bluuebunny/crossref_metadata_2025 huggingface dataset:
THE USE OF DUPLICATION-GENERATING REARRANGEMENTS FOR STUDYING HETEROKARYON INCOMPATIBILITY GENES IN NEUROSPORA
Authors: Perkins David D
Published: January 2021

Heterokaryon (vegetative) incompatibility, governing the fusion of somatic hyphal filaments to form stable heterokaryons, is of interest because of its widespread occurrence in fungi and its bearing on cellular recognition. Conventional investigations of the genetic basis of heterokaryon incompatibility in N. crassa are difficult because in commonly used stocks differences are present at several het loci, all with similar incompatibility phenotypes. This difficulty is overcome by using duplications (partial diploids) that are unlikely to contain more than one het locus. A phenotypically expressed incompatibility reaction occurs when unlike het alleles are present within the same somatic nucleus, and this parallels the heterokaryon incompatibility reaction that occurs when unlike alleles in different haploid nuclei are introduced into the same somatic hypha by mycelial fusion.—Nontandem duplications were used to confirm that the incompatibility reactions in heterokaryons and in duplications are alternate expressions of the same genes. This was demonstrated for three loci which had previously been established by conventional heterokaryon tests—het-e, het-c and mt. These were each obtained in duplications as recombinant meiotic segregants from crosses heterozygous for duplication-generating chromosome rearrangements. The particular method of producing the duplications is irrelevant so long as the incompatibility alleles are heterozygous.—The duplication technique has made it possible to determine easily the het-e and het-c genotypes of numerous laboratory and wild strains of unknown constitution. In laboratory strains both loci are represented simply by two alleles. Analysis of het-c is more complicated in some wild strains, where differences have been demonstrated at one or more additional het loci within the duplication used and multiple allelism is also possible.—The results show that the duplication method can be used to identify and map additional vegetative incompatibility loci, without the necessity of heterokaryon tests.
SELECTION FOR A LARGE GENETIC DUPLICATION IN SALMONELLA TYPHIMURIUM
Authors: Straus Daniel S , Hoffmann George R
Published: January 2021

Salmonella typhimurium strains containing a duplication of nearly a third of the genome have been isolated by a simple procedure involving selection for improved utilization of L-malate as sole carbon source. The duplication occurs at a very high spontaneous frequency. Strains containing the duplication can be isolated selectively on malate medium, or by a non-selective procedure involving Hfr conjugation. When strains containing the duplication are maintained on non-selective medium, the duplication is readily lost. Genetic evidence suggests that the duplication is chromosomal and tandem. The fact that the recA gene is included in the duplication has been used to obtain evidence that the recA1 marker is recessive to its wild-type allele. Unlike tandem duplications previously described in E. coli, the duplication described in this report appears to have unique endpoints
SERUM ANTIGENS OF CATTLE. IV. IMMUNOGENETICS OF A SOLUBLE ANTIGENIC DETERMINANT DERIVED FROM LYSED ERYTHROCYTES
Authors: Rapacz Jan , Korda Nancy , Stone W H
Published: January 2021

This paper describes an allotypic system in cattle called Ec (Erythrocyte cattle). The antigenic determinant is derived from red cells and is detected in lysates by gel precipitation with alloimmune serum. The Ecl specificity is controlled by an autosomal dominant gene and occurs with variable frequencies in different cattle breeds. The Ec system is a very useful genetic marker because the homozygous and heterozygous genotypes show a dosage effect and can be distinguished by the size of the precipitin rings in double diffusion gel precipitation tests.
ESTERASE GENE FREQUENCY DIFFERENCES AND LINKAGE EQUILIBRIUM IN DROSOPHILA VIRILIS POPULATIONS FROM DIFFERENT ECOLOGICAL HABITATS
Authors: Tsuno Kendo
Published: January 2021

In order to study the gene frequency and linkage equilibrium in natural populations, about 5740 chromosomes of Drosophila virilis flies from eight natural populations were analyzed by the null strain method for two linked esterase loci. The frequencies of these esterase genes appear to depend on habitat characteristics rather than on geographical distribution. The populations kept a high degree of linkage equilibrium among them (D =.005).
POLARITY IN SEGMENTS OF THE ESCHERICHIA COLI trp OPERON WITH DELETED INTRAOPERONIC TRANSLATIONAL INITIATION SIGNALS
Authors: Kano Yasunobu , Imamoto Fumio
Published: January 2021

The effect of deletion of the operator-distal genes of the trp operon, including the trpE-trpD intercistronic punctuation point, on the degree of transcriptional polarity (in this case the effect of a nonsense mutation on the level of mRNA from the distal part of the very gene where the mutation is located) was investigated. Double mutants which contain a nonsense mutation and a deletion in trpE were constructed, and the degree of transcriptional polarity was estimated by the decrease in messenger RNA for the operator-distal trpE beyond the nonsense mutation, as well as by the production of truncated messenger RNA for the region of trpE proximal to the nonsense mutation. The content of mRNA of operator-distal trpE and the size of the mRNA of operator-proximal trpE of the double mutants show that transcriptional polarity is not relaxed as a function of distance of the nonsense mutation from the operator-distal end of the trpE segment (at which the subsequent high efficiency translational initiation signal has been deleted). These findings are consistent with the conclusion that the degree of polarity depends on the distance of the nonsense mutation fro mthe subsequent translation initiation signal, but not on its distance from the operator-distal end, including possible translational or transcriptional termination signals
RECOMBINATION AS A REQUIREMENT FOR SEGREGATION OF A PARTIALLY DIPLOID MUTANT OF PNEUMOCOCCUS
Authors: Ledbetter Mary Lee S , Hotchkiss Rollin D
Published: January 2021

Conditions are described in which the pneumococcal mutant strain sulr-c, resistant to the drug sulfanilamide, gives rise to sensitive segregants resistant to nitrobenzoic acid at a frequency constant with time. This segregant frequency is markedly enhanced upon exposure of the cells to doses of ultraviolet light or mitomycin C that permit survival of 50% to 90% of the cells. Treatment with acridine orange diminishes the segregant frequency. From the known influences of these three agents on genetic recombination, we propose that a recombination event is necessary in the generation of segregants.—During a period of incubation following treatment with ultraviolet light or mitomycin C, cell division resumes and the original segregant frequency is restored. Thus potential segregants are either unable to replicate in the absence of selection, or they are under-represented among the cells dividing soon after treatment.—If the sulr-c mutation is introduced into a mutant pneumococcal strain lacking an ATP-dependent exonuclease activity and deficient in recombination with transforming DNA, segregant frequencies are unaffected. This fact may indicate limits upon the type of recombination event responsible for segregation.
High prevalence of blaCTX-M-15 and nosocomial transmission of hypervirulent epidemic clones of Klebsiella pneumoniae at a tertiary hospital in Ethiopia
Authors: Sewunet Tsegaye , Asrat Daniel , Woldeamanuel Yimtubezinash , Ny Sofia , Westerlund Fredrik , Aseffa Abraham , Giske Christian G
Published: January 2021

Background

Genomic epidemiology of antibiotic resistance is not sufficiently studied in low-income countries.

Objectives

To determine prevalence of ESBL production, and resistome and virulome profiles, of Klebsiella pneumoniae isolated at Jimma Medical Center, Ethiopia.

Methods

Strains isolated from patients with suspected infections between June and November 2016 were characterized by MALDI-TOF for species identification and disc diffusion for antimicrobial susceptibility testing. All K. pneumoniae isolates were characterized by double disc diffusion for ESBL production and all ESBL-producing strains (ESBL-KP) were subjected to WGS on the Illumina (HiSeq 2500) platform. DNA was extracted by automated systems (MagNA Pure 96). Genome assembly was performed using SPAdes (v. 3.9) and draft genomes were used for analysing molecular features of the strains. Maximum likelihood trees were generated using FastTree/2.1.8 based on SNPs in shared genomic regions to identify transmission clusters.

Results

Of the 146 K. pneumoniae strains isolated, 76% were ESBL-KP; 93% of the ESBL-KP strains showed resistance to multiple antimicrobial classes. blaCTX-M-15 (84.4%) was the most prevalent ESBL gene. Resistance genes for aminoglycosides and/or fluoroquinolones [aac(6′)-Ib-cr (65.1%)], phenicols [catB3 (28.4%)], sulphonamides [sul1 (61.2%) and sul2 (60.5%)], trimethoprim [dfrA27 (32.1%)], macrolides [mph(A) (12.8%)] and rifampicin [arr2/arr3 (39.4%)] were prevalent. Plasmids of the IncF and IncR families were prevalent among ST218, ST147, ST15 and ST39. KL64 and KL57 capsular types and O1 and O2 LPSs were prevalent. A high-risk clone, ST218-KL57 encoding rmpA1/rmpA2 and iutA, was detected. Phylogenetic analysis showed a cluster of clonally related strains from different units of the hospital.

Conclusions

Prevalence of ESBL-KP was high and blaCTX-M-15 was the predominant ESBL gene. ESBL genes had spread through both clonal and polyclonal expansion of high-risk and hypervirulent clones. Nosocomial transmission of MDR strains between different units of the hospital was observed.
Recurring Critical Results and Their Impact on the Volume of Critical Calls at a Tertiary Care Center
Authors: Karin Amir , Kulasingam Vathany , Chartier Lucas B , Ejumudo Angela , Wolff Talya , Brinc Davor
Published: January 2021

Background

When a test result is critically abnormal, laboratories notify the responsible caregivers immediately, usually with a phone call. If the same test was ordered repeatedly, our institution has a policy of not notifying the caregiver if the previous result was also critical and within 24 h. We compared our policy with those of several different laboratories in North America and estimated the impact of changing our current policy to calling for all critical results, regardless of the time interval.

Methods

Several North American laboratories (n = 15) were surveyed regarding their critical result notification policy. For our institution, we performed a retrospective analysis focusing on critical values in a 5-month period for common chemistry tests. We estimated the effect on volume of calls and the impact on workload with regard to changing the critical result notification policy and critical thresholds.

Results

A majority of surveyed laboratories had some form of restriction for calling about recurring critical results. In our institution, removing the restrictions would increase the average number of daily calls by 11%–155%, depending on the analyte. The choice of critical thresholds also has an effect on the number of calls, and the effect depends on the analyte and the threshold chosen.

Conclusions

Guidelines do not specify how recurring critical results should be communicated. Depending on the institutional resources, some laboratories call only the first critical result for one or more tests if certain criteria are met. Modification of these policies can lead to significant changes in the volume of calls made by the laboratory and can have numerous impacts related to workload, logistics, and patient care.
Body Composition Modifications Due to the “Search, Rescue and Survival Training” in Male Military Firefighter Cadets
Authors: de Barros Thiago Ramos , Salerno Verônica Pinto , Ponce Thalita , Mainenti Míriam Raquel Meira
Published: January 2021

Introduction

To train and prepare cadets for a career as firefighters in Rio de Janeiro, the second-year students of the Officers Training Course are submitted to a Search, Rescue, and Survival Training (SRST) course, which is characterized by long periods of high physical exertion and sleep restriction during a 9-day instruction module, and food restriction during a 7-day survival module. The present study investigated changes in the body composition of 39 male cadets submitted to SRST during training and 4 weeks of recovery with no restrictions in food consumption.

Materials and Methods

Each cadet was evaluated by anthropometric measurements at six time points: pre-SRST; after the first module; after the second module; and after 1, 2, and 4 weeks of recovery. Measurements included body girths and skinfolds, to estimate trunk (chest and waist) and limbs (arm and thigh) dimensions, as well as body composition. Repeated measures ANOVA and Friedman test were applied (depending on each data distribution).

Results

Statistically significant decreases in body weight (76.2; 69.8-87.2 to 63.9; 58.9-73.5 kg) and fat free mass (FFM, 69.2; 63.7-77.2 to 60.1; 56.2-68.0 kg) were observed following the second module of SRST. Following a single week of recovery, the FFM returned to pre-SRST values. Body weight returned to pre-training levels in 2 weeks. Body fat percentage and mass also significantly decreased during SRST (9.0; 7.7-12.3 to 6.5; 5.1-9.3% and 6.9; 5.6-10.0 to 6.9; 5.6-10.0 kg, respectively), which showed a slower and more gradual recovery that reached pre-SRST values after 4 weeks. The girths of arm, thigh, chest and waist significantly decreased due to SRST. The girths of the limbs (arm and thigh) returned to pre-training values after one month of recovery, while the girths of the trunk (chest and waist) did not return to pre-SRST values during the study period.

Conclusions

The findings suggest that men who experience periods of high energy demands and sleep restriction followed by a period of food restriction will endure unavoidable physical consequences that can be mostly reversed by a 1-month recovery.
Disease impact of rheumatoid arthritis in patients not treated with advanced therapies; survey findings from the National Rheumatoid Arthritis Society
Authors: Nikiphorou Elena , Jacklin Hannah , Bosworth Ailsa , Jacklin Clare , Kiely Patrick
Published: January 2021

Objective

The aim was to reveal the everyday impact of living with RA in people not treated with advanced therapies (i.e. biologic or targeted synthetic DMARDs).

Methods

People with RA, with disease duration >2 years, not currently treated with advanced therapies, completed an online survey promoted by the National Rheumatoid Arthritis Society. Items covered demographics, current treatment, RA flare frequency, the Rheumatoid Arthritis Impact of Disease (RAID) tool and questions reflecting work status and ability. Descriptive and multivariable regression analyses were performed.

Results

There were 612 responses from patients having a mean age of 59 years, 88% female, 37.7% with disease duration 2–5 years and 27.9% with disease duration 5–10 years. In the last year, 90% reported an RA flare, with more than six flares in 23%. A RAID patient acceptable state was recorded in 12.4%. Each of the seven domains was scored in the high range by >50% respondents; 74.3% scored sleep problems and 72% fatigue in the high range. A need to change working hours was reported by 70%. Multivariable analyses revealed that increasing difficulties with daily physical activities, reduced emotional and physical well-being in the past week were all significantly associated with pain, number of flares and ability to cope (P < 0.005). The RAID score was significantly predictive of the number of flares.

Conclusion

Patients not currently treated with advanced therapies experience profound difficulties in everyday living with RA, across a broad range of measures. We advocate that patient-reported measures be used to facilitate holistic care, addressing inflammation and other consequences of RA on everyday life.
Kicking the Can Down the Road: Government Interventions in the European Banking Sector
Authors: Acharya Viral V , Borchert Lea , Jager Maximilian , Steffen Sascha
Published: January 2021

We analyze government interventions in the eurozone banking sector during the 2008–2009 financial crisis. Using a novel data set, we document that fiscally constrained governments “kicked the can down the road” by providing banks with guarantees instead of fully-fledged recapitalizations. We econometrically address the endogeneity associated with bailout decisions in identifying their consequences. We find that forbearance prompted undercapitalized banks to shift their assets from loans to risky sovereign debt and engage in zombie lending, resulting in weaker credit supply, elevated risk in the banking sector, and, eventually, a greater reliance on liquidity support from the European Central Bank.
Glycolysis-derived acidic microenvironment as a driver of endothelial dysfunction in systemic sclerosis
Authors: Andreucci Elena , Margheri Francesca , Peppicelli Silvia , Bianchini Francesca , Ruzzolini Jessica , Laurenzana Anna , Fibbi Gabriella , Bruni Cosimo , Bellando-Randone Silvia , Guiducci Serena , Romano Eloisa , Manetti Mirko , Matucci-Cerinic Marco , Calorini Lido
Published: January 2021

Objectives

SSc is an autoimmune disease characterized by peripheral vasculopathy and skin and internal organ fibrosis. Accumulating evidence underlines a close association between a metabolic reprogramming of activated fibroblasts and fibrosis. This prompted us to determine the metabolism of SSc dermal fibroblasts and the effect on the vasculopathy characterizing the disease.

Methods

A Seahorse XF96 Extracellular Flux Analyzer was used to evaluate SSc fibroblast metabolism. In vitro invasion and capillary morphogenesis assays were used to determine the angiogenic ability of endothelial cells (ECs). Immunofluorescence, flow cytometry and real-time PCR techniques provided evidence of the molecular mechanism behind the impaired vascularization that characterizes SSc patients.

Results

SSc fibroblasts, compared with controls, showed a boosted glycolytic metabolism with increased lactic acid release and subsequent extracellular acidification that in turn was found to impair EC invasion and organization in capillary-like networks without altering cell viability. A molecular link between extracellular acidosis and endothelial dysfunction was identified as acidic ECs upregulated MMP-12, which cleaves and inactivates urokinase-type plasminogen activator receptor, impairing angiogenesis in SSc. Moreover, the acidic environment was found to induce the loss of endothelial markers and the acquisition of mesenchymal-like features in ECs, thus promoting the endothelial-to-mesenchymal transition process that contributes to both capillary rarefaction and tissue fibrosis in SSc.

Conclusion

This study showed the relationship of the metabolic reprogramming of SSc dermal fibroblasts, extracellular acidosis and endothelial dysfunction that may contribute to the impairment and loss of peripheral capillary networks in SSc disease.
Effects of Hybrid Susceptibility and Inoculation Timing on Goss’s Bacterial Wilt and Leaf Blight Severity and Corn Yield
Authors: Bauske Elizabeth C. , Friskop Andrew J.
Published: January 2021

Goss’s bacterial wilt and leaf blight (Goss’s wilt) of corn is the most important corn disease in North Dakota (ND), and yield loss due to the disease has not been reliably quantified in northern corn growing regions. To help quantify the amount of yield loss caused by Goss’s wilt, a total of six field experiments were conducted from 2015 to 2017. Experiments were designed in a randomized complete block with a split plot arrangement. Hybrids served as main plots and Clavibacter nebraskensis inoculation timings as subplots. Three hybrids were used and classified as susceptible, moderately susceptible, and resistant. Inoculation timings included a noninoculated control, six to 10 leaf collars (V6 to V10), reproductive silk stage (R1), or a sequential combination of V6 to V10 and R1. A high level of disease (greater than 50% on susceptible hybrid) occurred in three experiments, a low level of disease (less than 5% on susceptible hybrid) in one experiment, and no disease was reported in two experiments. A combined analysis of the high disease experiments indicated yield losses of 34 to 41% on the susceptible hybrid when C. nebraskensis inoculation occurred at V6 to V10. Yield losses of 22 to 25% occurred on the moderately susceptible hybrid when C. nebraskensis inoculation occurred at V6 to V10, and statistical differences in yield loss were not found among inoculations timings on the resistant hybrid. Correlation analyses suggest that for every 1% increase in R1 disease severity on the susceptible hybrid, yield was reduced by 117 kg/ha (1.9 bu/acre). The current study further demonstrates the importance of hybrid resistance and provides updated yield loss information on Goss’s wilt in a northern corn growing region.
Rapid Start of Antiretroviral Therapy in Youth Diagnosed with HIV Infection
Authors: Patel Nehali D. , Dallas Ronald H. , Knapp Katherine M. , Flynn Patricia M. , Gaur Aditya H.
Published: January 2021

Background:

Guidelines for the use of antiretroviral agents in adults and adolescents with HIV recommend that antiretroviral therapy (ART) be started as soon as possible. While rapid initiation of ART in adults with HIV has been well-described, there is relatively little information describing this approach for youth.

Methods:

On April 1, 2018, St. Jude Children’s Research Hospital began offering ART to youth with HIV infection at their first clinic visit. We report the results of a quality improvement initiative that compared patients who offered ART at their first visit to a historical cohort of patients who initiated ART at a subsequent visit. Demographic, HIV biomarker, and visit information were abstracted from medical records, described and compared using univariate statistical methods.

Results:

There were 124 ART-naive youth (median age 19 years, 91% male, 94% black) first seen during the indicated time period. A total of 54 patients were in the baseline cohort and 70 patients were in the rapid start cohort. 90% of youth in the rapid start cohort started ART on their first clinic visit. Time from first clinic visit to undetectable viral load was significantly higher in the baseline cohort compared with the rapid start cohort (median 54 vs. 41 days; P = 0.01). Retention in care 12 months following the first clinic visit was comparable and overall high (>80%).

Conclusions:

Starting ART-naïve youth with HIV infection on ART at their first clinic visit is feasible, has high acceptance, leads to faster viral load suppression, and is associated with high retention in care.
Impact of the Rotavirus Vaccination Program in Norway After Four Years With High Coverage
Authors: Bruun Tone , Salamanca Beatriz Valcarcel , Bekkevold Terese , Døllner Henrik , Gibory Moustafa , Gilje Ann Marit , Haarr Elisebet , Kran Anne-Marte Bakken , Leegaard Truls M. , Nakstad Britt , Nordbø Svein Arne , Rojahn Astrid , Størdal Ketil , Flem Elmira
Published: January 2021

Background:

Use of rotavirus vaccines worldwide since 2006 has led to a significant impact on the burden of rotavirus disease. However, only a third of European countries have introduced rotavirus vaccination in their immunization programs. In October 2014, rotavirus vaccination was introduced for Norwegian infants under strict age restrictions. Exclusive use of the monovalent rotavirus vaccine (RV1) and high vaccination coverage from the beginning enabled evaluation of the impact of this vaccine during the first 4 years after introduction.

Methods:

Prospective laboratory-based surveillance among children <5 years of age hospitalized for acute gastroenteritis at 5 Norwegian hospitals was used to assess the vaccine effectiveness of 2 vaccine doses against rotavirus hospitalization in a case-control study. We used community controls selected from the national population-based immunization registry, and test-negative controls recruited through hospital surveillance. We also assessed the vaccine impact by using time-series analysis of retrospectively collected registry data on acute gastroenteritis in primary and hospital care during 2009–2018.

Results:

Vaccine effectiveness against rotavirus-confirmed hospitalization was 76% (95% confidence interval [CI]: 34%–91%) using test-negative controls, and 75% (95% CI: 44%–88%) using community controls. In the postvaccine period, acute gastroenteritis hospitalizations in children <5 years were reduced by 45% compared with the prevaccine years (adjusted incidence rate ratios 0.55; 95% CI: 0.49–0.61). Reduction in hospitalizations was also seen in cohorts not eligible for vaccination. Rates in primary care decreased to a lesser degree.

Conclusions:

Four years after introduction of rotavirus vaccination in the national childhood immunization program, we recorded a substantial reduction in the number of children hospitalized for acute gastroenteritis in Norway, attributable to a high vaccine effectiveness.
From cloning to mutant in 5 days: rapid allelic exchange in Staphylococcus aureus
Authors: Monk Ian R. , Stinear Timothy P.
Published: January 2021

In the last 10 years, the barriers preventing the uptake of foreign DNA by clinical

Staphylococcus aureus

isolates have been identified and powerful mutagenesis techniques such as allelic exchange are now possible in most genotypes. However, these targeted approaches can still be cumbersome, and the construction of unmarked deletions/point mutations may take many weeks or months. Here, we introduce a streamlined allelic exchange protocol using IMxxB

Escherichia coli

and the plasmid pIMAY-Z. With this optimized approach, a site-specific mutation can be introduced into

S. aureus

in 5 days, from the start of cloning to isolation of genomic DNA for confirmatory whole-genome sequencing. This streamlined protocol considerably reduces the time required to introduce a specific, unmarked mutation in

S. aureus

and should dramatically improve the scalability of gene-function studies.
Natural variants in SARS-CoV-2 S protein pinpoint structural and functional hotspots: implications for prophylaxis and therapeutic strategies
Authors: Pokhrel Suman , Kraemer Benjamin R. , Burkholz Scott , Mochly-Rosen Daria
Published: January 2021

In December 2019, a novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of pneumonia with severe respiratory distress and outbreaks in Wuhan, China. The rapid and global spread of SARS-CoV-2 resulted in the coronavirus 2019 (COVID-19) pandemic. Earlier during the pandemic, there were limited genetic viral variations. As millions of people became infected, multiple single amino acid substitutions emerged. Many of these substitutions have no consequences. However, some of the new variants show a greater infection rate, more severe disease, and reduced sensitivity to current prophylaxes and treatments. Of particular importance in SARS-CoV-2 transmission are mutations that occur in the Spike (S) protein, the protein on the viral outer envelope that binds to the human angiotensin-converting enzyme receptor (hACE2). Here, we conducted a comprehensive analysis of 441,168 individual virus sequences isolated from humans throughout the world. From the individual sequences, we identified 3,540 unique amino acid substitutions in the S protein. Analysis of these different variants in the S protein pinpointed important functional and structural sites in the protein. This information may guide the development of effective vaccines and therapeutics to help arrest the spread of the COVID-19 pandemic.
Evaluation of at-home methods for N95 filtering facepiece respirator decontamination
Authors: Chen T.X. , Pinharanda A. , Steinemann N. A. , Yasuma-Mitobe K. , Lee E. , Hahn J. , Wu L. , Fanourakis S. , Peterka D. S. , Hillman E.M.C
Published: January 2021

N95 filtering facepiece respirators (FFRs) are essential for the protection of healthcare professionals and other high-risk groups against Coronavirus Disease of 2019 (COVID-19). In response to shortages in FFRs during the ongoing COVID-19 pandemic, the Food and Drug Administration issued an Emergency Use Authorization permitting FFR decontamination and reuse. However, although industrial decontamination services are available at some large institutions, FFR decontamination is not widely accessible.

To be effective, FFR decontamination must 1) inactivate the virus; 2) preserve FFR integrity, specifically fit and filtering capability; and 3) be non-toxic and safe. Here we identify and test at-home heat-based methods for FFR decontamination that meet these requirements using common household appliances. Our results identify potential protocols for simple and accessible FFR decontamination, while also highlighting unsuitable methods that may jeopardize FFR integrity.

One sentence summary

Survey of at-home methods for N95 respirator decontamination using heat and evaluation of their effects on N95 respirator integrity.
Binding and activation of serotonergic G-protein coupled receptors by the multimodal antidepressant vortioxetine
Authors: Ladefoged Lucy Kate , Koch Rebekka , Biggin Philip C. , Schiøtt Birgit
Published: January 2021

G-protein coupled receptors are important pharmacological targets. Despite substantial progress, important questions still remain concerning the details of activation: how can a ligand act as an agonist in one receptor, but as an antagonist in a homologous receptor, and how can agonists activate a receptor despite lacking polar functional groups able to interact with helix 5? Studying vortioxetine, an important multimodal antidepressant drug, may elucidate both questions. Herein, we present a thoroughin silicoanalysis of vortioxetine binding to 5-HT1A, 5-HT1B, and 5-HT7receptors and compare to available experimental data. We are able to rationalize the differential mode of action of vortioxetine at different receptors, but also, in the case of the 5-HT1Areceptor, we observe the initial steps of activation suggesting that interaction with helix 5 does not necessarily require a hydrogen bond as previously suggested. The results extend our current understanding of agonist and antagonist action at GPCRs.
eDNA metabarcoding vs metagenomics: an assessment of dietary competition in two estuarine pipefishes
Authors: Serite Conny P. , Emami-Khoyi Arsalan , Ntshudisane Ofentse K. , James Nicola C. , van Vuuren Bettine Jansen , Bodill Taryn , Cowley Paul D. , Whitfield Alan K. , Teske Peter R.
Published: January 2021

Understanding the dietary preferences of endangered species can be useful in implementing conservation strategies, including habitat restoration, translocation, and captive breeding. Environmental DNA (eDNA) from feces provides a non-invasive method for analyzing animal diets. Currently, metabarcoding, a PCR-based method, is the method of choice for analyzing such data. However, this method has limitations, specifically PCR bias, which can result in the overestimation of the importance of certain taxa and the failure to detect other taxa because they do not amplify. The present study compared metabarcoding with metagenomics, a marker-free method, to assess the diversity of prey items in the feces of a critically endangered South African estuarine pipefish,Syngnathus watermeyeri, and its widely distributed congener (S. temminckii) to investigate the potential dietary competition. Our results showed a distinct difference between the diets ofS. watermeyeriandS. temminckii, with the former mainly consuming calanoid copepods and the latter preferring caridean shrimp. Metagenomics produced more species identifications than metabarcoding; however, most of the species identified are not present in South Africa. The identifications made by both methods mostly belonged to the same families, but some prey items were identified only by either method. Both methods would benefit from a complete reference database of South African estuarine macroinvertebrates.
In-depth computational analysis of natural and artificial carbon fixation pathways
Authors: Löwe Hannes , Kremling Andreas
Published: January 2021

In the recent years, engineering new-to-nature CO2 and C1 fixing metabolic pathways made a leap forward. These new, artificial pathways promise higher yields and activity than natural ones like the Calvin-Benson-Bassham cycle. The question remains how to best predict theirin vivoperformance and what actually makes one pathway “better” than another.

In this context, we explore aerobic carbon fixation pathways by a computational approach and compare them based on their ATP-efficiency and specific activity considering the kinetics and thermodynamics of the reactions. Beside natural pathways, this included the artificial Reductive Glycine Pathway, the CETCH cycle and two completely new cycles with superior stoichiometry: The Reductive Citramalyl-CoA cycle and the 2-Hydroxyglutarate-Reverse Tricarboxylic Acid cycle. A comprehensive kinetic data set was collected for all enzymes of all pathways and missing kinetic data was sampled with the Parameter Balancing algorithm. Kinetic and thermodynamic data were fed to the Enzyme Cost Minimization algorithm to check for respective inconsistencies and calculate pathway specific activities.

We found that the Reductive Glycine Pathway, the CETCH cycle and the new Reductive Citramalyl-CoA cycle were predicted to have higher ATP-efficiencies and specific activities than the natural cycles. The Calvin Cycle performed better than previously thought, however. It can be concluded that the weaker overall characteristics in the design of the Calvin Cycle might be compensated by other benefits like robustness, low nutrient demand and a good compatibility with the host’s physiological requirements. Nevertheless, the artificial carbon fixation cycles hold great potential for future applications in Industrial Biotechnology and Synthetic Biology.
The spread of breathing air from wind instruments and singers using schlieren techniques
Authors: Becher Lia , Gena Amayu W. , Alsaad Hayder , Richter Bernhard , Spahn Claudia , Voelker Conrad
Published: January 2021

In this article, the spread of breathing air when playing wind instruments and singing was investigated and visualized using two methods: (1) schlieren imaging with a schlieren mirror and (2) background-oriented schlieren (BOS). These methods visualize airflow by visualizing density gradients in transparent media. The playing of professional woodwind and brass instrument players, as well as professional classical trained singers, were investigated to estimate the spread distances of the breathing air. For a better comparison and consistent measurement series, a single high and a single low note as well as an extract of a musical piece were investigated. Additionally, anemometry was used to determine the velocity of the spreading breathing air and the extent to which it was still quantifiable. The results presented in this article show there is no airflow escaping from the instruments, which is transported farther than 1.2 m into the room. However, differences in the various instruments have to be considered to assess properly the spread of the breathing air. The findings discussed below help to estimate the risk of cross-infection for wind instrument players and singers and to develop efficacious safety precautions, which is essential during critical health periods such as the current COVID-19 pandemic.
Meta-Analysis of Adenoviral p53 Gene Therapy Clinical Trials in Recurrent Head and Neck Squamous Cell Carcinoma
Authors: Sobol Robert E. , Menander Kerstin B. , Chada Sunil , Wiederhold Dora , Sellman Beatha , Talbott Max , Nemunaitis John J.
Published: January 2021

Background

We conducted a meta-analysis of previous adenoviral p53 (Ad-p53) treatment data in recurrent head and neck squamous cell carcinoma (HNSCC) patients to identify optimal Ad-p53 treatment methods for future clinical trials.

Methods

The meta-analysis involved recurrent HNSCC patients treated with Ad-p53 for whom p53 genotyping and immunohistochemistry tumor biomarker studies had been performed (n = 70). Ad-p53 tumor treatment responses defined by RECIST 1.1 criteria were correlated with Ad-p53 dose and tumor p53 biomarkers. Gene expression profiles induced by Ad-p53 treatment were evaluated using the Nanostring IO 360 panel.

Results

Ad-p53 dose based upon the injected tumor volume had a critical effect on tumor responses. All responders had received Ad-p53 doses greater than 7 × 1010viral particles/cm3of tumor volume. There was a statistically significant difference in tumor responses between patients treated with greater than 7 × 1010viral particles/cm3compared to patients treated at lower Ad-p53 doses (Tumor Response 31% (9/29) for Ad-p53 > 7 × 1010viral particles/cm3versus 0% (0/25) for Ad-p53 < 7 × 1010viral particles/cm3; p = 0.0023). All responders were found to have favorable p53 biomarker profiles defined by less than 20% p53 positive tumor cells by immunohistochemistry (IHC), wild type p53 gene sequence or p53 deletions, truncations, or frame-shift mutations without functional p53 tetramerization domains. Preliminary gene expression profiling results revealed that Ad-p53 treatment increased Type I Interferon signaling, decreased TGF-beta and beta-catenin signaling resulting in an increased CD8+ T cell signature which are associated with increased responses to immune checkpoint blockade.

Conclusions

Our findings have important implications for future p53 targeted cancer treatments and identify fundamental principles to guide Ad-p53 gene therapy. We discovered that previous Ad-p53 clinical trials were negatively impacted by the inclusion of patients with unfavorable p53 biomarker profiles and by under dosing of Ad-p53 treatment. Future Ad-p53 clinical trials should have favorable p53 biomarker profiles inclusion criteria and Ad-p53 dosing above 7 × 1010viral particles/cm3of injected tumor volume. Preliminary gene expression profiling identified p53 mechanisms of action associated with responses to immune checkpoint blockade supporting evaluation of Ad-p53 in combination with immune checkpoint inhibitors.
An engineered lactate responding promoter system operating in glucose-rich and anaerobic environments
Authors: Zúñiga Ana , Chang Hung-Ju , Fristot Elsa , Bonnet Jerome
Published: January 2021

Bacteria equipped with genetically-encoded lactate biosensors would support several applications in biopharmaceutical production, diagnosis, or therapeutics. However, many applications involve glucose-rich and anaerobic environments, in which current whole-cell lactate biosensors have low performance. Here we engineered a synthetic lactate biosensor system by repurposing the natural LldPRD promoter regulated by the LldR transcriptional regulator. We removed glucose catabolite repression by designing a hybrid promoter, containing LldR operators and tuned both regulator and reporter gene expression to optimize biosensor signal-to-noise ratio. The resulting lactate biosensor, termed ALPaGA (ALactatePromoter Operating inGlucose andAnaerobia) can operate in glucose rich, aerobic and anaerobic conditions. Our work provides a versatile lactate biosensing platform suitable for many environmental conditions.
Pixelwise H-score: a novel digital image analysis-based metric to quantify membrane biomarker expression from immunohistochemistry images
Authors: Ram Sripad , Vizcarra Pamela , Whalen Pamela , Deng Shibing , Painter CL , Jackson-Fisher Amy , Pirie-Shepherd Steven , Xia Xiaoling , Powell Eric L.
Published: January 2021

Immunohistochemistry (IHC) assays play a central role in evaluating biomarker expression in tissue sections for diagnostic and research applications. Manual scoring of IHC images, which is the current standard of practice, is known to have several shortcomings in terms of reproducibility and scalability to large scale studies. Here, by using a digital image analysis-based approach, we introduce a new metric called the pixelwise H-score (pix H-score) that quantifies biomarker expression from whole-slide scanned IHC images. The pix H-score is an unsupervised algorithm that only requires the specification of intensity thresholds for the biomarker and the nuclear-counterstain channels. We present the detailed implementation of the pix H-score in two different whole-slide image analysis software packages Visiopharm and HALO. We consider three biomarkers P-cadherin, PD-L1, and 5T4, and show how the pix H-score exhibits tight concordance to multiple orthogonal measurements of biomarker abundance such as the biomarker mRNA transcript and the pathologist H-score. We also compare the pix H-score to existing automated image analysis algorithms and demonstrate that the pix H-score provides either comparable or significantly better performance over these methodologies. We also present results of an empirical resampling approach to assess the performance of the pix H-score in estimating biomarker abundance from select regions within the tumor tissue relative to the whole tumor resection. We anticipate that the new metric will be broadly applicable to quantify biomarker expression from a wide variety of IHC images. Moreover, these results underscore the benefit of digital image analysis-based approaches which offer an objective, reproducible, and highly scalable strategy to quantitatively analyze IHC images.
Heterogeneity versus the COVID-19 Pandemic
Authors: Shanmugam Ramalingam , Ledlow Gerald , Singh Karan P.
Published: January 2021

In this paper, heterogeneity is formally defined, and its properties are explored. We define and distinguish observable versus non-observable heterogeneity. It is proposed thatheterogeneityamong the vulnerable is a significant factor in the contagion impact of COVID-19, as demonstrated with incidence rates on a Diamond Princess Cruise ship in February 2020. Given the nature of the disease, its heterogeneity and human social norms, pre-voyage and post-voyage quick testing procedures may become the new standard for cruise ship passengers and crew. The technological advances in testing available today would facilitate more humanistic treatment as compared to more archaic quarantine and isolation practices for all onboard ship. With quick testing, identification of those infected and thus not allowed to embark on a cruise or quarantining those disembarking and other mitigation strategies, the popular cruise adventure could be available safely again. Whatever the procedures implemented, the methodological purpose of this study should add valuable insight in the modeling of disease and specifically, the COVID-19 virus.
CTP-Synthase 2 fromArabidopsis thalianais required for complete embryo development
Authors: Hickl Daniel , Scheuring David , Möhlmann Torsten
Published: January 2021

Pyrimidine denovosynthesis is an essential pathway in all organisms. The final and rate limiting step in the synthesis of the nucleotide CTP is catalyzed by CTP-Synthase (CTPS) and Arabidopsis harbors five isoforms. Single knockouts of each of these do not show apparent phenotypical alterations with the exception ofCTPS2.T-DNA insertion lines for this isoform were unable to produce homozygous offspring. Here we show thatCTPS2exhibits a distinct expression pattern throughout embryo development and loss of function mutants were embryo lethal, as siliques from+/ctps2plants contained nearly 25 % aborted seeds. This phenotype was rescued by complementation withCTPS2under control of its endogenous promoter. Reporter lines revealedCTPS2expression only in the tip of columella cells in embryos of the heart and later stages. FurthermoreCTPS2expression in roots, most pronounced in the columella cells, shoots and vasculature tissue of young seedlings was observed. Filial generations of+/ctps2plants did not germinate properly, even under external cytidine supply. During embryo developmentCTPS2expression was similar to the well known auxin reporter DR5. Indeed, the cloned promoter region we used in this study possesses a repeat of an auxin response element. Thus, we conclude that CTPS2 is essential for CTP supply in the developing embryo and a knockout ofCTPS2is embryo lethal.
Genome-wide screening of circadian and non-circadian impact of Neat1 genetic deletion
Authors: Jacq Audrey , Becquet Denis , Bello-Goutierrez Maria-Montserrat , Boyer Bénédicte , Guillen Séverine , Franc Jean-Louis , François-Bellan Anne-Marie
Published: January 2021

The functions of the long non-coding RNA, Nuclear enriched abundant transcript 1 (Neat1), are poorly understood. Neat1 is required for the formation of paraspeckles, but its respective paraspeckle-dependent or independent functions are unknown. Several studies including ours reported that Neat1 is involved in the regulation of circadian rhythms. We characterized the impact of Neat1 genetic deletion in a rat pituitary cell line. The mRNAs whose circadian expression pattern or expression level is regulated by Neat1 were identified after high-throughput RNA sequencing of the circadian transcriptome of wild-type cells compared to cells in which Neat1 was deleted by CRISPR/Cas9. The numerous RNAs affected by Neat1 deletion were found to be circadian or non-circadian, targets or non-targets of paraspeckles, and to be associated with many key biological processes showing that Neat1, interacting or independently of the circadian system, could play crucial roles in key physiological functions through diverse mechanisms.
Integrin β3 Targeting Biomaterial Preferentially Promotes Secretion of bFGF and Proliferation of iPSC-Derived Vascular Smooth Muscle Cells
Authors: Dash Biraja C. , Duan Kaiti , Kyriakides Themis R. , Hsia Henry C.
Published: January 2021

Human-induced pluripotent stem cell-derived-vascular smooth muscle cells (hiPSC-VSMC) have been shown to promote angiogenesis and wound healing. However, there is a paucity of research on how the extracellular matrix (ECM) microenvironment may impact the hiPSC-VSMC’s function. In this study, our objective was to understand the effect of specific ECM ligand-integrin interaction on hiPSC- VSMC’s paracrine secretion, cell proliferation, and morphology. We here showed a precise modulation of hiPSC-VSMC in a fibronectin functionalized fibrillar collagen scaffold by targeting their integrin β3. The secretion of proangiogenic growth factor, basic fibroblast growth factor (bFGF) was found to be fibronectin dependent via αvβ3 integrin interactions. Also, our data indicate the possible role of a positive feedback loop between integrin β3, bFGF, and matrix metalloproteinase-2 in regulating hiPSC- VSMC’s morphology and cell proliferation. Finally, the secretome with improved proangiogenic activity shows potential for future regenerative applications.
The Ran pathway uniquely regulates cytokinesis in cells with different fates in the earlyC. elegansembryo
Authors: Ozugergin Imge , Mastronardi Karina , Law Chris , Piekny Alisa
Published: January 2021

Cytokinesis occurs at the end of mitosis and occurs due to the ingression of a contractile ring that cleaves the daughter cells. This process is tightly controlled to prevent cell fate changes or aneuploidy, and the core machinery is highly conserved among metazoans. Multiple mechanisms regulate cytokinesis, but their requirement in different cell types is not known. Here, we show that differently fated AB and P1cells in the earlyC. elegansembryo have unique cytokinesis kinetics supported by distinct levels and cortical patterning of myosin. Through perturbation of polarity regulators and the generation of stable tetraploid strains, we demonstrate that these differences depend on both cell fate and size. Additionally, these parameters could influence the Ran pathway, which coordinates the contractile ring with chromatin position, and controls cytokinesis differently in AB and P1cells. Our findings demonstrate the need to consider multiple parameters when modeling ring kinetics.