bluuebunny/crossref_metadata_2025 huggingface dataset:
Unbiased identification of nanoparticle cell uptake mechanism via a genome-wide CRISPR/Cas9 knockout screen
Authors: Nostrand Eric L. Van , Barnhill Sarah A. , Shishkin Alexander A. , Nelles David A. , Byeon Eric , Nguyen Thai , Wong Yiu Chueng Eric , Gianneschi Nathan C. , Yeo Gene W.
Published: October 2020

A major bottleneck in nanocarrier and macromolecule development for therapeutic delivery is our limited understanding of the processes involved in their uptake into target cells. This includes their active interactions with membrane transporters that co-ordinate cellular uptake and processing. Current strategies to elucidate the mechanism of uptake, such as painstaking manipulation of individual effectors with pharmacological inhibitors or specific genetic knockdowns, are limited in scope and biased towards previously studied pathways or the intuition of the investigators. Furthermore, each of these approaches present significant off-target effects, clouding the outcomes. We set out to develop and examine an unbiased whole-genome screening approach using pooled CRISPR/Cas9 libraries for its ability to provide a robust and rapid approach to identify novel effectors of material uptake. Enabling this, we developed a methodology termed fast-library of inserts (FLI)-seq for library preparation and quantitative readout of pooled screens that shows improved technical reproducibility and is easier to perform than existing methods. In this proof-of-concept study we use FLI-seq to identify a solute carrier protein family member, SLC18B1, as a transporter for polymeric micellar nanoparticles, confirming the viability for this approach to yield novel insights into uptake mechanisms.
Adsorption mechanism of bacteria onto a Na-montmorillonite surface with organic and inorganic calcium
Authors: Tang Guowang , Jia Cangqin , Wang Guihe , Yu Peizhi , Jiang Xihao
Published: October 2020

The adsorption of bacteria onto the Na-montmorillonite (Na-MMT) was studied as a function of time, bacterial concentration, temperature and pH with the introduction of the organic and inorganic calcium sources. The results indicated that albeit revealing the same adsorption mechanism, the organic calcium (i.e., Ca(CH3COO)2) proposed in this study is more beneficial and environmentally friendly than the inorganic calcium (i.e., CaCl2) in terms of the adsorption of bacteria onto the Na-MMT surface, which can be ascribed to the formation of the denser aggregates in the Na-MMT with Ca(CH3COO)2. Meanwhile, the adsorption kinetics and isotherms followed the pseudo-second-order kinetic model and Langmuir Equation for both two calcium sources. Meanwhile, the adsorption bands of the water molecules on the minerals were observed to shift significantly after the bacterial adsorption, showing that the hydrogen bonding on the Na-MMT surface played an important role during this process. A value of ΔH0> 0 indicated that the bacterial adsorption was affected by van der Waals force and hydrophobic interaction. Finally, the negative zeta potentials of the Na-MMT increased with the addition of Ca2+ions, and the experimental data also showed that the adsorption of bacteria onto the Na-MMT was mainly determined by the electrostatic and non-electrostatic forces.
The baculovirus promoter OpIE2 sequence has inhibitory effect on the activity of the Cytomegalovirus (CMV) promoter in HeLa and HEK-293T cells
Authors: Aladdin A , Sahly N , Faty R , Youssef MM , Salem TZ
Published: October 2020

Understanding how promoters work in non-host cells is complex. Nonetheless, understanding this process is crucial while performing gene expression modulation studies. In this study, inhibitory regions in the 5’ end of the OpIE2 insect viral promoter were found to be blocking the activity of the CMV promoter in mammalian cells. This finding was reached in the process of constructing a shuttle vector with CMV and OpIE2 promoters in a tandem arrangement to achieve gene expression in both mammalian and insect cells, respectively. OpIE2 promoter was cloned downstream of the CMV promoter and upstream of the EGFP reporter gene. After introducing the constructed shuttle vector to insect and mammalian cells, a significant drop in the CMV promoter activity in mammalian cells was observed. To enhance the CMV promoter activity, several modification were made to the shuttle vector including site-directed mutagenesis to remove all ATG codons from the downstream promoter (OpIE2), separating the two promoters to eliminate the effect of transcription interference between them, and finally, identifying some inhibitory regions in the OpIE2 promoter sequence. When these inhibitory regions were removed, high expression levels in insect and mammalian cells were restored. In conclusion, a shuttle vector was constructed that works efficiently in both mammalian and insect cell lines. This study showed that inserting 261 to 313 bp from the 3’ end of the OpIE2 promoter downstream of the CMV promoter maintains efficient gene expression in both Sf9 and mammalian cells.
TheDrosophilahistone demethylase KDM5 is required during early neurodevelopment for proper mushroom body formation and cognitive function
Authors: Hatch Hayden A. M. , Belalcazar Helen M. , Marshall Owen J. , Secombe Julie
Published: October 2020

Mutations in the lysine demethylase 5 (KDM5) family of transcriptional regulators are associated with intellectual disability, yet little is known regarding the spatiotemporal requirements or neurodevelopmental contributions of KDM5 proteins. Utilizing the mushroom body (MB), a major learning and memory center within theDrosophilabrain, we demonstrate that KDM5 is specifically required within ganglion mother cells and immature neurons for proper neurodevelopment and cognitive function. Within this cellular subpopulation, we identify a core network of KDM5-regulated genes that are critical modulators of neurodevelopment. Significantly, we find that a majority of these genes are direct targets of Prospero (Pros), a transcription factor with well-established roles in neurodevelopment in other neuronal contexts. We demonstrate that Pros is essential for MB development and functions downstream of KDM5 to regulate MB morphology. We therefore provide evidence for a KDM5-Pros axis that orchestrates a transcriptional program critical for proper axonal development and cognitive function.
Role of cell polarity dynamics and motility in pattern formation due to contact dependent signalling
Authors: Bajpai Supriya , Prabhakar Ranganathan , Chelakkot Raghunath , Inamdar Mandar M.
Published: October 2020

A key challenge in biology is to understand how spatiotemporal patterns and structures arise during the development of an organism. An initial aggregate of spatially uniform cells develops and forms the differentiated structures of a fully developed organism. On the one hand, contact-dependent cell-cell signalling is responsible for generating a large number of complex, self-organized, spatial patterns in the distribution of the signalling molecules. On the other hand, the motility of cells coupled with their polarity can independently lead to collective motion patterns that depend on mechanical parameters influencing tissue deformation, such as cellular elasticity, cell-cell adhesion and active forces generated by actin and myosin dynamics. Although modelling efforts have, thus far, treated cell motility and cell-cell signalling separately, experiments in recent years suggest that these processes could be tightly coupled. Hence, in this paper, we study how the dynamics of cell polarity and migration influence the spatiotemporal patterning of signalling molecules. Such signalling interactions can occur only between cells that are in physical contact, either directly at the junctions of adjacent cells or through cellular protrusional contacts. We present a vertex model which accounts for contact-dependent signalling between adjacent cells and between non-adjacent neighbours through long protrusional contacts that occur along the orientation of cell polarization. We observe a rich variety of spatiotemporal patterns of signalling molecules that is influenced by polarity dynamics of the cells, relative strengths of adjacent and non-adjacent signalling interactions, range of polarized interaction, signalling activation threshold, relative time scales of signalling and polarity orientation, and cell motility. Though our results are developed in the context of Delta-Notch signalling, they are sufficiently general and can be extended to other contact dependent morpho-mechanical dynamics.
Stable Protein Sialylation in Physcomitrella
Authors: Bohlender Lennard L. , Parsons Juliana , Hoernstein Sebastian N. W. , Rempfer Christine , Ruiz-Molina Natalia , Lorenz Timo , Rodriguez-Jahnke Fernando , Figl Rudolf , Fode Benjamin , Altmann Friedrich , Reski Ralf , Decker Eva L.
Published: October 2020

Recombinantly produced proteins are indispensable tools for medical applications. Since the majority of them are glycoproteins, theirN-glycosylation profiles are major determinants for their activity, structural properties and safety. For therapeutical applications, a glycosylation pattern adapted to product and treatment requirements is advantageous. Physcomitrella (Physcomitrium patens, moss) is able to perform highly homogeneous complex-typeN-glycosylation. Additionally, it has been glyco-engineered to eliminate plant-specific sugar residues by knock-out of the β1,2-xylosyltransferase and α1,3-fucosyltransferase genes (Δxt/ft). Furthermore,P. patensmeets wide-ranging biopharmaceutical requirements such as GMP compliance, product safety, scalability and outstanding possibilities for precise genome engineering. However, all plants, in contrast to mammals, lack the capability to performN-glycan sialylation. Since sialic acids are a common terminal modification on humanN-glycans, the property to performN-glycan sialylation is highly desired within the plant-based biopharmaceutical sector. In this study, we present the successful achievement of proteinN-glycan sialylation in stably transformedP. patens. The sialylation ability was achieved in a Δxt/ft moss line by stable expression of six mammalian coding sequences combined with targeted organelle-specific localization of the encoded enzymes responsible for synthesis, activation, transport and transfer of sialic acid. Production of free and (CMP)-activated sialic acid was proven. The glycosidic anchor for the attachment of terminal sialic acid was generated by the introduction of a chimeric human β1,4-galactosyltransferase gene under the simultaneous knock-out of the gene encoding the endogenous β1,3-galactosyltransferase. Functional complex-typeN-glycan sialylation was confirmed via mass spectrometric analysis of a stably co-expressed recombinant human protein.
Targeting host glycolysis as a strategy for antimalarial development
Authors: Jezewski Andrew J. , Lin Yu-Hsi , Reisz Julie A. , Culp-Hill Rachel , Barekatain Yasaman , Yan Victoria C. , D’Alessandro Angelo , Muller Florian L. , Odom John Audrey R.
Published: October 2020

Glycolysis controls cellular energy, redox balance, and biosynthesis. Antiglycolytic therapies are under investigation for treatment of obesity, cancer, aging, autoimmunity, and microbial diseases. Interrupting glycolysis is highly valued as a therapeutic strategy, because glycolytic disruption is generally tolerated in mammals. Unfortunately, anemia is a known dose-limiting side effect of these inhibitors and presents a major caveat to development of antiglycolytic therapies. We developed specific inhibitors of enolase – a critical enzyme in glycolysis – and validated their metabolic and cellular effects on human erythrocytes. Enolase inhibition increases erythrocyte susceptibility to oxidative damage and induces rapid and premature erythrocyte senescence, rather than direct hemolysis. We apply our model of red cell toxicity to address questions regarding erythrocyte glycolytic disruption in the context ofPlasmodium falciparummalaria pathogenesis. Our study provides a framework for understanding red blood cell homeostasis under normal and disease states and clarifies the importance of erythrocyte reductive capacity in malaria parasite growth.
HP1α is a chromatin crosslinker that controls nuclear and mitotic chromosome mechanics
Authors: Strom Amy R. , Biggs Ronald J. , Banigan Edward J. , Wang Xiaotao , Chiu Katherine , Herman Cameron , Collado Jimena , Yue Feng , Politz Joan C. Ritland , Tait Leah J. , Scalzo David , Telling Agnes , Groudine Mark , Brangwynne Clifford P. , Marko John F. , Stephens Andrew D.
Published: October 2020

Chromatin, which consists of DNA and associated proteins, contains genetic information and is a mechanical component of the nucleus. Heterochromatic histone methylation controls nucleus and chromosome stiffness, but the contribution of heterochromatin protein HP1α (CBX5) is unknown. We used a novel HP1α auxin-inducible degron human cell line to rapidly degrade HP1α. Degradation did not alter transcription, local chromatin compaction, or histone methylation, but did decrease chromatin stiffness. Single-nucleus micromanipulation reveals that HP1α is essential to chromatin-based mechanics and maintains nuclear morphology, separate from histone methylation. Further experiments with dimerization-deficient HP1αI165Eindicate that chromatin crosslinking via HP1α dimerization is critical, while polymer simulations demonstrate the importance of chromatin-chromatin crosslinkers in mechanics. In mitotic chromosomes, HP1α similarly bolsters stiffness while aiding in mitotic alignment and faithful segregation. HP1α is therefore a critical chromatin-crosslinking protein that provides mechanical strength to chromosomes and the nucleus throughout the cell cycle and supports cellular functions.
Pax7 pioneer factor action requires both paired and homeo DNA binding domains
Authors: Pelletier Audrey , Mayran Alexandre , Gouhier Arthur , Omichinski James G , Balsalobre Aurelio , Drouin Jacques
Published: October 2020

The pioneer transcription factor Pax7 contains two DNA binding domains (DBD), a paired and a homeo domain. Previous work on Pax7 and the related Pax3 had shown that each DBD can bind a cognate DNA sequence, thus defining two targets of binding and possibly modalities of action. Genomic targets of Pax7 pioneer action leading to chromatin opening are enriched for composite DNA target sites containing juxtaposed binding sites for both paired and homeo domains. The present work investigated the implication of both DBDs in pioneer action. We now show that the composite sequence is a higher affinity Pax7 binding site compared to either paired or homeo binding sites and that efficient binding to this site involves both DBDs. We also show that a Pax7 monomer binds composite sites and that methylation of cytosines within the binding site does not affect binding, which is consistent with pioneer action exerted at methylated DNA sites within nucleosomal heterochromatin. Finally, introduction of single amino acid mutations in either the paired or homeo domain that impair binding to cognate DNA sequences showed that both DBDs must be intact for pioneer action. In contrast, only the paired domain is required for low affinity binding of heterochromatin sites. Thus, Pax7 pioneer action on heterochromatin requires unique protein:DNA interactions that are more complex compared to its simpler DNA binding modalities at accessible enhancer target sites.

Significance Statement

Pioneer transcription factors have the unique ability to recognize DNA target sites within closed heterochromatin and to trigger chromatin opening. Only a fraction of the heterochromatin recruitment sites of pioneers are subject to chromatin opening. The molecular basis for this selectivity is unknown and the present work addressed the importance of DNA sequence affinity for selection of sites to open. The pioneering ability of the pioneer factor Pax7 is not strictly determined by affinity or DNA sequence of binding sites, nor by number or methylation status of DNA sites. Mutation analyses showed that recruitment to heterochromatin is primarily dependent on the Pax7 paired domain whereas the ability to open chromatin requires both paired and homeo DNA binding domains.
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

The 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 Statement

Optical 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

Voltage 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 Statement

In 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

Therapeutic 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

Adeno-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

The 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

To 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

Background

Transcranial 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.

Objectives

In this study we investigate how these two factors, WM structure and brain state, interact to shape the effect of tDCS on brain network activity.

Methods

We 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.

Results

We 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.

Conclusions

Our 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

Puddle 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.
Simulations of Lévy walk
Authors: Abhignan Venkat , Rajadurai Sinduja
Published: October 2020

We simulate stable distributions to study the ideal movement pattern for the spread of a virus using autonomous carrier. We observe Lévy walks to be the most ideal way to spread and further study how the parameters in Lévy distribution affects the spread.
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

Background

Type 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.

Methods

The 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.

Results

Result 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.

Conclusions

This 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

Tumor 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.
Evolution and origin of sliding clamp in bacteria, archaea and eukarya
Authors: Acharya Sandesh , Dahal Amol , Bhattarai Hitesh Kumar
Published: October 2020

Replication of DNA is an essential process in all domains of life. A protein often involved without exception in replication is the sliding clamp. The sliding clamp encircles the DNA and helps replicative polymerase stay attached to the replication machinery increasing the processivity of the polymerase. In eukaryotes and archaea the sliding clamp is called the Proliferating Cell Nuclear Antigen (PCNA) and consists of two domains. This PCNA forms a trimer encircling the DNA as a hexamer. In bacteria, the structure of the sliding clamp is highly conserved, but the protein itself, called beta clamp, contains three domains, which dimerize to form a hexamer. The bulk of literature touts a conservation of the structure of the sliding clamp, but fails to recognize conservation of protein sequence among sliding clamps. In this paper we have used PSI blast to the second interation in NCBI to show a statistically significant sequence homology betweenPyrococcus furiosusPCNA andKallipyga gabonensisbeta clamp. The last two domains of beta clamp align with the two domains of PCNA. This homology data demonstrates that PCNA and beta clamp arose from a common ancestor. In this paper, we have further used beta clamp and PCNA sequences from diverse bacteria, archaea and eukarya to build maximum likelihood phylogenetic tree. Most, but not all, species in different domains of life harbor one sliding clamp from vertical inheritance. Some of these species that have two or more sliding clamps have acquired them from gene duplication or horizontal gene transfer events.
Association between neurofibromatosis type 1 and cerebrovascular diseases in children: a systematic review
Authors: Barreto-Duarte Beatriz , Andrade-Gomes Fabiana H. , Arriaga María B. , Araújo-Pereira Mariana , Cubillos-Angulo Juan Manuel , Andrade Bruno B.
Published: October 2020

Background

Neurofibromatosis type 1 (NF-1) is an autosomal dominant disease that affects one in every 3000 individuals. This disease can present a wide range of clinical manifestations, ranging from skin abnormalities to severe vascular changes. Although little recognized, cerebrovascular diseases (CVD), often present since childhood and diagnosed late, may have clinical manifestations ranging from headache and cognitive deficits to aneurysm rupture causing death. Thus, the CVD play an important role in the clinical manifestations, the severity of the condition and the prognosis of patients with NF-1. This systematic review aims to summarize the body of evidence linking NF-1 and CVD the in children.

Methods

Two independent reviewers performed a systematic review on the PubMed and EMBASE search platforms, using the following key terms: “neurofibromatosis type 1”, “recklinghausen disease”, “children”, “adolescents”, “stroke”, “moyamoya disease”, “vascular diseases”, “cerebrovascular disorders”, “aneurysm” and “congenital abnormalities”. Studies focused on assessing the development of CVD in children with NF-1 were included.

Results

Seven studies met the inclusion criteria. Twelve different clinical manifestations have been associated with cerebrovascular changes in children with NF-1; 44,5% of diagnosed patients were asymptomatic.

Conclusion

The available evidence suggests that cerebrovascular diseases are related with the progression of NF-1, even in the absence of a clear clinical manifestation. In addition, better prognosis was observed when imaging tests were performed to screen for cerebrovascular changes. This generated early interventions and consequently more favorable outcomes.
Complete mitogenome of endemic Plum-headed parakeetPsittacula cyanocephala– characterization and phylogenetic analysis
Authors: Dey Prateek , Sharma Sanjeev Kumar , Sarkar Indrani , Ray Swapna Devi , Pramod Padmnabhan , Sastry Kochiganti Venkata Hanumat , Quadros Goldin , Singh Renu , Singh Ram Pratap
Published: October 2020

Psittacula cyanocephalais a parakeet endemic to the Indian sub-continent, widespread in the illegal bird trade. Previous studies onPsittaculaparakeets have highlighted taxonomic ambiguities, warranting further studies to resolve such issues. Since the mitochondrial genome provides useful information about a species concerning its evolution and phylogenetics, we sequenced the complete mitogenome ofP. cyanocephalausing NGS, validated 38.86% of the mitogenome using Sanger Sequencing and compared it with other available whole mitogenomes ofPsittacula. The complete mitogenome of the species was 16814 bp in length with 54.08% AT composition.P. cyanocephala mitogenome comprises of 13 protein-coding genes, 2 rRNAs and 22 tRNAs.P. cyanocephalamitogenome organization was consistent with otherPsittaculamitogenomes. Comparative codon usage analysis indicated the role of natural selection onPsittaculamitogenomes. Strong purifying selection pressure was observed maximum onnad1andnad4lgenes. The mitochondrial control region of allPsittaculaspecies displayed the ancestral avian CR gene order. Phylogenetic analyses revealed thePsittaculagenus as paraphyletic nature, containing at least 4 groups of species within the same genus, suggesting its taxonomic reconsideration. Our results provide useful information for developing forensic tests to control the illegal trade of the species, scientific basis for phylogenetic revision of genusPsittacula.
Unravelling the evolutionary relationships of hepaciviruses within and across rodent hosts
Authors: Bletsa Magda , Vrancken Bram , Gryseels Sophie , Boonen Ine , Fikatas Antonios , Li Yiqiao , Laudisoit Anne , Lequime Sebastian , Bryja Josef , Makundi Rhodes , Meheretu Yonas , Akaibe Benjamin Dudu , Mbalitini Sylvestre Gambalemoke , Van de Perre Frederik , Van Houtte Natalie , Těšíková Jana , Wollants Elke , Van Ranst Marc , Drexler Jan Felix , Verheyen Erik , Leirs Herwig , de Bellocq Joelle Gouy , Lemey Philippe
Published: October 2020

Hepatitis C virus (HCV; genusHepacivirus) represents a major public health problem, infecting about 3 % of the human population (±185,000,000 people). Because no plausible animal reservoir carrying closely related hepaciviruses has been identified, the zoonotic origins of HCV still remain elusive. Motivated by recent findings of divergent hepaciviruses in rodents and a plausible African origin of HCV genotypes, we have screened a comprehensive collection of small mammals samples from seven sub-Saharan African countries. Out of 4,303 samples screened, 80 were found positive for the presence of hepaciviruses in 29 different host species. We here report 56 novel genomes that considerably increase the diversity of three divergent rodent hepacivirus lineages, which previously were almost exclusively represented by New World and European hepaciviruses. Further-more, we provide undisputable evidence for hepacivirus co-infections in rodents, which remarkably, we exclusively but repeatedly found in four sampled species of brush-furred mice. We also point at hepacivirus co-infections indirectly in different animal hosts by demonstrating evidence for recombination within specific host lineages. Our study considerably expands the available hepacivirus genomic data and elucidates the relatively deep evolutionary history that these pathogens have in rodents compared to other mammalian hosts. Overall, our results emphasize the importance of rodents as a potential hepacivirus reservoir and as models for investigating HCV infection dynamics.
Post-metamorphic skeletal growth in the sea urchinParacentrotus lividusand implications for body plan evolution
Authors: Thompson Jeffrey R. , Paganos Periklis , Benvenuto Giovanna , Arnone Maria Ina , Oliveri Paola
Published: October 2020

Understanding the molecular and cellular processes that underpin animal development are crucial for understanding the diversity of body plans found on the planet today. Because of their abundance in the fossil record, and tractability as a model system in the lab, skeletons provide an ideal experimental model to understand the origins of animal diversity. We herein use molecular and cellular markers to understand the growth and development of the juvenile sea urchin (echinoid) skeleton. We developed a detailed staging scheme based off of the first ∼four weeks of post-metamorphic life of the regular echinoidParacentrotus lividus. We paired this scheme with immunohistochemical staining for neuronal, muscular, and skeletal tissues, and fluorescent assays of skeletal growth and cell proliferation to understand the molecular and cellular mechanisms underlying skeletal growth and development of the sea urchin body plan. Our experiments highlight the role of skeletogenic proteins in accretionary skeletal growth and cell proliferation in the addition of new metameric tissues. Furthermore, our work provides a framework for understanding the developmental evolution of sea urchin body plans on macroevolutionary timescales.