking place to study and analyse the dynamics of the pandemic. In the present work, we firstly present a deep overview of the main forecasting models to predict the new cases of COVID-19. In this context, we focus on univariate time series models in order to analyze the dynamic change of this pandemic through time. We secondly shed light on multivariate time series forecasting models using weather and daily tests data, to study the impact of exogenous features on the progression of COVID-19. In the final stage of this paper, we present our proposed approach based on LSTM and GRU ensemble learning model and evaluate the results using the MAE, RMSE and MAPE for the prediction of new cases. The results of our experiments using the Canadian dataset show that the ensemble model performs well in comparison to other models. In addition, this research provides us with a new outcome regarding the dynamic correlation between temperature, humidity and daily test data and its impact on the new contaminated cases.
Signaling through FcγRIIA and the C5a-C5aR pathway mediates platelet hyperactivation in COVID-19
Authors: Apostolidis Sokratis A. , Sarkar Amrita , Giannini Heather M. , Goel Rishi R. , Mathew Divij , Suzuki Aae , Baxter Amy E. , Greenplate Allison R. , Alanio Cécile , Abdel-Hakeem Mohamed , Oldridge Derek A. , Giles Josephine , Wu Jennifer E. , Chen Zeyu , Huang Yinghui Jane , Pattekar Ajinkya , Manne Sasikanth , Kuthuru Oliva , Dougherty Jeanette , Weiderhold Brittany , Weisman Ariel R. , Ittner Caroline A. G. , Gouma Sigrid , Dunbar Debora , Frank Ian , Huang Alexander C. , Vella Laura A. ,   The UPenn COVID Processing Unit, Reilly John P. , Hensley Scott E. , Rauova Lubica , Zhao Liang , Meyer Nuala J. , Poncz Mortimer , Abrams Charles S. , Wherry E. John
Published: May 2021

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibit higher basal levels of activation measured by P-selectin surface expression, and have a poor functional reserve upon in vitro stimulation. Correlating clinical features to the ability of plasma from COVID-19 patients to stimulate control platelets identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions, thus identifying these potentially actionable pathways as central for platelet activation and/or vascular complications in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect. These studies have implications for the role of platelet hyperactivation in complications associated with SARS-CoV-2 infection.

Cover illustration

One-sentence summary

The FcγRIIA and C5a-C5aR pathways mediate platelet hyperactivation in COVID-19
ACE2 expression in rat brain: implications for COVID-19 associated neurological manifestations
Authors: Hernández Vito S , Zetter Mario A , Guerra Enrique C. , Hernández-Araiza Ileana , Karuzin Nikita , Hernández-Pérez Oscar R. , Eiden Lee E , Zhang Limei
Published: May 2021

We examined cell type-specific expression and distribution of rat brain angiotensin converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, in rodent brain. ACE2 is ubiquitously present in brain vasculature, with the highest density of ACE2 expressing capillaries found in the olfactory bulb, the hypothalamic paraventricular, supraoptic and mammillary nuclei, the midbrain substantia nigra and ventral tegmental area, and the hindbrain pontine nucleus, pre-Bötzinger complex, and nucleus oftractus solitarius. ACE2 was expressed in astrocytes and astrocytic foot processes, pericytes and endothelial cells, key components of the blood-brain-barrier. We found discrete neuronal groups immunopositive for ACE2 in brainstem respiratory rhythm generating centers including the pontine nucleus, the parafascicular/retrotrapezoid nucleus, the parabrachial nucleus, the Bötzinger and pre-Bötzinger complex and the nucleus of tractus solitarius; in arousal-related pontine reticular nucleus and in gigantocellular reticular nuclei; in brainstem aminergic nuclei, including substantia nigra, ventral tegmental area, dorsal raphe, and locus coeruleus; in the epithalamic habenula, hypothalamic paraventricular and suprammamillary nuclei; and in the hippocampus. Identification of ACE2-expressing neurons in rat brain within well-established functional circuits facilitates prediction of possible neurological manifestations of brain ACE2 dysregulation during and after COVID-19 infection.

Highlights

ACE2 is present in astrocytes, pericytes, and endothelia of the blood brain barrier.

Neuronal ACE2 expression is shown in discrete nuclei through the brain.

Brainstem breathing, arousal-related, hypothalamic and limbic nuclei express ACE2.

ACE2 is expressed in circuits potentially involved in COVID-19 pathophysiology.
Inhibiting LSD1 suppresses coronavirus-induced inflammation but spares innate antiviral activity
Authors: Mazzarella Luca , Santoro Fabio , Ravasio Roberto , Massa Paul E. , Rodighiero Simona , Gavilán Elena , Romanenghi Mauro , Duso Bruno Achutti , Bonetti Emanuele , Pallavi Rani , Trastulli Deborah , Pallavicini Isabella , Gentile Claudia , Leonardi Tommaso , Pasqualato Sebastiano , Buttinelli Gabriele , Martino Angela Di , Fedele Giorgio , Schiavoni Ilaria , Stefanelli Paola , Meroni Giuseppe , Steinkuhler Christian , Fossati Gianluca , Minucci Saverio , Pelicci Pier Giuseppe
Published: May 2021

Tissue-resident macrophages exert critical but conflicting effects on the progression of coronavirus infections by secreting both anti-viral type I Interferons and tissue-damaging inflammatory cytokines. Steroids, the only class of host-targeting drugs approved for Covid19, indiscriminately suppress both responses, possibly impairing viral clearance, and provide limited clinical benefit. Here we set up a mousein vitroco-culture system that reproduces the macrophage response to SARS-CoV2 seen in patients and allows quantitation of inflammatory and antiviral activities. We show that the NFKB-dependent inflammatory response can be selectively inhibited by ablating the lysine-demethylase LSD1, which additionally unleashed interferon-independent ISG activation and blocked viral egress through the lysosomal pathway. These results provide a rationale for repurposing LSD1 inhibitors, a class of drugs extensively studied in oncology, for Covid-19 treatment.

One-Sentence Summary

Targeting a chromatin-modifying enzyme in coronavirus infections curbs tissue-damage without affecting antiviral response
Vitamin C Binds to SARS Coronavirus-2 Main Protease Essential for Viral Replication
Authors: Malla Tek Narsingh , Pandey Suraj , Aldama Luis , Feliz Dennisse , Noda Moraima , Poudyal Ishwor , Phillips George N. , Stojković Emina A. , Schmidt Marius
Published: May 2021

There is an urgent need for anti-viral agents that treat and/or prevent Covid-19 caused by SARS-Coronavirus (CoV-2) infections. The replication of the SARS CoV-2 is dependent on the activity of two cysteine proteases, a papain-like protease, PL-pro, and the 3C-like protease known as main protease Mpro or 3CLpro. The shortest and the safest path to clinical use is the repurposing of drugs with binding affinity to PLpro or 3CLpro that have an established safety profile in humans. Several studies have reported crystal structures of SARS-CoV-2 main protease in complex with FDA approved drugs such as those used in treatment of hepatitis C. Here, we report the crystal structure of 3CLpro in complex Vitamin C (L-ascorbate) bound to the protein’s active site at 2.5 Ångstrom resolution. The crystal structure of the Vitamin C 3CLpro complex may aid future studies on the effect of Vitamin C not only on the coronavirus main protease but on related proteases of other infectious viruses.
City-wide school-located influenza vaccination: a retrospective cohort study
Authors: Benjamin-Chung Jade , Arnold Benjamin F. , Mishra Kunal , Kennedy Chris J. , Nguyen Anna , Pokpongkiat Nolan N. , Djajadi Stephanie , Seth Anmol , Klein Nicola P. , Hubbard Alan E. , Reingold Arthur , Colford John M.
Published: May 2021

Background

We measured the effectiveness of a city-wide school-located influenza vaccination (SLIV) program implemented in over 102 elementary schools in Oakland, California.

Methods

We conducted a retrospective cohort study among Kaiser Permanente Northern California (KPNC) members of all ages residing in either the intervention or a multivariate-matched comparison site from September 2011 - August 2017. Outcomes included medically attended acute respiratory illness (MAARI), influenza hospitalization, and Oseltamivir prescriptions. We estimated difference-in-differences (DIDs) in 2014-15, 2015-16, and 2016-17 using generalized linear models and adjusted for race, ethnicity, age, sex, health plan, and language.

Results

Pre-intervention member characteristics were similar between sites. Among school-aged children, SLIV was associated with lower Oseltamivir prescriptions per 1,000 (DIDs: −3.5 (95% CI −5.5, −1.5) in 2015-16; −4.0 (95% CI −6.5, −1.6) in 2016-17) but not with other outcomes. SLIV was associated with lower MAARI per 1,000 in adults 65+ years (2014-15: −13.2, 95% CI −23.2, −3.2; 2015-16: −21.5, 95% CI −31.1, −11.9; 2016-17: −13.0, 95% CI −23.2, −2.9). There were few significant associations with other outcomes among adults.

Conclusions

A city-wide SLIV intervention was associated with lower Oseltamivir prescriptions in school-aged children and lower MAARI among people over 65 years, suggesting possible indirect effects of SLIV among older adults.
Semi-supervised identification of SARS-CoV-2 molecular targets
Authors: Beck Kristen L. , Seabolt Ed , Agarwal Akshay , Nayar Gowri , Bianco Simone , Krishnareddy Harsha , Mukherjee Vandana , Kaufman James H.
Published: May 2021

SARS-CoV-2 genomic sequencing efforts have scaled dramatically to address the current global pandemic and aid public health. In this work, we analyzed a corpus of 66,000 SARS-CoV-2 genome sequences. We developed a novel semi-supervised pipeline for automated gene, protein, and functional domain annotation of SARS-CoV-2 genomes that differentiates itself by not relying on use of a single reference genome and by overcoming atypical genome traits. Using this method, we identified the comprehensive set of known proteins with 98.5% set membership accuracy and 99.1% accuracy in length prediction compared to proteome references including Replicase polyprotein 1ab (with its transcriptional slippage site). Compared to other published tools such as Prokka (base) and VAPiD, we yielded an 6.4- and 1.8-fold increase in protein annotations. Our method generated 13,000,000 molecular target sequences— some conserved across time and geography while others represent emerging variants. We observed 3,362 non-redundant sequences per protein on average within this corpus and describe key D614G and N501Y variants spatiotemporally. For spike glycoprotein domains, we achieved greater than 97.9% sequence identity to references and characterized Receptor Binding Domain variants. Here, we comprehensively present the molecular targets to refine biomedical interventions for SARS-CoV-2 with a scalable high-accuracy method to analyze newly sequenced infections.
Optimization of AAV6 transduction enhances site-specific genome editing of primary human lymphocytes
Authors: Rogers Geoffrey L. , Huang Chun , Clark Robert , Seclen Eduardo , Chen Hsu-Yu , Cannon Paula M.
Published: May 2021

Adeno-associated virus serotype 6 (AAV6) is a valuable reagent for genome editing of hematopoietic cells due to its ability to serve as a homology donor template. However, a comprehensive study of AAV6 transduction of hematopoietic cells in culture, with the goal of maximizingex vivogenome editing, has not been reported. Here, we evaluated how the presence of serum, culture volume, transduction time, and electroporation parameters could influence AAV6 transduction. Based on these results, we identified an optimized protocol for genome editing of human lymphocytes based on a short, highly concentrated AAV6 transduction in the absence of serum, followed by electroporation with a targeted nuclease. In human CD4+T cells and B cells, this protocol improved editing rates up to 7-fold and 21-fold respectively, when compared to standard AAV6 transduction protocols described in the literature. As a result, editing frequencies could be maintained using 50-100-fold less AAV6, which also reduced cellular toxicity. Our results highlight the important contribution of cell culture conditions forex vivogenome editing with AAV6 vectors and provide a blueprint for improving AAV6-mediated homology-directed editing of human T and B cells.
Metagenomic and Metatranscriptomic Analysis Reveals Enrichment for Xenobiotic-Degrading Bacterial Specialists and Xenobiotic-Degrading Genes in a Canadian Prairie Two-Cell Biobed System
Authors: Russell J. N. , Perry B. J. , Bergsveinson J. , Freeman C. N. , Sheedy C. , Nilsson D. , Braul L. , Yost C. K.
Published: May 2021

Biobeds are agriculture-based bioremediation tools used to safely contain and microbially degrade on-farm pesticide waste and rinsate, thereby reducing the negative environmental impacts associated with pesticide use. While these engineered ecosystems demonstrate efficient pesticide removal, their microbiome dynamics remain largely understudied both taxonomically and functionally. As such, further characterization of these parameters may aid in the optimization of biobed management and deployment. This study used metagenomic and metatranscriptomic techniques to characterize the microbial community in a two-cell Canadian biobed system before and after a field season of pesticide application. These culture-independent approaches identified an enrichment of xenobiotic-degrading bacteria, such asAfipia,SphingopyxisandPseudomonas, and enrichment and transcription of xenobiotic-degrading genes, such as peroxidases, oxygenases, and hydroxylases, among others; we were able to directly link the transcription of these genes toPseudomonas,Oligotropha,Mesorhizobium,Rhodopseudomonas, andStenotrophomonastaxa.
Structures of the active HER2/HER3 receptor complex reveal dynamics at the dimerization interface induced by binding of a single ligand
Authors: Diwanji Devan , Trenker Raphael , Thaker Tarjani M. , Wang Feng , Agard David A. , Verba Kliment A. , Jura Natalia
Published: May 2021

The Human Epidermal Growth Factor Receptor 2 (HER2) and HER3 form a potent pro-oncogenic heterocomplex upon binding of growth factor neuregulin-1β (NRG1β)1–3. The mechanism by which HER2 and HER3 interact remains unknown in the absence of any structures of the complex. We isolated the NRG1β-bound near full-length HER2/HER3 dimer and obtained a 2.9Å cryo-electron microscopy (cryo-EM) reconstruction of the extracellular domain module which reveals unexpected dynamics at the HER2/HER3 dimerization interface. We show that the dimerization arm of NRG1β-bound HER3 is unresolved likely because the apo HER2 monomer fails to undergo a ligand-induced conformational change needed to establish a HER3 dimerization arm binding pocket. In a second structure of an oncogenic extracellular domain mutant of HER2, S310F, we observe a compensatory interaction with the HER3 dimerization arm that stabilizes the dimerization interface. We show that both HER2/HER3 and HER2-S310F/HER3 retain the capacity to bind to the HER2-directed therapeutic antibody, trastuzumab, but the mutant complex does not bind to pertuzumab. Our 3.5Å structure of the HER2-S310F/HER3/NRG1β/trastuzumab Fragment antigen binding (Fab) complex shows that the receptor dimer undergoes a conformational change to accommodate trastuzumab. Thus, like oncogenic mutations, therapeutics exploit the intrinsic dynamics of the HER2/HER3 heterodimer. The unique features of a singly liganded HER2/HER3 heterodimer underscore the allosteric sensing of the ligand occupancy by the dimerization interface and explain why extracellular domains of HER2 do not homo-associate via canonical active dimer interface.
Increased alpha-helicity of a supercharged coiled-coil protein increases siRNA delivery efficiency of protein-lipid hybrid vehicle
Authors: Thomas Joseph , Monkovic Julia , Frezzo Joseph A. , Katyal Priya , Punia Kamia , Montclare Jin K.
Published: May 2021

Gene therapy has the potential to treat various diseases and has recently gained new interest due to the deployment nucleic acid based vaccines for COVID-19. Despite these developments, there still remains a need for further development of gene delivery vehicles to increase their safety and efficacy.. We have recently developed a lipoproteoplex (LPP) consisting of a super-charged coiled-coil protein (CSP) and a cationic liposomal carrier, that has the ability to condense nucleic acids and deliver themin vivo. The LPP is distinct from other liposomal gene delivery systems in that it utilizes a modular protein component to drive transfection activity as opposed to relying on the passive effects of the cationic lipids. A CSP library has been rationally designed to improve the efficacy of the LPP compared to the parent protein via improved alpha-helical structure and increased nucleic acid binding through the use of extended histidine tags and increased positive charge. The secondary structure and nucleic acid binding ability of each library member was assessed, then compared to functional transfection data in NIH-3T3 mouse fibroblasts. Structural and functional data suggests that increasing alpha-helicity of the protein component of the LPP compared to the parent sequence doubles nucleic acid binding affinity and increases transfection activity almost 3-fold with a favorable safety profile.
The method utilized to purify the SARS-CoV-2 N protein can affect its molecular properties
Authors: Tarczewska Aneta , Kolonko-Adamska Marta , Zarębski Mirosław , Dobrucki Jurek , Ożyhar Andrzej , Greb-Markiewicz Beata
Published: May 2021

One of the main structural proteins of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nucleocapsid protein (N). The basic function of this protein is to bind genomic RNA and to form a protective nucleocapsid in the mature virion. The intrinsic ability of the N protein to interact with nucleic acids makes its purification very challenging. Therefore, typically employed purification methods appear to be insufficient for removing nucleic acid contamination. In this study, we present a novel purification protocol that enables the N protein to be prepared without any bound nucleic acids. We also performed comparative structural analysis of the N protein contaminated with nucleic acids and free of contamination and showed significant differences in the structural and phase separation properties of the protein. These results indicate that nucleic-acid contamination may severely affect molecular properties of the purified N protein. In addition, the notable ability of the N protein to form condensates whose morphology and behaviour suggest more ordered forms resembling gel-like or solid structures is described.
River phosphorus cycling constrains lake cyanobacteria blooms
Authors: King Whitney M. , Curless Susan E. , Hood James M.
Published: May 2021

Bioavailable phosphorus exports from rivers during high flow often fuel downstream harmful cyanobacterial blooms; yet whether river phosphorus cycles affect these exports is unclear. Here, we examined river phosphorus cycling during high flow events in a large agricultural watershed that drives cyanobacterial blooms in Lake Erie. We show that between 2003 and 2019 river phosphorus cycles, through phosphorus sorption, reduced bioavailable phosphorus exports by 24%, potentially constraining Lake Erie cyanobacterial blooms by 61%. Over the last 45-years, phosphorus sorption has declined with suspended sediment exports due to increases in soil-erosion-minimizing agricultural practices, likely contributing to recent cyanobacterial blooms. In this, and likely other agricultural watersheds, rivers perform an unrecognized ecosystem service during high flow creating field-river-lake linkages that need to be incorporated into phosphorus management.
Generation of SIV resistant T cells and Macrophages from Nonhuman Primate Induced Pluripotent Stem Cells with Edited CCR5 locus
Authors: D’Souza Saritha S. , Kumar Akhilesh , Weinfurter Jason , Park Mi Ae , Maufort John , Tao Lihong , Kang HyunJun , Golos Thaddeus , Thomson James A , Reynolds Matthew , Slukvin Igor
Published: May 2021

Adoptive therapies with genetically modified somatic T cells rendered HIV resistant have shown promise for AIDS therapy. A renewable source of HIV resistant human T cells from induced pluripotent stem cells (iPSCs) would further facilitate and broaden the applicability of these therapies. Here, we report successful targeting of the CCR5 locus in iPSCs generated from peripheral blood T cells (T-iPSCs) or fibroblasts (fib-iPSCs) from Mauritian Cynomolgus macaques (MCM), using CRISPR/Cas9 technology. We found that CCR5 editing does not affect pluripotency or hematopoietic and T cell differentiation potentials of fib-iPSCs. However, deletion of CCR5 in T-iPSCs leads to selective loss of their T cell redifferentiation potential without affecting myeloid development. T cells and macrophages produced from CCR5-edited MCM- iPSCs did not support replication of the CCR5-tropic simian immunodeficiency viruses SIVmac239 (T-cell tropic) and SIVmac316 (macrophage-tropic). Overall, these studies provide a platform for further exploration of AIDS therapies based on gene-edited iPSCs in a nonhuman primate preclinical model.
Misregulation of MYB16 causes stomatal cluster formation by disrupting polarity in asymmetric cell division
Authors: Yang Shao-Li , Tran Ngan , Tsai Meng-Ying , Ho Chin-Min Kimmy
Published: May 2021

Stomata and leaf cuticle regulate water evaporation from the plant body and balance the trade-off between photosynthesis and water loss. We identified MYB16, a key transcription factor controlling cutin biosynthesis, from previous stomatal lineage ground cell (SLGC)-enriched transcriptome study. The preferential localization of MYB16 in SLGCs but not meristemoids suggests a link between cutin synthesis and stomatal development. Here, we showed that downregulation of MYB16 in meristemoids was directly mediated by the stomatal master transcription factor, SPEECHLESS (SPCH). The suppression of MYB16 before asymmetric division was crucial for stomatal patterning because overexpression or ectopic expression of MYB16 in meristemoids increased impermeability and elevated stomatal density and clusters. The aberrant pattern of stomata was due to reduced and disrupted establishment of polarity during asymmetric cell division. Manipulating polarity by growing seedlings on hard agar rescued stomatal clusters and polarity defects in MYB16 ectopic lines. By expressing a cutinase in MYB16 ectopic lines, stomatal clustering was reduced, which suggests that the ectopic accumulation of cuticle affects the polarity in asymmetrically dividing cells and causes clustered stomata. Taken together, inhibiting MYB16 expression by SPCH in early stomatal lineage is required to correctly place the polarity complex for proper stomatal patterning during leaf morphogenesis.
Predicting the effects of drug combinations using probabilistic matrix factorization
Authors: Nafshi Ron , Lezon Timothy R.
Published: May 2021

Drug development is costly and time-consuming, and developing novel practical strategies for creating more effective treatments is imperative. One possible solution is to prescribe drugs in combination. Synergistic drug combinations could allow lower doses of each constituent drug, reducing adverse reactions and drug resistance. However, it is not feasible to sufficiently test every combination of drugs for a given illness to determine promising synergistic combinations. Since there is a finite amount of time and resources available for finding synergistic combinations, a model that can identify synergistic combinations from a limited subset of all available combinations could accelerate development of therapeutics. By applying recommender algorithms, such as the low-rank matrix completion algorithm Probabilistic Matrix Factorization (PMF), it may be possible to identify synergistic combinations from partial information of the drug interactions. Here, we use PMF to predict the efficacy of two-drug combinations using the NCI ALMANAC, a robust collection of pairwise drug combinations of 104 FDA-approved anticancer drugs against 60 common cancer cell lines. We find that PMF is able predict drug combination efficacy with high accuracy from a limited set of combinations and is robust to changes in the individual training data. Moreover, we propose a new PMF-guided experimental design to detect all synergistic combinations without testing every combination.