bluuebunny/crossref_metadata_2025 huggingface dataset:
Daily Salt Intake, its Discretionary Use and Validation of Methods for Estimation using Spot Urine – Findings from Islamabad, Pakistan
Authors: Nadeem Saqib Muhammad Arif , Rafique Ibrar , Ansar Muhammad , Rahat Tayyaba
Published: July 2020

Background

The study was designed to estimate daily salt intake, its discretionary use in healthy individuals and to validate three common methods for salt estimation in Pakistani population.

Methods

Information on demography and discretionary salt use was collected healthy adults (>18 years) along with a blood sample, spot urine sample and 24 hours urine samples. Sodium, chloride, potassium levels and serum creatinine were measured using standard methods. For daily salt estimation, three common methods i.e. INTERSALT, Tanaka and Kawasaki were validated for their applicability in local settings.

Results

Overall 24 h sodium excretion was 158 mmol/l indicating intake of 8.64 (±4.43) grams salt per day which was significantly associated with male gender (p. <0.004) and adding salt during cooking (p. <0.0001). Most (73%) of the participants know about hazardous effects of high salt intake, however, only 25% consider important to lower salt intake. Although, there is insignificant difference (p. 0.09) between measured and estimated 24 hour urine however none of three methods i.e. INTERSALT(bias: -19.64; CCC -0.79), Tanaka(bias: 167.35; CCC -0.37) and Kawasaki (bias: -42.49, CCC -0.79) showed any agreement between measured and estimated 24 hour sodium.

Conclusion

Daily intake of salt was high than recommended by the WHO. Findings showed that none of the three methods could be used for estimating daily intake of salt in local settings of Pakistan.
Saliva offers a sensitive, specific and non-invasive alternative to upper respiratory swabs for SARS-CoV-2 diagnosis
Authors: Byrne Rachel L , Kay Grant A , Kontogianni Konstantina , Brown Lottie , Collins Andrea M , Cuevas Luis E. , Ferreira Daniela M , Fraser Alice J , Garrod Gala , Hill Helen , Menzies Stefanie , Mitsi Elena , Owen Sophie I , Williams Christopher T , Hyder-Wright Angela , Adams Emily R , Cubas-Atienzar Ana I
Published: July 2020

RT-qPCR utilising upper respiratory swabs are the diagnostic gold standard for SARS-CoV-2 despite reported low sensitivity and limited scale up due to global shortages. Saliva is a non-invasive, equipment independent alternative to swabs.

We collected 145 paired saliva and nasal/throat (NT) swabs at diagnosis (day 0) and repeated on day 2 and day 7 dependent on inpatient care and day 28 for study follow up. Laboratory cultured virus was used to determine the analytical sensitivity of spiked saliva and swabs containing amies preservation media.

Self-collected saliva samples were found to be consistent, and in some cases superior when compared to healthcare worker collected NT swabs from COVID-19 suspected participants. We report for the first time the analytical limit of detection of 10−2and 100 pfu/ml for saliva and swabs respectively.

Saliva is a easily self-collected, highly sensitive specimen for the detection of SARS-CoV-2.
Undocumented infectives in the Covid-19 pandemic
Authors: Melis Maurizio , Littera Roberto
Published: July 2020

Background

A crucial role in epidemics is played by the number of undetected infective individuals who continue to circulate and spread the disease. Epidemiological investigations and mathematical models have revealed that the rapid diffusion of Covid-19 can mostly be attributed to the large percentage of undocumented infective individuals who escape testing.

Methods

The dynamics of an infection can be described by the SIR model, which divides the population into susceptible (S), infective (I) and removed (R) subjects. In particular, we exploited the Kermack and McKendrick epidemic model which can be applied when the population is much larger than the fraction of infected subjects.

Results

We proved that the fraction of undocumented infectives, in comparison to the total number of infected subjects, is given by , where R0 is the basic reproduction number. The mean value R0 = 2.10 (2.09 − 2.11) for the Covid-19 epidemic in three Italian regions yielded a percentage of undetected infectives of 52.4% (52.2% - 52.6%) compared to the total number of infectives.

Conclusions

Our results, straightforwardly obtained from the SIR model, highlight the role played by undetected carriers in the transmission and spread of the SARS-CoV-2 infection. Such evidence strongly recommends careful monitoring of the infective population and ongoing adjustment of preventive measures for disease control until a vaccine becomes available.
Development of a Notch pathway assay and quantification of functional Notch pathway activity in T-cell acute lymphoblastic leukemia
Authors: Canté-Barrett Kirsten , Holtzer Laurent , van Ooijen Henk , Hagelaar Rico , Cordo Valentina , Verhaegh Wim , van de Stolpe Anja , Meijerink Jules P.P.
Published: July 2020

The Notch signal transduction pathway is pivotal for various physiological processes including immune responses, and has been implicated in the pathogenesis of many diseases including T-cell acute lymphoblastic leukemia. Various targeted drugs are available that inhibit Notch pathway signaling, but their effectiveness varies due to variable Notch pathway activity among individual patients. Quantitative measurement of Notch pathway activity is therefore essential to identify patients who could benefit from targeted treatment. We here describe a new assay that infers a quantitative Notch pathway activity score from mRNA levels of conserved direct NOTCH target genes. Following biological validation, we assessed Notch pathway activity in a cohort of TALL patient samples and related it to biological and clinical parameters including outcome. High Notch pathway activity was not limited to T-ALL samples harbouring strongNOTCH1mutations, including juxtamembrane domain mutations or hetero-dimerization combined with PEST-domain orFBXW7mutations, indicating that additional mechanisms may activate NOTCH signaling. The measured Notch pathway activity related to intracellular NOTCH levels, indicating that the pathway activity score more accurately reflects Notch pathway activity than predicted on the basis ofNOTCH1mutations. Importantly, patients with low Notch pathway activity had a significantly shorter event-free survival compared to patients showing higher activity.
Interest of circulating tumor DNA as a biomarker for canine cancers: illustration in histiocytic sarcoma, oral malignant melanoma and multicentric lymphoma
Authors: Prouteau Anaïs , Denis Jérôme Alexandre , De Fornel Pauline , Cadieu Edouard , Derrien Thomas , Kergal Camille , Botherel Nadine , Ulvé Ronan , Rault Mélanie , Bouzidi Amira , François Romain , Dorso Laetitia , Lespagnol Alexandra , Devauchelle Patrick , Abadie Jérôme , André Catherine , Hédan Benoît
Published: July 2020

Circulating tumor DNA (ctDNA) has become an attractive biomarker in human oncology and may be informative in cancer-affected dogs. By performing ddPCR or PARR methods, we detected tumor-specific point mutations, copy number alterations and chromosomal rearrangements in the plasma of cancer-affected dogs. It allowed the detection of ctDNA in 2/8 (25%) oral malignant melanoma cases, 12/13 (92.3%) lymphoma cases and 21/23 (91.3%) histiocytic sarcoma (HS) cases. The value of ctDNA to diagnose HS was explored in 133 dogs including 49 with HS. In this cohort, screening recurrentPTPN11mutations in plasma had a specificity of 98.8%, and a sensitivity between 42.8-77% according to HS clinical presentation, being higher in internal forms, especially with pulmonary location. Regarding lymphoma, the follow-up of four dogs showed that the minimal residual disease detection by targeting lymphoma-specific antigen receptor rearrangement in the plasma was concordant with the clinical evaluation. Moreover, ctDNA analysis appeared interesting to assess treatment response and to predict relapse.

This study shows that ctDNA is detectable in the plasma of cancer-affected dogs and is a relevant biomarker for diagnosis and clinical follow-up. With a growing interest in integrating natural canine tumors to explore new therapies, this biomarker appears promising in comparative oncology research.
JAK/STAT pathway promotesDrosophilaneuroblast proliferation via the directCycEregulation
Authors: Du Lijuan , Wang Jian
Published: July 2020

How neural stem cells regulate their proliferative potential and lineage diversity is a central problem in developmental neurobiology.DrosophilaMushroom bodies (MBs), centers of olfactory learning and memory, are generated by a specific set of neuroblasts (Nbs) that are born in the embryonic stage and continuously proliferate till the end of the pupal stage. Although MB presents an excellent model for studying neural stem cell proliferation, the genetic and molecular mechanisms that control the unique proliferative characteristics of the MB Nbs are largely unknown. Further, the signaling cues controlling cell cycle regulators to promote cell cycle progression in MB Nbs remain poorly understood. Here, we report that JAK/STAT signaling pathway is required for the proliferation activity and maintenance of MB Nbs. Loss of JAK/STAT activity severely reduces the later-born MB neuron types and leads to premature neuroblast termination, which can be rescued by tissue-specific overexpression ofCycEanddiap1. Higher JAK/STAT pathway activity in MB results in more neurons, without producing supernumerary Nbs. Furthermore, we show that JAK/STAT signaling effector Stat92E directly regulatesCycEtranscription in MB Nbs. Finally, MB Nb clones of loss or excessCycEphenocopy those of decreased or increased JAK/STAT signaling pathway activities. We conclude that JAK/STAT signaling controls MB Nb proliferative activity through directly regulatingCycEexpression to control cell cycle progression.
Associations of Mitochondrial Genomic Variation with Corticobasal Degeneration, Progressive Supranuclear Palsy, and Neuropathological Tau Measures
Authors: Valentino Rebecca R. , Tamvaka Nikoleta , Heckman Michael G. , Johnson Patrick W. , Soto-Beasley Alexandra I. , Walton Ronald L. , Koga Shunsuke , Uitti Ryan J. , Wszolek Zbigniew K. , Dickson Dennis W. , Ross Owen A.
Published: July 2020

Mitochondrial health is important in ageing and dysfunctional oxidative phosphorylation (OXPHOS) accelerates ageing and influences neurodegeneration. Mitochondrial DNA (mtDNA) codes for vital OXPHOS subunits and mtDNA background has been associated with neurodegeneration; however, no study has characterised mtDNA variation in Progressive supranuclear palsy (PSP) or Corticobasal degeneration (CBD) risk or pathogenesis. In this case-control study, 916 (42.5% male) neurologically-healthy controls, 1051 (54.1% male) pathologically-confirmed PSP cases, and 173 (51.4% male) pathologically-confirmed CBD cases were assessed to determine how stable mtDNA polymorphisms, in the form of mtDNA haplogroups, were associated with risk of PSP, risk of CBD, age of PSP onset, PSP disease duration, and neuropathological tau pathology measures for neurofibrillary tangles (NFT), neuropil threads (NT), tufted astrocytes (TA), and oligodendroglial coiled bodies (CB). 767 PSP cases and 152 CBD cases had quantitative tau pathology scores. mtDNA was genotyped for 39 unique SNPs using Agena Bioscience iPlex technologies and mitochondrial haplogroups were defined to mitochondrial phylogeny. After adjustment for multiple testing, we observed a significant association with risk of CBD for mtDNA sub-haplogroup H4 (OR=4.49, P=0.001) and the HV/HV0a haplogroup was associated with a decreased severity of NT tau pathology in PSP cases (P=0.0023). Our study reports that mitochondrial genomic background may be associated with risk of CBD and may be influencing tau pathology measures in PSP. Replication of these findings will be important.
Modelling co-translational dimerisation for programmable nonlinearity in synthetic biology
Authors: Stoof Ruud , Goñi-Moreno Ángel
Published: July 2020

Nonlinearity plays a fundamental role in the performance of both natural and synthetic biological networks. Key functional motifs in living microbial systems, such as the emergence of bistability or oscillations, rely on nonlinear molecular dynamics. Despite its core importance, the rational design of nonlinearity remains an unmet challenge. This is largely due to a lack of mathematical modelling that accounts for the mechanistic basics of nonlinearity. We introduce a model for gene regulatory circuits that explicitly simulates protein dimerization—a well-known source of nonlinear dynamics. Specifically, our approach focusses on modelling co-translational dimerization: the formation of protein dimers during—and not after—translation. This is in contrast to the prevailing assumption that dimer generation is only viable between freely diffusing monomers (i.e., post-translational dimerization). We provide a method for fine-tuning nonlinearity on demand by balancing the impact of co- versus post-translational dimerization. Furthermore, we suggest design rules, such as protein length or physical separation between genes, that may be used to adjust dimerization dynamics in-vivo. The design, build and test of genetic circuits with on-demand nonlinear dynamics will greatly improve the programmability of synthetic biological systems.
The variation among sites of protein structure divergence is shaped by mutation and scaled by selection
Authors: Marcos María Laura , Echave Julian
Published: July 2020

Protein structures do not evolve uniformly, but the degree of structure divergence varies among sites. The resulting site-dependent structure divergence patterns emerge from a process that involves mutation and selection, which may both, in principle, influence the emergent pattern. In contrast with sequence divergence patterns, which are known to be mainly determined by selection, the relative contributions of mutation and selection to structure divergence patterns is unclear. Here, studying 6 protein families with a mechanistic biophysical model of protein evolution, we untangle the effects of mutation and selection. We found that even in the absence of selection, structure divergence varies from site to site because the mutational sensitivity is not uniform. Selection scales the profile, increasing its amplitude, without changing its shape. This scaling effect follows from the similarity between mutational sensitivity and sequence variability profiles.
Nogo-A Beyond the RhoA/ROCK Pathway – Novel Components of Intracellular Nogo-A Signaling Cascades
Authors: Maibach Martina A. , Piovesana Ester , Kaiser Julia , Holm Mea M. , Risic Zorica , Maurer Michael , Schwab Martin E.
Published: July 2020

Nogo-A is a well-characterized myelin-associated membrane protein that restricts fibre growth and the regenerative capacity of the adult central nervous system after injury. To date Nogo-A post-receptor signalling pathway research focused on the RhoA/ROCK cascade, which can lead to growth cone collapse and neurite retraction. Much less is known about continued intracellular Nogo-A signalling mediating long-term neurite outgrowth inhibition resulting from transcriptional and translational changes. Here, we propose a simple but highly reproduciblein vitroassay to study Nogo-A related signaling and neurite outgrowth inhibition in general. Furthermore, we identified ERK1/2 as downstream effector of Nogo-A, partially mediating its neurite outgrowth inhibition. We describe ERK1/2 dependent changes of translational events such as elevation of RhoA levels within the growth cone, which may potentiate the cells’ responses to Nogo-A. We also observed Nogo-A dependent upregulation of the JAK/STAT pathway inhibitors SOCS3 and KLF4 and downregulation of insulin mediated phosphorylation of AKT, indicating direct negative crosstalk between Nogo-A signalling and the growth promoting JAK/STAT and AKT/mTORC1 pathways.
YAP/TAZ and EZH2 synergize to impair tumor suppressor activity of TGFBR2 in non-small cell lung cancer
Authors: Sardo Federica Lo , Pulito Claudio , Sacconi Andrea , Korita Etleva , Sudol Marius , Strano Sabrina , Blandino Giovanni
Published: July 2020

Lung cancer is the leading cause of cancer-related deaths, worldwide. Non–small cell lung cancer (NSCLC) is the most prevalent lung cancer subtype. YAP and TAZ have been implicated in lung cancer by acting as transcriptional co-activators of oncogenes or as transcriptional co-repressors of tumor suppressor genes. Previously we reported that YAP and TAZ regulate microRNAs expression in NSCLC. Among the set of regulated miRNAs, the oncogenic miR-25, 93, and 106b, clustering within the MCM7 gene were selected for further studies. We firstly identified Transforming Growth Factor-β (TGF-β) Receptor 2 (TGFBR2), a member of the TGF-β signaling, as a target of the miRNA cluster, which exhibited prognostic value because of its tumor suppressor activity. We found that YAP/TAZ-mediated repression of TGFBR2 occurs both: post-transcriptionally through the miR-106b-25 cluster and transcriptionally by engaging the EZH2 epigenetic repressor that we reported here as a novel target gene of YAP/TAZ. Furthermore, we document that YAP/TAZ and EZH2 cooperate in lung tumorigenesis by transcriptionally repressing a specific subset of tumor suppressor genes, including TGFBR2. Our findings point to YAP/TAZ and EZH2 as potential therapeutic targets for NSCLC treatment.
Induction of a SALL4-dependency for targeted cancer therapy
Authors: Yang Junyu , Gao Chong , Liu Miao , Chen Zhiyuan , Liu Yao-Chung , Kwon Junsu , Qi Jun , Tian Xi , Stein Alicia , Liu Yanjing , Kong Nikki R. , Wu Yue , Yin Shenyi , Xi Jianzhong Jeff , Luo Hongbo , Silberstein Leslie E. , Thoms Julie A. I. , Unnikrishnan Ashwin , Pimanda John E. , Tenen Daniel G. , Chai Li
Published: July 2020

Oncofetal protein SALL4 is critical for tumor cell survival, making it a promising target in cancer therapy. However, it is detectable only in a subset of cancer patients, which limits the therapeutic impact of a SALL4 targeted therapy. Here we report that SALL4 can be activated and/or upregulated pharmacologically by hypomethylating agents, such as 5-Aza-2’-deoxycytidine (DAC), which are used clinically, and that SALL4 negative cancer cells become SALL4 dependent following exogenous expression of SALL4. In addition, the histone deacetylase inhibitor Entinostat (ENT) negatively regulates SALL4 expression by upregulating miR-205. Both ENT and miR-205 treatment induced cell apoptosis, rescuable by SALL4 expression or miR-205 inhibition. Finally, DAC pre-treatment sensitizes SALL4 negative cancer cell lines to ENT both in culture andin vivoby upregulating SALL4. Overall, we propose a framework whereby the scope of targeted therapy can be expanded by sensitizing cancer cells to treatment by target induction and engineered dependency.

Significance

This proof of concept study demonstrates that targeted cancer therapy can be achieved by inducing a targetable gene establishing a survival-dependency for cancer cells. For SALL4, sequential treatment of DAC and ENT could expand the scope of SALL4 targeted cancer therapy.
The DNA methyltransferase 1 (DNMT1) acts on neurodegeneration by modulating proteostasis-relevant intracellular processes
Authors: Bayer Cathrin , Pitschelatow Georg , Hannemann Nina , Linde Jenice , Reichard Julia , Pensold Daniel , Zimmer-Bensch Geraldine
Published: July 2020

The limited regenerative capacity of neuronal cells requires tight orchestration of cell death and survival regulation in the context of longevity, as well as age-associated and neurodegenerative diseases. Subordinate to genetic networks, epigenetic mechanisms, like DNA methylation and histone modifications, are involved in the regulation of neuronal functionality, and emerge as key contributors to the pathophysiology of neurodegenerative diseases. DNA methylation, a dynamic and reversible process, is executed by DNA methyltransferases (DNMTs). DNMT1 was previously shown to regulate neuronal survival in the aged brain, whereby a DNMT1-dependent modulation of processes relevant for protein degradation was proposed as underlying mechanism. Functional proteostasis networks are a mandatory prerequisite for the functionality and long-term survival of neurons. Malfunctioning proteostasis is found, inter alia, in neurodegenerative contexts. Here, we investigated whether DNMT1 affects critical aspects of the proteostasis network by a combination of expression studies, life cell imaging and biochemical analyses. We found that DNMT1 negatively impacts retrograde trafficking and autophagy, both being involved in the clearance of aggregation-prone proteins by the aggresome-autophagy pathway. In line with this, we found that the transport of GFP-labeled mutant HTT to perinuclear regions, proposed to by cytoprotective, also depends on DNMT1. Depletion ofDnmt1accelerated HTT perinuclear HTT aggregation and improved the survival of cells transfected with mutant HTT. This suggests that mutant HTT-induced cytotoxicity is at least in part mediated by DNMT1-dependent modulation of degradative pathways.
DNA damage alters nuclear mechanics through chromatin reorganisation
Authors: dos Santos Ália , Cook Alexander W. , Gough Rosemarie E , Schilling Martin , Olszok Nora Aleida , Brown Ian , Wang Lin , Aaron Jesse , Martin-Fernandez Marisa L. , Rehfeldt Florian , Toseland Christopher P.
Published: July 2020

DNA double-strand breaks (DSBs) drive genomic instability. For efficient and accurate repair of these DNA lesions, the cell activates DNA damage repair pathways. However, it remains unknown how these processes may affect the biomechanical properties of the nucleus and what role nuclear mechanics play in DNA damage and repair efficiency.

Here, we used Atomic Force Microscopy (AFM) to investigate nuclear mechanical changes, arising from externally induced DNA damage. We found that nuclear stiffness is significantly reduced after cisplatin treatment, as a consequence of DNA damage signalling. This softening was linked to global chromatin decondensation, which improves molecular diffusion within the organelle. We propose that this can increase recruitment for repair factors. Interestingly, we also found that reduction of nuclear tension, through cytoskeletal relaxation, has a protective role to the cell and reduces accumulation of DNA damage. Overall, these changes protect against further genomic instability and promote DNA repair. We propose that these processes may underpin the development of drug resistance.
The genotype-phenotype landscape of an allosteric protein
Authors: Tack Drew S. , Tonner Peter D. , Pressman Abe , Olson Nathanael D. , Levy Sasha F. , Romantseva Eugenia F. , Alperovich Nina , Vasilyeva Olga , Ross David
Published: July 2020

Allostery is a fundamental biophysical mechanism that underlies cellular sensing, signaling, and metabolism. Quantitative methods to characterize the genotype-phenotype relationships for allosteric proteins would provide data needed to improve engineering of biological systems, to uncover the role of allosteric mis-regulation in disease, and to develop allosterically targeted drugs1. Here we report the large-scale measurement of the genotype-phenotype landscape for an allosteric protein: thelacrepressor fromEscherichia coli, LacI. Using a method that combines long-read and short-read DNA sequencing, we quantitatively determine the dose-response curves for nearly 105variants of the LacI sensor. With the resulting data, we train a deep neural network (DNN) capable of predicting the dose-response curves for additional LacI genotypesin silico.We then map the impact of amino acid substitutions on the allosteric function of LacI. Additionally, we demonstrate engineering of allosteric function with unprecedented accuracy by identifying LacI variants from the measured landscape with quantitatively specified dose-response curves. Finally, we discover sensors with previously unreported band-stop dose-response curves. Overall, our results provide the first high-coverage, quantitative view of genotype-phenotype relationships for an allosteric protein, revealing a surprising diversity of phenotypes and showing that each phenotype is accessible via multiple distinct genotypes.
Development of conformational BRET biosensors that monitor Ezrin, Radixin and Moesin activation in real-time
Authors: Leguay Kévin , Decelle Barbara , He Yu Yan , Hogue Mireille , Kobayashi Hiroyuki , Gouill Christian Le , Bouvier Michel , Carréno Sébastien
Published: July 2020

Ezrin, Radixin and Moesin compose the family of ERM proteins. They link actin and microtubule filaments to the plasma membrane to control signaling and cell morphogenesis. Importantly, their activity promotes invasive properties of metastatic cells from different cancer origins. Therefore, a precise understanding of how these proteins are regulated is important for the understanding of the mechanism controlling cell shape as well as providing new opportunities for the development of innovative cancer therapies. ERMs are believed to exist in two main conformational states: a close-inactive conformation in the cytoplasm that is recruited at the plasma membrane to convert into an open-active conformation. Here, we developed and characterized novel BRET-based conformational biosensors, compatible with high throughput screening, that monitor Ezrin, Radixin or Moesin individual activation in living cells. We showed that these biosensors faithfully monitor ERM activation and can be used to quantify the impact of small molecules, mutation of regulatory amino acids or depletion of upstream regulators on their activity. The use of these biosensors allowed us to uncover a novel aspect of ERM activation process involving a pool of closed-inactive ERMs stably associated with the plasma membrane. Upon stimulation, this pool serves as a cortical reserve that is rapidly activated before the recruitment of cytoplasmic ERMs.
Polymeric Pathogen-like Particles-Based Combination Adjuvants Elicit Potent Mucosal T Cell Immunity to Influenza A Virus
Authors: Kingstad-Bakke Brock , Toy Randall , Lee Woojong , Pradhan Pallab , Vogel Gabriela , Marinaik Chandranaik , Larsen Autumn , Kawaoka Yoshihoro , Roy Krishnendu , Suresh M
Published: July 2020

Eliciting durable and protective T cell-mediated immunity in the respiratory mucosa remains a significant challenge. Polylactic-co-glycolic acid (PLGA)-based cationic pathogen-like particles (PLPs) loaded with TLR agonists mimic biophysical properties of microbes and hence, simulate pathogen-pattern recognition receptor interactions to safely and effectively stimulate innate immune responses. We generated micro particle PLPs loaded with TLR4 (glucopyranosyl lipid adjuvant, GLA) or TLR9 (CpG) agonists, and formulated them with and without a mucosal delivery enhancing carbomer-based nanoemulsion adjuvant (ADJ). These adjuvants delivered intranasally to mice elicited high numbers of influenza nucleoprotein (NP)-specific CD8+/ CD4+ effector and tissue-resident memory T cells (TRMs) in lungs and airways. PLPs delivering TLR4 versus TLR9 agonists drove phenotypically and functionally distinct populations of effector and memory T cells. While PLPs loaded with CpG or GLA provided immunity, combining the adjuvanticity of PLP-GLA and ADJ synergistically enhanced the development of airway and lung TRMs and protective immunity to pathogenic influenza A virus. Further, balanced CD8 (Tc1/Tc17) and CD4 (Th1/Th17) recall responses were linked to effective influenza virus control in the lungs. These studies provide mechanistic insights into vaccine-induced T cell immunity in the respiratory tract and pave the way for the development of a universal influenza vaccine.
Effect of sunlight on the efficacy of commercial antibiotics used in agriculture
Authors: Khan Sebastian , Osborn Amanda , Eswara Prahathees J.
Published: July 2020

Antibiotic stewardship is of paramount importance to limit the emergence of antibiotic-resistant bacteria in not only hospital settings, but also in animal husbandry, aquaculture, and agricultural sectors. Currently, large quantities of antibiotics are applied to treat agricultural diseases like citrus greening disease (CGD). The two commonly used antibiotics approved for this purpose are streptomycin and oxytetracycline. Although investigations are ongoing to understand how efficient this process is to control the spread of CGD, to our knowledge, there have been no studies that evaluate the effect of environmental factors such as sunlight on the efficacy of the above-mentioned antibiotics. We conducted a simple disc-diffusion assay to study the efficacy of streptomycin and oxytetracycline after exposure to sunlight for 7- or 14-day periods usingEscherichia coliandBacillus subtilisas the representative strains of Gram-negative and Gram-positive organisms respectively. Freshly prepared discs and discs stored in the dark for 7 or 14 days served as our controls. We show that the antibiotic potential of oxytetracycline exposed to sunlight dramatically decreases over the course of 14 days against bothE. coliandB. subtilis. However, the effectiveness of streptomycin was only moderately impacted by sunlight. It is important to note that antibiotics that last longer in the environment may play a deleterious role in the rise and spread of antibiotic-resistant bacteria. Further studies are needed to substantively analyze the safety and efficacy of antibiotics used for broader environmental applications.

IMPORTANCE

Although antibiotics have been used for agricultural purposes for decades, due to the rapid rise in antibiotic resistance this usage needs to be revisited. Questions remain on the appropriate mode of application of antibiotics and the actual benefits of using antibiotics for treating the infections caused by plant pathogens, especially for the ones that are intracellular in nature. Here we show that the two commonly used commercial antibiotics, oxytetracycline and streptomycin, lose their efficacy at different rates in the presence of sunlight. While the former loses its potency within days the latter remains active for many days. Thus, oxytetracycline may not be active long enough to produce desired effect and streptomycin may persist in the environment and as a side effect due to its selective pressure, may force the rise of streptomycin-resistant pathogens.
EP3 signaling is decoupled from regulation of glucose-stimulated insulin secretion in β-cells compensating for obesity and insulin resistance
Authors: Schaid Michael D. , Harrington Jeffrey M. , Kelly Grant M. , Sdao Sophia M. , Merrins Matthew J. , Kimple Michelle E.
Published: July 2020

Of the β-cell signaling pathways altered by non-diabetic obesity and insulin resistance, some are adaptive while others actively contribute to β-cell failure and demise. Cytoplasmic calcium (Ca2+) and cyclic AMP (cAMP), which control the timing and amplitude of insulin secretion, are two important signaling intermediates that can be controlled by stimulatory and inhibitory G protein-coupled receptors. Previous work has shown the importance of the cAMP-inhibitory EP3 receptor in the beta-cell dysfunction of type 2 diabetes. To examine alterations in β-cell cAMP during diabetes progression we utilized a β-cell specific cAMP biosensor in tandem with islet Ca2+recordings and insulin secretion assays. Three groups of C57BL/6J mice were used as a model of the progression from metabolic health to type 2 diabetes: wildtype, normoglycemicLeptinOb, and hyperglycemicLeptinOb. Here, we report robust increases in β-cell cAMP and insulin secretion responses in normoglycemicLeptinobmice as compared to wild-type: an effect that was lost in islets from hyperglycemicLeptinobmice, despite elevated Ca2+duty cycle. Yet, the correlation of EP3 expression and activity to reduce cAMP levels and Ca2+duty cycle with reduced insulin secretion only held true in hyperglycemicLeptinObmice. Our results suggest alterations in beta-cell EP3 signaling may be both adaptive and maladaptive and define β-cell EP3 signaling as much more nuanced than previously understood.
A simple protein-based surrogate neutralization assay for SARS-CoV-2
Authors: Abe Kento T. , Li Zhijie , Samson Reuben , Samavarchi-Tehrani Payman , Valcourt Emelissa J. , Wood Heidi , Budylowski Patrick , Dupuis Alan P. , Girardin Roxie C. , Rathod Bhavisha , Wang Jenny H. , Barrios-Rodiles Miriam , Colwill Karen , McGeer Allison J , Mubareka Samira , Gommerman Jennifer L. , Durocher Yves , Ostrowski Mario , McDonough Kathleen A. , Drebot Michael A. , Drews Steven J. , Rini James M. , Gingras Anne-Claude
Published: July 2020

Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin converting-enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of two viral based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus, and a spike pseudotyped viral-vector-based assay.
Parallelized engineering of mutational models using piggyBac transposon delivery of CRISPR libraries
Authors: Nuttle Xander , Burt Nicholas D. , Currall Benjamin , Moysés-Oliveira Mariana , Mohajeri Kiana , Yadav Rachita , Tai Derek J. C. , Gusella James F. , Talkowski Michael E.
Published: July 2020

Novel gene and variant discoveries have reached unprecedented scale with the emergence of exome and genome sequencing studies across a spectrum of human disease initiatives. Highly parallelized functional characterization of these variants is now paramount to deciphering disease mechanisms, and approaches that facilitate editing of induced pluripotent stem cells (iPSCs) to derive otherwise inaccessible tissues of interest (e.g., brain) have become critical in genomics research. Here, we sought to facilitate scalable editing of multiple genes and variants by developing a genome engineering approach that incorporates libraries of CRISPR/Cas9 guide RNAs (gRNAs) into a piggyBac (PB) transposon system. To test the efficiency of inducing small indels, targeted deletions, and large reciprocal copy number variants (CNVs), we simultaneously delivered to human iPSCs bothCas9and a library including 59 single gRNAs targeting segmental duplications, 70 paired gRNAs flanking particular genic regions, and three single gRNAs targeting the coding sequence of an individual gene,MAGEL2. After editing, we isolated single cells, expanded resultant colonies, and genotyped their gRNA contents and mutational outcomes. We observed that 97.7% of gRNA constructs were integrated into at least one colony, with 85.6% of colonies containing three or fewer PB integrations. This PB editing method generated 354 cell lines with 57.8% of sequenced gRNA cleavage sites modified in at least one line, 14.4% of these lines altered at multiple targets, and single-copy indel mutagenesis predominating. Among the edits generated were eight targeted genomic deletions, including pathogenic microdeletions at chromosome 15q11-q13 (∼5.3 Mbp), chromosome 16p11.2 (∼740 kbp), and chromosome 17q11.2 (∼1.4 Mbp). These data highlight PB editing as a powerful platform for gene inactivation and testify to its strong potential for oligogenic modeling. The ability to rapidly establish high-quality mutational models at scale will facilitate the development of near-isogenic cellular collections and catalyze comparative functional genomic studies, better positioning us to investigate the roles of hundreds of genes and mutations in development and disease.
Dual cooperation between HSP70 and the 26S proteasome in co-translational protein quality control
Authors: Tian Guiyou , Hu Cheng , Yun Yun , Yang Wensheng , Dubiel Wolfgang , Cheng Yabin , Wolf Dieter A.
Published: July 2020

Co-translational degradation via the ubiquitin-proteasome system mediates quality control of 15 – 25% of nascent proteins, a proportion that is known to increase dramatically as a result of proteotoxic stress. Whereas the ubiquitylation machinery involved has been characterized, mechanisms coordinating the proteasomal destruction of ribosome-attached nascent proteins remain poorly defined. In pursuit of such mechanisms, we discovered dual cooperation of the HSP70 family member HSPA1 with the 26S proteasome: First, in response to proteotoxic stress, HSPA1 promotes proteasome recruitment to translating 80S ribosomes in a manner independent of nascent chain ubiquitylation. Secondly, HSPA1, in association with its cognate nucleotide exchange factor HSPH1, maintains co-translationally ubiquitylated proteins in a soluble state required for efficient proteasomal degradation. Both mechanisms conspire to confer thermotolerance to cells and to promote the growth of esophageal cancer cells in vitro and in animals. Consistent with these observations, HSPH1 knockout impedes tumor growth in vitro and in animals and correlates with favorable prognosis in digestive tract cancers, thus nominating HSPH1 as a cancer drug target.

Highlights

Proteotoxic stress causes translational arrest, co-translational protein ubiquitylation, and proteasome recruitment to ribosomes

Co-translational proteasome recruitment is independent of nascent chain ubiquitylation but is augmented by HSPA1

HSPA1-HSPH1 disaggregase confers thermotolerance by maintaining the solubility and proteasomal clearance of ubiquitylated proteins

Low HSPH1 impedes co-translational thermotolerance and tumor growth and correlates with favorable prognosis in various cancers
Functional study of the AMD-associated geneTMEM97in retinal pigmented epithelium using CRISPR interference
Authors: Wang Jiang-Hui , Urrutia-Cabrera Daniel , Mora Santiago Mesa , Nguyen Tu , Hung Sandy , Hewitt Alex W. , Edwards Thomas L. , Wong Raymond C.B.
Published: July 2020

Age-related macular degeneration (AMD) is a blinding disease characterised by dysfunction of the retinal pigmented epithelium (RPE) which culminates in disruption or loss of the neurosensory retina. Genome-wide association studies have identified >65 genetic risk factors for AMD, including theTMEM97locus.TMEM97encodes the Sigma-2 receptor which is involved in apoptosis and cytotoxicity across a range of neurodegenerative diseases. However, the expression pattern ofTMEM97in the human retina and its functional role in retinal cells has remained elusive. Here we utilised CRISPR interference (CRISPRi) to investigate the functional role ofTMEM97in the retina. Transcriptome analysis of all major cell types within the human retina showed thatTMEM97is expressed in the RPE, retinal ganglion cells (RGCs) and amacrine cells. Using CRISPRi, we performed loss-of-function study ofTMEM97in the human RPE cell line, ARPE19. We generated a stable ARPE19 cell line expressing dCas9-KRAB which facilitated knockdown ofTMEM97using specific sgRNAs. Our results show that knockdown ofTMEM97in ARPE19 exerts a protective effect against oxidative stress-induced cell death. This work provides the first functional study ofTMEM97in RPE and supports the role ofTMEM97in AMD pathobiology. Our study highlights the potential for using CRISPRi to study AMD genetics, and the CRISPRi cell line generated here provided an usefulin vitrotool for functional studies of other AMD-associated genes.
Aedes aegyptiSNAP and a calcium transporter ATPase influence dengue virus dissemination
Authors: Marin-Lopez Alejandro , Jiang Junjun , Wang Yuchen , Cao Yongguo , MacNeil Tyler , Hastings Andrew K , Fikrig Erol
Published: July 2020

Dengue virus (DENV) is a flavivirus that causes marked human morbidity and mortality worldwide, being transmitted to humans byAedes aegyptimosquitoes. Habitat expansion ofAedes, mainly due to climate change and increasing overlap between urban and wild habitats, places nearly half of the world’s population at risk for DENV infection. After a bloodmeal from a DENV-infected host, the virus enters the mosquito midgut. Next, the virus migrates to, and replicates in, other tissues, like salivary glands. Successful viral transmission occurs when the infected mosquito takes another blood meal on a susceptible host and DENV is released from the salivary gland via saliva into the skin. During viral dissemination in the mosquito and transmission to a new mammalian host, DENV interacts with a variety of vector proteins, which are uniquely important during each phase of the viral cycle. Our study focuses on the interaction between DENV particles and protein components in theA. aegyptivector. We performed a mass spectrometry assay where we identified a set ofA aegyptisalivary gland proteins which potentially interact with the DENV virion. Using dsRNA to silence gene expression, we analyzed the role of these proteins in viral infectivity. Two of these candidates, a synaptosomal-associated protein (AeSNAP) and a calcium transporter ATPase (ATPase) appear to play a role in viral replication bothin vitroandin vivo. These findings suggest that AeSNAP plays a protective role during DENV infection of mosquitoes and that ATPase protein is required for DENV during amplification within the vector.

Importance

Aedes aegyptimosquitoes are the major vector of different flaviviruses that cause human diseases, including dengue virus. There is a great need for better therapeutics and preventive vaccines against flaviviruses. Flaviviruses create complex virus-host and virus-vector interactions. The interactions between viral particles and protein components in the vector is not completely understood. In this work we characterize how two mosquito proteins, “AeSNAP” and “ATPase”, influence DENV viral dissemination withinA. aegypti, using bothin vitroandin vivomodels. These results elucidate anti-vector measures that may be potentially be used to control dengue virus spread in the mosquito vector.
The Ndr/LATS kinase Cbk1 regulates a specific subset of Ace2 functions and suppresses the hyphae-to-yeast transition inCandida albicans
Authors: Wakade Rohan S. , Ristow Laura C. , Stamnes Mark A. , Kumar Anuj , Krysan Damian J.
Published: July 2020

TheRegulation ofAce2 andMorphogenesis (RAM) pathway is an important regulatory network in the human fungal pathogenCandida albicans. The RAM pathway’s two most well-studied components, the NDR/Lats kinase Cbk1 and its putative substrate, the transcription factor Ace2, have a wide range of phenotypes and functions. It is not clear, however, which of these functions are specifically due to the phosphorylation of Ace2 by Cbk1. To address this question, we first compared the transcriptional profiles ofCBK1andACE2deletion mutants. This analysis indicates that, of the large number of genes whose expression is affected by deletion ofCBK1andACE2, only 5.5% of those genes are concordantly regulated. Our data also suggest that Ace2 directly or indirectly represses a large set of genes during hyphal morphogenesis. Second, we generated strains containingACE2alleles with alanine mutations at the Cbk1 phosphorylation sites. Phenotypic and transcriptional analysis of theseace2mutants indicates that, as inSaccharomyces cerevisiae, Cbk1 regulation is important for daughter cell localization of Ace2 and cell separation during yeast phase growth. In contrast, Cbk1 phosphorylation of Ace2 plays a minor role inC. albicansyeast-to-hyphae transition. We have, however, discovered a new function for the Cbk1-Ace2 axis. Specifically, Cbk1 phosphorylation of Ace2 prevents the hyphae-to-yeast transition. To our knowledge, this is one of the first regulators of theC. albicanshyphae-to-yeast transition to be described. Finally, we present an integrated model for the role of Cbk1 in the regulation of hyphal morphogenesis inC. albicans.

Importance

Regulation ofAce2 andMorphogenesis (RAM) pathway is a key regulatory network that plays a role in many aspects ofC. albicanspathobiology. In addition to characterizing the transcriptional effects of this pathway, we discovered that Cbk1 and Ace2, a key RAM pathway regulator-effector pair, mediate a specific set of the overall functions of the RAM pathway. We have also discovered a new function for the Cbk1-Ace2 axis; suppression of the hyphae-to-yeast transition. Very few regulators of this transition have been described and our data indicate that maintenance of hyphal morphogenesis requires suppression of yeast phase growth by Cbk1-regulated Ace2.
Antibody responses to a suite of novel serological markers for malaria surveillance demonstrate strong correlation with clinical and parasitological infection across seasons and transmission settings in The Gambia
Authors: Wu Lindsey , Mwesigwa Julia , Affara Muna , Bah Mamadou , Correa Simon , Hall Tom , Beeson James G. , Tetteh Kevin K.A. , Kleinschmidt Immo , D’Alessandro Umberto , Drakeley Chris
Published: July 2020

Background

As malaria transmission declines, sensitive diagnostics are needed to evaluate interventions and monitor transmission. Serological assays measuring malaria antibody responses offer a cost-effective detection method to supplement existing surveillance tools.

Methods

A prospective cohort study was conducted from 2013 to 2015 in 12 villages across five administrative regions in The Gambia. Serological analysis included samples from the West Coast Region at the start and end of the season (July and December 2013) and from the Upper River Region in July and December 2013, and April and December 2014. Antigen-specific antibody responses to eight Plasmodium falciparum (P.falciparum) antigens – Etramp5.Ag1, GEXP18, HSP40.Ag1, Rh2.2030, EBA175 RIII-V, PfMSP119, PfAMA1, PfGLURP.R2 - were quantified using a multiplexed bead-based assay. The association between antibody responses and clinical and parasitological endpoints was estimated at the individual, household, and population level.

Results

Strong associations were observed between clinical malaria and concurrent sero-positivity to Etramp5.Ag1 (aOR 4.60 95%CI 2.98 - 7.12), PfMSP119 (aOR 4.09 95%CI 2.60 - 6.44), PfAMA1 (aOR 2.32 95%CI 1.40 - 3.85) and PfGLURP.R2 (aOR 3.12, 95%CI 2.92 – 4.95), while asymptomatic infection was associated with sero-positivity to all antigens. Village-level sero-prevalence amongst children 2-10 years against Etramp5.Ag1, HSP40.Ag1, and PfMSP119 showed the highest correlations with clinical and P.falciparum infection incidence rates. For all antigens, there was increased odds of asymptomatic P.falciparum infection in subjects residing in a compound with greater than 50% sero-prevalence, with a 2- to 3-fold increase in odds of infection associated with Etramp5.Ag1, GEXP18, Rh2.2030, PfMSP119 and PfAMA1. For individuals residing in sero-positive compounds, the odds of clinical malaria were reduced, suggesting a protective effect.

Conclusions

At low transmission, long-lived antibody responses could indicate foci of malaria transmission that have been ongoing for several seasons or years. In settings where sub-patent infections are prevalent and fluctuate below the detection limit of polymerase chain reaction (PCR), the presence of short- lived antibodies may indicate recent infectivity, particularly in the dry season when clinical cases are rare. Serological responses may reflect a persistent reservoir of infection, warranting community- targeted interventions if individuals are not clinically apparent but have the potential to transmit. Therefore, serological surveillance at both the individual and household level may be used to target interventions where there are foci of asymptomatically infected individuals.