bluuebunny/crossref_metadata_2025 huggingface dataset:
Validation of a Novel Molecular Assay to the Diagnostic of COVID-19 Based on Real Time PCR with High Resolution Melting
Authors: Ferreira Beatriz Iandra da Silva , Gomes Natália Lins da Silva , Coelho Wagner Luis da Costa Nunes Pimentel , Costa Vanessa Duarte da , Carneiro Vanessa Cristine de Souza , Kader Rafael Lopes , Amaro Marisa Pimentel , Villar Lívia Melo , Miyajima Fábio , Alves-Leon Soniza Vieira , de Paula Vanessa Salete , Leon Luciane Almeida Amado , Moreira Otacilio Cruz
Published: July 2021

With the emergence of the Covid-19 pandemic, the world faced an unprecedented need for RT-qPCR-based molecular diagnostic tests, leading to a lack of kits and inputs, especially in developing countries. Hence, the costs for commercial kits and inputs were overrated, stimulating the development of alternative methods to detect SARS-CoV-2 in clinical specimens. The availability of the complete SARS-CoV-2 genome at the beginning of the pandemic facilitated the development of specific primers and standardized laboratory protocols for Covid-19 molecular diagnostic. High-sensitive and cost-effective molecular biology technique based on the Melting Temperature differences between purine and pyrimidine bases can be used to the detection and genotyping of pathogens in clinical specimens. Here, a RT-qPCR assays with High Resolution Melting (HRM-RTqPCR) was developed for different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states in Brazil, in comparison to a commercial TaqMan RT-qPCR assay, as gold standard. The sensitivity of the HRM-RTqPCR assays targeting N, RdRp and E were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 targets, and diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, the HRM-RTqPCR emerges as an alternative and low-cost methodology to increase the molecular diagnostic of patients suspicious for Covid-19, especially in restricted-budget laboratories.
Comparison of kinetics of immune responses to SARS-CoV-2 proteins in individuals with varying severity of infection and following a single dose of the AZD1222
Authors: Jayathilaka Deshni , Jeewandara Chandima , Gomes Laksiri , Jayadas Tibutius Thanesh Pramanayagam , Kamaladasa Achala , Guruge Dinuka , Darshana Pradeep , Ranasinghe Thushali , Abyrathna Inoka Sepali , Danasekara Saubhagya , Gunathilaka Buddini , Kuruppu Heshan , Wijewickrama Ananda , Wijayamuni Ruwan , Schimanski Lisa , Tan T.K. , Ogg Graham S. , Townsend Alain , Malavige Gathsaurie Neelika
Published: July 2021

Background

While there have been many studies characterizing the IgG and IgA responses to different SARS-CoV-2 proteins in individuals with natural infection, the induction of IgG and IgA to different viral proteins in vaccinees have not been extensively studied. Therefore, we sought to investigate the antibody responses to SARS-CoV-2 following natural infection and following a single dose of AZD2221, in Sri Lankan individuals.

Methods

Using Luminex assays, we characterized the IgG and IgA responses in patients with varying severity of illness and following a single dose of the vaccine at 4 weeks and 12 weeks since onset of illness or following vaccination. Haemagglutination test (HAT) was used to assess the antibodies to the receptor binding domain of SARS-CoV-2 wild type (WT), B.1.1.7, B.1.351 and B.1.617.2 (VOCs) and surrogate neutralizing test to measure ACE2 receptor blocking antibodies.

Results

Those with mild illness and in vaccinees, the IgG responses to S1, S2, RBD and N protein increased from 4 weeks to 12 weeks, while it remained unchanged in those with moderate/severe illness. Those who had a febrile illness in 2017 and 2018 (controls) also gave IgG and IgA high responses to the S2 subunit. In the vaccinees, the most significant rise was seen for the IgG antibodies to the S2 subunit (p<0.0001). Vaccinees had several fold lower IgA antibodies to all the SARS-CoV-2 proteins tested than those with mild and moderate/severe illness at 4 weeks and 12 weeks. At 12 weeks the HAT titres were significantly lower to the B.1.1.7 in vaccinees and significantly lower in those with mild illness, and in vaccinees to B.1.351 and for B.1.617.2. No such difference was seen in those with moderate/severe illness.

Conclusions

Vaccinees had significantly less IgA to SARS-CoV-2, but comparable IgG responses to those with natural infection. However, following a single dose, vaccinees had reduced antibody levels to the variants of concern (VOC), which further declined with time, compared to natural infection.
The DiSCover Project: Protocol and Baseline Characteristics of a Decentralized Digital Study Assessing Chronic Pain Outcomes and Behavioral Data
Authors: Lee Jennifer L. , Cerrada Christian J. , Ying Vang Mai Ka , Scherer Kelly , Tai Caroline , Tran Jennifer L.A. , Juusola Jessie L. , Sang Christine N.
Published: July 2021

Background

Chronic pain affects approximately 50 million adults in the United States and impacts mood, everyday functioning, and quality of life. The challenges of analgesic clinical trials and, therefore, the approval of new non-opioid analgesics, are based in part on a fundamental lack of understanding of those outcomes that are relevant to an individual’s overall functioning.

Objectives

To determine the behaviors and health outcomes associated with chronic pain. This manuscript presents an overview of the study design, baseline health and behavioral characteristics of our sample, and preliminary findings of how behavioral characteristics differ between individuals with and without chronic pain.

Methods

The study is a decentralized digital longitudinal cohort study of 10,036 individuals (5,832 with chronic pain [CP] and 4,204 with no chronic pain [NCP]), age 18 years or older, living in the United States. The study period was one year. Data were collected from wearable activity trackers and health or fitness mobile applications to capture passively collected behavioral data including steps, sleep, and heart rate. Patient-reported outcomes on mood and pain, including the BPI-SF, PHQ-9, and GAD-7, were collected at various timepoints during the study.

Results

The data suggest greater levels of depression and anxiety, lower quality of life, less physical activity, more variable sleep, and higher resting heart rate are associated with CP.

Conclusions

The longitudinal data from the larger study will yield substantial contributions to the body of literature in chronic pain, particularly in delineating relational and causal factors relevant to the impact of chronic pain, and potential development of a digital biomarker to assess and monitor patients’ everyday experience with chronic pain.
A Poisson binomial based statistical testing framework for comprehensive comorbidity discovery across massive Electronic Health Record datasets
Authors: Lemmon Gordon , Wesolowski Sergiusz , Henrie Alex , Tristani-Firouzi Martin , Yandell Mark
Published: July 2021

Discovery of comorbidities (the concomitant occurrence of distinct medical conditions in the same patient) is a prerequisite for creating forecasting tools for downstream outcomes research. Current comorbidity discovery applications are designed for small datasets and use stratification to control for confounding variables such as age, sex, or ancestry. Stratification lowers false positive rates, but reduces power, as the size of the study cohort is decreased. Here, we describe a Poisson Binomial based approach to comorbidity discovery (PBC) designed for big-data applications that circumvents the need for stratification. PBC adjusts for confounding demographic variables on a per-patient basis, and models temporal relationships. We benchmark PBC using two datasets, the publicly available MIMIC-IV; and the entire Electronic Health Record (EHR) corpus of the University of Utah Hospital System, encompassing over 1.6 million patients, to compute comorbidity statistics on 4,623,841 pairs of potentially comorbid medical terms. The results of this computation are provided as a searchable web resource. Compared to current methods, the PBC approach reduces false positive associations, while retaining statistical power to discover true comorbidities.
Trans Cohorts Metabolomic Modulation Following Long-Term Successful Therapy in HIV-Infection
Authors: Mikaeloff Flora , Svensson-Akusjärvi Sara , Ikomey George Mondinde , Krishnan Shuba , Sperk Maike , Gupta Soham , Vega Magdaleno Gustavo Daniel , Escós Alejandra , Lyonga Emilia , Okomo Marie Claire , Tagne Claude Tayou , Babu Hemalatha , Lorson Christian L , Végvári Ákos , Banerjea Akhil C. , Kele Julianna , Hanna Luke Elizabeth , Singh Kamal , de Magalhães João Pedro , Benfeitas Rui , Neogi Ujjwal
Published: July 2021

Despite successful combination antiretroviral therapy (cART), persistent low-grade immune activation together with inflammation and toxic antiretroviral drugs can lead to long-lasting metabolic adaptation in people living with HIV (PLWH). The successful short-term cART reported abnormalities in the metabolic reprogramming in PLWH, but the long-term consequences are unknown. This study investigated alterations in the plasma metabolic profiles by comparing PLWH and matched HIV-negative controls (HC) from Cameroon and India. We used untargeted and targeted LC-MS/MS-based metabolic profiling in PLWH with long-term (>5years) successful therapy in a trans cohorts’ approach. Advanced statistical and bioinformatics analyses showed altered amino acid metabolism, more specifically to glutaminolysis in PLWH with therapy than HIV-negative controls that can lead to excitotoxicity in both the cohorts. A significantly lower level of neurosteroids was observed in both cohorts and could potentiate neurological impairments in PLWH. The modulation of cellular glutaminolysis promoted increased cell death and latency reversal in pre-monocytic HIV-1 latent cell model U1, which may be essential for the clearance of the inducible reservoir in HIV-integrated cells. Our patient-based metabolomics andin vitrostudy, therefore, highlight the importance of altered glutaminolysis in PLWH that can be linked accelerated neurocognitive aging and metabolic reprogramming in latently infected cells.
Predicting Infectivity: Comparing Four PCR-based Assays to Detect Culturable SARS-CoV-2 in Clinical Samples
Authors: Bruce Emily A. , Mills Margaret G. , Sampoleo Reigran , Perchetti Garrett A. , Huang Meei-Li , Despres Hannah W. , Shirley David J. , Jerome Keith R. , Greninger Alexander L. , Botten Jason W.
Published: July 2021

With the COVID-19 pandemic caused by SARS-CoV-2 now in its second year, there remains an urgent need for diagnostic testing that can identify infected individuals, particularly those who harbor infectious virus. Various RT-PCR strategies have been proposed to identify specific viral RNA species that may predict the presence of infectious virus, including detection of transcriptional intermediates (e.g. subgenomic RNA [sgRNA]) and replicative intermediates (e.g. negative-strand RNA species). Using a novel primer/probe set for detection of subgenomic (sg)E transcripts, we successfully identified 100% of specimens containing culturable SARS-CoV-2 from a set of 126 clinical samples (total sgE CT values ranging from 12.3-37.5). This assay showed superior performance compared to a previously published sgRNA assay and to a negative-strand RNA assay, both of which failed to detect target RNA in a subset of samples from which we isolated live virus. In addition, total levels of viral RNA (genome, negative-strand, and sgE) detected with the WHO/Charité primer-probe set correlated closely with levels of infectious virus. Specifically, infectious virus was not detected in samples with a CT above 31.0. Clinical samples with higher levels of viral RNA also displayed cytopathic effect (CPE) more quickly than those with lower levels of viral RNA. Finally, we found that the infectivity of SARS-CoV-2 samples is significantly dependent on the cell type used for viral isolation, as Vero E6 cells expressing TMRPSS2 extended the analytical sensitivity of isolation by more than 3 CT compared to parental Vero E6 cells and resulted in faster isolation. Our work shows that using a total viral RNA Ct cut-off of >31 or specifically testing for sgRNA can serve as an effective rule-out test for viral infectivity.
Cortical function and sensorimotor plasticity predict future low back pain after an acute episode: the UPWaRD prospective cohort study
Authors: Jenkins Luke C , Chang Wei-Ju , Buscemi Valentina , Liston Matthew , Humburg Peter , Nicholas Michael , Graven-Nielsen Thomas , Hodges Paul W , McAuley James H , Schabrun Siobhan M
Published: July 2021

Predicting the development of chronic low back pain (LBP) at the time of an acute episode remains challenging. The Understanding persistent Pain Where it ResiDes (UPWaRD) study aimed to identify neurobiological and psychological risk factors for chronic LBP. Individuals with acute LBP (N=120) participated in a prospective cohort study with six-month follow-up. Candidate predictors were selected from the neurobiological (e.g. sensorimotor cortical excitability assessed by sensory and motor evoked potentials, Brain Derived Neurotrophic Factor genotype), psychological (e.g. depression and anxiety), symptom-related (e.g. LBP history) and demographic domains. Analyses involved multivariable linear regression models with pain intensity or disability degree as continuous variables. Secondary analyses involved a multivariable logistic model with presence of low back pain at six months (thresholding pain intensity and disability degree) as a dichotomous variable. Lower sensory cortex and corticomotor excitability, higher baseline pain intensity, higher depression, stress and pain catastrophizing were the strongest predictors (R2=0.47) of pain intensity at six months. Older age and higher pain catastrophizing were the strongest predictors (R2=0.30) of disability at six months. When LBP outcome was dichotomised, sensory cortex and corticomotor excitability, BDNF genotype, depression and anxiety, LBP history and baseline pain intensity, accurately discriminated those who did and did not report LBP at six months (c-statistic 0.91). This study identifies novel risk factors for future LBP after an acute episode that can predict an individual’s pain intensity and level of disability at six-month follow-up, and accurately discriminate between those who will and will not report LBP at six months.
Potential applications of the rapid COVID-19 antibody test kit screening in comparison to the RT-PCR in patients and personnel at the Department of Obstetrics and Gynecology
Authors: Narkwichean Amarin , Jomoui Wittaya , Boonpradit Rarinthip , Laosooksathit Wipada , Nopsopon Tanawin , Pongpirul Krit
Published: July 2021

Objective

To explore potential applications of the rapid antibody test for COVID-19 screening, in comparison to RT-PCR, for emergency obstetric and gynecological procedures, and medical personnel in the Department of Obstetrics and Gynecology.

Methods

A cross-sectional study was conducted in expected 290 participants: 230 patients and 60 medical staff, during the four-month national COVID-19 outbreak period (Aug – Sep 2020, and Dec 2020 - Jan 2021). All participants underwent both rapid antibody tests and RT-PCR (at admission for patients).

Results

A total of 270 participants completed the study. Fever and URI symptoms were present in 6/210 patients (2.8%) while one patient (0.5%) had a history of traveling to a high-risk area. However, only two (1%) asymptomatic patients had positive IgM results. Concerning the medical personnel, 10% fell into the ‘patient under investigation (PUI)’ category. 4/60 (6.7%) IgM positive was observed in the staff cohort in which 3/4 came from non-PUI participants. Neither participant had RT-PCR positive demonstrating a 1.9% total false positive rate.

Conclusion

Rapid point-of-care antibody test can be used to screen either a pregnant coming for delivery, a patient who requires urgent/emergency operative procedures, or medical personnel, at least in the defined lower-prevalence COVID-19 situation.

Trial registration

This study was approved by the Institutional Review Board of Srinakharinwirot University (IRB SWUEC119/2563F) and was registered at the Thai Clinical Trials Registry (TCTR20210613001).
Modelling infectious disease transmission potential as a function of human behaviour
Authors: Walters Caroline E. , Dunbar Maria Bekker-Nielsen , Weston Dale , Hall Ian M.
Published: July 2021

Mathematical modelling is an important public health tool for aiding understanding the spread of respiratory infectious diseases, such as influenza or COVID-19, and for quantifying the effects of behavioural interventions. However, such models rarely explicitly appeals to theories of human behaviour to justify model assumptions. Here we propose a novel mathematical model of disease transmission via fomites (luggage trays) at airport security screening during an outbreak. Our model incorporates the self-protective behaviour of using hand sanitiser gel in line with the extended parallel processing model (EPPM) of behaviour.

We find that changing model assumptions of human behaviour in line with the EPPM gives qualitatively different results on the optimal placement of hand sanitiser gels within an airport compared to the model with naive behavioural assumptions. Specifically, that it is preferable to place hand sanitiser gels after luggage screening in most scenarios, however in situations where individuals perceive high threat and low efficacy this strategy may need to be reviewed. This model demonstrates how existing behavioural theories can be incorporated into mathematical models of infectious disease.
SARS-CoV-2 seroprevalence in Chattogram, Bangladesh before the Delta surge, March-June 2021
Authors: Bhuiyan Taufiqur Rahman , Dent Hulse Juan , Hegde Sonia T. , Akhtar Marjahan , Islam Md. Taufiqul , Hasan Khan Zahid , Khan Ishtiakul Islam , Ahmed Shakeel , Rashid Md Mamunur , Rashid Rumana , Gurley Emily S. , Shirin Tahmina , Khan Ashraful Islam , Azman Andrew S. , Qadri Firdausi
Published: July 2021

In a representative serosurvey conducted March–June 2021, 64.1% (95%CrI 60.0– 68.1%) of Sitakunda subdistrict (Bangladesh) had anti-SARS-CoV-2 IgG antibodies after adjusting for age, sex, household clustering and test performance. Before the surge of Delta, most of the population had been infected despite low incidence of virologically-confirmed COVID-19.
Tobacco and Nicotine Use in the United States Army Trainee Soldier’s Environment
Authors: Lang Adam Edward , Yakhkind Aleksandra , Prom-Wormley Elizabeth
Published: July 2021

Tobacco and nicotine use rates within the United States military population are high, and rates among young trainee soldiers are not improving over time. Trainee soldiers are heavily influenced by role models in their environment, and nicotine product use rates in this environment has not previously been evaluated. This study found that over 30% of non-trainee U.S. Army soldiers, known as cadre, within the training environment use nicotine products. For the young trainee soldier, this creates an initial Army experience that continues to foster a culture of tobacco and nicotine use. We urge changes in Army tobacco and nicotine policy to protect the vulnerable trainee soldier population.
Correlations Between Community-Level HIV Preexposure Prophylaxis Coverage and Individual-Level Sexual Behaviors among US Men Who Have Sex with Men
Authors: Mann Laura M. , Guillou Adrien Le , Goodreau Steven M. , Marcus Julia L. , Sanchez Travis , Weiss Kevin M. , Jenness Samuel M.
Published: July 2021

Background

HIV preexposure prophylaxis (PrEP) has been associated with changes in sexual behavior after PrEP initiation. However, behavioral differences may also emerge among PrEP non-users in communities with high PrEP coverage.

Methods

We used demographic, behavioral, and sexual network data from ARTnet, a cross-sectional study of US men who have sex with men conducted during 2017–2019. Multivariable regression models with a Bayesian modeling framework in which individuals were nested within their residential geographic areas were used to estimate associations between area-level PrEP coverage and five sexual behavior outcomes (number of total, main, and casual male partners [network degree]; count of one-time partnerships; and consistent condom use in one-time partnerships), controlling for individual PrEP use.

Results

PrEP coverage ranged from 10.3% (Philadelphia) to 38.9% (San Francisco). Total degree was highest in Miami (1.35) and lowest in Denver (0.78), while the count of one-time partners was highest in San Francisco (11.7/year) and lowest in Detroit (1.5/year). Adjusting for individual PrEP use and demographics, community PrEP coverage was associated with higher total degree (adjusted incidence rate ratio [aIRR]=1.73; 95% CrI, 0.92–3.44), casual degree (aIRR=2.05; 95% CrI, 0.90–5.07), and count of one-time partnerships (aIRR=1.90; 95% CrI, 0.46–8.54). Without adjustment for individual PrEP use, these associations were amplified. There were weaker associations with main degree (aIRR=1.21; 95% CrI, 0.48–3.20) and consistent condom use in one-time partnerships (aIRR=1.68; 95% CrI, 0.86–3.35).

Conclusions

Most of the associations between community PrEP coverage and sexual behavior were explained by individual PrEP use. However, there were residual associations after controlling for individual PrEP use, suggesting that PrEP coverage may partially drive community-level changes in sexual behavior.
Structure-guided bifunctional molecules hit a DEUBAD-lacking hRpn13 species upregulated in multiple myeloma
Authors: Lu Xiuxiu , Sabbasani Venkata R. , Osei-Amponsa Vasty , Evans Christine N. , King Julianna C. , Tarasov Sergey G. , Dyba Marzena , Chan King C. , Schwieters Charles D. , Choudhari Sulbha , Fromont Caroline , Zhao Yongmei , Tran Bao , Chen Xiang , Matsuo Hiroshi , Andresson Thorkell , Chari Raj , Swenson Rolf E. , Tarasova Nadya I. , Walters Kylie J.
Published: July 2021

Proteasome substrate receptor hRpn13 is a promising anti-cancer target. By integrated in silico and biophysical screening, we identified a chemical scaffold that binds hRpn13 with non-covalent interactions that mimic the proteasome and a weak electrophile for Michael addition. hRpn13 Pru domain binds proteasomes and ubiquitin whereas its DEUBAD domain binds deubiquitinating enzyme UCHL5. NMR revealed lead compound XL5 to interdigitate into a hydrophobic pocket created by lateral movement of a Pru β-hairpin with an exposed end for Proteolysis Targeting Chimeras (PROTACs). Implementing XL5-PROTACs as chemical probes identified a DEUBAD-lacking hRpn13 species (hRpn13Pru) present naturally with cell type-dependent abundance. XL5-PROTACs preferentially target hRpn13Pru, causing its ubiquitination. Gene-editing and rescue experiments established hRpn13 requirement for XL5-PROTAC-triggered apoptosis and increased p62 levels. These data establish hRpn13 as an anti-cancer target for multiple myeloma and introduce an hRpn13-targeting scaffold that can be optimized for preclinical trials against hRpn13Pru-producing cancer types.
Novel Hendra virus variant detected by sentinel surveillance of Australian horses
Authors: Annand Edward J. , Horsburgh Bethany A. , Xu Kai , Reid Peter A. , Poole Ben , de Kantzow Maximillian C. , Brown Nicole , Tweedie Alison , Michie Michelle , Grewar John D. , Jackson Anne E. , Singanallur Nagendrakumar B. , Plain Karren M. , Kim Karan , Tachedjian Mary , van der Heide Brenda , Crameri Sandra , Williams David T. , Secombe Cristy , Laing Eric D. , Sterling Spencer , Yan Lianying , Jackson Louise , Jones Cheryl , Plowright Raina K. , Peel Alison J. , Breed Andrew C. , Diallo Ibrahim , Dhand Navneet K. , Britton Philip N. , Broder Christopher C. , Smith Ina , Eden John-Sebastian
Published: July 2021

A novel Hendra virus (HeV) variant, not detected by routine testing, was identified and isolated from a Queensland horse that suffered acute, fatal disease consistent with HeV infection. Whole genome sequencing and phylogenetic analysis demonstrated the variant to have ~83% nucleotide identity to the prototype HeV strain. An updated RT-qPCR assay was designed for routine HeV surveillance. In silico and in vitro comparison of the receptor-binding protein with prototypic HeV showed that the human monoclonal antibody m102.4 used for post-exposure prophylaxis, as well as the current equine vaccine, should be effective against this variant. Genetic similarity of this virus to sequences detected from grey-headed flying-foxes suggests the variant circulates at-least in this species. Studies determining infection kinetics, pathogenicity, reservoir-species associations, viral–host co-evolution and spillover dynamics for this virus are urgently needed. Surveillance and biosecurity practices should be updated to appreciate HeV spillover risk across all regions frequented by flying foxes.
Interpreting Polygenic Score Effects in Sibling Analysis
Authors: Fletcher Jason , Wu Yuchang , Li Tianchang , Lu Qiongshi
Published: July 2021

Researchers often claim that sibling analysis can be used to separate causal genetic effects from the assortment of biases that contaminate most downstream genetic studies. Indeed, typical results from sibling models show large (>50%) attenuations in the associations between polygenic scores and phenotypes compared to non-sibling models, consistent with researchers’ expectations about bias reduction. This paper explores these expectations by using family (quad) data and simulations that include indirect genetic effect processes and evaluates the ability of sibling models to uncover direct genetic effects. We find that sibling models, in general, fail to uncover direct genetic effects; indeed, these models have both upward and downward biases that are difficult to sign in typical data. When genetic nurture effects exist, sibling models create “measurement error” that attenuate associations between polygenic scores and phenotypes. As the correlation between direct and indirect effect changes, this bias can increase or decrease. Our findings suggest that interpreting results from sibling analysis aimed at uncovering direct genetic effects should be treated with caution.
A quantitative approach to easily characterize and predict cell death and escape to β-lactam treatments
Authors: Andreani Virgile , Gross Viktoriia , You Lingchong , Glaser Philippe , El Meouche Imane , Batt Gregory
Published: July 2021

Commensal and pathogenicE. colistrains are increasingly found to be resistant to β-lactams, one of the most widely prescribed classes of antibiotics. Understanding escape to such treatments is complex since β-lactams have several cellular targets and since several mechanisms might be involved in treatment escape in a combined manner. Surprisingly, the accumulated knowledge has not yet proven effective enough to predict the bacterial response to antibiotic treatments at both cellular and population levels with quantitative accuracy for -producing bacteria. Here, we propose a mathematical model that captures in a comprehensive way key phenomena happening at the molecular, cellular, and population levels, as well as their interactions. Our growth-fragmentation model gives a central role to cellular heterogeneity and filamentation as a way for cells to gain time until the degradation of the antibiotic by the β-lactamases released by the dead cell population. Importantly, the model can account for the observed temporal evolution of the total (live and dead) biomass and of the live cell numbers for various antibiotic concentrations. To our knowledge, this is the first model able to quantitatively reconciliate these two classical views on cell death (OD and CFUs) for clinical isolates expressing extended-spectrum beta-lactamases (ESBL). Moreover, our model has a strong predictive power. When calibrated using a slight extension of OD-based data that we propose here, it can predict the CFU profiles in initial and delayed treatments despite inoculum effects, and suggest non-trivial optimal treatments. Generating quality data in quantity has been essential for model development and validation on non-model E. coli strains. We developed protocols to increase the reproducibility of growth kinetics assays and to increase the throughput of CFU assays.
Pathogenic potential in catheter-associated Escherichia coli is associated with separable biofilm and virulence gene determinants
Authors: Zou Zongsen , Potter Robert F. , McCoy William H. , Katumba George L. , Mucha Peter J. , Dantas Gautam , Henderson Jeffrey P.
Published: July 2021

Urinary catheterization facilitates Escherichia coli colonization of the urinary tract and increases infection risk. While specific pathotypes are well-recognized for some E. coli infections, it is unclear whether strain-specific characteristics among E. coli are associated with infection risk in catheterized patients. Here we used comparative genomics and a simulated catheter biofilm model to compare strains associated with catheter-associated urinary tract infection (CAUTI) and catheter-associated asymptomatic bacteriuria (CAASB). CAUTI was associated with a phylotype B2 sub-clade dominated by the multidrug resistant ST131 lineage, while CAASB isolates were genetically more diverse. Catheter-associated biofilm formation was widespread but quantitatively variable among isolates. Network community analysis resolved distinct groups of genes associated with infection or biofilm formation, with iron acquisition-associated genes prominent throughout. Using a reporter construct and targeted mutagenesis, we detected a biofilm phenotype for the ferric citrate transport (Fec) system, the most prominent correlate of high catheter biofilm formation in these patients. In mixed cultures, catheter biofilms formed by some CAASB strains suppressed catheter colonization by ST131 CAUTI isolates. These results are consistent with a paradigm in which catheter biofilm-associated genes increase infection risk in strains with a high pathogenic potential and decrease infection risk through niche exclusion in strains with low pathogenic potential.
The M2 Gene Is a Determinant of Reovirus-Induced Myocarditis
Authors: Zita Marcelle Dina , Phillips Matthew B. , Stuart Johnasha D. , Kumarapeli Asangi R. , Snyder Anthony J. , Paredes Amairani , Sridharan Vijayalakshmi , Boerma Marjan , Danthi Pranav , Boehme Karl W.
Published: July 2021

Although a broad range of viruses cause myocarditis, the mechanisms that underlie viral myocarditis are poorly understood. Here, we report that the M2 gene is a determinant of reovirus myocarditis. The M2 gene encodes outer capsid protein μ1, which mediates host membrane penetration during reovirus entry. We infected newborn C57BL/6 mice with reovirus strain type 1 Lang (T1L) or a reassortant reovirus in which the M2 gene from strain type 3 Dearing (T3D) was substituted into the T1L genetic background (T1L/T3DM2). T1L was non-lethal in wild-type mice, whereas greater than 90% of mice succumbed to T1L/T3DM2 infection. T1L/T3DM2 produced higher viral loads than T1L at the site of inoculation. In secondary organs, T1L/T3DM2 was detected with more rapid kinetics and reached higher peak titers than T1L. We found that hearts from T1L/T3DM2-infected mice were grossly abnormal, with large lesions indicative of substantial inflammatory infiltrate. Lesions in T1L/T3DM2-infected mice contained necrotic cardiomyocytes with pyknotic debris, and extensive lymphocyte and histiocyte infiltration. In contrast, T1L induced the formation of small purulent lesions in a small subset of animals, consistent with T1L being mildly myocarditic. Finally, more activated caspase-3-positive cells were observed in hearts from animals infected with T1L/T3DM2 compared to T1L. Together, our findings indicate that substitution of the T3D M2 allele into an otherwise T1L genetic background is sufficient to change a non-lethal infection into a lethal infection. Our results further indicate that T3D M2 enhances T1L replication and dissemination in vivo, which potentiates the capacity of reovirus to cause myocarditis.

IMPORTANCE

Reovirus is a non-enveloped virus with a segmented double-stranded RNA genome that serves as a model for studying viral myocarditis. The mechanisms by which reovirus drives myocarditis development are not fully elucidated. We found that substituting the M2 gene from strain type 3 Dearing (T3D) into an otherwise type 1 Lang (T1L) genetic background (T1L/T3DM2) was sufficient to convert the non-lethal T1L strain into a lethal infection in neonatal C57BL/6 mice. T1L/T3DM2 disseminated more efficiently and reached higher maximum titers than T1L in all organs tested, including the heart. T1L is mildly myocarditic and induced small areas of cardiac inflammation in a subset of mice. In contrast, hearts from mice infected with T1L/T3DM2 contained extensive cardiac inflammatory infiltration and more activated caspase-3-positive cells, which is indicative of apoptosis. Together, our findings identify the reovirus M2 gene as a new determinant of reovirus-induced myocarditis.
Increased microbial diversity and decreased prevalence of common pathogens in the gut microbiomes of wild turkeys compared to domestic turkeys
Authors: Craft Julia , Edington Hyrum , Christman Nicholas D. , Chaston John , Erickson David , Wilson Eric
Published: July 2021

Turkeys (Meleagris gallopavo) provide a globally important source of protein and constitute the second most important source of poultry meat in the world. Bacterial diseases are common in commercial poultry production causing significant production losses for farmers. Due to the increasingly recognized problems associated with large-scale/indiscriminant antibiotic use in agricultural settings, poultry producers need alternative methods to control common bacterial pathogens. In this study we compared the cecal microbiota of wild and domestic turkeys, hypothesizing that environmental pressures faced by wild birds may select for a disease-resistant microbial community. Sequence analysis of 16S rRNA genes amplified from cecal samples indicate that free-roaming wild turkeys carry a rich and variable microbiota compared to domestic turkeys raised on large-scale poultry farms. Wild turkeys also had very low levels of Staphylococcus, Salmonella and E. coli when compared to domestic turkeys. E. coli strains isolated from wild or domestic turkey cecal samples also belong to distinct phylogenetic backgrounds and differ in their propensity to carry virulence genes. E. coli strains isolated from factory-raised turkeys were far more likely to carry genes for capsule (kpsII, kpsIII) or siderophore (iroN, fyuA) synthesis than those isolated from wild turkeys. These results suggest that the microbiota of wild turkeys may provide colonization resistance against common poultry pathogens.

Importance

Due to the increasingly recognized problems associated with antibiotic use in agricultural settings, poultry producers need alternative methods to control common bacterial pathogens. In this study we compare the microbiota of wild and domestic turkeys. Results suggest that free ranging wild turkeys carry a distinct microbiome when compared to farm raised turkeys. The microbiome of wild birds contains very low levels of poultry pathogens compared to farm raised birds. The microbiomes of wild turkeys may be used to guide development of new ways to control disease in large scale poultry production.
A vast evolutionarily transient translatome contributes to phenotype and fitness
Authors: Wacholder Aaron , Parikh Saurin Bipin , Coelho Nelson Castilho , Acar Omer , Houghton Carly , Chou Lin , Carvunis Anne-Ruxandra
Published: July 2021

Translation is the process by which ribosomes synthesize proteins. Ribosome profiling recently revealed that many short sequences previously thought to be noncoding are pervasively translated. To identify protein-coding genes in this noncanonical translatome, we combine an integrative framework for extremely sensitive ribosome profiling analysis, iRibo, with high-powered selection inferences tailored for short sequences. We construct a reference translatome forSaccharomyces cerevisiaecomprising 5,400 canonical and almost 19,000 noncanonical translated elements. Only 14 noncanonical elements were evolving under detectable purifying selection. Surprisingly, a representative subset of translated elements lacking signatures of selection demonstrated involvement in processes including DNA repair, stress response and post-transcriptional regulation. Our results suggest that most translated elements are not conserved protein-coding genes and contribute to genotype-phenotype relationships through fast-evolving molecular mechanisms.
DNA sliding and loop formation by E. coli SMC complex: MukBEF
Authors: Zhou Man
Published: July 2021

SMC (structural maintenance of chromosomes) complexes share conserved architectures and function in chromosome maintenance via an unknown mechanism. Here we have used single-molecule techniques to study MukBEF, the SMC complex in Escherichia coli. Real-time movies show MukB alone can compact DNA and ATP inhibits DNA compaction by MukB. We observed that DNA unidirectionally slides through MukB, potentially by a ratchet mechanism, and the sliding speed depends on the elastic energy stored in the DNA. MukE, MukF and ATP binding stabilize MukB and DNA interaction, and ATP hydrolysis regulates the loading/unloading of MukBEF from DNA. Our data suggests a new model for how MukBEF organizes the bacterial chromosome in vivo; and this model will be relevant for other SMC proteins.
Understanding transfer of learning from an online self-directed learning and clinical decision support resource (BMJ Best Practice) for health professions education: a capability approach perspective
Authors: Zhang Da , Xiao Li , Duan Jingqi , Chang Xinxin , Walsh Kieran , Sandars John , Brown Jeremy , Dang Xiaorong , Shen Wei , Du Junjie , Cao Yanjie
Published: July 2021

Objective

To understand transfer of learning of an online self-directed learning and clinical decision support resource for health professionals (BMJ Best Practice) informed by Sen’s capability approach.

Design

A semi-structured questionnaire was used to identify the extent to which BMJ Best Practice enabled participants to achieve their valued outcomes and the factors that enabled and constrained their achievement.

Setting

This study was carried out in a single centre, which is a tertiary care medical institution containing around 1500 inpatient beds.

Participants

200 physicians at Air Force Medical Center were invited to take part in this study. 184 physicians completed the online questionnaire.

Results

78 percent of physicians felt that BMJ Best Practice enabled them to achieve their valued outcomes and to apply their new knowledge to inform their practice. The main factors that constrained their achievement were technological.

Conclusion

Sen’s capability approach offers an innovative and useful model to further understand health professions education since it highlights the importance of the learner perspective of valued outcomes, including the difficulties associated with the effective transfer of learning in CPD.

Strengths and limitations of this study

This is a real-life study based on the experiences of physicians who are directly providing care to patients.

The study has strong theoretical foundations – being based as it is on Sen’s capability approach.

The study covers the vital subject of the application of medical knowledge in actual practice.

The research design is a cross-sectional survey; the study did not investigate whether physicians’ reflections on their valued outcomes and achievements might have changed over time.

This study was carried out in a single centre.
Defining the Sensitivity Landscape of 74,389 EGFR Variants to Tyrosine Kinase Inhibitors
Authors: An Lei , Chen Shuqing , Wu Guangyao , Liu Chang , Wang Zhenxing , Wang Chunli , Shi Zeyuan , Niu Chenguang , Li Xiaodong , Tang Wenxue , Xu Hongen , Wang Yueqiang
Published: July 2021

Background

Tyrosine kinase inhibitors (TKIs) therapy is a standard treatment for patients with advanced non-small-cell lung carcinoma (NSCLC) when activating epidermal growth factor receptor (EGFR) mutations are detected. However, except for the well-studiedEGFRmutations, mostEGFRmutations lack treatment regimens.

Methods

We constructed twoEGFRvariant libraries containing substitutions, deletions, or insertions using the saturation mutagenesis method. All the variants were located in theEGFRmutation hotspot (exons 18–21). The sensitivity of these variants to afatinib, erlotinib, gefitinib, icotinib, and osimertinib was systematically studied by determining their enrichment in massively parallel cytotoxicity assays using an endogenous EGFR-depleted cell line, PC9.

Results

A total of 3,914 and 70,475 variants were detected in the constructedEGFRSubstitution-Deletion (Sub-Del) and exon 20 Insertion (Ins) libraries, accounting for 99.3% and 55.8% of the designed variants, respectively. Of the 3,914 Sub-Del variants, 813 were highly enriched in the reversible TKI (erlotinib, gefitinib, icotinib) cytotoxicity assays and 51 were enriched in the irreversible TKI (afatinib, osimertinib) cytotoxicity assays. For the 70,475 Ins variants, insertions at amino acid positions 770–774 were highly enriched in all the five TKI cytotoxicity assays. Moreover, the top 5% of the enriched insertion variants included a glycine or serine insertion at high frequency.

Conclusions

We present a comprehensive reference for the sensitivity ofEGFRvariants to five commonly used TKIs. The approach used here should be applicable to other genes and targeted drugs.
From the Modern Synthesis to the Molecular Synthesis: updating how we teach and assess evolution by natural selection
Authors: Sievers Matt , Reemts Connor , Dickinson Katherine J. , Mukerji Joya , Beltran Ismael Barreras , Theobald Elli J. , Velasco Vicente , Freeman Scott
Published: July 2021

Evolution by natural selection is recognized as both the most important concept in undergraduate biology and the most difficult to teach. Unfortunately, teaching and assessment of evolution have been impaired by legacy approaches that focus on Darwin’s original insights and the Modern Synthesis’ integration of Mendelian genetics, but ignore or downplay advances from what we term the Molecular Synthesis. To create better alignment between instructional approaches and contemporary research in the biosciences, we propose that the primary learning goal in teaching evolution should be for students to connect genotypes, phenotypes, and fitness. To support this approach, we developed and tested assessment questions and scoring rubrics called the Extended Assessing Conceptual Reasoning of Natural Selection (E-ACORNS) instrument. Initial E-ACORNS data suggest that after traditional instruction, few students recognize the molecular synthesis—prompting us to propose that introductory course sequences be re-organized with the molecular synthesis as their central theme.
ENKD1 is a centrosomal and ciliary microtubule-associated protein important for primary cilium assembly and Hedgehog signaling
Authors: Tiryaki Fatmanur , Deretic Jovana , Firat-Karalar Elif Nur
Published: July 2021

Centrioles and cilia are conserved, microtubule-based structures critical for cell function and development. Their structural and functional defects cause cancer and developmental disorders. How microtubules are organized into ordered structures by microtubule-associated proteins (MAPs) and tubulin modifications is best understood during mitosis but is largely unexplored for the centrioles and the ciliary axoneme, which are composed of remarkably stable microtubules that maintain their length at steady state. In particular, we know little about the identity of the centriolar and ciliary MAPs and how they work together during the assembly and maintenance of the cilium and centriole. Here, we identified Enkurin domain containing 1 (ENKD1) as a component of the centriole wall and the axoneme in mammalian cells, and showed that it has extensive proximity interactions with these compartments and MAPs. Using in vitro and cellular assays, we found that ENKD1 is a new MAP that promotes microtubule polymerization and regulates microtubule organization and stability. Consistently, overexpression of ENKD1 increased tubulin polymerization and acetylation and disrupted microtubule organization. Cells depleted for ENKD1 were defective in ciliary length and content regulation and failed to respond to Hedgehog pathway activation. Together, our results establish ENKD1 as a new centriolar and ciliary MAP that regulate primary cilium structure and function, and advances our understanding of the functional and regulatory relationship between MAPs and the primary cilium.
Structural insight into the stabilization of microtubules by taxanes
Authors: Prota Andrea E. , Lucena-Agell Daniel , Ma Yuntao , Estévez-Gallego Juan , Li Shuo , Bargsten Katja , Josa-Prado Fernando , Altmann Karl-Heinz , Gaillard Natacha , Kamimura Shinji , Mühlethaler Tobias , Gago Federico , Oliva María A. , Steinmetz Michel O. , Fang Wei-Shuo , Díaz J. Fernando
Published: July 2021

Paclitaxel (Taxol®) is a taxane and a first-line chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the current lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect the drug’s mechanism of action. Here, we solved the crystal structure of the core baccatin III moiety of paclitaxel lacking the C13 side chain in complex with tubulin at 1.9 Å resolution. Based on this information, we engineered two tailor-made taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects along with those of paclitaxel, docetaxel, and baccatin III on the microtubule lattice by X-ray fiber diffraction. We then compared high-resolution structures of ligand-bound tubulin and microtubule complexes with apo forms and used molecular dynamics simulations to understand the consequences of taxane binding to tubulin as well as to simplified protofilament and microtubule-lattice models. Our combined approach sheds light on three mechanistic questions. Firstly, taxanes bind better to microtubules as compared to unassembled tubulin due to a dual structural mechanism: Tubulin assembly is linked to a conformational reorganization of the βM loop, which otherwise occludes ligand access to the taxane site, while the bulky C13 side chains preferentially recognize the microtubule-assembled over the unassembled conformational state of tubulin. Second, the occupancy of the taxane site by a ligand has no influence on the straightness of tubulin protofilaments. Finally, binding of the taxane core to the taxane site displaces the S9-S10 loop of β-tubulin resulting in microtubule expansion. Our results provide detailed new insights into the mechanism of microtubule-stabilization by taxanes.