bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis
Authors: de Neves Manta Fernanda Saloum , Jacomasso Thiago , de Cássia Pontello Rampazzo Rita , Maria Moreira Suelen Justo , Zahra Najua , Ribeiro-Alves Marcelo , Krieger Marco Aurélio , Costa Alexandre Dias Tavares , Ozório Moraes Milton 
Published: October 2021

AbstractLeprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and trained health professionals. Furthermore, adequate care and transmission control depend on early and reliable pathogen detection. Here, we describe a qPCR test for routine diagnosis of leprosy-suspected patients. The reaction simultaneously amplifies two specific Mycobacterium leprae targets (16S rRNA and RLEP), and the human 18S rRNA gene as internal control. The limit of detection was estimated to be 2.29 copies of M. leprae genome. Analytical specificity was evaluated using a panel of 20 other skin pathogenic microorganisms and Mycobacteria, showing no cross-reactivity. Intra- and inter-operator Cp variation was evaluated using dilution curves of M. leprae DNA or a synthetic gene, and no significant difference was observed between three operators in two different laboratories. The multiplex assay was evaluated using 97 patient samples with clinical and histopathological leprosy confirmation, displaying high diagnostic sensitivity (91%) and specificity (100%). Validation tests in an independent panel of 50 samples confirmed sensitivity and specificity of 97% and 98%, respectively. Importantly, assay performance remained stable for at least five months. Our results show that the newly developed multiplex qPCR effectively and specifically detects M. leprae DNA in skin samples, contributing to an efficient diagnosis that expedites the appropriate treatment.Author SummaryLeprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Disease diagnosis is currently performed on skin examinations for clinical signs, bacilli staining in skin smears and invasive skin biopsies. However, the spectrum of clinical manifestations and the low bacterial load can hinder accurate diagnosis, which is critical for providing proper intervention and adequate care as well as for establishing transmission control. Quantitative PCR (qPCR) methods for detecting bacterial DNA are more sensitive and could aid in differentially diagnosing leprosy from other dermatological conditions. In this work, we present a new multiplex qPCR that detects two bacterial genes for the diagnosis and a human gene as an internal reaction control. The new qPCR, developed using GMP-grade reagents, is highly sensitive, specific, reproducible, and stable. The results presented here are the basis of a novel and robust tool with potential to increase the accuracy of leprosy diagnosis in routine or reference laboratories.
T cell stimulation remodels the latently HIV-1 infected cell population by differential activation of proviral chromatin
Authors: Lindqvist Birgitta , Assi Wlaa , Roux Julie , Love Luca , Jütte Bianca B. , Sönnerborg Anders , Tezil Tugsan , Verdin Eric , Svensson J. Peter 
Published: October 2021

AbstractThe reservoir of latently HIV-1 infected cells is heterogeneous. To achieve an HIV-1 cure, the reservoir of activatable proviruses should be eliminated while permanently silenced proviruses may be tolerated. We have developed a method to assess the proviral nuclear microenvironment in single cells. In latently HIV-1 infected cells, a zinc finger protein tethered to the HIV-1 promoter produced a fluorescent signal as a protein of interest came in its proximity, such as the viral transactivator Tat when recruited to the nascent RNA. Tat is essential for viral replication. In these cells we assessed the proviral activation and chromatin composition. By linking Tat recruitment to proviral activity, we dissected the mechanisms of HIV-1 latency reversal and the consequences of HIV-1 production. A pulse of promoter-associated Tat was identified that contrasted to the continuous production of viral proteins. As expected, promoter H3K4me3 led to substantial expression of the provirus following T cell stimulation. However, the activation-induced cell cycle arrest and death led to a surviving cell fraction with proviruses encapsulated in repressive chromatin. Further, this cellular model was used to reveal mechanisms of action of small molecules. In a proof-of-concept study we determined the effect of an enhancer specific CBP/P300-inhibitor on HIV-1 latency reversal. Only proviruses resembling active enhancers, associated with H3K4me1 and H3K27ac, efficiently recruited Tat. Tat-independent HIV-1 latency reversal of unknown significance still occurred. We present a method for single cell assessment of the microenvironment of the latent HIV-1 proviruses, used here to reveal how T cell stimulation modulates the proviral activity and how the subsequent fate of the infected cell depends on the chromatin context.
Highly Reversible Tunable Thermal-repressible Split-T7 RNA polymerases (Thermal-T7RNAPs) for dynamic gene regulation
Authors: Chee Wai Kit David , Yeoh Jing Wui , Dao Viet Linh , Poh Chueh Loo 
Published: October 2021

SUMMARYTemperature is a physical cue that is easy to apply, allowing cellular behaviors to be controlled in a contactless and dynamic manner via heat-inducible/repressible systems. However, existing heat-repressible systems are limited and rely on thermal sensitive mRNA or transcription factors which function at low temperatures, lack tunability, suffer delays or overly-complex. To provide an alternative mode of thermal regulation, we developed a library of compact, reversible and tunable thermal-repressible split-T7 RNA polymerase systems (Thermal-T7RNAPs) which fuses temperature-sensitive domains of Tlpa protein with split-T7RNAP to enable direct thermal control of the T7RNAP activity between 30 – 42 °C. We generated a large mutant library with varying thermal performances via automated screening framework to extend temperature tunability. Lastly, using the mutants, novel thermal logic circuitry was implemented to regulate cell growth and achieve active thermal control of the cell proportions within co-cultures. Overall, this technology expands avenues for thermal control in biotechnology applications.
Metabolic Regulation of Mitochondrial Morphologies in Pancreatic Beta Cells: Bioenergetics-Mitochondrial Dynamics Coupling
Authors: Tseng Wen-Wei , Chu Ching-Hsiang , Chang Chen , Lee Yi-Ju , Zhao Shirui , Ho Yi-Ping , Wei An-Chi 
Published: October 2021

AbstractCellular bioenergetics and mitochondrial dynamics are crucial for the secretion of insulin by pancreatic beta cells in response to elevated blood glucose concentrations. To obtain better insights into the interactions between energy production and mitochondrial fission/fusion dynamics, we combine live-cell mitochondria imaging with biophysical-based modeling and network analysis to elucidate the principle regulating mitochondrial morphology to match metabolic demand in pancreatic beta cells. A minimalistic differential equation-based model for beta cells was constructed to include glycolysis, oxidative phosphorylation, simple calcium dynamics, and graph-based fission/fusion dynamics controlled by ATP synthase flux and proton leak flux. The model revealed that mitochondrial fission occurs in response to hyperglycemia, starvation, ATP synthase inhibition, uncoupling, and diabetic condition, in which the rate of proton leak exceeds the rate of mitochondrial ATP synthesis. Under these metabolic challenges, the propensities of tip-to-tip fusion events simulated from the microscopic images of the mitochondrial networks were lower than those in the control group and prevented mitochondrial network formation. The modeling and network analysis could serve as the basis for further detailed research on the mechanisms of bioenergetics and mitochondrial dynamics coupling.
Structure of an ant-myrmecophile-microbe community
Authors: Perry Elena K. , Siozios Stefanos , Hurst Gregory D. D. , Parker Joseph 
Published: October 2021

AbstractSuperorganismal ant colonies play host to a menagerie of symbiotic arthropods, termed myrmecophiles, which exhibit varying degrees of social integration into colony life. Such systems permit examination of how animal community interactions influence microbial assemblages. Here, we present an ecologically and phylogenetically comprehensive characterization of an ant-myrmecophile-microbe community in Southern California. Using 16S rRNA profiling, we find that microbiotas of the velvety tree ant (Liometopum occidentale) and its cohort of myrmecophiles are distinguished by species-specific characteristics but nevertheless bear signatures of their behavioral interactions. We found that the host ant microbiome was diverse at all taxonomic levels; that of a myrmecophilous cricket was moderately diverse, while microbiotas of three myrmecophilous rove beetles (Staphylinidae), which have convergently evolved symbiosis withLiometopum, were dominated by intracellular endosymbionts. Yet, despite these compositional differences, similarities between ant and myrmecophile microbiotas correlated with the nature and intimacy of their behavioral relationships. Physical interactions such as grooming and trophallaxis likely facilitate cross-species extracellular microbial sharing. Further, phylogenetic comparisons of microbiotas from myrmecophile rove beetles and outgroups revealed a lack of co-cladogenesis of beetles and intracellular endosymbionts, and limited evidence for convergence among the myrmecophiles’ intracellular microbiotas. Comparative genomic analyses of the dominantRickettsiaendosymbiont of the most highly socially integrated myrmecophile imply possible functions unrelated to nutrient-provisioning in the host beetle’s specialized lifestyle. Our findings indicate that myrmecophile microbiotas evolve largely independently of the constraints of deep evolutionary history, and that the transition to life inside colonies, including social interactions with hosts, plays a significant role in structuring bacterial assemblages of these symbiotic insects.
Emergence of rare carbapenemases (FRI, GES-5, IMI, SFC, and SFH-1) in Enterobacterales isolated from surface waters in Japan
Authors: Gomi Ryota , Matsumura Yasufumi , Tanaka Michio , Ihara Masaru , Sugie Yoshinori , Matsuda Tomonari , Yamamoto Masaki 
Published: October 2021

ABSTRACTObjectivesCarbapenemase-producing Enterobacterales (CPE) pose serious threats to public health. Compared with clinical CPE, the genetic characteristics of environmental CPE are not well understood. This study aimed to characterize the genetic determinants of carbapenem resistance in CPE isolated from environmental waters in Japan.MethodsEighty-five water samples were collected from rivers and a lake in Japan. CPE were identified using selective media, and genome sequencing was performed for the obtained isolates (n = 21).ResultsVarious rare/novel carbapenemases were identified: GES-5 in Raoultella planticola (n = 1), FRI-8 and FRI-11 in Enterobacter spp. (n = 8), IMI-22 and IMI-23 in Serratia ureilytica (n = 3), and SFC-1, SFC-2 and SFH-1 in Serratia fonticola (n = 9). Genomes of 11 isolates could be closed, allowing the elucidation of the genetic contexts of the carbapenemase genes. The blaGES-5 gene was located within a class 1 integron, In2071 (cassette array, blaGES-5-aacA3-aadA16), on a 33 kb IncP6 plasmid. The blaFRI-8 genes were carried on IncFII(Yp) plasmids ranging in size from 191 kb to 244 kb, and the blaFRI-11 genes were carried on 70 kb and 74 kb IncFII(pECLA)/IncR plasmids. The blaIMI-22 and blaIMI-23 genes were colocated on a 107 kb plasmid. The blaSFC and blaSFH-1 genes were found on putative genomic islands inserted at tRNA-Phe genes in chromosomes.ConclusionsThis study revealed the presence of rare/novel carbapenemases among CPE in aquatic environments, suggesting that the environment may act as a potential reservoir of these minor carbapenemases.
Imputation-free reconstructions of three-dimensional chromosome architectures in human diploid single-cells using allele-specified contacts
Authors: Hirata Yoshito , Oda Arisa H. , Motono Chie , Shiro Masanori , Ohta Kunihiro 
Published: October 2021

AbstractThe sparseness of chromosomal contact information and the presence of homologous chromosomes with very similar nucleotide sequences make Hi-C analysis difficult. We propose a new algorithm using allele-specific single-nucleotide variations (SNVs) to reconstruct the three-dimensional (3D) chromosomal architectures from the Hi-C dataset of single diploid cells. Our algorithm has a function to discriminate SNVs specifically found between homologous chromosomes to our “recurrence plot”-based algorithm to estimate the 3D chromosome structure, which does not require imputation for ambiguous segment information. The new algorithm can efficiently reconstruct 3D chromosomal structures in single human diploid cells by employing only Hi-C segment pairs containing allele-specific SNVs. The datasets of the remaining pairs of segments without allele-specific SNVs are used to validate the estimated chromosome structure. This approach was used to reconstruct the 3D structures of human chromosomes in single diploid cells at a 1-Mb resolution. Introducing a subsequent mathematical measure further improved the resolution to 40-kb or 100-kb. The reconstruction data reveals that human chromosomes form chromosomal territories and take fractal structures where the mean dimension is a non-integer value. We also validate our approach by estimating 3D protein/polymer structures.
Machine learning-based modulation of Ca2+-binding affinity in EF-hand proteins and comparative structural insights into site-specific cooperative binding
Authors: Mazumder Mohit , Kumar Sanjeev , Kumar Devbrat , bhattacharya Alok , Gourinath S. 
Published: October 2021

AbstractCa2+-binding proteins are present in almost all living organisms and different types display different levels of binding affinities for the cation. Here, we applied two new scoring schemes enabling the user to manipulate the binding affinities of such proteins. We specifically designed a unique EF-hand loop capable of binding calcium with high affinity by altering five residues of the loop based on the scoring scheme. We worked on the N-terminal domain of Entamoeba histolytica calcium-binding protein1 (NtEhCaBP1), and used site-directed mutagenesis to incorporate the designed loop sequence into the second EF hand motif of this protein. The binding isotherms calculated using ITC calorimetry showed a ∼500-fold greater association constant (Ka) for the mutant. The crystal structure of the mutant was also determined, and displayed more compact Ca2+-coordination spheres in both of its EF loops than did the structure of the wildtype protein, consistent with the greater calcium-binding affinities of the mutant. The NtEhCaBP1 mutant was also shown to form a hexamer rather than just a trimer, and this hexamer formation was attributed to the position of the last helix of the mutant having been changed as a result of the strong calcium coordination. Further dynamic correlation analysis revealed that the mutation in the second EF loop changed the entire residue network of the monomer, resulting in a stronger coordination of Ca2+ even in other EF hand loop.
A particle-based computational model to analyse remodelling of the red blood cell cytoskeleton during malaria infections
Authors: Jäger Julia , Patra Pintu , Sanchez Cecilia P. , Lanzer Michael , Schwarz Ulrich S. 
Published: October 2021

AbstractRed blood cells can withstand the harsh mechanical conditions in the vasculature only because the bending rigidity of their plasma membrane is complemented by the shear elasticity of the underlying spectrin-actin network. During an infection by the malaria parasite Plasmodium falciparum, the parasite mines host actin from the junctional complexes and establishes a system of adhesive knobs, whose main structural component is the knob-associated histidine rich protein (KAHRP) secreted by the parasite. Here we aim at a mechanistic understanding of this dramatic transformation process. We have developed a particle-based computational model for the cytoskeleton of red blood cells and simulated it with Brownian dynamics to predict the mechanical changes resulting from actin mining and KAHRP-clustering. Our simulations include the three-dimensional conformations of the semi-flexible spectrin chains, the capping of the actin protofilaments and several established binding sites for KAHRP. For the healthy red blood cell, we find that incorporation of actin protofilaments leads to two regimes in the shear response. Actin mining decreases the shear modulus, but knob formation increases it. We show that dynamical changes in KAHRP binding affinities can explain the experimentally observed relocalization of KAHRP from ankyrin to actin complexes and demonstrate good qualitative agreement with experiments by measuring pair cross-correlations both in the computer simulations and in super-resolution imaging experiments.Author summaryMalaria is one of the deadliest infectious diseases and its symptoms are related to the blood stage, when the parasite multiplies within red blood cells. In order to avoid clearance by the spleen, the parasite produces specific factors like the adhesion receptor PfEMP1 and the multifunctional protein KAHRP that lead to the formation of adhesive knobs on the surface of the red blood cells and thus increase residence time in the vasculature. We have developed a computational model for the parasite-induced remodelling of the actin-spectrin network to quantitatively predict the dynamical changes in the mechanical properties of the infected red blood cells and the spatial distribution of the different protein components of the membrane skeleton. Our simulations show that KAHRP can relocate to actin junctions due to dynamical changes in binding affinities, in good qualitative agreement with super-resolution imaging experiments. In the future, our simulation framework can be used to gain further mechanistic insight into the way malaria parasites attack the red blood cell cytoskeleton.
A unique genomic island-governed cannibalism in Bacillus enhanced biofilm formation through a novel regulation mechanism
Authors: Huang Rong , Li Qing , Wang Dandan , Feng Haichao , Zhang Nan , Shao Jiahui , Shen Qirong , Xu Zhihui , Zhang Ruifu 
Published: October 2021

AbstractCannibalism is a differentiation strategy and social multicellular behavior in biofilms. The novel factors and mechanisms to trigger the bacterial cannibalism remain scarce. Here, we report a novel bacillunoic acids-mediated strategy for manipulating cannibalism in Bacillus velezensis SQR9 biofilm formation. A subfraction of cells differentiate into cannibals that secrete toxic bacillunoic acids to lyse a fraction of their sensitive siblings, and the released nutrients enhance biofilm formation. Meanwhile, the self-immunity of cannibal cells was induced by bacillunoic acids. A two-component system, the OmpS-OmpR signal-transduction pathway, controls the expression of the ABC transporter BnaAB for self-immunity. Specifically, bacillunoic acids activate the autophosphorylation of OmpS, a transmembrane histidine kinase, which then transfers a phosphoryl group to its response regulator OmpR. The phosphorylation of OmpR activates the transcription of the transporter gene bnaAB by binding its promoter. Thus, bacillunoic acids are pumped out of cells by the ABC transporter BnaAB. Moreover, we discovered that strain SQR9 could use the bacillunoic acids-mediated cannibalism to optimize its community to produce more bacillunoic acids for bacterial competition. This study revealed that bacillunoic acids play a previously undiscovered dual role in both cannibalism during biofilm formation and interspecies competition, which has an important biological significance.
Models of the cardiac L-type calcium current: a quantitative review
Authors: Agrawal Aditi , Wang Ken , Polonchuk Liudmila , Cooper Jonathan , Hendrix Maurice , Gavaghan David J. , Mirams Gary R. , Clerx Michael 
Published: October 2021

AbstractThe L-type calcium current (ICaL) plays a critical role in cardiac electrophysiology, and models of ICaL are vital tools to predict arrhythmogenicity of drugs and mutations. Five decades of measuring and modelling ICaL have resulted in several competing theories (encoded in mathematical equations). However, the introduction of new models has not typically been accompanied by a data-driven critical comparison with previous work, so that it is unclear which model is best suited for any particular application. In this review, we describe and compare 73 published mammalian ICaL models, and use simulated experiments to show that there is a large variability in their predictions, which is not substantially diminished when grouping by species or other categories. We provide model code for 60 models, list major data sources, and discuss experimental and modelling work that will be required to reduce this huge list of competing theories and ultimately develop a community consensus model of ICaL.
Phenotypic diversity created by a transposable element increases productivity and resistance to competitors in plant populations
Authors: Latzel Vít , Puy Javier , Thieme Michael , Bucher Etienne , Götzenberger Lars , de Bello Francesco 
Published: October 2021

AbstractAn accumulating body of evidence indicates that natural plant populations harbour a large diversity of transposable elements (TEs). TEs provide genetic and epigenetic variation that can substantially translate into changes in plant phenotypes. Despite the wealth of data on the ecological and evolutionary effects of TEs on plant individuals, we have virtually no information on the role of TEs on populations and ecosystem functioning. On the example of Arabidopsis thaliana, we demonstrate that TE-generated variation creates differentiation in ecologically important functional traits. In particular, we show that Arabidopsis populations with increasing diversity of individuals differing in copy numbers of the ONSEN retrotransposon had higher phenotypic and functional diversity. Moreover, increased diversity enhanced population productivity and reduced performance of interspecific competitors. We conclude that TE-generated diversity can have similar effects on ecosystem as usually documented for other biological diversity effects.
URMC-099 Prophylaxis Prevents Hippocampal Vascular Vulnerability and Synaptic Damage in an Orthopedic Model of Delirium Superimposed on Dementia
Authors: Miller-Rhodes Patrick , Li Herman , Velagapudi Ravikanth , Terrando Niccolò , Gelbard Harris A 
Published: October 2021

AbstractSystemic perturbations can drive a neuroimmune cascade after surgical trauma, including affecting the blood-brain barrier (BBB), activating microglia, and contributing to cognitive deficits such as delirium. Delirium superimposed on dementia (DSD) is a particularly debilitating complication that renders the brain further vulnerable to neuroinflammation and neurodegeneration, albeit these molecular mechanisms remain poorly understood. Here we have used an orthopedic model of tibial fracture/fixation in APPSwDI/mNos2-/- AD (CVN-AD) mice to investigate relevant pathogenetic mechanisms underlying DSD. We conducted the present study in 6 months-old CVN-AD mice, an age at which we speculated amyloid-β pathology had not saturated BBB and neuroimmune functioning. We found that URMC-099, our brain-penetrant anti-inflammatory neuroprotective drug, prevented inflammatory endothelial activation, breakdown of the BBB, synapse loss, and microglial activation in our DSD model. Taken together, our data link post-surgical endothelial activation, microglial MafB immunoreactivity, and synapse loss as key substrates for DSD, all of which can be prevented by URMC-099.