bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optical spike detection and connectivity analysis with a far-red voltage-sensitive fluorophore reveals changes to network connectivity in development and disease
Authors: Walker Alison S. , Raliski Benjamin K. , Karbasi Kaveh , Zhang Patrick , Sanders Kate , Miller Evan W. 
Published: October 2020

AbstractThe ability to optically record dynamics of neuronal membrane potential promises to revolutionize our understanding of neurobiology. In this study, we show that the far-red voltage sensitive fluorophore, Berkeley Red Sensor of Transmembrane potential −1, or BeRST 1, can be used to monitor neuronal membrane potential changes across dozens of neurons at a sampling rate of 500 Hz. Notably, voltage imaging with BeRST 1 can be implemented with affordable, commercially available illumination sources, optics, and detectors. BeRST 1 is well-tolerated in cultures of rat hippocampal neurons and provides exceptional optical recording fidelity, as judged by dual fluorescence imaging and patch-clamp electrophysiology. We developed a semi-automated spike-picking program to reduce user bias when calling action potentials and used this in conjunction with BeRST 1 to develop an optical spike and connectivity analysis workflow (OSCA) for high-throughput dissection of neuronal activity dynamics in development and disease. The high temporal resolution of BeRST 1 enables dissection of firing rate changes in response to acute, pharmacological interventions with commonly used inhibitors like gabazine and picrotoxin. Over longer periods of time, BeRST 1 also tracks chronic perturbations to neurons exposed to amyloid beta (Aβ1-42), revealing modest changes to spiking frequency but profound changes to overall network connectivity. Finally, we use OSCA to track changes in neuronal connectivity during development, providing a functional readout of network assembly. We envision that use of BeRST 1 and OSCA described here will be of use to the broad neuroscience community.Significance StatementOptical methods to visualize membrane potential dynamics provide a powerful complement to Ca2+imaging, patch clamp electrophysiology, and multi-electrode array recordings. However, modern voltage imaging strategies often require complicated optics, custom-built microscopes, or genetic manipulations that are impractical outside of a subset of model organisms. Here, we describe the use of Berkeley Red Sensor of Transmembrane potential, or BeRST 1, a far-red voltage-sensitive fluorophore that can directly visualize membrane potential changes with millisecond resolution across dozens of neurons. Using only commercially available components, voltage imaging with BeRST 1 reveals profound changes in neuronal connectivity during development, exposes changes to firing rate during acute pharmacological perturbation, and illuminates substantial increases in network connectivity in response to chronic exposure to amyloid beta.
Imaging voltage in complete neuronal networks within patterned microislands reveals preferential wiring of excitatory hippocampal neurons
Authors: Walker Alison S. , Raliski Benjamin K. , Nguyen Dat Vinh , Zhang Patrick , Sanders Kate , Karbasi Kaveh , Miller Evan W. 
Published: October 2020

AbstractVoltage imaging with fluorescent dyes affords the opportunity to map neuronal activity in both time and space. One limitation to imaging is the inability to image complete neuronal networks: some fraction of cells remains outside of the observation window. Here, we combine voltage imaging,post hocimmunocytochemistry, and patterned microisland hippocampal culture to provide imaging of complete neuronal networks. The patterned microislands completely fill the field of view of our high-speed (500 Hz) camera, enabling reconstruction of the spiking patterns of every single neuron in the network. Cultures raised on microislands develop similarly to neurons grown on coverslips and display similar composition of inhibitory and excitatory cell types. The principal excitatory cell types (CA1, CA3, and dentate granule cells, or DGC) are also present in similar proportions in both preparations. We calculate the likelihood that action potential firing in one neuron to trigger action potential firing in a downstream neuron in a spontaneously active network to construct a functional connection map of these neuronal ensembles. Importantly, this functional map indicates preferential connectivity between DGC and CA3 neurons and between CA3 and CA1 neurons, mimicking the neuronal circuitry of the intact hippocampus. We envision that patterned microislands, in combination with voltage imaging and methods to classify cell types, will be a powerful method for exploring neuronal function in both healthy and disease states. Additionally, because the entire neuronal network is sampled simultaneously, this strategy has the power to go further, revealing all functional connections between all cell types.Significance StatementIn vitromodel systems provide unsurpassed control and access for exploring the molecular and cellular details of neurobiology. We developed a patterned microisland system for culturing rat hippocampal neurons that recapitulates the features of bulk hippocampal cultures, but with the added benefit of allowing access to high-speed imaging of entire neuronal ensembles using voltage imaging. By using far-red voltage-sensitive fluorophores, we map the functional connections across all cells in the neuronal ensemble, revealing that several important functional synapses present in the intact hippocampus are recapitulated in this microisland system. We envision the methods described here will be a powerful complement to ongoing research into basic neurobiological mechanisms and the search for therapies to treat diseases arising from their dysfunction.
Restructuring of the Immune Contexture Improves Checkpoint Blockade Efficacy in Murine Lung Cancer
Authors: Ebelt Nancy D. , Zuniga Edith , Marzagalli Monica , Zamloot Vic , Blazar Bruce R. , Salgia Ravi , Manuel Edwin R. 
Published: October 2020

ABSTRACTTherapeutic options for non-small cell lung cancer (NSCLC) treatment have changed dramatically in recent years with the advent of novel immunotherapeutic approaches. Among these, immune checkpoint blockade (ICB), using monoclonal antibodies, has shown tremendous promise in a small proportion of patients. In order to better predict patients that will respond to ICB treatment, biomarkers such as tumor-associated CD8+T cell frequency, tumor checkpoint protein status and mutational burden have been utilized, however, with mixed success. In this study, we hypothesized that significantly altering the suppressive tumor immune landscape in NSCLC could potentially improve ICB efficacy. Using sub-therapeutic doses of ourSalmonella typhimurium-based therapy targeting the suppressive molecule indoleamine 2,3-dioxygenase (shIDO-ST) in tumor-bearing mice, we observed dramatic changes in immune subset phenotypes that included increases in antigen presentation markers, decreased regulatory T cell frequency and overall reduced checkpoint protein expression. Combination shIDO-ST treatment with anti-PD-1/CTLA-4 antibodies enhanced tumor growth control, compared to either treatment alone, which was associated with a significant intratumoral influx of CD8+and CD4+T lymphocytes. These results suggest that the success of ICB therapy may be more accurately predicted by taking into account multiple factors such as potential for antigen presentation and frequency of suppressive immune subsets in addition to markers already being considered. Alternatively, combination treatment with agents such as shIDO-ST could be used to create a more conducive tumor microenvironment for improving response rates to immunotherapy.
Adeno-associated virus capsid assembly is divergent and stochastic
Authors: Wörner Tobias P. , Bennett Antonette , Habka Sana , Snijder Joost , Friese Olga , Powers Thomas , Agbandje-McKenna Mavis , Heck Albert J.R. 
Published: October 2020

AbstractAdeno-associated viruses (AAVs) are small, non-enveloped, and have a T=1 icosahedral capsid. They belong to the Parvoviridae, genus Dependoparvovirus. Interest in AAVs has grown over recent years as they have emerged as promising gene therapy vectors. The AAV capsid, encapsulating the transgene, consists, in total, of 60 subunits made up from three distinct viral proteins (VPs) originating from the same cap gene (VP1, VP2, and VP3), which vary only in their N-terminus. While all three VPs play a crucial and specific role in cell-entry and transduction, their exact stoichiometry and organization in AAV capsids has, despite the availability of several high-resolution structures remained elusive. Here we obtained a set of native mass spectra of intact AAV capsids (Mw ≈ 3.8 MDa) that display both highly resolved regions and regions wherein interferences occur. Through spectrum simulation we resolved and elucidated this spectral complexity, allowing us to directly assess the VP stoichiometries in a panel of serotypes from different production platforms. The data reveals an extremely heterogeneous population of capsids of variable composition. The relative abundance for each of the hundreds of co-occurring capsid compositions is accurately described by a model based upon stochastic assembly from a mixed pool of expressed VP1, VP2, and VP3. We show that even the single-most abundant VP stoichiometry represents only a few percent of the total AAV population. We estimate that virtually every AAV capsid in a particular preparation has a unique composition and arrangement, i.e. no particle is identical. The systematic scoring of the stochastic assembly model against experimental high-resolution native MS data offers a sensitive and accurate new method to characterize these exceptionally heterogeneous gene-delivery vectors.
Screening of tomato seed bacterial endophytes for antifungal activity reveals lipopeptide producing Bacillus siamensis as a potential bio-control agent
Authors: Sharma Ayushi , Kaushik Nutan , Sharma Abhishek , Bajaj Abhay , Rasane Mandar , Shouche Yogesh S. , Marzouk Takwa , Djébali Naceur 
Published: October 2020

ABSTRACTThe current study investigates the diversity pattern and fungicidal potential of bacterial endophytes isolated from two different organic varieties of tomato plants (V1 and V2). A total of seventy-four bacterial isolates identified by 16S rRNA sequencing revealed a single genus Bacillus with 16 different species. The Shannon diversity H’ (1.45), Simpson’s index of diversity (0.9), Magalef’ index (2.1), Evenness (0.96), and Species richness (8) indicated the high endophytic bacterial diversity in the V1 variety of the tomato. Bacterial endophytes isolated from both the varieties were screened for their antifungal activity against five economically critical fungal pathogens (viz., Botrytis cinerea, Rhizoctonia solani, Fusarium solani, Verticillium lateritium, and Alternaria solani) of tomato crop through dual culture assay. The data revealed B. siamensis KCTC 13613(T) as the most potent antagonist significantly (p < 0.05), inhibiting the mycelial growth between 75 to 90% against selected fungal pathogens. High bioactivity of lipopeptide extract of B. siamensis was recorded against R. solani with IC50 value of 72 ppm. The UPLC-HDMS analysis of this lipopeptide extract revealed the presence of, Surfactin and Bacillomycin D.
Estimating axial diffusivity in the NODDI model
Authors: Howard Amy FD , Cottaar Michiel , Drakesmith Mark , Fan Qiuyun , Huang Susie Y , Jones Derek K , Lange Frederik J , Mollink Jeroen , Rudrapatna Suryanarayana Umesh , Tian Qiyuan , Miller Karla L , Jbabdi Saad 
Published: October 2020

AbstractTo estimate microstructure-related parameters from diffusion MRI data, biophysical models make strong, simplifying assumptions about the underlying tissue. The extent to which many of these assumptions are valid remains an open research question. This study was inspired by the disparity between the estimated intra-axonal axial diffusivity from literature and that typically assumed by the Neurite Orientation Dispersion and Density Imaging (NODDI) model (d║= 1.7μm2/ms). We first demonstrate how changing the assumed axial diffusivity results in considerably different NODDI parameter estimates. Second, we illustrate the ability to estimate axial diffusivity as a free parameter of the model using high b-value data and an adapted NODDI framework. Using both simulated andin vivodata we investigate the impact of fitting to either real-valued or magnitude data, with Gaussian and Rician noise characteristics respectively, and what happens if we get the noise assumptions wrong in this high b-value and thus low SNR regime. Our results from real-valued human data estimate intra-axonal axial diffusivities of ~ 2 – 2.5μm2/ms, in line with current literature. Crucially, our results demonstrate the importance of accounting for both a rectified noise floor and/or a signal offset to avoid biased parameter estimates when dealing with low SNR data.
Investigating the interaction between white matter and brain state on tDCS-induced changes in brain network activity
Authors: Kurtin Danielle L. , Violante Ines R. , Zimmerman Karl , Leech Robert , Hampshire Adam , Patel Maneesh C. , Carmichael David W. , Sharp David J. , Li Lucia M. 
Published: October 2020

AbstractBackgroundTranscranial direct current stimulation (tDCS) is a form of noninvasive brain stimulation whose potential as a cognitive therapy is hindered by our limited understanding of how participant and experimental factors influence its effects. Using functional MRI to study brain networks, we have previously shown in healthy controls that the physiological effects of tDCS are strongly influenced by brain state. We have additionally shown, in both healthy and traumatic brain injury (TBI) populations, that the behavioral effects of tDCS are positively correlated with white matter (WM) structure.ObjectivesIn this study we investigate how these two factors, WM structure and brain state, interact to shape the effect of tDCS on brain network activity.MethodsWe applied anodal, cathodal and sham tDCS to the right inferior frontal gyrus (rIFG) of healthy (n=22) and TBI participants (n=34). We used the Choice Reaction Task (CRT) performance to manipulate brain state during tDCS. We acquired simultaneous fMRI to assess activity of cognitive brain networks and used Fractional Anisotropy (FA) as a measure of WM structure.ResultsWe find that the effects of tDCS on brain network activity in TBI participants are highly dependent on brain state, replicating findings from our previous healthy control study in a separate, patient cohort. We then show that WM structure further modulates the brain-state dependent effects of tDCS on brain network activity. These effects are not unidirectional – in the absence of task with anodal and cathodal tDCS, FA is positively correlated with brain activity in several regions of the default mode network. Conversely, with cathodal tDCS during CRT performance, FA is negatively correlated with brain activity in a salience network region.ConclusionsOur results show that experimental and participant factors interact to have unexpected effects on brain network activity, and that these effects are not fully predictable by studying the factors in isolation.
Gene flow in phylogenomics: Sequence capture resolves species limits and biogeography of Afromontane forest endemic frogs from the Cameroon Highlands
Authors: Dolinay Matej , Nečas Tadeáš , Zimkus Breda M. , Schmitz Andreas , Fokam Eric B. , Lemmon Emily Moriarty , Lemmon Alan R. , Gvoždík Václav 
Published: October 2020

ABSTRACTPuddle frogs of thePhrynobatrachus steindachnerispecies complex are a useful group for investigating speciation and phylogeography in Afromontane forests of the Cameroon Highlands (Cameroon Volcanic Line) in western Central Africa. The species complex is represented by six morphologically relatively cryptic mitochondrial DNA lineages, with only two of them distinguished at the species level – southernP. jimzimkusiand Lake Oku endemicP. njiomock, leaving the remaining four lineages with a pooled identification as ‘P. steindachneri’. In this study, the six mtDNA lineages are subjected to genomic sequence capture analyses to delimit species (together with morphology) and to study biogeography. Nuclear DNA data (387 loci; 571,936 aligned base pairs) distinguished all six mtDNA lineages, but the splitting pattern and depths of divergences supported only four main clades—besidesP. jimzimkusiandP. njiomock, only two from the four ‘P. steindachneri’ mtDNA lineages. One is here described as a new species,P. sp. nov. Reticulate evolution (hybridization) was detected within the species complex with morphologically intermediate hybrid individuals placed between the parental species in phylogenomic analyses, forming a phylogenetic artefact – a ladder-like tree pattern. The presence of hybrids is undesirable in standard phylogenetic analyses, but is essential and beneficial in the network multispecies coalescent. This latter approach allowed us an insight into the reticulate evolutionary history of these endemic frogs. Introgressions likely occurred during the Middle and Late Pleistocene climatic oscillations, due to the cyclic connections (likely dominating during cold glacials) and separations (warm interglacials) of montane forests. The genomic phylogeographic pattern supports the earliest division between southern (Mt. Manengouba to Mt. Oku) and northern mountains at the onset of the Pleistocene. Further subdivisions occurred in the Early Pleistocene separating populations from the northernmost (Tchabal Mbabo, Gotel Mts.) and middle mountains (Mt. Mbam, Mt. Oku, Mambilla Plateau), as well as the microendemic lineage restricted to Lake Oku (Mt. Oku). Mount Oku harboring three species is of particular conservation importance. This unique model system is highly threatened as all the species within the complex have exhibited severe population declines in the past decade, placing them on the brink of extinction. We therefore urge for conservation actions in the Cameroon Highlands to preserve their diversity before it is too late.
Control of Blood Glucose Level for Type 1 Diabetes Mellitus using Improved Hovorka Equations: Comparison between Clinical and In-Silico Works
Authors: Mohd Sohadi Nur’Amanina , Som Ayub Md , Mohd Nor Noor Shafina , Mohd Yusof Nur Farhana , Ali Sherif Abdulbari , Pacana Noor Dyanna Andres 
Published: October 2020

AbstractBackgroundType 1 diabetes mellitus (T1DM) occurs due to inability of the body to produce sufficient amount of insulin to regulate blood glucose level (BGL) at normoglycemic range between 4.0 to 7.0 mmol/L. Thus, T1DM patients require to do self-monitoring blood glucose (SMBG) via finger pricks and depend on exogenous insulin injection to maintain their BGL which is very painful and exasperating. Ongoing works on artificial pancreas device nowadays focus primarily on a computer algorithm which is programmed into the controller device. This study aims to simulate so-called improved equations from the Hovorka model using actual patients’ data through in-silico works and compare its findings with the clinical works.MethodsThe study mainly focuses on computer simulation in MATLAB using improved Hovorka equations in order to control the BGL in T1DM. The improved equations can be found in three subsystems namely; glucose, insulin and insulin action subsystems. CHO intakes were varied during breakfast, lunch and dinner times for three consecutive days. Simulated data are compared with the actual patients’ data from the clinical works.ResultsResult revealed that when the patient took 36.0g CHO during breakfast and lunch, the insulin administered was 0.1U/min in order to maintain the blood glucose level (BGL) in the safe range after meal; while during dinner time, 0.083U/min to 0.1 U/min of insulins were administered in order to regulate 45.0g CHO taken during meal. The basal insulin was also injected at 0.066U/min upon waking up time in the early morning. The BGL was able to remain at normal range after each meal during in-silico works compared to clinical works.ConclusionsThis study proved that the improved Hovorka equations via in-silico works can be employed to model the effect of meal disruptions on T1DM patients, as it demonstrated better control as compared to the clinical works.
Functional diversity of PFKFB3 splice variants in glioblastomas
Authors: Heydasch Ulli , Kessler Renate , Warnke Jan-Peter , Eschrich Klaus , Scholz Nicole , Bigl Marina 
Published: October 2020

AbstractTumor cells tend to metabolize glucose through aerobic glycolysis instead of oxidative phosphorylation in mitochondria. One of the rate limiting enzymes of glycolysis is 6-phosphofructo-1-kinase, which is allosterically activated by fructose 2,6-bisphosphate which in turn is produced by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2 or PFKFB). Mounting evidence suggests that cancerous tissues overexpress the PFKFB isoenzyme, PFKFB3, being causing enhanced proliferation of cancer cells.Initially, six PFKFB3 splice variants with different C-termini have been documented in humans. More recently, additional splice variants with varying N-termini were discovered the functions of which are to be uncovered.Glioblastoma is one of the deadliest forms of brain tumors. Up to now, the role of PFKFB3 splice variants in the progression and prognosis of glioblastomas is only partially understood. In this study, we first re-categorized the PFKFB3 splice variant repertoire to simplify the denomination. We investigated the impact of increased and decreased levels of PFKFB3-4 (former UBI2K4) and PFKFB3-5 (former variant 5) on the viability and proliferation rate of glioblastoma U87 and HEK-293 cells. The simultaneous knock-down of PFKFB3-4 and PFKFB3-5 led to a decrease in viability and proliferation of U87 and HEK-293 cells as well as a reduction in HEK-293 cell colony formation. Overexpression of PFKFB3-4 but not PFKFB3-5 resulted in increased cell viability and proliferation. This finding contrasts with the common notion that overexpression of PFKFB3 enhances tumor growth, but instead suggests splice variant-specific effects of PFKFB3, apparently with opposing effects on cell behaviour. Strikingly, in line with this result, we found that in human IDH-wildtype glioblastomas, the PFKFB3-4 to PFKFB3-5 ratio was significantly shifted towards PFKFB3-4 when compared to control brain samples. Our findings indicate that the expression level of distinct PFKFB3 splice variants impinges on tumorigenic properties of glioblastomas and that splice pattern may be of important diagnostic value for glioblastoma.