bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypoxic Preconditioning Promotes Survivals of Human Adipocyte Mesenchymal Stem Cell via Expression of Prosurvival and Proangiogenic Biomarkers
Authors: Suryawan I Gde Rurus , Pikir Budi Susetyo , Rantam Fedik Abdul , Ratri Anudya Kartika , Nugraha Ricardo Adrian
Published: January 2021

Background

Contributing factors for improved survival of human adipocytes mesenchymal stem cells (h-AMSCs) cultured through hypoxia preconditioning, in example apoptosis inhibition involving BCL2 and HSP27 expression, trigger signal expression (VEGF), SCF expression, OCT-4 expression, and CD44+ expression.

Objective

To explain the mechanism and role of hypoxic preconditioning and the optimal duration of hypoxic preconditioning exposure to improve survival of h-AMSCs so that could it could be used as a benchmark for h-AMSCs culture strategy before transplantation.

Methods

This study was an experimental laboratory explorative study (in vitro study) with hypoxic preconditioning in human-adipose mesenchymal stem cells (h-AMSCs) cultures. This research was conducted through 4 stages ;First, Isolation and h-AMSCs culture from adipose tissue of patient (human). Second is the characterization of h-AMSCs from adipose tissue by phenotype (Flowcytometry) through CD44+, CD90+ and CD45-expression before being pre-conditioned for hypoxic treatment. Third, the hypoxic preconditioning in h-AMSCs culture (in vitro) was performed with an oxygen concentration of 1% for 24, 48 and 72 hours. Fourth, observation of survival from h-AMSCs culture was tested on the role of CD44 +, VEGF, SCF, OCT-4, BCL2, HSP27 with Flowcytometry method and apoptotic inhibition by Tunnel Assay method.

Results

The result of regression test showed that time difference had an effect on VEGF expression (p=0,000;β=−0,482) and hypoxia condition also influenced VEGF expression (p= 0,000;β=0,774). The result of path analysis showed that SCF had an effect on OCT-4 expression (p=0,000;β=0,985). The regression test results showed that time effects on HSP27 expression (p=0.000;β=0.398) and hypoxia precondition also affects HSP27 expression (p=0.000;β=0.847). Pathway analysis showed that BCL2 expression inhibited apoptosis (p=0.030;β=−0.442) and HSP27 expression also inhibited apoptosis (p=0,000;β=−0.487).

Conclusion

In conclusion, hypoxic preconditioning of h-AMSC culture has proven to increase the expression of VEGF, SCF, OCT-4, and BCL2 and HSP27. This study demonstrated and explained the existence of a new mechanism of increased h-AMSC survival in cultures with hypoxic preconditioning (O2 1%) via VEGF, SCF, OCT-4, BCL2, and HSP 27. But CD 44+ did not play a role in the mechanism of survival improvement of human AMSC survival.
Fluorescence-based 3D targeting of FIB-SEM acquisition of small volumes in large samples
Authors: Ronchi Paolo , Machado Pedro , D’Imprima Edoardo , Mizzon Giulia , Best Benedikt T. , Cassella Lucia , Schnorrenberg Sebastian , Montero Marta G. , Jechlinger Martin , Ephrussi Anne , Leptin Maria , Mahamid Julia , Schwab Yannick
Published: January 2021

Cells are three dimensional objects. Therefore, 3D electron microscopy is often crucial for correct interpretation of ultrastructural data. Today samples are frequently imaged in 3D at ultrastructural resolution using volume Scanning Electron Microscopy (SEM) methods such as Focused Ion Beam (FIB) SEM and Serial Block face SEM. While these imaging modalities allow for automated data acquisition, precise targeting of (small) volumes of interest within a large sample remains challenging. Here, we provide an easy and reliable approach to target FIB-SEM acquisition of fluorescently labelled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeting based on confocal acquisition of the fluorescence signal in the resin block. Targeted trimming of the block exposes the cell of interest and laser branding of the surface after trimming creates landmarks to precisely position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as a 3D culture of mouse primary mammary gland organoids, tracheal terminal cells inDrosophila melanogasterlarvae and ovarian follicular cells in adultDrosophila, discovering ultrastructural details that could not be appreciated before.

Ronchi et al. present a workflow to facilitate the precise targeting of three-dimensional (3D) Electron Microscopy acquisitions, guided by fluorescence. This method allows ultrastructural visualization of single cells within a millimeter-range large specimen, based on molecular identity characterized by fluorescence.
All-optical electrophysiology in hiPSC-derived neurons with synthetic voltage sensors
Authors: Puppo Francesca , Sadegh Sanaz , Trujillo Cleber A. , Thunemann Martin , Campbell Evan , Vandenberghe Matthieu , Shan Xiwei , Akkouh Ibrahim A , Miller Evan W. , Bloodgood Brenda L. , Silva Gabriel A. , Dale Anders M. , Einevoll Gaute T. , Djurovic Srdjan , Andreassen Ole A. , Muotri Alysson R. , Devor Anna
Published: January 2021

Voltage imaging and “all-optical electrophysiology” in human induced pluripotent stem cell (hiPSC)-derived neurons have opened unprecedented opportunities for high-throughput phenotyping of activity in neurons possessing unique genetic backgrounds of individual patients. While prior all-optical electrophysiology studies relied on genetically encoded voltage indicators, viral transduction of human neurons with large or multiple expression vectors can impact cell function and often lead to massive cell death. Here, we demonstrate an alternative protocol using a synthetic voltage sensor and genetically encoded optogenetic actuator that generate robust and reproducible results. We demonstrate the functionality of this method by measuring spontaneous and evoked activity in three independent hiPSC-derived neuronal cell lines with distinct genetic backgrounds.
Analysis of phloem trajectory links tissue maturation to cell specialization
Authors: Roszak Pawel , Heo Jung-ok , Blob Bernhard , Toyokura Koichi , de Luis Balaguer Maria Angels , Lau Winnie W. Y. , Hamey Fiona , Cirrone Jacopo , Wang Xin , Ursache Robertas , Tavares Hugo , Verstaen Kevin , Wendrich Jos , Melnyk Charles W. , Shasha Dennis , Ahnert Sebastian E. , Saeys Yvan , De Rybel Bert , Heidstra Renze , Scheres Ben , Mähönen Ari Pekka , Göttgens Berthold , Sozzani Rosangela , Birnbaum Kenneth D. , Helariutta Yrjö
Published: January 2021

The mechanisms that allow cells in the plant meristem to coordinate tissue-wide maturation gradients with specialized cell networks are critical for indeterminate growth. Here, we reconstructed the protophloem developmental trajectory of 19 cells from cell birth to terminal differentiation at single cell resolution in the Arabidopsis root. We found that cellular specification is mediated near the stem cell niche by PHLOEM EARLY DOF (PEAR) transcription factors. However, the PEAR dependent differentiation program is repressed by a broad gradient of PLETHORA (PLT) transcription factors, which directly inhibit PEARs’ own direct targetALTERED PHLOEM DEVELOPMENT (APL). The dissipation of PLT gradient around 7 cells from the stem cell activates APL expression, and a subsequent transitional network that results in a “seesaw” pattern of mutual inhibition over developmental time. Together, we provide a mechanistic understanding of how morphogen-like maturation gradients interface with cell-type specific transcriptional regulators to stage cellular differentiation.
The chromatin, topological and regulatory properties of pluripotency-associated poised enhancers are conservedin vivo
Authors: Crispatzu Giuliano , Rehimi Rizwan , Pachano Tomas , Bleckwehl Tore , de la Cruz Molina Sara , Xiao Cally , Mahabir-Brenner Esther , Bazzi Hisham , Rada-Iglesias Alvaro
Published: January 2021

Poised enhancers (PEs) represent a limited and genetically distinct set of distal regulatory elements that control the induction of developmental genes in a hierarchical and non-redundant manner. Before becoming activated in differentiating cells, PEs are already bookmarked in pluripotent cells with unique chromatin and topological features that could contribute to their privileged regulatory properties. However, since PEs were originally identified and subsequently characterized using embryonic stem cells (ESC) as anin vitrodifferentiation system, it is currently unknown whether PEs are functionally conservedin vivo. Here, we generate and mine various types of genomic data to show that the chromatin and 3D structural features of PEs are conserved among mouse pluripotent cells bothin vitroandin vivo. We also uncovered that, in mouse pluripotent cells, the interactions between PEs and their bivalent target genes are globally controlled by the combined action of Polycomb, Trithorax and architectural proteins. Moreover, distal regulatory sequences located close to developmental genes and displaying the typical genetic (i.e. proximity to CpG islands) and chromatin (i.e. high accessibility and H3K27me3 levels) features of PEs are commonly found across vertebrates. These putative PEs show high sequence conservation, preferentially within specific vertebrate clades, with only a small subset being evolutionary conserved across all vertebrates. Lastly, by genetically disrupting evolutionary conserved PEs in mouse and chicken embryos, we demonstrate that these regulatory elements play essential and non-redundant roles during the induction of major developmental genesin vivo.
Potent but transient immunosuppression of T-cells is a general feature of CD71+erythroid cells
Authors: Grzywa Tomasz M. , Sosnowska Anna , Rydzynska Zuzanna , Lazniewski Michal , Plewczynski Dariusz , Klicka Klaudia , Malecka-Gieldowska Milena , Rodziewicz-Lurzynska Anna , Ciepiela Olga , Justyniarska Magdalena , Pomper Paulina , Grzybowski Marcin M. , Blaszczyk Roman , Wegrzynowicz Michal , Tomaszewska Agnieszka , Basak Grzegorz , Golab Jakub , Nowis Dominika
Published: January 2021

CD71+erythroid cells (CECs) have been recently recognized in both neonates and cancer patients as potent immunoregulatory cells. Here, we show that in mice early-stage CECs expand in anemia, have high levels of arginase 2 (ARG2) and reactive oxygen species (ROS). In the spleens of anemic mice, CECs expansion-inducedL-arginine depletion suppresses T-cell responses. In humans with anemia, CECs expand and express ARG1 and ARG2 that suppress T-cells IFN-γ production. Moreover, bone marrow CECs from healthy human donors suppress T-cells proliferation. CECs differentiated from peripheral blood mononuclear cells potently suppress T-cell activation, proliferation, and IFN-γ production in an ARG- and ROS-dependent manner. These effects are the most prominent for early-stage CECs (CD71highCD235adimcells). The suppressive properties disappear during erythroid differentiation as more differentiated CECs and mature erythrocytes lack significant immunoregulatory properties. Our studies provide a novel insight into the role of CECs in the immune response regulation.
Development and validation of an LC-MS/MS method for determination of B vitamins and some its derivatives in whole blood
Authors: Kahoun David , Fojtíková Pavla , Vácha František , Nováková Eva , Hypša Václav
Published: January 2021

Obligate symbiotic bacteria associated with the insects feeding exclusively on vertebrate blood are supposed to complement B vitamins presumably lacking in their diet, vertebrate blood. Recent genomic analyses revealed considerable differences in biosynthetic capacities across different symbionts, indicating that levels of B vitamins vary across different vertebrate hosts. However, a rigorous determination of B vitamins content in blood of various vertebrates has not yet been approached. A reliable analytical method focused on B vitamin complex in blood and hemolymph can provide valuable informative background and understanding of general principles of insect symbiosis.

In this work chromatographic separation of a mixture of eight B vitamins (B1 – thiamine, B2 – riboflavin, B3 – niacin, B5 – pantothenic acid, B6 – pyridoxine, B7 – biotin, B9 – folic acid and B12 – cyanocobalamine), four B vitamin derivatives (B3 – niacinamide, B6 – pyridoxal-5-phosphate, B6 – 4-pyridoxic acid and B9 – tetrahydrofolic acid) and 3 stable isotope labelled internal standards (B3 – niacin-13C6, B5 – pantothenic acid (di-β-alanine-13C6,15N2) and B7 – biotin-(ring-6,6-d2)) on C30 column was developed. Detection was carried out using dual-pressure linear ion trap mass spectrometer in FullScan MS/MS and SIM mode. Matching internal standards with analytes was done according to the results of linearity, accuracy and precision. Except for vitamin B9 (tetrahydrofolic acid) instrument quantitation limits of all analytes were ranging from 0.42 to 5.0 μg/L, correlation coefficients from 0.9997 to 1.0000 and QC coefficients from 0.53 to 3.2 %.

Optimization of whole blood sample preparation step was focused especially on evaluation of two types of protein-precipitation agents: trichloroacetic acid and zinc sulphate in methanol. Samples of whole blood prepared in six independent replicates were spiked at 10 μg/L and 100 μg/L level. The best results were obtained for zinc sulphate in methanol, but only nine analytes (B1 – thiamine, B2 – riboflavin, B3 – niacin, B3 – niacinamide, B5 – pantothenic acid, B6 – pyridoxine, B6 – 4-pyridoxic acid, B7 – biotin and B12 – cyanocobalamine) were successfully validated. Accuracy of the procedure using this protein-precipitating agent was ranging from 89 to 120 %, precision from 0.5 to 13 % and process efficiency from 65 to 108 %.

Highlights

LC-MS/MS method for quantitation of eight B vitamins and four B vitamin derivatives was developed.

Deproteinization agents trichloroacetic acid and ZnSO4/methanol were tested for protein precipitation of whole blood.

Accuracy, precision and process efficiency were evaluated.

Successful method validation for seven B vitamins and two B vitamin derivatives in whole blood.
Impact of an alpha helix and a cysteine-cysteine disulfide bond on the resistance of bacterial adhesion pili to stress
Authors: Baker Joseph L. , Dahlberg Tobias , Bullitt Esther , Andersson Magnus
Published: January 2021

Escherichia coliexpress adhesion pili that mediate attachment to host cell surfaces that are exposed to body fluids in the urinary and gastrointestinal tracts. Pilin subunits are organized into helical polymers, with a tip adhesin for specific host binding. Pili can elastically unwind when exposed to fluid flow force, reducing the adhesin load, thereby facilitating sustained attachment. Here we investigate biophysical and structural differences of pili commonly expressed on bacteria that inhabit the urinary and intestinal tracts. Optical tweezers measurements reveal that Class 1 pili of uropathogenicE. coli(UPEC), as well as Class 1b of enterotoxigenicE. coli(ETEC), undergo an additional conformational change beyond pilus unwinding, providing significantly more elasticity to their structure than ETEC Class 5 pili. Looking comprehensively at structural and steered molecular dynamics simulation data, we find this difference in Class 1 pili subunit behavior originates from anα-helical motif that can unfold when exposed to force. A disulfide bond cross-linkingβ-strands in Class 1 pili stabilizes subunits, allowing them to tolerate higher forces than Class 5 pili that lack this covalent bond. We suggest that these extra contributions to pilus resiliency are relevant for the UPEC niche since resident bacteria are exposed to stronger, more transient shear forces compared to those experienced by ETEC bacteria in the mucosa of the intestinal tract. Interestingly, Class 1b ETEC pili include the same structural features seen in UPEC pili, while requiring lower unwinding forces that are more similar to those of Class 5 ETEC pili.

Significance Statement

Adhesion pili are often essential virulence factors for attachment of pathogenic bacteria in specific environmental niches. We provide mechanistic details of structural differences impacting the biophysical properties of pili found on bacteria in the urinary and intestinal tracts. We see that pili from urinary tract bacteria are composed of subunits optimized for their microenvironment. First, they can tolerate higher forces than intestinal pili due to a disulfide bond that limits subunit unfolding. Second, their greater flexibility is due to anα-helical motif that can unfold, absorbing force that could otherwise lead to bacteria detachment. Our work provides insight into the central role of pilus structural and biophysical properties for the sustained bacterial adherence necessary to initiate disease.
Empirical abundance distributions are more uneven than expected given their statistical baseline
Authors: Diaz Renata M. , Ye Hao , Ernest S. K. Morgan
Published: January 2021

Exploring and accounting for the emergent properties of ecosystems as complex systems is a promising horizon in the search for general processes to explain common ecological patterns. For example, the ubiquitous hollow-curve form of the species abundance distribution is frequently assumed to reflect ecological processes structuring communities, but can also emerge as a statistical phenomenon from the mathematical definition of an abundance distribution. Although the hollow curve may be a statistical artefact, ecological processes may induce subtle deviations between empirical species abundance distributions and their statistically most probable forms. These deviations may reflect biological processes operating on top of mathematical constraints and provide new avenues for advancing ecological theory. Examining ∼22,000 communities, we found that empirical SADs are highly uneven and dominated by rare species compared to their statistical baselines. Efforts to detect deviations may be less informative in small communities – those with few species or individuals – because these communities have poorly-resolved statistical baselines. The uneven nature of many empirical SADs demonstrates a path forward for leveraging complexity to understand ecological processes governing the distribution of abundance, while the issues posed by small communities illustrate the limitations of using this approach to study ecological patterns in small samples.
Simultaneous single cell measurements of intranuclear proteins and gene expression
Authors: Chung Hattie , Parkhurst Christopher N. , Magee Emma M. , Phillips Devan , Habibi Ehsan , Chen Fei , Yeung Bertrand , Waldman Julia , Artis David , Regev Aviv
Published: January 2021

Identifying gene regulatory targets of nuclear proteins in tissues remains a challenge. Here we describeintranuclearCellularIndexing ofTranscriptomes andEpitopes (inCITE-seq), a scalable method for measuring multiplexed intranuclear protein levels and the transcriptome in parallel in thousands of cells, enabling joint analysis of TF levels and gene expressionin vivo. We apply inCITE-seq to characterize cell state-related changes upon pharmacological induction of neuronal activity in the mouse brain. Modeling gene expression as a linear combination of quantitative protein levels revealed the genome-wide effect of each TF and recovered known targets. Cell type-specific genes associated with each TF were co-expressed as distinct modules that each corresponded to positive or negative TF levels, showing that our approach can disentangle relative contributions of TFs to gene expression and add interpretability to gene networks. InCITE-seq can illuminate how combinations of nuclear proteins shape gene expression in native tissue contexts, with direct applications to solid or frozen tissues and clinical specimens.
Low cost and sustainable hyaluronic acid production in a manufacturing platform based onBacillus subtilis3NA strain
Authors: Cerminati Sebastián , Leroux Mélanie , Anselmi Pablo , Peirú Salvador , Alonso Juan C. , Priem Bernard , Menzella Hugo G.
Published: January 2021

Hyaluronic acid (HA) is a high value glycosaminoglycan mostly used in health and cosmetic applications. Commercial HA is produced from animal tissues or in toxigenic bacteria of the genusStreptococcusgrown in complex media, which are expensive and raise environmental concerns due to the disposal of large amounts of broth with high organic loads. Other microorganisms were proposed as hosts for the heterologous production of HA, but the methods are still costly. The extraordinary capacity of this biopolymer to bind and retain water attracts interest for large scale applications where biodegradable materials are needed, but its high cost and safety concerns are barriers for its adoption.

Bacillus subtilis3NA strain is prototrophic, amenable for genetic manipulation, GRAS, and can rapidly reach high cell densities in salt-based media. These phenotypic traits were exploited to create a platform for biomolecule production using HA as a proof of concept. First, the 3NA strain was engineered to produce HA; second, a chemically defined medium was formulated using commodity-priced inorganic salts combined at the stoichiometric ratios needed to build the necessary quantities of biomass and HA; and third, a scalable fermentation process, where HA can be produced at the maximum volumetric productivity (VP), was designed.

A comparative economic analysis against other methods indicates that the new process may increase the operating profit of a manufacturing plant by more than 100 %. The host, the culture medium, and the rationale employed to develop the fermentation process described here, introduce an IP free platform that could be adaptable for production of other biomolecules.

Key Points

A platform for the production of biomolecules was designed based onB. subtilis3NA, a chemically defined medium and a fermentation process.

As proof of concept, high quality hyaluronic acid was produced with an environmentally friendly process.

A techno-economic analysis indicates that the process is more that 100% profitable than current methods.
Fundamental identifiability limits in molecular epidemiology
Authors: Louca Stilianos , McLaughlin Angela , MacPherson Ailene , Joy Jeffrey B. , Pennell Matthew W.
Published: January 2021

Viral phylogenies provide crucial information on the spread of infectious diseases, and many studies fit mathematical models to phylogenetic data to estimate epidemiological parameters such as the effective reproduction ratio (Re) over time. Such phylodynamic inferences often complement or even substitute for conventional surveillance data, particularly when sampling is poor or delayed. It remains generally unknown, however, how robust phylodynamic epidemiological inferences are, especially when there is uncertainty regarding pathogen prevalence and sampling intensity. Here we use recently developed mathematical techniques to fully characterize the information that can possibly be extracted from serially collected viral phylogenetic data, in the context of the commonly used birth-death-sampling model. We show that for any candidate epidemiological scenario, there exist a myriad of alternative, markedly different and yet plausible “congruent” scenarios that cannot be distinguished using phylogenetic data alone, no matter how large the dataset. In the absence of strong constraints or rate priors across the entire study period, neither maximum-likelihood fitting nor Bayesian inference can reliably reconstruct the true epidemiological dynamics from phylogenetic data alone; rather, estimators can only converge to the “congruence class” of the true dynamics. We propose concrete and feasible strategies for making more robust epidemiological inferences from viral phylogenetic data.
Python-Microscope: A new open source Python library for the control of microscopes
Authors: Pinto David Miguel Susano , Phillips Mick A , Hall Nicholas , Mateos–Langerak Julio , Stoychev Danail , Pinto Tiago Susano , Booth Martin J , Davis Ilan , Dobbie Ian M
Published: January 2021

Custom built microscopes often require control of multiple hardware devices and precise hardware coordination. It is also desirable to have a solution that is scalable to complex systems and translatable between components from different manufacturers. Here we report Python-Microscope, a free and open source Python library for high performance control of arbitrarily complex and scalable custom microscope systems. Python-Microscope offers simple to use Python-based tools, abstracting differences between physical devices by providing a defined interface for different device types. Concrete implementations are provided for a range of specific hardware and a framework exists for further expansion. Python-Microscope supports the distribution of devices over multiple computers while maintaining synchronisation via highly precise hardware triggers. We discuss the architecture choices of Python-Microscope that overcome the performance problems often raised against Python and demonstrate the different use cases that drove its design: its integration in user facing projects, namely in the Microscope-Cockpit project; in controlling complex microscopes at high speed while using the Python programming language; and as a microscope simulation tool for software development.
Experimental re-infected cats do not transmit SARS-CoV-2
Authors: Gaudreault Natasha N. , Carossino Mariano , Morozov Igor , Trujillo Jessie D. , Meekins David A. , Madden Daniel W. , Cool Konner , Artiaga Bianca Libanori , McDowell Chester , Bold Dashzeveg , Balaraman Velmurugan , Kwon Taeyong , Ma Wenjun , Henningson Jamie , Wilson Dennis W. , Wilson William C. , Balasuriya Udeni B. R. , García-Sastre Adolfo , Richt Juergen A.
Published: January 2021

SARS-CoV-2 is the causative agent of COVID-19 and responsible for the current global pandemic. We and others have previously demonstrated that cats are susceptible to SARS-CoV-2 infection and can efficiently transmit the virus to naïve cats. Here, we address whether cats previously exposed to SARS-CoV-2 can be re-infected with SARS-CoV-2. In two independent studies, SARS-CoV-2-infected cats were re-challenged with SARS-CoV-2 at 21 days post primary challenge (DPC) and necropsies performed at 4, 7 and 14 days post-secondary challenge (DP2C). Sentinels were co-mingled with the re-challenged cats at 1 DP2C. Clinical signs were recorded, and nasal, oropharyngeal, and rectal swabs, blood, and serum were collected and tissues examined for histologic lesions. Viral RNA was transiently shed via the nasal, oropharyngeal and rectal cavities of the re-challenged cats. Viral RNA was detected in various tissues of re-challenged cats euthanized at 4 DP2C, mainly in the upper respiratory tract and lymphoid tissues, but less frequently and at lower levels in the lower respiratory tract when compared to primary SARS-CoV-2 challenged cats at 4 DPC. Histologic lesions that characterized primary SARS-CoV-2 infected cats at 4 DPC were absent in the re-challenged cats. Naïve sentinels co-housed with the re-challenged cats did not shed virus or seroconvert. Together, our results indicate that cats previously infected with SARS-CoV-2 can be experimentally re-infected with SARS-CoV-2; however, the levels of virus shed was insufficient for transmission to co-housed naïve sentinels. We conclude that SARS-CoV-2 infection in cats induces immune responses that provide partial, non-sterilizing immune protection against reinfection.
mmSIM: An open toolbox for accessible structured illumination microscopy
Authors: Russell Craig T. , Shaw Michael
Published: January 2021

Since the first practical super-resolution structured illumination fluorescence microscopes (SIM) were demonstrated more than two decades ago the method has become increasingly popular for a wide range of bioimaging applications. The high cost and relative inflexibility of commercial systems, coupled with the conceptual simplicity of the approach and the desire to exploit and customise existing hardware, have led to the development of a large number of home-built systems. Several detailed hardware designs are available in the scientific literature, complemented by open-source software tools for SIM image validation and reconstruction. However, there remains a lack of simple open-source software to control these systems and manage the synchronization between hardware components, which is critical for effective SIM imaging. This article describes a new suite of software tools based on the popular Micro-Manager package, which enable the keen microscopist to develop and run a SIM system. We use the software to control two custom-built, high-speed, spatial light modulator-based SIM systems, evaluating their performance by imaging a range of fluorescent samples. By simplifying the process of SIM hardware development, we aim to support wider adoption of the technique.
ΔFBA: Predicting metabolic flux alterations using genome-scale metabolic models and differential transcriptomic data
Authors: Ravi Sudharshan , Gunawan Rudiyanto
Published: January 2021

Genome-scale metabolic models (GEMs) provide a powerful framework for simulating the entire set of biochemical reactions occurring in a cell. Constraint-based modeling tools like flux balance analysis (FBA) developed for the purposes of predicting metabolic flux distribution using GEMs face considerable difficulties in estimating metabolic flux alterations between experimental conditions. Particularly, the most appropriate metabolic objective for FBA is not always obvious, likely context-specific, and not necessarily the same between conditions. Here, we propose a new method, called ΔFBA (deltaFBA), that employs constraint-based modeling, in combination with differential gene expression data, to evaluate changes in the intracellular flux distribution between two conditions. Notably, ΔFBA does not require specifying the cellular objective to produce the flux change predictions. We showcased the performance of ΔFBA through several case studies involving the prediction of metabolic alterations caused by genetic and environmental perturbations inEscherichia coliand caused by Type-2 diabetes in human muscle.
Sterically-Confined Rearrangements of SARS-CoV-2 Spike Protein Control Cell Invasion
Authors: Dodero-Rojas Esteban , Onuchic José N. , Whitford Paul C.
Published: January 2021

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious, and transmission involves a series of processes that may be targeted by vaccines and therapeutics. During transmission, host cell invasion is controlled by a large-scale conformational change of the Spike protein. This conformational rearrangement leads to membrane fusion, which creates transmembrane pores through which the viral genome is passed to the host. During Spike-protein-mediated fusion, the fusion peptides must be released from the core of the protein and associate with the host membrane. Interestingly, the Spike protein possesses many post-translational modifications, in the form of branched glycans that flank the surface of the assembly. Despite the large number of glycosylation sites, until now, the specific role of glycans during cell invasion has been unclear. Here, we propose that glycosylation is needed to provide sufficient time for the fusion peptides to reach the host membrane, otherwise the viral particle would fail to enter the host. To understand this process, an all-atom model with simplified energetics was used to perform thousands of simulations in which the protein transitions between the prefusion and postfusion conformations. These simulations indicate that the steric composition of the glycans induces a pause during the Spike protein conformational change. We additionally show that this glycan-induced delay provides a critical opportunity for the fusion peptides to capture the host cell. This previously-unrecognized role of glycans reveals how the glycosylation state can regulate infectivity of this pervasive pathogen.
Drosophilare-zero their path integrator at the center of a fictive food patch
Authors: Behbahani Amir H. , Palmer Emily H. , Corfas Román A. , Dickinson Michael H.
Published: January 2021

The ability to keep track of one’s location in space is a critical behavior for animals navigating to and from a salient location, and its computational basis is now beginning to be unraveled. Here, we tracked flies in a ring-shaped channel as they executed bouts of search triggered by optogenetic activation of sugar receptors. Unlike experiments in open field arenas, which produce highly tortuous search trajectories, our geometrically constrained paradigm enabled us to monitor flies’ decisions to move toward or away from the fictive food. Our results suggest that flies use path integration to remember the location of a food site even after it has disappeared, and that flies can remember the location of a former food site even after walking around the arena one or more times. To determine the behavioral algorithms underlyingDrosophilasearch, we developed multiple state transition models and found that flies likely accomplish path integration by combining odometry and compass navigation to keep track of their position relative to the fictive food. Our results indicate that whereas flies re-zero their path integrator at food when only one feeding site is present, they adjust their path integrator to a central location between sites when experiencing food at two or more locations. Together, this work provides a simple experimental paradigm and theoretical framework to advance investigations of the neural basis of path integration.
Molecular programs of fibrotic change in aging human lung
Authors: Lee Jasmine , Islam Mohammad Naimul , Boostanpour Kaveh , Aran Dvir , Christenson Stephanie , Matthay Michael A. , Eckalbar Walter , DePianto Daryle J. , Arron Joseph R. , Magee Liam , Bhattacharya Sunita , Matsumoto Rei , Kubota Masaru , Farber Donna L. , Bhattacharya Jahar , Wolters Paul J. , Bhattacharya Mallar
Published: January 2021

Aging is associated with both overt and subclinical lung fibrosis, which increases risk for mortality from viruses and other respiratory pathogens. The molecular programs that induce fibrosis in the aging lung are not well understood. To overcome this knowledge gap, we undertook multimodal profiling of distal lung samples from healthy human donors across the lifespan. Telomere shortening, a cause of cell senescence and fibrosis, was progressive with age in a sample of 86 lungs and was associated with foci of DNA damage. Bulk RNA sequencing confirmed activation of cellular senescence and pro-fibrotic pathways as well as genes necessary for collagen processing with increasing age. These findings were validated in independent datasets for lung and sun-exposed skin, but not other organs including heart, liver and kidney. Cell type deconvolution analysis revealed a progressive loss of lung epithelial cells and an increasing proportion of fibroblasts. Consistent with the observed pro-fibrotic transcriptional profile, second harmonic imaging demonstrated increased density of interstitial collagen in aged human lungs. Furthermore, regions of parenchymal fibrosis were associated with decreased alveolar expansion and surfactant secretion. These findings reveal the transcriptional and structural features of fibrosis and associated physiologic impairments in normal lung aging.
Biceps femoris long head sarcomere and fascicle length adaptations after three weeks of eccentric exercise training
Authors: Pincheira Patricio A. , Boswell Melissa A. , Franchi Martino V. , Delp Scott L. , Lichtwark Glen A.
Published: January 2021

Purpose

Eccentric exercise is widely used to increase muscle fascicle lengths and thus decrease the risk of muscle strain injuries. However, the mechanisms behind this protection are still unknown. The aim of this study was to determine whether Biceps femoris long head (BFlh) fascicle length increases in response to three weeks of eccentric exercise training are the result of addition of in-series sarcomeres within muscle fibres.

Methods

Ten recreationally active participants (age: 27 ± 3 years, mass: 70 ± 14 kg, height: 174 ± 9 cm) completed three weeks of Nordic hamstring exercise (NHE) training. We collected in vivo sarcomere and muscle fascicle images of the BFlh in two regions (central and distal), utilising microendoscopy and 3D ultrasonography. These images allowed us to estimate sarcomere length, sarcomere number and fascicle lengths before and after the training intervention.

Results

Eccentric knee flexion strength increased after the training (15%, P < 0.001, ηp2= 0.75). Further, we found a significant increase in fascicle (21%, P < 0.001, ηp2= 0.81) and sarcomere (17%, P < 0.001, ηp2= 0.9) lengths in the distal but not in the central portion of the muscle. The estimated number of in series sarcomeres did not change in either region.

Conclusion

Fascicle length adaptations appear to be heterogeneous in the BFlf in response to three weeks of NHE training. An increase in sarcomere length, rather than the addition of sarcomeres in series, appears to be underlying this adaptation. The mechanism driving regional increases in fascicle and sarcomere length remain unknown, but we speculate it may be driven by regional changes in the passive tension of muscle or connective tissue adaptations.
DBPOM: a comprehensive database of pharmaco-omics for cancer precision medicine
Authors: Liu Yijun , Song Fuhu , Li Zhi , Chen Liang , Xu Ying , Sun Huiyan , Chang Yi
Published: January 2021

During the course of cancer treatment, both efficacy and the adverse effects of drugs on patients should be taken into account. Although some public databases and modeling frameworks have been developed through studies on drug response, the negative effects of drugs are always neglected. Furthermore, most of them only considered the ramifications of the drug on the cell line, but the effects on the patient still require a huge amount of work to integrate data from various databases and calculations, especially in relation to precision treatment. In order to address these issues, we developed the DBPOM (http://www.dbpom.net/, a comprehensive database of pharmaco-omics for cancer precision medicine), which explores various drugs’ efficacy levels by calculating their potency in reverse, or enhancing cancer-associated gene expression changes. When compared with existing databases, the DBPOM could estimate the effectiveness of a drug on individual patients through the mapping of various cell lines to each person according to their genetic mutation similarities. The DBPOM is an easy-to-use and one-stop database for clinicians and drug researchers to search and analyze the overall effect of a drug or a drug combination on cancer patients as well as the biological functions that they target. We anticipate that DBPOM will become an important resource and analysis platform for drug development, drug mechanism studies and the discovery of new therapies.
Free energy and kinetics of cAMP permeation through connexin26 hemichannel with and without voltage
Authors: Jiang Wenjuan , Lin Yi-Chun , Botello-Smith Wesley , Contreras Jorge , Harris Andrew L. , Maragliano Luca , Luo Yun Lyna
Published: January 2021

The connexin family is a diverse group of highly regulated non-β-barrel wide-pore channels permeable to biological signaling molecules. Despite their critical roles in mediating selective molecular signaling in health and disease, the molecular basis of permeation through these pores remains unclear. Here, we report the thermodynamics and kinetics of binding and transport of a second messenger, adenosine-3’,5’-cyclophosphate (cAMP), through a connexin26 hemichannel. Inward and outward fluxes of cAMP were first obtained from 4 μs simulations with voltages and multiple cAMPs in solution. The results are compared with the intrinsic potential of mean force (PMF) and the mean first passage times (MFPTs) of a single cAMP in the absence of voltage, obtained from a total of 16.5 μs of multi-replica Voronoi-tessellated Markovian milestoning simulations. The computed transit times through the pore correspond well to existing experimental data. Both voltage simulations and milestoning simulations revealed two cAMP binding sites with binding constants and dissociation rates computed from PMF and MFPTs. The protein dipole inside the pore produces an asymmetric PMF, reflected in unequal cAMP MFPTs in each direction once within the pore. The free energy profiles under voltages derived from intrinsic PMF provided a unified understanding of directional transition rates with/without voltage, and revealed the unique role of channel polarity and the mobile electrolyte within a wide pore on the total free energy. In addition, we show how these factors influence the cAMP dipole vector during permeation, and how cAMP affects the local and non-local pore diameter in a position-dependent manner.

Significance Statement

Connexins are wide-pore channels permeable to cellular signaling molecules. They mediate molecular signaling crucial in physiology, pathology, and development; mutations in connexins cause human pathologies. However, the fundamental structural, thermodynamic, and kinetic determinants of molecular permeability properties are unknown. Using multiple molecular dynamics simulation techniques, we report, for the first time, an in-depth investigation of the free energy and the directional transition rates of an important biological signaling molecule, cAMP, through a connexin channel. We reveal the energetics and binding sites that determine the cAMP flux, and the effects of mobile ions and external electrical field on the process. The results provide a basis for understanding the unique features of molecular flux through connexins and other non-β-barrel wide-pore channels.
Enhancing top-down proteomics of brain tissue with FAIMS
Authors: Fulcher James M. , Makaju Aman , Moore Ronald J. , Zhou Mowei , Bennett David A. , De Jager Philip L. , Qian Wei-Jun , Paša-Tolić Ljiljana , Petyuk Vladislav A.
Published: January 2021

Proteomic investigations of Alzheimer’s and Parkinson’s disease have provided valuable insights into neurodegenerative disorders. Thus far, these investigations have largely been restricted to bottom-up approaches, hindering the degree to which one can characterize a protein’s “intact” state. Top-down proteomics (TDP) overcomes this limitation, however it is typically limited to observing only the most abundant proteoforms and of a relatively small size. Therefore, offline fractionation techniques are commonly used to reduce sample complexity, limiting throughput. A higher throughput alternative is online fractionation, such as gas phase high-field asymmetric waveform ion mobility spectrometry (FAIMS). Utilizing a high complexity sample derived from Alzheimer’s disease brain tissue, we describe how the addition of FAIMS to TDP can robustly improve the depth of proteome coverage. For example, implementation of FAIMS at −50 compensation voltage (CV) more than doubled the mean number of non-redundant proteoforms observed (1,833 ± 17, n = 3), compared to without (754 ± 35 proteoforms). We also found FAIMS can influence the transmission of proteoforms and their charge envelopes based on their size. Importantly, FAIMS enabled the identification of intact amyloid beta (Aβ) proteoforms, including the aggregation-prone Aβ1-42variant which is strongly linked to Alzheimer’s disease.
Parvalbumin Interneuron Activity Underlies Vulnerability to Post-Traumatic Stress Disorder in Autism
Authors: Al Abed A. Shaam , Allen Tiarne V. , Ahmed Noorya Y. , Sellami Azza , Sontani Yovina , Marighetto Aline , Desmedt Aline , Dehorter Nathalie
Published: January 2021

A rising concern in Autism Spectrum Disorder (ASD) is the heightened sensitivity to stress and trauma, the potential consequences of which have been overlooked, particularly upon the severity of the ASD traits. This study investigated the predisposition to, and impact of, Post-Traumatic Stress Disorder (PTSD) in ASD. We first demonstrated a reciprocal relationship between the two disorders and revealed that exposure to a mild stressful event induces PTSD-like memory in four mouse models of ASD. We also establish an unanticipated consequence of stress in this condition, showing that the formation of PTSD-like memory leads to the aggravation of the core traits associated with ASD. Such a susceptibility to developing PTSD-like memory in ASD stemmed from hyperactivation of the prefrontal cortex and altered fine-tuning of parvalbumin interneuron firing. We show that this traumatic memory can be treated by recontextualization, reducing the deleterious effects on the core symptoms of ASD. Overall, this study reveals multi-level neurobiological mechanisms that explain the increased vulnerability to develop PTSD in ASD. It provides a framework for future examination of the impact of PTSD-like memory in autism and offers new directions toward behavioral therapeutic interventions targeting traumatic memory in ASD.
Evolution of irreversible somatic differentiation
Authors: Gao Yuanxiao , Park Hye Jin , Traulsen Arne , Pichugin Yuriy
Published: January 2021

A key innovation emerging in complex animals is irreversible somatic differentiation: daughters of a vegetative cell perform a vegetative function as well, thus, forming a somatic lineage that can no longer be directly involved in reproduction. Primitive species use a different strategy: vegetative and reproductive tasks are separated in time rather than in space. Starting from such a strategy, how is it possible to evolve life forms which use some of their cells exclusively for vegetative functions? Here, we developed an evolutionary model of development of a simple multicellular organism and found that three components are necessary for the evolution of irreversible somatic differentiation: (i) costly cell differentiation, (ii) vegetative cells that significantly improve the organism’s performance even if present in small numbers, and (iii) large enough organism size. Our findings demonstrate how an egalitarian development typical for loose cell colonies can evolve into germ-soma differentiation dominating metazoans.
Surface GluA1 and glutamatergic transmission are increased in cortical neurons of a VPS35 D620N knock-in mouse model of parkinsonism and altered by LRRK2 kinase inhibition
Authors: Kadgien Chelsie A , Kamesh Anusha , Khinda Jaskaran , Cao Li Ping , Fox Jesse , Farrer Matthew J , Milnerwood Austen J
Published: January 2021

Vacuolar protein sorting 35 (VPS35) regulates receptor recycling from endosomes. A missense mutation in VPS35 (D620N) leads to autosomal-dominant, late-onset Parkinson’s disease. Here, we use a VPS35 D620N knock-in mouse to study the neurobiology of this mutation. In brain tissue, we confirm previous findings that the mutation results in reduced binding of VPS35 with WASH-complex member FAM21, and robustly elevated phosphorylation of the LRRK2 kinase substrate Rab10. In cultured cortical neurons, the mutation results in increased endosomal recycling protein density (VPS35-FAM21 co-clusters and Rab11 clusters), glutamate release, and GluA1 surface expression. LRRK2 kinase inhibition exerted genotype-specific effects on GluA1 surface expression, but did not impact glutamate release phenotypes. These results improve our understanding of the early effects of the D620N mutation on cellular functions that are specific to neurons. These observations provide candidate pathophysiological pathways that may drive eventual transition to late-stage parkinsonism in VPS35 families, and support a synaptopathy model of neurodegeneration.