bluuebunny/crossref_metadata_2025 huggingface dataset:
Regressing SARS-CoV-2 sewage measurements onto COVID-19 burden in the population: a proof-of-concept for quantitative environmental surveillance
Authors: Bar-Or Itay , Yaniv Karin , Shagan Marilou , Ozer Eden , Erster Oran , Mendelson Ella , Mannasse Batya , Shirazi Rachel , Kramarsky-Winter Esti , Nir Oded , Abu-Ali Hala , Ronen Zeev , Rinott Ehud , Lewis Yair E. , Friedler Eran , Bitkover Eden , Paitan Yossi , Berchenko Yakir , Kushmaro Ariel
Published: May 2020

SARS-CoV-2 is an RNA virus, a member of the coronavirus family of respiratory viruses that includes SARS-CoV-1 and MERS. COVID-19, the clinical syndrome caused by SARSCoV-2, has evolved into a global pandemic with more than 2,900,000 people infected. It has had an acute and dramatic impact on health care systems, economies, and societies of affected countries within these few months. Widespread testing and tracing efforts are employed in many countries in order to contain and mitigate this pandemic. Recent data has indicated that fecal shedding of SARS-CoV-2 is common, and that the virus can be detected in wastewater. This indicates that wastewater monitoring is a potentially efficient tool for epidemiological surveillance of SARS-CoV-2 infection in large populations at relevant scales. Collecting raw sewage data, representing specific districts, and crosslinking this data with the number of infected people from each location, will enable us to derive and provide quantitative surveillance tools. In particular, this will provide important means to (i) estimate the extent of outbreaks and their spatial distributions, based primarily on in-sewer measurements (ii) manage the early-warning system quantitatively and efficiently (and similarly, verify disease elimination). Here we report the development of a virus concentration method using PEG or alum, providing an important a tool for detection of SARS-CoV-2 RNA in sewage and relating it to the local populations and geographic information. This will provide a proof of concept for the use of sewage associated virus data as a reliable epidemiological tool.
Diagnosing SARS-CoV-2 infection: the danger of over-reliance on positive test results
Authors: Cohen Andrew N. , Kessel Bruce , Milgroom Michael G.
Published: May 2020

Contrary to the practice during previous epidemics, with COVID-19 health authorities have treated a single positive result from a PCR-based test as confirmation of infection, irrespective of signs, symptoms and exposure. This is based on a widespread belief that positive results in these tests are highly reliable. However, evidence from external quality assessments and real-world data indicate enough a high enough false positive rate to make positive results highly unreliable over a broad range of scenarios. This has clinical and case management implications, and affects an array of epidemiological statistics, including the asymptomatic ratio, prevalence, and hospitalization and death rates, as well as epidemiologic models. Steps should be taken to raise awareness of false positives and reduce their frequency. The most important immediate action is to check positive results with additional tests, at least when prevalence is low.

Key messages

The high specificities (usually 100%) reported in PCR-based tests for SARS-CoV-2 infection do not represent the real-world use of these tests, where contamination and human error produce significant rates of false positives.

Widespread lack of awareness of the real-world false positive rates affects an array of clinical, case management and health policy decisions. Similarly, health authorities’ guidance on interpreting test results is often wrong.

Steps should be taken immediately to reduce the frequency and impacts of false positive results, including checking positive results with additional tests at least when prevalence is low.
Mathematical model of COVID-19 intervention scenarios for São Paulo-Brazil
Authors: Neto Osmar Pinto , Reis José Clark , Brizzi Ana Carolina Brisola , Zambrano Gustavo José , de Souza Joabe Marcos , Pedroso Wellington Amorim , de Mello Pedreiro Rodrigo Cunha , de Matos Brizzi Bruno , Abinader Ellysson Oliveira , Kennedy Deanna M. , Zângaro Renato Amaro
Published: May 2020

An epidemiological compartmental model was used to simulate social distancing strategies to contain the COVID-19 pandemic and prevent a second wave in São Paulo, Brazil. Optimization using genetic algorithm was used to determine the optimal solutions. Our results suggest the best-case strategy for São Paulo is to maintain or increase the current magnitude of social distancing for at least 60 more days and increase the current levels of personal protection behaviors by a minimum of 10% (e.g., wearing facemasks, proper hand hygiene and avoid agglomeration). Followed by a long-term oscillatory level of social distancing with a stepping-down approach every 80 days over a period of two years with continued protective behavior.
Hydroxychloroquine application is associated with a decreased mortality in critically ill patients with COVID-19
Authors: Yu Bo , Li Chenze , Chen Peng , Zhou Ning , Wang Luyun , Li Jia , Jiang Hualiang , Wang Dao Wen
Published: May 2020

Importance

Coronavirus disease 2019 (COVID-19) is a pandemic with no specific drugs and high mortality. The most urgent thing is to find effective treatments.

Objective

To determine whether hydroxychloroquine application may be associated with a decreased risk of death in critically ill COVID-19 patients and what is potential mechanism.

Design, Setting and Patients

This retrospective study included all 568 critically ill COVID-19 patients who were confirmed by pathogen laboratory tests despite antiviral treatment and had severe acute respiratory distress syndrome, PAO2/FIO2 <300 with need of mechanical ventilation in Tongji Hospital, Wuhan, between February 1 of 2020 to April 8 of 2020. All 568 patients received comparable basic treatments including antiviral drugs and antibiotics, and 48 of them additionally received oral hydroxychloroquine (HCQ) treatment (200 mg twice a day for 7-10 days). Primary endpoint is mortality of patients, and inflammatory cytokines levels were compared between hydroxychloroquine and non-hydroxychloroquine (NHCQ) treatments.

MAIN OUTCOMES AND MEASURES

In-hospital death and hospital stay time (day) were obtained, level of inflammatory cytokine (IL-6) was measured and compared between HCQ and NHCQ treatments.

RESULTS

The median age of 568 critically ill patients is 68 (57, 76) years old with 37.0% being female. Mortalities are 18.8% (9/48) in HCQ group and 45.8% (238/520) in NHCQ group (p<0.001). The time of hospital stay before patient death is 15 (10-21) days and 8 (4 - 14) days for the HCQ and NHCQ groups, respectively (p<0.05). The level of inflammatory cytokine IL-6 was significantly lowered from 22.2 (8.3-118.9) pg/mL at the beginning of the treatment to 5.2 (3.0-23.4) pg/ml (p<0.05) at the end of the treatment in the HCQ group but there is no change in the NHCQ group.

CONCLUSIONS AND RELEVANCE

Hydroxychloroquine treatment is significantly associated with a decreased mortality in critically ill patients with COVID-19 through attenuation of inflammatory cytokine storm. Therefore, hydroxychloroquine should be prescribed for treatment of critically ill COVID-19 patients to save lives.

Key Points

Question

Could hydroxychloroquine administration be beneficial in the treatment of critically ill patients with COVID-19?

Findings

In this retrospective study, a total of 568 critically ill patients with COVID-19 all received basic therapy and additionally 48 of them received hydroxychloroquine for 7–10 days (200 mg twice per day). Hydroxychloroquine treatment is significantly associated with a decreased mortality in critically ill COVID-19 patients and attenuated inflammatory cytokine IL-6 level.

Meaning

Our data suggest that hydroxychloroquine could be used to treat critically ill patients with COVID-19 which may save a lot of lives.
Social Distancing Has Merely Stabilized COVID-19 in the US
Authors: Wagner Aaron B. , Hill Elaine L. , Ryan Sean E. , Sun Ziteng , Deng Grace , Bhadane Sourbh , Martinez Victor Hernandez , Wu Peter , Li Dongmei , Anand Ajay , Acharya Jayadev , Matteson David S.
Published: May 2020

Social distancing measures, with varying degrees of restriction, have been imposed around the world in order to stem the spread of COVID-19. In this work we analyze the effect of current social distancing measures in the United States. We quantify the reduction in doubling rate, by state, that is associated with social distancing. We find that social distancing is associated with a statistically-significant reduction in the doubling rate for all but three states. At the same time, we do not find significant evidence that social distancing has resulted in a reduction in the number of daily confirmed cases. Instead, social distancing has merely stabilized the spread of the disease. We provide an illustration of our findings for each state, including point estimates of the effective reproduction number, R, both with and without social distancing. We also discuss the policy implications of our findings.
Thiocyanate and organic carbon inputs drive convergent selection for specific autotrophicAfipiaandThiobacillusstrains within complex microbiomes
Authors: Huddy Robert J. , Sachdeva Rohan , Kadzinga Fadzai , Kantor Rose , Harrison Susan T.L. , Banfield Jillian F.
Published: May 2020

Thiocyanate (SCN-) contamination threatens aquatic ecosystems and pollutes vital fresh water supplies. SCN-degrading microbial consortia are commercially deployed for remediation, but the impact of organic amendments on selection within SCN-degrading microbial communities has not been investigated. Here, we tested whether specific strains capable of degrading SCN-could be reproducibly selected for based on SCN-loading and the presence or absence of added organic carbon. Complex microbial communities derived from those used to treat SCN-contaminated water were exposed to systematically increased input SCN concentrations in molasses-amended and -unamended reactors and in reactors switched to unamended conditions after establishing the active SCN-degrading consortium. Five experiments were conducted over 790 days and genome-resolved metagenomics was used to resolve community composition at the strain level. A singleThiobacillusstrain proliferated in all reactors at high loadings. Despite the presence of manyRhizobialesstrains, a singleAfipiavariant dominated the molasses-free reactor at moderately high loadings. This strain is predicted to breakdown SCN-using a novel thiocyanate dehydrogenase, oxidize resulting reduced sulfur, degrade product cyanate (OCN−) to ammonia and CO2via cyanase, and fix CO2via the Calvin-Benson-Bassham cycle. Removal of molasses from input feed solutions reproducibly led to dominance of this strain. Neither thisAfipiastrain nor the thiobacilli have the capacity to produce cobalamin, a function detected in low abundance community members. Although sustained by autotrophy, reactors without molasses did not stably degrade SCN-at high loading rates, perhaps due to loss of biofilm-associated niche diversity. Overall, convergence in environmental conditions led to convergence in the strain composition, although reactor history also impacted the trajectory of community compositional change.
Do l’Hoest’s monkeys show sensitivity to conspecifics’ informational state?
Authors: Rivoal Lola , Dezecache Guillaume , Berthet Mélissa , Maille Audrey
Published: May 2020

A series of field studies in chimpanzees have shown that the examination of communicative behaviour (e.g. alarm calling) could be particularly fruitful to help uncover mind-reading abilities in non-human animals. In this study, we sought to extend communication-based protocols to one species of Cercopithecids. Specifically, we looked at whether social gazing behaviour of L’Hoest’s monkeys (Allochrocebus lhoesti) is sensitive to the informational state of conspecifics using an original experimental design, thereby focusing on their ability to represent conspecifics’ rather than human experimenters’ mental states. We presented a group of 11 zoo-housed monkeys with a box that contained either an appetitive (mealworms), aversive (raptor stuffed toy) or neutral stimulus (wood bark chips). The discoverer (the first individual that inspected the content of the box) emitted more social gazes in the aversive condition than in the neutral and appetitive conditions. Besides, social gazing was related to the informational state of the conspecifics in the appetitive and neutral conditions, with more social gazes directed towards informed individuals (who had inspected the box) rather than uninformed ones (who had not inspected the box yet). Additional examination of the behaviour of the discoverers revealed that they were likely addressing social gazes to individuals who were in close proximity to them, suggesting that they preferentially gaze at informed conspecifics because the latter remained in proximity. Our study calls for a more widespread assessment of mind-reading capacities in primates, to further reveal the evolutionary history of traits that were thought to be uniquely human until fairly recently.
An engineered 4-1BBL fusion protein with “activity-on-demand”
Authors: Mock Jacqueline , Stringhini Marco , Villa Alessandra , Neri Dario
Published: May 2020

Engineered cytokines are gaining importance for cancer therapy but those products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein (termed F8-4-1BBL), which does not exhibit cytokine activity in solution but regains biological activity upon antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively-spliced EDA domain of fibronectin, and selectively localized to tumorsin vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent anti-tumor activity in various mouse models of cancer, without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display “activity-on-demand” properties at the site of disease upon antigen binding and reduce toxicity to normal tissues.
Phylogeny and Biogeography of South American Marsh Pitcher Plant GenusHeliamphora(Sarraceniaceae) Endemic to the Guiana Highlands
Authors: Liu Sukuan , Smith Stacey D.
Published: May 2020

Heliamphorais a genus of carnivorous pitcher plants endemic to the Guiana Highlands with fragmented distributions. We presented a well resolved, time-calibrated, and nearly comprehensiveHeliamphoraphylogeny estimated using Bayesian inference and maximum likelihood based on nuclear genes (26S, ITS, andPHYC) and secondary calibration. We used stochastic mapping to infer ancestral states of morphological characters and ecological traits. Our ancestral state estimations revealed that the pitcher drainage structures characteristic of the genus transformed from a hole to a slit in single clade, while other features (scape pubescence and hammock-like growth) have been gained and lost multiple times. Habitat was similarly labile inHeliamphora, with multiple transitions from the ancestral highland habitats into the lowlands. Using Mantel test, we found closely related species tend to be geographically closely distributed. Placing our phylogeny in a historical context, major clades likely emerged through both vicariance and dispersal during Miocene with more recent diversification driven by vertical displacement during the Pleistocene glacial-interglacial thermal oscillations. Despite the dynamic climatic history experienced byHeliamphora, the temperature changes brought by global warming pose a significant threat, particularly for those species at the highest elevations.
Structural and functional analysis of pyocin S8 fromPseudomonas aeruginosa: requirement of a glutamate in the H-N-H motif for the DNase activity
Authors: Turano Helena G. , Gomes Fernando , Domingos Renato M. , Degenhardt Maximilia F. S. , Oliveira Cristiano L. P. , Garratt Richard C. , Lincopan Nilton , Netto Luis E. S.
Published: May 2020

Multi-drug resistance (MDR) is a serious threat to global public health, making the development of new antimicrobials an urgent necessity. Pyocins are protein antibiotics produced byPseudomonas aeruginosastrains to kill closely related cells during intraspecific competition. Here, we report an in depth biochemical, microbicidal and structural characterization of a new S-type pyocin, named S8. Initially, we described the domain organization and secondary structure of S8. Subsequently, we observed that a recombinant S8 composed of the killing subunit in complex with the immunity (Im) protein killed the strain PAO1. Furthermore, mutation of a highly conserved glutamic acid to alanine (Glu100Ala) completely inhibited this antimicrobial activity. Probably the integrity of the H-N-H motif is essential in the killing activity of S8, as Glu100 is a highly conserved component of this structure. Next, we observed that S8 is a metal-dependent endonuclease, as EDTA treatment abolished its ability to cleave supercoiled pUC18 plasmid. Supplementation of apo S8 with Ni2+strongly induced this DNase activity, whereas Mn2+and Mg2+exhibited moderate effects and Zn2+was inhibitory. Additionally, S8 bound Zn2+with a higher affinity than Ni2+and the Glu100Ala mutation decreased the affinity of S8 for these metals as shown by isothermal titration calorimetry (ITC). Finally, we describe the crystal structure of the Glu100Ala pyocin-S8DNase-Im complex at 1.38 Å, which gave us new insights into the endonuclease activity of S8. Our results reinforce the possible use of S8 as an alternative antibiotic for MDRPseudomonas aeruginosastrains, while leaving commensal human microbiota intact.
Dopamine neurons promotes wakefulness via the DopR receptor in the Drosophila mushroom body
Authors: Driscoll Margaret , Buchert Steven , Coleman Victoria , McLaughlin Morgan , Nguyen Amanda , Sitaraman Divya
Published: May 2020

Neural circuits involved in regulation of sleep play a critical role in sleep-wake transition and ability of an organism to engage in other behaviors critical for survival. The fruit fly, Drosophila melanogaster is a powerful system for the study of sleep and circuit mechanisms underlying sleep and co-regulation of sleep with other behaviors. InDrosophila, two neuropils in the central brain, mushroom body (MB) and central complex (CX) have been shown to influence sleep homeostasis and receive neuromodulator input critical to sleep-wake switch.

Dopamine neurons (DANs) are the primary neuromodulator inputs to the MB but the mechanisms by which they regulate sleep- and wake-promoting neurons within MB are unknown. Here we investigate the role of subsets of DANs that signal wakefulness and project to wake-promoting compartments of the MB. We find that inhibition of specific subsets of PAM and PPL1 DANs projecting to the MB increase sleep in the presence of strong wake-inducing stimuli that reduces GABA transmission, although activity of these neurons is not directly modulated by GABA signaling. Of these subsets we find that DANs innervating the γ5 and β’2 MB compartments require both DopR1 and DopR2 receptors located in downstream Kenyon cells and mushroom body output neurons (MBONs). Further, we report that unlike the activity of wake-promoting MBONs and KCs, whose activity is modulated by sleep-need and PAM-DAN activity is independent of sleep-need. We have characterized a dopamine mediated sleep-circuit providing an inroad into understanding how common circuits within MB regulate sleep, wakefulness and behavioral arousal.
α-Synuclein facilitates clathrin assembly in synaptic vesicle endocytosis
Authors: Vargas Karina J. , Colosi P. L. , Girardi Eric , Park Jae-Min , Chandra Sreeganga S.
Published: May 2020

α-Synuclein and family members β-, and γ-synuclein, are presynaptic proteins that sense and generate membrane curvature, properties important for synaptic vesicle (SV) cycling. αβγ-synuclein triple knockout (KO) neurons exhibit SV endocytosis (SVE) deficits. Here, we investigate how SVE is regulated by α-synuclein. Immuno-electron microscopy (EM) of synaptosomes reveals that α-synuclein relocalizes from SVs to the synaptic membrane upon stimulation, allowing α-synuclein to function on presynaptic membranes during or after stimulation. On cell membranes, we observe that α-synuclein is colocalized with clathrin and its adaptor AP180. Clathrin patches that contain both α-synuclein and AP180 were significantly larger than clathrin patches containing either protein alone. We also find that recruitment of clathrin and AP180 recruitment to membranes are altered in the absence of synucleins. Visualizing clathrin assembly on membranes using anin vitroendocytosis reconstitution system reveals that α-synuclein increases clathrin patch size and enhances clathrin lattice curvature, facilitating normal clathrin coated pit maturation. Thus, α-synuclein is an endocytic accessory protein that acts at early stages of SVE to controls the size and curvature of clathrin structures on the membrane.
Mesophyll conductance in two cultivars of wheat (Triticum aestivum) grown in glacial to super-elevated [CO2]
Authors: Jahan Eisrat , Thomson Peter C. , Tissue David T.
Published: May 2020

Mesophyll conductance (gm) is an important factor limiting photosynthesis. However,gmresponse to long-term growth in variable [CO2] is not well understood, particularly in crop plants. Here, we grew two cultivars of wheat (Halberd and Cranbrook), known to differ ingmunder current environmental conditions, in four [CO2] treatments: glacial (180 μmol mol−1), pre-industrial (280 μmol mol−1), current ambient (450 μmol mol−1) and super-elevated (1000 μmol mol−1) in well-watered and moderate water limitation conditions, to develop an evolutionary and future climate perspective ongmcontrol of photosynthesis and water use efficiency (WUE). In the two wheat genotypes,gmincreased with rising [CO2] from glacial to ambient [CO2], but declined at super-elevated [CO2]; however, the specific mechanism ofgmresponse to [CO2] remains unclear. Althoughgmandgm/gsc(mesophyll conductance/stomatal conductance) were strongly associated with the variability ofAand WUE, we found that plants with highergmmay increaseAwithout increasinggsc, which increased WUE. These results may be useful to inform plant breeding programs and cultivar selection for Australian wheat under future environmental conditions.

Highlight

Mesophyll conductance increased with increasing [CO2] from glacial to ambient CO2levels, then declined at super-elevated CO2for both well-watered and water-limited treatments. These responses of mesophyll conductance with varying [CO2] have a physiological basis.
AKI during COVID-19 infection: low incidence, high risk of death
Authors: Mou Zhixiang , Zhang Xu
Published: May 2020

Background

Coronavirus disease 2019 (COVID-19) has spread rapidly worldwide and seriously threatens human health since December 2019. However, information about acute kidney injury (AKI) during COVID-19 infection is limited. Some studies thought that presence of AKI is very common and cannot be ignored, while some studies pointed that AKI only is a rare incident during COVID-19 infection. This meta-analysis aimed to find out the truth of COVID-19 related AKI and to provide stronger evidence.

Methods

Two authors independently performed a literature search using PubMed, Web of Science, Embase, and Cochrane Library fulfilled the pre-specified criteria until April 25, 2020 to include studies reported the necessary clinic characteristics, then the incidence of AKI, incidence of required RRT, the mortality with AKI and the death risk with AKI during COVID-19 infection were pooled for statistical analysis by Open Meta-Analyst software to get conclusions.

Findings

It was found that the incidence of AKI in hospitalized patients with COVID-19 infection is low, only about 3.8%; the in-hospital mortality with AKI in COVID-19 infected patients is reach up to 32.6%; the death risk with AKI in COVID-19 infected patients is about 16.1 times higher than those without AKI.

Conclusions

This meta-analysis indicated AKI during COVID-19 infection should be pay more attention, it maybe a strong red flag to death risk. It is still need additional studies to support the conclusions and to explore the AKI mechanism during COVID-19 infection.
Genome-wide mutagenesis identifies factors involved inEnterococcus faecalisvaginal adherence and persistence
Authors: Alhajjar Norhan , Chatterjee Anushila , Spencer Brady L. , Burcham Lindsey R. , Willett Julia L. E. , Dunny Gary M. , Duerkop Breck A. , Doran Kelly S.
Published: May 2020

Enterococcus faecalisis a Gram-positive commensal bacterium native to the gastrointestinal tract and an opportunistic pathogen of increasing clinical concern.E. faecalisalso colonizes the female reproductive tract and reports suggest vaginal colonization increases following antibiotic treatment or in patients with aerobic vaginitis. Currently, little is known about specific factors that promoteE. faecalisvaginal colonization and subsequent infection. We modified an established mouse vaginal colonization model to exploreE. faecalisvaginal carriage and demonstrate that both vancomycin resistant and sensitive strains colonize the murine vaginal tract. Following vaginal colonization, we observedE. faecalisin vaginal, cervical and uterine tissue. A mutant lacking endocarditis- and biofilm-associated pili (Ebp) exhibited a decreased ability to associate with human vaginal and cervical cellsin vitro, but did not contribute to colonizationin vivo. Thus, we screened a low-complexity transposon (Tn) mutant library to identify novel genes important forE. faecaliscolonization and persistence in the vaginal tract. This screen revealed 383 mutants that were underrepresented during vaginal colonization at 1, 5 and 8 days post-inoculation compared to growth in culture medium. We confirmed that mutants deficient in ethanolamine catabolism or in the type VII secretion system were attenuated in persisting during vaginal colonization. These results reveal the complex nature of vaginal colonization and suggest that multiple factors contribute toE. faecalispersistence in the reproductive tract.

IMPORTANCE

Despite increasing prevalence and association ofE. faecaliswith aerobic vaginitis, essentially nothing is known about the bacterial factors that influenceE. faecalisvaginal colonization. We have adapted an animal model of vaginal colonization that supports colonization of multipleE. faecalisstrains. Additionally, we determined that ethanolamine utilization and type VII secretion system genes contribute to vaginal colonization and persistence. Identification of factors important for vaginal colonization and persistence provides potential targets for the development of therapeutics. This study is the first to identify key determinants that promote vaginal colonization byE. faecalis, which may represent an important reservoir for antibiotic resistant enterococci.
Structural basis of norepinephrine recognition and transport inhibition in neurotransmitter transporters
Authors: Pidathala Shabareesh , Mallela Aditya Kumar , Joseph Deepthi , Penmatsa Aravind
Published: May 2020

Norepinephrine is a biogenic amine neurotransmitter that has widespread effects on cardiovascular tone, alertness and sensation of pain. As a consequence, blockers of norepinephrine uptake have served as vital tools to treat depression and chronic pain. Here, we employ a modifiedDrosophila melanogasterdopamine transporter as a surrogate for the human norepinephrine transporter and determine the X-ray structures of the transporter in its substrate-free and norepinephrine-bound forms. We also report structures of the transporter in complex with inhibitors of chronic pain including duloxetine, milnacipran and a synthetic opioid, tramadol. When compared to dopamine, we observe that norepinephrine binds in a different pose, in the vicinity of subsite C within the primary binding site. Our experiments reveal that this region is the binding site for chronic pain inhibitors and a determinant for norepinephrine-specific reuptake inhibition, thereby providing a paradigm for the design of specific inhibitors for catecholamine neurotransmitter transporters.

Highlights

X-ray structures of theDrosophiladopamine transporter in substrate-free and norepinephrine bound forms.

Norepinephrine and dopamine bind in distinct conformations within the binding pocket.

Chronic pain inhibitors S-duloxetine, milnacipran and tramadol bind in the primary binding site and overlap with the norepinephrine-binding pose.

Selective norepinephrine reuptake inhibition occurs through specific interactions at the subsite C in the primary binding pocket.
Spatial Regulation of MCAK Promotes Cell Polarization and Focal Adhesion Turnover to Drive Robust Cell Migration
Authors: Zong Hailing , Hazelbaker Mark , Moe Christina , Ems-McClung Stephanie C. , Hu Ke , Walczak Claire E.
Published: May 2020

The asymmetric distribution of microtubule (MT) dynamics in migrating cells is important for cell polarization, yet the underlying regulatory mechanisms remain underexplored. Here, we addressed this question by studying the role of the MT depolymerase, MCAK, in the highly persistent migration of RPE-1 cells. MCAK knockdown leads to slowed migration and poor directional movement. Fixed and live cell imaging revealed that MCAK knockdown results in excessive membrane ruffling as well as defects in cell polarization and the maintenance of a major protrusive front. Additionally, loss of MCAK increases the lifetime of focal adhesions by decreasing their disassembly rate. These defects are due in part to the loss of the spatial distribution of MCAK activity, wherein activity is higher in the trailing edge of cells compared to the leading edge. Overexpression of Rac1 has a dominant effect over MCAK activity, placing it downstream or in a parallel pathway to MCAK function in migration. Together, our data support a model that places MCAK at a key nexus of a feedback loop, in which polarized distribution of MCAK activity and subsequent differential regulation of MT dynamics contributes to cell polarity and directional migration.
Origin of imported SARS-CoV-2 strains in The Gambia identified from Whole Genome Sequences
Authors: Kanteh Abdoulie , Manneh Jarra , Jabang Sona , Kujabi Mariama A. , Sanyang Bakary , Oboh Mary A. , Bojang Abdoulie , Jallow Haruna S. , Nwakanma Davis , Secka Ousman , Roca Anna , Amambua-Ngwa Alfred , Antonio Martin , Baldeh Ignatius , Forrest Karen , Samateh Ahmadou Lamin , D’Alessandro Umberto , Sesay Abdul Karim
Published: May 2020

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a positive-sense single stranded RNA virus with high human transmissibility. This study generated Whole Genome data to determine the origin and pattern of transmission of SARS-CoV-2 from the first six cases tested in The Gambia. Total RNA from SARS-CoV-2 was extracted from inactivated nasopharyngeal-oropharyngeal swabs of six cases and converted to cDNA following the ARTIC COVID-19 sequencing protocol. Libraries were constructed with the NEBNext ultra II DNA library prep kit for Illumina and Oxford Nanopore Ligation sequencing kit and sequenced on Illumina MiSeq and Nanopore GridION, respectively. Sequencing reads were mapped to the Wuhan reference genome and compared to eleven other SARS-CoV-2 strains of Asian, European and American origins. A phylogenetic tree was constructed with the consensus genomes for local and non-African strains. Three of the Gambian strains had a European origin (UK and Spain), two strains were of Asian origin (Japan). In The Gambia, Nanopore and Illumina sequencers were successfully used to identify the sources of SARS-CoV-2 infection in COVID-19 cases.
Insurgence and worldwide diffusion of genomic variants in SARS-CoV-2 genomes
Authors: Comandatore Francesco , Chiodi Alice , Gabrieli Paolo , Biffignandi Gherard Batisti , Perini Matteo , Ricagno Stefano , Mascolo Elia , Petazzoni Greta , Ramazzotti Matteo , Rimoldi Sara Giordana , Gismondo Maria Rita , Micheli Valeria , Sassera Davide , Gaiarsa Stefano , Bandi Claudio , Brilli Matteo
Published: May 2020

The SARS-CoV-2 pandemic that we are currently experiencing is exerting a massive toll both in human lives and economic impact. One of the challenges we must face is to try to understand if and how different variants of the virus emerge and change their frequency in time. Such information can be extremely valuable as it may indicate shifts in aggressiveness, and it could provide useful information to trace the spread of the virus in the population. In this work we identified and traced over time 7 amino acid variants that are present with high frequency in Italy and Europe, but that were absent or present at very low frequencies during the first stages of the epidemic in China and the initial reports in Europe. The analysis of these variants helps defining 6 phylogenetic clades that are currently spreading throughout the world with changes in frequency that are sometimes very fast and dramatic. In the absence of conclusive data at the time of writing, we discuss whether the spread of the variants may be due to a prominent founder effect or if it indicates an adaptive advantage.
PolyA-modulating antisense oligonucleotides reveal opposing functions for long non-coding RNA NEAT1 isoforms in neuroblastoma
Authors: Naveed Alina , Cooper Jack , Li Ruohan , Hubbard Alysia , Liu Tao , Wilton Steve , Fletcher Sue , Fox Archa
Published: May 2020

Many long non-coding RNAs (lncRNA) are highly dysregulated in cancer and are emerging as therapeutic targets. One example is NEAT1, which consists of two overlapping lncRNA isoforms, NEAT1_1 (3.7kb) and NEAT1_2 (23kb), that are functionally distinct. The longer NEAT1_2 is responsible for scaffolding gene-regulatory nuclear bodies termed paraspeckles, whereas NEAT1_1 is involved in paraspeckle-independent function. The NEAT1 isoform ratio is dependent on the efficient cleavage and polyadenylation of NEAT1_1 at the expense of NEAT1_2. Here we developed a targeted antisense oligonucleotide (ASO) approach to sterically block NEAT1_1 polyadenylation processing, achieving upregulation of NEAT1_2 and abundant paraspeckles. We have applied these ASOs to cells of the heterogeneous infant cancer, neuroblastoma, as we found higher NEAT1_1:NEAT1_2 ratio and lack of paraspeckles in high-risk neuroblastoma cells. These ASOs decrease NEAT1_1 levels, increase NEAT1_2/paraspeckles and concomitantly reduce cell viability in high-risk neuroblastoma specifically. In contrast, overexpression of NEAT1_1 has the opposite effect, increasing cell-proliferation. Transcriptomic analyses of high-risk neuroblastoma cells with altered NEAT1 ratios and increased paraspeckle abundance after ASO treatment showed an upregulation of differentiation pathways, as opposed to the usual aggressive neuroblastic phenotype. Thus, we have developed potential anti-cancer ASO drugs that can transiently increase growth-inhibiting NEAT1_2 RNA at the expense of growth-promoting NEAT1_1 RNA. These ASOs, unlike others that degrade lncRNAs, provide insights into the importance of altering lncRNA polyadenylation events to suppress tumorigenesis as a strategy to combat cancer.
Cryo-EM of human Arp2/3 complexes provides structural insights into actin nucleation modulation by ARPC5 isoforms
Authors: von Loeffelholz Ottilie , Purkiss Andrew , Cao Luyan , Kjaer Svend , Kogata Naoko , Romet-Lemonne Guillaume , Way Michael , Moores Carolyn A.
Published: May 2020

The Arp2/3 complex regulates many cellular processes by stimulating formation of branched actin filament networks. Because three of its seven subunits exist as two different isoforms, mammals produce a family of Arp2/3 complexes with different properties that may be suited to different physiological contexts. To shed light on how isoform diversification affects Arp2/3 function, we determined a 4.2 Å resolution cryo-EM structure of the most active human Arp2/3 complex containing ARPC1B and ARPC5L, and compared it with the structure of the least active ARPC1A-ARPC5-containing complex. The architecture of each isoform-specified Arp2/3 is the same. Strikingly, however, the N-terminal half of ARPC5L is partially disordered compared to ARPC5, suggesting that this region of ARPC5/ARPC5L is an important determinant of complex activity. Confirming this idea, the nucleation activity of Arp2/3 complexes containing hybrid ARPC5/ARPC5L subunits is higher when the ARPC5L N-terminus is present, thereby explaining activity differences between the different Arp2/3 complexes.
α-Synuclein aggregation intermediates form fibril polymorphs with distinct prion-like properties
Authors: Mehra Surabhi , Ahlawat Sahil , Kumar Harish , Singh Nitu , Navalkar Ambuja , Patel Komal , Kadu Pradeep , Kumar Rakesh , Jha Narendra N. , Udgaonkar Jayant B. , Agarwal Vipin , Maji Samir K.
Published: May 2020

α-Synuclein (α-Syn) amyloid fibrils in synucleinopathies (such as Parkinson’s disease (PD), multiple system atrophy (MSA)) are structurally and functionally different, reminiscent of prion-like strains. However, how a single protein can form different fibril polymorphs in various synucleinopathies is not known. Here, we demonstrate the structure-function relationship of two distinct α-Syn fibril polymorphs, the pre-matured fibrils (PMF) and helix-matured fibrils (HMF) based on α-Syn aggregation intermediates. These polymorphs not only display the structural differences, including their fibril core structure as demonstrated by solid-state nuclear magnetic resonance (NMR) spectroscopy and H/D-exchange coupled with mass spectrometry but also possess different cellular activities such as seeding, cellular internalization, and cell-to-cell transmission. The HMF with a compact core structure exhibits low seeding potencyin cellsbut readily internalizes and transmits from one cell to another. Whereas the less structured PMF lacks the cell-to-cell transmission ability but induces abundant α-Syn pathology and triggers the formation of aggresomesin cells. Overall, the study highlights how the conformational heterogeneity in the aggregation pathway may lead to fibril polymorphs with distinct prion-like behavior in PD.
Genome-wide off-rates reveal how DNA binding dynamics shape transcription factor function
Authors: de Jonge Wim J. , Brok Mariël , Lijnzaad Philip , Kemmeren Patrick , Holstege Frank C.P.
Published: May 2020

Protein-DNA interactions are dynamic and these dynamics are an important aspect of chromatin-associated processes such as transcription or replication. Due to a lack of methods to study on- and off-rates across entire genomes, protein-DNA interaction dynamics have not been studied extensively. Here we determinein vivooff-rates for theSaccharomyces cerevisiaechromatin organising factor Abf1, at 191 sites simultaneously across the yeast genome. Average Abf1 residence times span a wide-range, varying between 4.5 and 37 minutes. Sites with different off-rates are associated with different functional characteristics. This includes their transcriptional dependency on Abf1, nucleosome positioning and the size of the nucleosome-free region, as well as the ability to roadblock RNA polymerase II for termination. The results show how off-rates contribute to transcription factor function and that DIVORSEQ (DeterminingIn VivoOff-Rates by SEQuencing) is a meaningful way of investigating protein-DNA binding dynamics genome-wide.
Cellular Stress Signaling Activates Type-I IFN Response Through FOXO3-regulated Lamin Posttranslational Modification
Authors: Hwang Inah , Dai Ziwei , Li Fei , Sanchez Teresa , Locasale Jason W , Cantley Lewis L , Zheng Hongwu , Paik Jihye
Published: May 2020

Neural stem/progenitor cells (NSPCs) persist over the lifespan while encountering constant challenges from age or injury related brain environmental changes, including elevated oxidative stress. A time-dependent stress response that regulates the dynamic balance between quiescence and differentiation is thus essential to preserve NSPC long-term regenerative potential. Here we report that acutely elevated cellular oxidative stress in NSPCs suppresses neurogenic differentiation through induction of FOXO3-mediated cGAS/STING and type I interferon (IFN-I) responses. We show that oxidative stress activates FOXO3 promoting upregulation of its transcriptional target glycine-N-methyltransferase (GNMT) and thus depletion of s-adenosylmethionine (SAM), a key co-substrate involved in methyl group transfer reactions. Mechanistically, we demonstrate that reduced intracellular SAM availability disrupts carboxymethylation and maturation of nuclear lamin, which trigger cytosolic release of chromatin fragments and subsequent activation of the cGAS/STING/IFN-I cascade. Together, our findings suggest the FOXO3-GNMT/SAM-lamin-cGAS/STING-IFN-I signaling cascade as a critical stress response program that preserves its long-term regenerative potential.
Evolution of chemotactic hitchhiking
Authors: Uppal Gurdip , Hu Weiyi , Vural Dervis Can
Published: May 2020

Bacteria typically reside in heterogeneous environments with various chemogradients where motile cells can gain an advantage over non-motile cells. Since motility is energetically costly, cells must optimize their swimming speed and behavior to maximize their fitness. Here we investigate how cheating strategies might evolve where slow or non-motile microbes exploit faster ones by sticking together and hitching a ride. Starting with physical and biological first-principles we computationally study the effects of sticking on the evolution of motility in a controlled chemostat environment. We find stickiness allows slow cheaters to dominate when nutrients are dispersed at intermediate distances. Here, slow microbes exploit faster ones until they consume the population, leading to a tragedy of commons. For long races, slow microbes do gain an initial advantage from sticking, but eventually fall behind. Here, fast microbes are more likely to stick to other fast microbes, and cooperate to increase their own population. We therefore find the nature of the hitchhiking interaction, parasitic or mutualistic, depends on the nutrient distribution.
Sensitive Determination of Infectious Titer of Recombinant Adeno-Associated Viruses (rAAVs) Using TCID50 End-Point Dilution and Quantitative Polymerase Chain Reaction (qPCR)
Authors: Lock Martin , Wilson James , Sena-Esteves Miguel , Gao Guangping
Published: May 2020

Adeno-associated virus (AAV) recombinants are currently the vector of choice for many gene therapy applications. As experimental therapies progress to clinical trials, the need to characterize recombinant adeno-associated viruses (rAAVs) accurately and reproducibly increases. Accurate determination of rAAV infectious titer is important for determining the activity of each vector lot and for ensuring lot-to-lot consistency. The following protocol developed in our laboratory uses a 96-well TCID50 format and quantitative polymerase chain reaction (qPCR) detection for the determination of rAAV infectious titer.
Determinants of non-performing loans in the banking sector in developing state
Authors: Khan Muhammad Asif , Siddique Asima , Sarwar Zahid
Published: May 2020

Purpose

The size of non-performing loans (NPLs) plays a key role in the stability of the banking sector of a country. The factors that explain the NPLs contain very important information for banks. Studies in this regard with respect to developing states such as Pakistan have received little attention. This study aimed to scrutinize the determinants of NPLs observing a case of the banking sector in Pakistan over the period from 2005 to 2017.

Design/methodology/approach

The sample consists of the banking sector (i.e., commercial banks) listed in Pakistan Stock Exchange over the period of 2005–2017. The banking factors, including profitability, operating efficiency, capital adequacy and income diversification, were evaluated. The estimations were done by regression modeling using random and fixed effects through STATA software.

Findings

Results show that the operating efficiency and profitability indicators have a negative association with NPLs but were statistically significant, while capital adequacy and income diversification have a negative association with NPLs but were statistically insignificant.

Research limitations/implications

The present study has considered limited banking indicators as determinants of NPLs and was limited to a specific time period from 2005 to 2017.

Originality/value

The study is an attempt to investigate various banking factors that affect the NPLs with respect to developing economies such as Pakistan.
Inadequately educated workforce and financing obstacles: international evidence from SMEs in developing countries
Authors: Jabbouri Imad , Farooq Omar
Published: May 2020

Purpose

This paper aims to document the impact of inadequately educated workforce on the extent of financing obstacles experienced by firms.

Design/methodology/approach

The authors use the data provided by the World Bank's Enterprise Surveys to test our arguments. The data were collected during the period between 2008 and 2018 in 141 developing countries. A pooled ordered logit regression analysis is performed to arrive at the results.

Findings

The study’s results show that firms with inadequately educated workforce are more likely to experience financing obstacles than other firms. The authors argue that poor performance and lack of technical expertise required to access finance are some of the reasons behind greater financing obstacles experienced by these firms. The study’s results are robust across different geographic regions. The authors also show that firms with inadequately educated workforce are more likely to seek informal credit for financing their short-term (working capital) and long-term (capital expenditures) capital requirements.

Practical implications

Understanding the factors that affect the financing constraints faced by small and medium enterprises (SMEs) should be valuable to managers of SMEs and policy-makers. By removing these constraints, managers can improve their access to financing, and policy-makers can facilitate higher economic growth and better economic conditions.

Originality/value

Prior studies have largely been silent on the impact of inadequately educated workforce on the access to finance. This paper draws attention to this issue within the context of SMEs in an international setting. SMEs are the drivers of economic growth in any country. However, their contributions to economic growth cannot materialize without fulfilling their capital needs.