bluuebunny/crossref_metadata_2025 huggingface dataset:
Community-level characteristics of COVID-19 vaccine hesitancy in England: A nationwide cross-sectional study
Authors: Georges Bucyibaruta , Marta Blangiardo , Garyfallos Konstantinoudis
Published: March 2022

One year after the start of the COVID-19 vaccination programme in England, more than 43 million people older than 12 years old had received at least a first dose. Nevertheless, geographical differences persist, and vaccine hesitancy is still a major public health concern; understanding its determinants is crucial to managing the COVID-19 pandemic and preparing for future ones. In this cross-sectional population-based study we used cumulative data on the first dose of vaccine received by 01-01-2022 at Middle Super Output Area level in England. We used Bayesian hierarchical spatial models and investigated if the geographical differences in vaccination uptake can be explained by a range of community-level characteristics covering socio-demographics, political view, COVID-19 health risk awareness and targeting of high risk groups and accessibility. Deprivation is the covariate most strongly associated with vaccine uptake (Odds Ratio 0.55, 95%CI 0.54-0.57; most versus least deprived areas). The most ethnically diverse areas have a 38% (95%CI 36-40%) lower odds of vaccine uptake compared with those least diverse. Areas with the highest proportion of population between 12 and 24 years old had lower odds of vaccination (0.87, 95%CI 0.85-0.89). Finally increase in vaccine accessibility is associated with higher COVID-19 uptake (OR 1.07, 95%CI 1.03-1.12). Our results suggest that one year after the start of the vaccination programme, there is still evidence of inequalities in uptake, affecting particularly minorities and marginalised groups. Strategies including prioritising active outreach across communities and removing practical barriers and factors that make vaccines less accessible are needed to level up the differences.
Plasma alkaline phosphatase is associated to mortality risk in aortic valve stenosis patients
Authors: Nunes José Pedro L.
Published: March 2022

Background

aortic valve stenosis is an important clinical condition, with a significant mortality rate in the elderly. Plasma values of alkaline phosphatase (ALP) have been shown to act as a marker of prognosis in different clinical conditions and in the general population.

Methods

Plasma levels of alkaline phosphatase were studied in a cohort of patients with aortic valve stenosis, and a five-year survival evaluation was carried out.

Results

24 patients were under study, of which 12 were dead at 5-year follow-up. The median age at baseline evaluation was 79 years (interquartile range, 72-85 years), and 11 patients were of the female sex (13 were male). The median value of ALP, of 83 IU/L, was used to separate patients into two groups; 2 patients died in the group with low ALP values versus 10 patients who died in the group with high ALP values. Using ALP with the same cut-off level, Kaplan Meyer study with log-rank analysis showed a significance level <0.01. Cox regression analysis showed an overall significant result, with a significant level for plasma ALP (significance level 0.03), but not for age, sex or transvalvular gradient (assessed by echocardiography).

Conclusions

Elevated plasma ALP is associated to increased mortality risk in aortic valve stenosis patients. This finding may merit evaluation in studies with a larger number of patients.
High Dimensional Mediation Analysis: a new method applied to maternal smoking, placental DNA methylation and birth outcomes
Authors: Jumentier Basile , Barrot Claire-Cécile , Estavoyer Maxime , Tost Jorg , Heude Barbara , François Olivier , Lepeule Johanna
Published: March 2022

Background

High-dimensional mediation analysis is an extension of unidimensional mediation analysis that includes multiple mediators, and is increasingly used to evaluate the indirect omics-layer effects of environmental exposures on health outcomes. Analyses involving high-dimensional mediators raise several statistical issues. While many methods have recently been developed, no consensus has been reached about the optimal combination of approaches to high-dimensional mediation analyses.

Objectives

We developed and validated a method for high-dimensional mediation analysis (HDMAX2) and applied it to evaluate the causal role of placental DNA methylation in the pathway between exposure to maternal smoking (MS) during pregnancy and gestational age (GA) and weight (BW) of the baby at birth.

Methods

HDMAX2 combines latent factor regression models for epigenome-wide association studies with max-squared tests for mediation, and considers CpGs and aggregated mediator regions (AMR). HDMAX2 was carefully evaluated on simulated data, and compared to state-of-the-art multi-dimensional epigenetic mediation methods. Then HDMAX2 was applied on data from 470 women of the EDEN cohort.

Results

HDMAX2 demonstrated increased power compared to state-of-the-art multi-dimensional mediation methods, and identified several AMRs not identified in previous mediation analyses of exposure to MS on BW and GA. The results provided evidence for a polygenic architecture of the mediation pathway with an overall indirect effect of CpGs and AMRs of 44.5 g lower BW (32.1% of the total effect). HDMAX2 also identified AMRs having simultaneous effects both on GA and on BW. Among the top hits of both GA and BW analyses, regions located inCOASY, BLCAPandESRP2also mediated the relationship between GA on BW, suggesting a reverse causality in the relationship between GA and the methylome.

Discussion

HDMAX2 outperformed existing approaches and revealed an unsuspected complexity of the causal relationships between exposure to MS and BW at the epigenome-wide level. HDMAX2 is applicable to a wide range of tissues and omic layers.
IgG N-glycans are associated with prevalent and incident complications of type 2 diabetes
Authors: Memarian Elham , Heijmans Ralph , Slieker Roderick C. , Sierra Adriana , Gornik Olga , Beulens Joline WJ , Hanic Maja , Elders Petra , Pascual Julio , Sijbrands Eric , Lauc Gordan , Dotz Viktoria , Barrios Clara , M’t Hart Leen , Wuhrer Manfred , van Hoek Mandy
Published: March 2022

Aims/hypothesis

Inflammation is important in development of type 2 diabetes complications. The N-glycosylation of IgG influences its role in inflammation. Until now, the association of IgG N-glycosylation with type 2 diabetes complications has not been extensively investigated. We hypothesized that N-glycosylation of IgG may be related to development of complications of type 2 diabetes.

Methods

In three independent type 2 diabetes cohorts, IgG N-glycosylation was measured by UPLC (DiaGene n=1815, GenodiabMar n=640) and mass spectrometry (DCS n=1266). We investigated the associations of IgG N-glycosylation (fucosylation, galactosylation, sialylation and bisection) with incident and prevalent nephropathy, retinopathy and macrovascular disease using Cox- and logistic regression, followed by meta-analyses. The models were adjusted for age, sex and additionally for clinical risk factors.

Results

IgG galactosylation was negatively associated with prevalent and incident nephropathy after adjustment for clinical risk factors. Sialylation was negatively associated with incident diabetic nephropathy. For retinopathy, similar associations were found for galactosylation in the basic model. For macrovascular complications, negative associations with galactosylation and sialylation were confined to the cross-sectional analyses.

Conclusions

We showed that IgG N-glycosylation traits are associated with higher prevalence and future development of nephropathy, after correction for clinical risk factors. For other complications, IgG N-glycosylation was associated with their prevalence only, possibly reflecting ongoing vascular inflammation. These findings indicate the predictive potential of IgG N-glycosylation in nephropathy.
“Integration of multimodal data in the developing tooth reveals candidate dental disease genes”
Authors: Winchester Emma Wentworth , Hardy Alexis , Cotney Justin
Published: March 2022

Dental malformations range from rare syndromes to common nonsyndromic phenotypes. These malformations can predispose individuals to dental disease, which can in turn affect systemic health. While many dental phenotypes are heritable, most cases have not been linked to deleterious mutations in single genes. We demonstrate that human and conserved mouse craniofacial enhancers show enrichment of dental phenotype-associated variants. Given these findings in bulk craniofacial tissues, we looked to determine the role of tooth enhancers in this phenomenon. We used ChIP-seq and machine learning to identify enhancers of E13.5 mouse incisors. Multi-tissue comparisons of human and mouse enhancers revealed that putative tooth enhancers had the strongest enrichment of dental phenotype-associated variants, suggesting a role for dysregulation of tooth development in dental phenotypes. To uncover novel dental phenotype-driving genes in the developing tooth we performed coexpression analysis and annotated the contributing cell types of gene modules using scRNAseq. Through integration of chromatin state, bulk gene coexpression, and cell type resolved gene expression we prioritized a list of candidate novel dental disease genes for future investigations in mouse models and human studies.
Metabolite interactions in the bacterial Calvin cycle and implications for flux regulation
Authors: Sporre Emil , Karlsen Jan , Schriever Karen , Samuelsson Johannes Asplund , Janasch Markus , Strandberg Linnéa , Kotol David , Zeckey Luise , Piazza Ilaria , Syrén Per-Olof , Edfors Fredrik , Hudson Elton P.
Published: March 2022

Metabolite-level regulation of enzyme activity is important for microbes to cope with environmental shifts. Knowledge of such regulations can also guide strain engineering to improve industrial phenotypes. Recently developed chemoproteomics workflows allow for genome-wide detection of metabolite-protein interactions that may regulate pathway activity. We applied limited proteolysis small molecule mapping (LiP-SMap) to identify and compare metabolite-protein interactions in the proteomes of two cyanobacteria and two lithoautotrophic bacteria that fix CO2using the Calvin cycle. Clustering analysis of the hundreds of detected interactions showed that some metabolites interacted in a species-specific manner, such as interactions of glucose-6-phosphate inCupriavidus necatorand of glyoxylate inSynechocystis spPCC 6803. These are interpreted in light of the different central carbon conversion pathways present. Metabolites interacting with the Calvin cycle enzymes fructose-1,6/sedoheptulose-1,7-bisphosphatase (F/SBPase) and transketolase were tested for effects on catalytic activityin vitro. The Calvin cycle intermediate glyceraldehyde-3-phosphate activated bothSynechocystisandCupriavidusF/SBPase, which suggests a feed-forward activation of the cycle in both photoautotrophs and chemolithoautotrophs. In contrast to the stimulating effect in reduced conditions, glyceraldehyde-3-phosphate inactivated theSynechocystisF/SBPase in oxidized conditions by accelerating protein aggregation. Thus, metabolite-level regulation of the Calvin cycle is more prevalent than previously appreciated and may act in addition to redox regulation.
Tripartite extended amygdala - basal ganglia CRH circuit drives arousal and avoidance behaviour
Authors: Chang Simon , Fermani Federica , Lao Chu-Lan , Huang Lianyun , Jakovcevski Mira , Giaimo Rossella Di , Gagliardi Miriam , Menegaz Danusa , Hennrich Alexander Adrian , Ziller Michael , Eder Matthias , Klein Rüdiger , Cai Na , Deussing Jan M.
Published: March 2022

An adaptive stress response involves various mediators and circuits orchestrating a complex interplay of physiological, emotional and behavioural adjustments. We identified a population of corticotropin-releasing hormone (CRH) neurons in the lateral part of the interstitial nucleus of the anterior commissure (IPACL) – a subdivision of the extended amygdala, which exclusively innervate the substantia nigra (SN). Specific stimulation of this circuit elicits arousal and avoidance behaviour contingent on CRH receptor 1 (CRHR1) located at axon terminals in the SN, which originate from external globus pallidus (GPe) neurons. The neuronal activity prompting the observed behaviour is shaped by IPACLCRH and GPeCRHR1 neurons coalescing in the SN. These results delineate a novel tripartite CRH circuit functionally connecting extended amygdala and basal ganglia nuclei to drive arousal and avoidance behaviour.

One-Sentence Summary

Brain centres involved in emotional and motor control are connected through a stress peptide promoting arousal and avoidance behaviour
Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum supports social novelty preference
Authors: de León Reyes Noelia Sofia , Díaz Paula Sierra , Nogueira Ramon , Ruiz-Pino Antonia , Nomura Yuki , de Solis Christopher , Schulkin Jay , Asok Arun , Leroy Félix
Published: March 2022

Social preference, the decision to interact with one member of the same species over another, is a key feature of optimizing social interactions. In rodents, social preference relies on both extrinsic factors, such as sex, strain and kinship, and intrinsic ones, such as the memory of previous encounters, which favors interactions with novel compared to familiar animals (social novelty preference). At present, it is unclear which neuronal circuits guide social preferences and whether such circuits promote social interactions with the preferred individuals or suppress interactions with the non-preferred ones. Although both the infra-limbic area of the pre-frontal cortex (ILA) and the lateral septum (LS) have been shown to support social novelty preference, the neuronal circuits and molecular mechanisms by which these brain regions interact to regulate social interactions are unknown. Here, we identify a population of inhibitory neurons in ILA that express the neuropeptide corticotropin releasing hormone (CRH) and project to the rostro-dorsal region of LS (rdLS). Release of CRH from ILA in rdLS during interactions with familiar mice disinhibits rdLS neurons, thereby suppressing interactions with familiar mice and contributing to social novelty preference. We further demonstrate how the maturation of CRH expression during the first two post-natal weeks enables the developmental shift from a preference for littermates in juveniles to a preference for novel mice in adults.
Induction of cortical Par complex polarity by designed proteins causes cytoskeletal symmetry breaking in unpolarized mammalian cells
Authors: Watson Joseph L. , Ben-Sasson Ariel J. , Bittleston Alice , Manton James D. , Baker David , Derivery Emmanuel
Published: March 2022

Polarized cells rely on a polarized cytoskeleton for polarized trafficking, oriented migration and spindle orientation during asymmetric cell division. While cytoskeleton remodeling machineries have been extensively characterized at the molecular level, how polarity signaling at the cortex controls remodeling of the cytoskeleton in the cytosol remains elusive. In particular, how the Par complex, the conserved mastermind of polarity during asymmetric cell division, gets assembled and functions is not understood at the molecular level. Here, we dissected the logic of the Par complex pathway by capitalizing on designed proteins able to induce spontaneous symmetry breaking of the cortex in populations of naïve, unpolarized cells. We found that the primary kinetic barrier to Par complex assembly is the relief of Par6 autoinhibition, and that inducing Par complex cortical polarity was sufficient to induce two key hallmarks of asymmetric cell division in unpolarized cells: spindle orientation and central spindle asymmetry. These two outputs of the Par complex are separately controlled: spindle orientation is determined by Par3 and does not require the kinase activity of aPKC, while central spindle asymmetry solely depends on an asymmetric activity of aPKC at the cortex. Our work shows how polarity information flows between the cortex and the cytosol despite its diffusive nature, and paves the way towards induction of asymmetric cell division in cultured cells.
Expressed therapeutic protein yields are predicted by transiently transfected mammalian cell population
Authors: Porosk Ly , Nebogatova Jekaterina , Härk Heleri Heike , Vunk Birgit , Arukuusk Piret , Toots Urve , Ustav Mart , Langel Ülo , Kurrikoff Kaido
Published: March 2022

Therapeutic proteins are currently at the hotspot of innovation in the pharmaceutical medicine. However, their industrial production is technically challenging and improved methods for transcriptional manipulation of mammalian industrial cell cultures are needed. In this work we show that some of the most frequently used lab scale transfection efficacy assays fail to predict performance in the protein production settings. We compare the efficacies of a number of transfection reagents using adherent and suspension mammalian cell cultures and assessment based on several assays that utilize reporter protein quantitation, transfected cell population and post-transfection viability of cells. We validate reporter assays for assessing transfection methods in the lab that predict protein production in industrial settings. We also demonstrate that cell penetrating peptide-based transfection achieve significantly higher protein yields compared to PEI and lipoplex methods in both CHO and HEK293 producer cell lines. Availability of fast lab scale screening methods allows future development of improved transfection methods for protein production. One such potentially effective transient transfection method is the CPP-based approach presented currently.
Targeting A-Kinase Anchoring Protein 12 Phosphorylation in Hepatic Stellate Cells Regulates Liver Injury and Fibrosis in Mouse Models
Authors: Ramani Komal , Mavila Nirmala , Abeynayake Aushinie , Tomasi Maria Lauda , Wang Jiaohong , Matsuda Mitchitaka , Seki Ekihiro
Published: March 2022

Trans-differentiation of hepatic stellate cells (HSCs) to activated state potentiates liver fibrosis through release of extracellular matrix (ECM) components, distorting the liver architecture. Since limited antifibrotics are available, pharmacological intervention targeting activated HSCs may be considered for therapy. A-kinase anchoring protein 12 (AKAP12) is a scaffolding protein that directs protein kinases A/C (PKA/PKC) and cyclins to specific locations spatiotemporally controlling their biological effects. It has been shown that AKAP12’s scaffolding functions are altered by phosphorylation. In previously published work, observed an association between AKAP12 phosphorylation and HSC activation. In this work we demonstrate that AKAP12’s scaffolding activity towards the endoplasmic reticulum (ER)-resident collagen chaperone, heat-shock protein 47 (HSP47) is strongly inhibited by AKAP12’s site-specific phosphorylation in activated HSCs. CRISPR-directed gene editing of AKAP12’s phospho- sites restores its scaffolding towards HSP47, inhibiting HSP47’s collagen maturation functions and HSC activation. AKAP12 phospho-editing dramatically inhibits fibrosis, ER stress response, HSC inflammatory signaling and liver injury in mice. Our overall findings suggest a pro-fibrogenic role of AKAP12 phosphorylation that may be targeted for therapeutic intervention in liver fibrosis.
Mechanistic insight into the ATP hydrolysis cycle of tick-borne encephalitis virus helicase
Authors: Anindita Paulina Duhita , Halbeisen Marco , Řeha David , Tůma Roman , Franta Zdeněk
Published: March 2022

Helicase domain of nonstructural protein 3 (NS3H) unwinds double-stranded RNA replication intermediate during flavivirus life cycle in ATP-dependent manner. While the helicases mechanism of Dengue and Zika viruses has been extensively studied, little is known about helicase activity of the tick-borne encephalitis virus NS3. In the current study, we demonstrated that ATP hydrolysis cycle of NS3H is strongly stimulated by ssRNA but not ssDNA, suggesting that NS3H is an RNA-specific helicase. However, ssDNA binding inhibits ATPase activity in a non-competitive manner. We also captured several structural snapshots of key ATP hydrolysis stages. An intermediate, in which the inorganic phosphate (Pi) and ADP resulted from ATP hydrolysis and remain trapped inside the ATPase site, suggests that Pi release is the rate-limiting step and is accelerated by RNA binding and/or translocation. Based on these structures, we modeled NS3H-ssRNA and -ssDNA binding and performed MD simulations. Our model suggests that NS3H-ssRNA binding triggers conformational changes, revealing the coupling between helicase and ATPase activities. The structural models revealed that ssDNA inhibition may occur via non-specific ssDNA binding to several positively charged surface patches which in turn causes repositioning of ATP molecule within the ATPase site.
Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain
Authors: Wörle Elisabeth , Newman Anthony , Burgio Gaetan , Grohmann Dina
Published: March 2022

Nucleases derived from the prokaryotic defense system CRISPR-Cas are frequently re-purposed for gene editing and molecular diagnostics. Hence, an in-depth understanding of the molecular mechanisms of these enzymes is of crucial importance. We focused on Cas12a from Francisella novicida (FnCas12a) and investigated the functional role of helix 1, a structural element that together with the bridge helix (BH) connects the recognition and the nuclease lobes of FnCas12a. Helix 1 is structurally connected to the lid domain that opens upon DNA target loading thereby activating the active site of FnCas12a. We probed the structural states of FnCas12a variants altered in helix 1 and/or the BH using single-molecule FRET measurements and assayed the pre-crRNA processing, cis- and trans-DNA cleavage activity. We show that helix 1 and not the BH is the predominant structural element that confers conformational stability of FnCas12a. Even small perturbations in helix 1 lead to a decrease in DNA cleavage activity while the structural integrity is not affected. Our data, therefore, implicate that the concerted remodeling of helix 1 and the BH upon DNA binding is structurally linked to the opening of the lid and therefore involved in the allosteric activation of the active site.
Strain stiffening of Ndc80 complexes attached to microtubule plus ends
Authors: Schwietert Felix , Volkov Vladimir A. , Huis in ’t Veld Pim J. , Dogterom Marileen , Musacchio Andrea , Kierfeld Jan
Published: March 2022

In the mitotic spindle, microtubules attach to chromosomes via kinetochores. The microtubule-binding Ndc80 complex is an integral part of kinetochores, and is essential for kinetochores to attach to microtubules and to transmit forces from dynamic microtubule ends to the chromosomes. The Ndc80 complex has a rod-like appearance with globular domains at its ends that are separated by a long coiled coil. Its mechanical properties are considered important for the dynamic interaction between kinetochores and microtubules. Here, we present a novel method that allows us to time-trace the effective stiffness of Ndc80 complexes following shortening microtubule ends against applied force in optical trap experiments. Applying this method to wild type Ndc80 and three variants (CH-domains mutated or Hec1-tail unphosphorylated, phosphorylated, or truncated), we reveal that each variant exhibits strain stiffening, i.e., the effective stiffness increases under tension that is built up by a depolymerizing microtubule. The strain stiffening relation is roughly linear and independent of the state of the microtubule. We introduce structure-based models, which show that the strain stiffening can be traced back to the specific architecture of the Ndc80 complex with a characteristic flexible kink, to thermal fluctuations of the microtubule, and to the bending elasticity of flaring protofilaments, which exert force to move the Ndc80 complexes. Our model accounts for changes in the amount of load-bearing attachments at various force levels and reproduces the roughly linear strain stiffening behavior, highlighting the importance of force-dependent binding affinity.

SIGNIFICANCE

By time-tracing the stiffness of microtubule end-tracking Ndc80 complexes in optical trap experiments, we detect strain stiffening, and, thereby, provide new insights into the elastic properties of the Ndc80 complex. The strain stiffening is robust against mutations in the Ndc80 complex. We relate strain stiffening to the structure of the Ndc80 complex by means of a simple polymer model, to thermal fluctuations of the microtubule, and to the flexibility of force-generating flaring protofilaments at the tip of the microtubule. Since Ndc80 complexes play a major role for transmitting force from microtubule ends to the kinetochore, their elastic properties are of great interest for a deeper understanding of chromosome dynamics in the mitotic spindle.
Neonate personality affects early-life resource acquisition in a large social mammal
Authors: Amin Bawan , Jennings Dómhnall J. , Norman Alison , Ryan Andrew , Ioannidis Vasiliki , Magee Alice , Haughey Hayley-Anne , Haigh Amy , Ciuti Simone
Published: March 2022

Current debate in the field of animal personality revolves around whether personality is reflecting individual differences in resource allocation or acquisition. Despite the large body of literature, the proximate relationships between personality, resource allocation, and acquisition are still unclear, especially during early stages of development. Here we studied how among-individual differences in behaviour develop over the first 6 months of life, and their potential association with resource acquisition in a free-ranging population of fallow deer (Dama dama). We related proxies of neonate personality – i.e. neonate physiological (heart rate) and behavioural (latency to leave at release) anti-predator responses to human handling – to the proportion of time fawns allocated to scanning during their first summer and autumn of life. We then investigated whether there was a trade-off between scanning time and foraging time in these juveniles, and how it developed over their first 6 months of life. We found that neonates with longer latencies at capture (i.e. risk-takers) allocated less time scanning their environment, but that this relationship was only present when fawns were 3-6 months old during autumn, but not when fawns were only 1-2 months old during summer. We also found that time spent scanning was negatively related to time spent foraging – a relationship rarely tested in juveniles of large mammals - and that this relationship becomes stronger over time, as fawns gradually switch from a nutrition rich (milk) to a nutrition poor (grass) diet. Our results highlight a potential mechanistic pathway in which neonate personality may drive differences in early-life resource acquisition, through allocation, of a large social mammal.
OnRamp: rapid nanopore plasmid validation
Authors: Mumm Camille , Drexel Melissa L. , McDonald Torrin L. , Diehl Adam G. , Switzenberg Jessica A. , Boyle Alan P.
Published: March 2022

Recombinant plasmid vectors are versatile tools which have facilitated discoveries in molecular biology, genetics, proteomics, and many other fields. As the enzymatic and bacterial processes used to create recombinant DNA can introduce errors, sequence validation is an essential step in plasmid assembly. Sanger sequencing is the current standard for plasmid validation, however this method is limited by an inability to sequence through complex secondary structure and lacks scalability when applied to full-plasmid sequencing of multiple plasmids due to read-length limits. While next-generation sequencing (NGS) does provide full-plasmid sequencing at scale, it is impractical and costly when utilized outside of library-scale validation. Here we present OnRamp (Oxford nanopore-based Rapid Analysis of Multiplexed Plasmids), an alternative method for routine plasmid validation which combines the advantages of NGS’s full plasmid coverage and scalability with Sanger’s affordability and accessibility by leveraging nanopore’s novel long-read sequencing technology. We include customized wet-lab protocols for plasmid preparation along with a pipeline designed for analysis of read data obtained using these protocols. This analysis pipeline is built into the OnRamp webapp (http://OnRamp.BoyleLab.org), which generates alignments between actual and predicted plasmid sequences, quality scores, and read-level views in a user-friendly manner, precluding the need for programming experience in analyzing nanopore results. Here we describe the OnRamp protocols and pipeline, and demonstrate our ability to obtain full sequences from pooled plasmids while detecting sequence variation even in regions of high secondary structure, at less than half the cost of equivalent Sanger sequencing.
Reproducibility problem with a proposed standard method to measure disinfection efficacy
Authors: Hilgren John , Srikanth Mrudula , Brutofsky Milady , Mainquist Keith , Prew Paul , King William , Jones Rhonda , Hellickson Lisa , Luedtke Josh , Restrepo Kristie , Brill Florian , Quinn Patrick
Published: March 2022

A collaborative study was carried out in four laboratories to determine the reproducibility of a proposed ASTM International standard method for quantitatively evaluating the efficacy of disinfectants on hard, non-porous surfaces against bacteria. The method, known as the Quantitative Method, has also been suggested as a future regulatory standard for the United States and internationally. The multi-lab study was carried out using Pseudomonas aeruginosa and an alkyl dimethyl benzyl ammonium chloride antimicrobial product diluted in hard water. Results of the study showed acceptable repeatability in log10 reductions within each laboratory, but unacceptable reproducibility across laboratories despite careful analyst training and standardization of test conditions. A follow-up study ruled out analyst-to-analyst differences as the cause of the poor reproducibility. As it currently exists, the Quantitative Method is not sufficiently reproducible. Ruggedness testing to assess the sensitivity of the method to small changes in operational factors is recommended.
Production of novel biosurfactant by a new yeast species isolated from Prunus mume Sieb. et Zucc
Authors: Kim Jeong-Seon , Lee Miran , Ki Dae-Won , Kwon Soon-Wo , Ko Young-Joon , Kim Jong-Shik , Yun Bong-Sik , Kim Soo-Jin
Published: March 2022

Biosurfactants reduce surface and interfacial tension due to their amphiphilic properties, and are an eco-friendly alternative for chemical surfactants. In this study, a novel yeast strain JAF-11 that produces biosurfactant was selected using drop collapse method, and the properties of the material were investigated. The nucleotide sequences of the strain were compared with closely related strains and identified based on the D1/D2 domain of the large-subunit rDNA (LSU) and internal transcribed spacer (ITS) regions. Neodothiora populina CPC 39399T, the closest species with strain JAF-11 in the phylogenetic tree, showed a sequence similarity of 97.75% for LSU and 94.27% for ITS, respectively. The result suggests that the strain JAF-11 represent a distinct species which cannot be assigned to any existing genus or species in the family Dothideaceae. Strain JAF-11 was able to produce biosurfactant reducing the surface tension of medium to 34.5 mN/m on the 6th day of culture and the result of measuring the critical micelle concentration (CMC) by extracting the crude biosurfactant was found to be 24 mg l-1. The molecular weight 502 of the purified biosurfactant was confirmed by measuring the fast atom bombardment mass spectrum (FAB-MS). The chemical structure was analyzed by measuring 1H nuclear magnetic resonance (NMR), 13C NMR, two-dimensional NMRs of the compound. The molecular formula was C26H46O9, and it was composed of one octanoyl group and two hexanoyl group to myo-inositol moiety. The new biosurfactant is the first report of a compound produced by a novel yeast strain JAF-11. This new biosurfactant is proposed as potential candidate for use in a variety field.
Growth inhibition of Akkermansia muciniphila by a secreted pathobiont sialidase
Authors: van Muijlwijk Guus H. , Bröring Emma , van Mierlo Guido , Jansen Pascal W.T.C. , Vermeulen Michiel , Aerts Piet C. , van Putten Jos P.M. , Palm Noah W. , de Zoete Marcel R.
Published: March 2022

Akkermansia muciniphila is considered a key constituent of a healthy gut microbiota. In inflammatory bowel disease (IBD), A. muciniphila has a reduced abundance while other, putative pathogenic, mucus colonizers bloom. We hypothesized that interbacterial competition may contribute to this observation. By screening the supernatants of a panel of enteric bacteria, we discovered that a previously uncharacterized Allobaculum species potently inhibits the growth of A. muciniphila. Mass spectrometry analysis identified a secreted Allobaculum sialidase as inhibitor of A. muciniphila growth. The sialidase targets sialic acids on casein O-glycans, thereby altering the accessibility of nutrients critical for A. muciniphila. The altered glycometabolic niche results in distorted A. muciniphila cell division and efficiently arrests its growth. The identification of a novel mechanism of A. muciniphila growth inhibition by a competing bacterial pathobiont may provide a rationale for interventions aimed at restoring and maintaining a healthy microbiota symbiosis in patients with intestinal disease.
Decorating chromatin for enhanced genome editing using CRISPR-Cas9
Authors: Chen Evelyn , Lin-Shiao Enrique , Trinidad Marena , Doost Mohammad Saffari , Colognori David , Doudna Jennifer A.
Published: March 2022

CRISPR-associated (Cas) enzymes have revolutionized biology by enabling RNA-guided genome editing. Homology-directed repair (HDR) in the presence of donor templates is currently the most versatile method to introduce precise edits following CRISPR-Cas-induced double-stranded DNA cuts, but HDR efficiency is generally low relative to end-joining pathways that lead to insertions and deletions (indels). We tested the hypothesis that HDR could be increased using a Cas9 construct fused to PRDM9, a chromatin remodeling factor that deposits histone methylations H3K36me3 and H3K4me3 to mediate homologous recombination in human cells. Our results show that the fusion protein contacts chromatin specifically at the Cas9 cut site in the genome to increase the observed HDR efficiency by three-fold and HDR:indel ratio by five-fold compared to that induced by unmodified Cas9. HDR enhancement occurred in multiple cell lines with no increase in off-target genome editing. These findings underscore the importance of chromatin features for the balance between DNA repair mechanisms during CRISPR-Cas genome editing and provide a new strategy to increase HDR efficiency.

Significance Statement

CRISPR-Cas-mediated homology-directed repair (HDR) enables precision genome editing for diverse research and clinical applications, but HDR efficiency is often low due to competing end-joining pathways. Here, we describe a simple strategy to influence DNA repair pathway choice and improve HDR efficiency by engineering CRISPR-Cas9-methyltransferase fusion proteins. This strategy highlights the impact of histone modifications on DNA repair following CRISPR-Cas-induced double-stranded breaks and adds to the CRISPR genome editing toolbox.
Dual role of an essential HtrA2/Omi protease in the human malaria parasite: maintenance of mitochondrial homeostasis and induction of apoptosis like cell death under cellular stress
Authors: Singh Shweta , Datta Gaurav , Jain Shaifali , Thakur Vandana , Muneer Azhar , Asad Mohd , Ali Shakir , Mohmmed Asif
Published: March 2022

HtrA family of serine protease is known to play role in mitochondrial homeostasis as well as in the programmed cell death. Mitochondrial homeostasis and metabolism are crucial for survival and propagation of malaria parasite within the host. Here we have functionally characterized a Plasmodium falciparum HtrA2 (PfHtrA2) protein, which harbours trypsin like protease activity that can be inhibited by its specific inhibitor, ucf-101. Transgenic parasite line was generated, using HA-glmS C-terminal tagging approach, for localization as well as for inducible knockdown of PfHtrA2. The PfHtrA2 was localized in the parasite mitochondrion during asexual life cycle. Genetic ablation of PfHtrA2 caused significant parasite growth inhibition, decreased replication of mtDNA, increased mitochondrial ROS production, caused mitochondrial fission/fragmentation, and hindered parasite development. However, ucf-101 treatment had no effect on the parasite growth, suggesting non-protease/chaperone role of PfHtrA2 in the parasite. Under cellular stress conditions, inhibition of PfHtrA2 by ucf-101 reduced activation of the caspase like protease as well as parasite cell death, suggesting involvement of protease activity of PfHtrA2 in apoptosis like cell death in the parasite. Under these cellular stress conditions, the PfHtrA2 gets processed but remains localized in the mitochondrion, suggesting that it acts within the mitochondrion by cleaving intra-mitochondrial substrate(s). This was further supported by trans-expression of PfHtrA2 protease domain in the parasite cytosol, which was unable to induce any cell death in the parasite. Overall, we show specific role of PfHtrA2 in maintaining mitochondrial homeostasis as well as in regulating the stress induced cell death.
Artificial Hsp100-mediated systems for re-localizing protein aggregates
Authors: Fischbach Arthur , Johns Angela , Hao Xinxin , Schneider Kara L. , Nyström Thomas
Published: March 2022

Spatial Protein Quality Control (sPQC) sequesters misfolded proteins into specific, organelle-associated inclusions within the cell to harness their toxicity. To approach the role of sPQC in cellular fitness, neurodegenerative diseases and aging, we report on the construction of Hsp100-based systems in yeast cells, which can artificially target protein aggregates to non-canonical locations. We demonstrated that aggregates of mutant Huntingtin (mHtt), the disease-causing agent of Huntington’s disease can be artificially targeted to daughter cells as well as to eisosomes and endosomes with this approach. Removing aggregates from mother cells did not significantly affect their lifespan and targeting mHtt to multiple smaller aggregates rather than one large inclusion did not alter its toxicity. We demonstrated that this approach is able to manipulate mHtt inclusion formation also in human cells and has the potential to be a useful complementation to present therapeutic approaches aimed at alleviating age-related neurodegenerative diseases.
Investigation of wind effect on different quadrotors
Authors: Ercan Hamdi , Ulucan Hamdi , Can Muharrem Selim
Published: March 2022

Purpose

Although windy weather conditions have a significant effect on the flight safety and stability of any aircraft, the fact that quadrotors are lighter than other aircraft makes them more sensitive to the wind. This study aims to examine the extent to which quadrotors and their sensors, which are used in many fields and whose use is expected to increase significantly in the future, are affected by wind.

Design/methodology/approach

Flight experiments were carried out on different routes assigned by using Pixhawk Holybro 4 and Radiolink flight controllers. In these flight experiments, quadrotors were exposed to winds at different speeds and directions.

Findings

In the flight experiments, the deviation amounts in the quadrotor’s route at which wind speed was determined, and it was seen that these deviations were very serious and affected the safe flight at high wind speeds. According to the sensor information obtained from both different quadrotors’ flight experiments at different wind speeds, it was determined that the wind decreased the sensors’ accuracy.

Practical implications

It is foreseen that the data obtained in this study will be a source to be used in the design of quadrotors to be used in public areas in the future and to take the wind into account for safe flight.

Originality/value

In this study, numerous flight tests were carried out experimentally at various speeds from low speeds to high speeds on different routes using different flight controllers. The deviation data on the obtained routes and the effect of the wind on the sensors are experienced in real atmospheric conditions.
Horizontal take-off of an insect-like FMAV based on stroke plane modulation
Authors: Chen Zihao , Zhang Weiping , Mou Jiawang , Zheng Kexin
Published: March 2022

Purpose

Vertical take-off is commonly adopted in most insect-mimicking flapping-wing micro air vehicles (FMAV) while insects also adopt horizontal take-off from the ground. The purpose of this paper is to study how insects adjust their attitude in such a short time during horizontal take-off by means of designing and testing an FMAV based on stroke plane modulation.

Design/methodology/approach

An FMAV prototype based on stroke plane rotating modulation is built to test the flight performance during horizontal take-off. Dynamic gain and decoupling mixer is added to compensate for the nonlinearity during the rotation angle of the stroke plane getting too large at the beginning of take-off. Force/torque test based on a six-axis sensor validates the change of aerodynamic force and torque at different rotation angles. High-speed camera and motion capture system test the flight performance of horizontal take-off.

Findings

Stroke plane modulation can provide a great initial pitch toque for FMAV to realize horizontal take-off. But the large range of rotation angles causes nonlinearity and coupling of roll and yaw. A dynamic gain and a mixer are added in the controller, and the FMAV successfully achieves horizontally taking off in less than 1 s.

Originality/value

The research in this paper shows stroke plane modulation is suitable for insect’s horizontal take-off
Relationship between environmental indoor conditions of a classroom and the performance of undergraduate students
Authors: Paschoalin Filho João Alexandre , Guerner Dias Antonio Jose , Storopoli João Henrique , Ghermandi Andrea , de Carvalho Hendrio Chaves
Published: March 2022

Purpose

This research aims at studying the influence of a classroom’s inner environmental conditions on undergraduate students’ performance using an experimental methodology.

Design/methodology/approach

The Uchida-Kraepelin test (U-K test) was applied to measure the performance of a group of 47 students in a selected classroom that was arranged according to the following experimental conditions: air-conditioning on, and doors and windows closed (D1); doors and windows open, and air-conditioning off (D2); air-conditioning off, and doors and windows closed (D3). After completing the tests, questionnaires were distributed to evaluate the students’ assessment of each set of environmental conditions.

Findings

On-site measurements of humidity and carbon dioxide levels stress the importance of ensuring good natural ventilation through open doors and windows, independently of whether the air-conditioning system is operated or not. Also, the authors find that the students’ self-assessment regarding the inner environmental conditions for each studied set was entirely accurate, with set D3 being assessed as the worst. The U-K test scores for each environmental set did not show statistically significant differences, which means that, in the studied conditions, the student’s performance in the tests was not affected by the inner environmental conditions.

Practical implications

There is a direct relationship between the building’s indoor conditions and an occupant’s health. Factors such as poor maintenance, bad indoor environmental quality (IEQ) and building age will worsen the building’s condition and negatively impact the occupant’s health. Educational buildings with poor IEQ can reduce the concentration and performance of occupants.

Social implications

School is an important place to help students grow in their various capabilities. They spend approximately 30% of their daily lives in schools for their educational activities. Since most of their activities are performed indoors, indoor environmental attributes, such as light, heat, air and sound, should be maintained as required. In general, schools are not thermally comfortable. The extreme thermal environment of classrooms affects students’ concentration. Thermal discomfort may also cause irritation. In addition to reduced concentration, such an environment could also cause tiredness, sluggishness and health problem.

Originality/value

Despite the importance of the issue, scientific investigations of the correlations between students’ performance and the quality of scholar buildings’ inner environmental conditions are still relatively recent. In this context, this research further explores the effect of a classroom’s different environmental inner conditions on the performance of undergraduate students at a university in São Paulo/Brazil.
Nutritional knowledge, eating habits and quality of life of coeliac disease patients
Authors: Ainsworth Dawn , Soon Jan Mei
Published: March 2022

Purpose

Coeliac disease (CD) is a lifelong autoimmune disorder and is managed with a strict gluten-free (GF) diet. At diagnosis, an individual's nutritional status is affected by how long CD has been active, their dietary intake, intestinal inflammation and degree of malabsorption. This study explores if age and time since diagnosis affect nutritional knowledge, eating habits and emotional wellbeing of participants.

Design/methodology/approach

An online survey using Qualtrics was conducted. The survey consists of 4 sections exploring (1) demographics, (2) nutritional knowledge, (3) eating habits and (4) quality of life (QoL). A total of 162 valid questionnaires were completed.

Findings

Those who'd been diagnosed for more than 5 years demonstrated better knowledge about GF or gluten containing products. Social interactions are limited by concerns about becoming ill, unwanted attention and increased financial costs. Eight-eight % of participants would go hungry at social events. Those aged between 40–59 and above 60 years felt more financially restricted compared to younger adults (χ2(4) = 10.73, p = 0.01). Strong emotions were experienced by participants since diagnosed with CD. Anxiety, feelings of concern, sadness, depression and fear have declined and happiness, confidence and being accepting of CD have increased since diagnosis across all years.

Originality/value

This study is one of the first few studies to investigate time since diagnosis and age-related differences in nutritional knowledge, eating habits and QoL of adults diagnosed with CD. Over time, negative emotions could potentially be alleviated with improved knowledge and experience.