bluuebunny/crossref_metadata_2025 huggingface dataset:
The Hsp90 chaperone system from the African trypanosome,Trypanosoma brucei
Authors: Jamabo Miebaka , Bentley Stephen J. , Macucule-Tinga Paula , Edkins Adrienne L. , Boshoff Aileen
Published: March 2021

African Trypanosomiasis is a neglected tropical disease caused byTrypanosoma brucei(T. brucei) and is spread by the tsetse fly in sub-Saharan Africa. The disease is fatal if left untreated and the currently approved drugs for treatment are toxic and difficult to administer. The trypanosome must survive in the insect vector and its mammalian host, and to adapt to these different conditions, the parasite relies on molecular chaperones called heat shock proteins. Heat shock proteins mediate the folding of newly synthesized proteins as well as prevent misfolding of proteins under normal conditions and during stressful conditions. Heat shock protein 90 (Hsp90) is one of the major molecular chaperones of the stress response at the cellular level. It functions with other chaperones and co-chaperones and inhibition of its interactions is being explored as a potential therapeutic target for numerous diseases. This study provides an in-silico overview of Hsp90 and its co-chaperones in bothT. brucei brucei and T. brucei gambiensein relation to human and other kinetoplastid parasites. The evolutionary, functional, and structural analyses of Hsp90 were also shown. The updated information on Hsp90 and its co-chaperones from recently published proteomics onT. bruceiwas examined for the different life cycle stages and subcellular localisations. The results show a difference betweenT. b. brucei and T. b. gambiensewithT. b. bruceiencoding 12 putativeHsp90genes, 10 of which are cytosolic and located on a single chromosome whileT. gambienseencodes 5Hsp90genes, 3 of which are located in the cytosol. Eight putative co-chaperones were identified in this study, 6 TPR-containing and 2 non-TPR-containing co-chaperones. This study provides an updated context for studying the biology of the African trypanosome and evaluating Hsp90 and its interactions as potential drug targets.
Functionally distinct T-helper cell phenotypes predict resistance to different types of parasites in a wild mammal
Authors: Corripio-Miyar Yolanda , Hayward Adam , Lemon Hannah , Sweeny Amy R. , Bal Xavier , Kenyon Fiona , Pilkington Jill G , Pemberton Josephine M. , Nussey Daniel H , McNeilly Tom N
Published: March 2021

The adaptive immune system is critical to an effective, long-lasting ability to respond to infection in vertebrates and T-helper (Th) cells play a key role in orchestrating the adaptive immune response. Laboratory studies show that functionally distinct Th responses provide protection against different kinds of parasites (i.e., Th1 responses against microparasites and Th2 against macroparasites).

Natural populations must deal with challenges from a wide range of infectious agents and co-infection with different types of parasite is the norm, so different Th responses are likely to play an important and dynamic role in maintaining host health and fitness. However, the relationship between T helper immune phenotypes and infection with different types of parasites remains poorly understood in wild animals.

In this study, we characterised variation in functionally distinct Th responses (Th1, Th2, Th17 and regulatory responses) in a wild population of Soay sheep using flow cytometry to detect Th-subset specific transcription factors, andex vivolymphocyte stimulation to quantify release of Th-associated cytokines. We specifically tested the prediction that raised Th1 and Th2 responses should predict reduced apicomplexan (coccidian) and helminth (nematode) parasite burdens, respectively.

Cell counts of different Th subsets measured by flow cytometry did not vary with age or sex. However, all measures of Th-associatedex vivocytokine production increased with age, and Th17- and regulatory Th-associated cytokine production increased more rapidly with age in males than females.

Independent of age and sex, Th2-associated immune measures negatively predicted gastro-intestinal strongyle nematode faecal egg count, while production of the Th1-associated cytokine IFN-γ negatively predicted coccidian faecal oocyst count.

Our results provide important support from outside the laboratory that Th1 and Th2 responses confer resistance to different kinds of parasites (micro- and macro-parasites, respectively). They also add to mounting evidence from wild populations that Th1/Th2 trade-offs often observed in controlled laboratory experiments may not readily translate to more complex natural systems.

Our study illustrates that harnessing more specific reagents and tools from laboratory immunology has the potential to illuminate our understanding of epidemiology and host-parasite co-evolution in the wild.
Open source timed pressure control hardware and software for delivery of air mediated distensions in animal models
Authors: Patel Trishna , Hendren Jamie , Lee Nathan , Mickle Aaron D
Published: March 2021

Studying the visceral sensory component of peripheral nervous systems can be challenging due to limited options for consistent and controlled stimulation. One method for mechanical stimulation of hollow organs, including colon and bladder, are controlled distensions mediated by compressed air. For example, distension of the bladder can be used as an assay for bladder nociception. Bladder distension causes a corresponding increase in abdominal electromyography, which increases with distension pressure and is attenuated with analgesics. However, the hardware used to control these distensions are primarily all one-off custom builds, without clear directions how to build your own. This has made it difficult for these methods to be fully utilized and replicated as not everyone has access, knowledge and resources required to build this controller. Here we show an open-source Arduino based system for controlling a solenoid valve to deliver timed pressure distensions in the experimental model. This device can be controlled by one of two methods through direct TTL pulses from the experimenters data acquisition software (ex. CED Spike2) or by a graphical user interface, where the user can set the time before, during, and after distension as well as the number of cycles. This systems low cost and relative ease to build will allow more groups to utilize timed pressure distensions in their experiments.

Specifications table
Late Anisian microbe-metazoan build-ups (“stromatolites”) in the Germanic Basin – aftermath of the Permian – Triassic Crisis
Authors: Pei Yu , Duda Jan-Peter , Schönig Jan , Luo Cui , Reitner Joachim
Published: March 2021

The so-called Permian – Triassic mass extinction was followed by a prolonged period of ecological recovery that lasted until the Middle Triassic. Triassic stromatolites from the Germanic Basin seem to be an important part of the puzzle, but have barely been investigated so far. Here we analyzed late Anisian (upper Middle Muschelkalk) stromatolites from across the Germanic Basin by combining petrographic approaches (optical microscopy, micro X-ray fluorescence, Raman imaging) and geochemical analyses (sedimentary hydrocarbons, stable carbon and oxygen isotopes). Paleontological and sedimentological evidence, such asPlacunopsisbivalves, intraclasts and disrupted laminated fabrics, indicate that the stromatolites formed in subtidal, shallow marine settings. This interpretation is consistent with δ13Ccarbof about −2.1‰ to −0.4‰. Occurrences of calcite pseudomorphs after gypsum suggest slightly evaporitic environments, which is well in line with the relative rarity of fossils in the host strata. Remarkably, the stromatolites are composed of microbes (perhaps cyanobacteria and sulfate reducing bacteria) and metazoans such as non-spicular demosponges,Placunopsisbivalves, and/orSpirobis-likeworm tubes. Therefore, these “stromatolites” should more correctly be referred to as microbe-metazoan build-ups. They are characterized by diverse lamination types, including planar, wavy, domal and conical ones. Microbial mats likely played an important role in forming the planar and wavy laminations. Domal and conical laminations commonly show clotted to peloidal features and mesh-like fabrics, attributed to fossilized non-spicular demosponges. Our observations not only point up that non-spicular demosponges are easily overlooked and might be mistakenly interpreted as stromatolites, but also demonstrate that microbe-metazoan build-ups were widespread in the Germanic Basin during Early to Middle Triassic times. In the light of our findings, it appears plausible that the involved organisms benefited from elevated salinities. Another (not necessarily contradictory) possibility is that the mutualistic relationship between microbes and non-spicular demosponges enabled these organisms to fill ecological niches cleared by the Permian – Triassic Crisis. If that is to be the case, it means that such microbe-metazoan associations maintained their advantage until the Middle Triassic.
Allele frequency divergence reveals ubiquitous influence of positive selection inDrosophila
Authors: Bertram Jason
Published: March 2021

Resolving the role of natural selection is a basic objective of evolutionary biology. It is generally difficult to detect the influence of selection because ubiquitous non-selective stochastic change in allele frequencies (genetic drift) degrades evidence of selection. As a result, selection scans typically only identify genomic regions that have undergone episodes of intense selection. Yet it seems likely such episodes are the exception; the norm is more likely to involve subtle, concurrent selective changes at a large number of loci. We develop a new theoretical approach that uncovers a previously undocumented genome-wide signature of selection in the collective divergence of allele frequencies over time. Applying our approach to temporally resolved allele frequency measurements from laboratory and wildDrosophilapopulations, we quantify the selective contribution to allele frequency divergence and find that selection has substantial effects on much of the genome. We further quantify the magnitude of the total selection coefficient (a measure of the combined effects of direct and linked selection) at a typical polymorphic locus, and find this to be large (of order 1%) even though most mutations are not directly under selection. We find that selective allele frequency divergence is substantial at intermediate allele frequencies, which we argue is most parsimoniously explained by positive — not purifying — selection. Thus, in these populations most mutations are far from evolving neutrally in the short term (tens of generations), including mutations with neutral fitness effects, and the result cannot be explained simply as a purging of deleterious mutations.

Author summary

Natural selection is the process fundamentally driving evolutionary adaptation; yet the specifics of how natural selection molds the genome are contentious. A prevailing neutralist view holds that the evolution of most mutations is essentially random. Here, we develop new theory that looks past the stochasticity of individual mutations and instead analyzes the behavior of mutations across the genome as a collective. We find that selection has a strong non-random influence on most of theDrosophilagenome over short timescales (tens of generations), including the bulk of mutations that are not themselves directly targeted by selection. We show that this likely involves ongoing positive selection.
Genomic Surveillance ofAcinetobacter baumanniiin the Philippines, 2013-2014
Authors: Chilam Jeremiah , Argimón Silvia , Limas Marilyn T. , Masim Melissa L. , Gayeta June M. , Lagrada Marietta L. , Olorosa Agnettah M. , Cohen Victoria , Hernandez Lara T. , Jeffrey Benjamin , Abudahab Khalil , Hufano Charmian M. , Sia Sonia B. , Holden Matthew T.G. , Stelling John , Aanensen David M. , Carlos Celia C.
Published: March 2021

Acinetobacter baumanniiis an opportunistic nosocomial pathogen that has increasingly become resistant to carbapenems worldwide. In the Philippines, carbapenem resistance and multi-drug resistance (MDR) rates are above 50%. We undertook a genomic study of carbapenem resistantA. baumanniiin the Philippines to characterize the population diversity and antimicrobial resistance (AMR) mechanisms.

We sequenced the whole genomes of 117A. baumanniiisolates recovered by 16 hospitals in the Philippines between 2013 and 2014. We determined the multi-locus sequence type (MLST), presence of acquired AMR determinants and relatedness between isolates from the genome sequences. We also compared the phenotypic and genotypic resistance results.

Carbapenem resistance was mainly explained by the acquisition of class-D beta-lactamase geneblaOXA-23. The concordance between phenotypic and genotypic resistance to imipenem was 98.15% and 94.97% overall for the seven antibiotics analysed. Twenty-two different sequence types (ST) were identified, including 7 novel STs. The population was dominated by high-risk international clone 2 (i.e., clonal complex 92), in particular by ST195 and ST208 and their single locus variants. With WGS we identified local clusters representing potential undetected nosocomial outbreaks, as well as multi-hospital clusters indicating inter-hospital transmission. Comparison with global genomes suggested that the establishment of carbapenem-resistant IC2 clones in the Philippines is likely the result of clonal expansion and geographical dissemination and at least partly explained by inadequate hospital infection control and prevention.

This study is the first extensive genomic study of carbapenem-resistantA. baumanniiin the Philippines and underscores the importance of hospital infection control and prevention to contain high-risk clones.
Genetically Encoded CRISPR components Yield Efficient Gene Editing in the Invasive Pest,Drosophila suzukii
Authors: Kandul Nikolay P. , Belikoff Esther J. , Liu Junru , Buchman Anna , Li Fang , Yamamoto Akihiko , Yang Ting , Shriner Isaiah , Scott Maxwell J. , Akbari Omar S.
Published: March 2021

Originally from Asia,Drosophila suzukii(Matsumura, 1931, Diptera:Drosophilidae) is presently a global pest of economically important soft-skinned fruits. Also commonly known as spotted wingDrosophila(SWD), it is largely controlled through repeated applications of broad-spectrum insecticides. There is a pressing need for a better understanding of SWD biology and for developing alternative environmentally-friendly methods of control. The RNA-guided Cas9 nuclease has revolutionized functional genomics and is an integral component of several recently developed genetic strategies for population control of insects. Here we have developed transgenic strains that encode three different terminators and four different promoters to express Cas9 in both the soma and/or germline of SWD. The Cas9 lines were evaluated through genetic crossing to transgenic lines that encode single guide RNAs targeting the conserved X-linkedyellowbody andwhiteeye genes. We find that several Cas9/gRNA lines display very high editing capacity. Going forward, these tools will be instrumental for evaluating gene function in SWD and may provide tools useful for the development of new genetic strategies for control of this invasive species.
Identification of Japanese Encephalitis Virus Genotype V and Other Mosquito-borne Viruses in Camp Humphreys, Republic of Korea, using Metagenomic Analysis
Authors: Sanborn Mark A. , Wuertz Kathryn McGuckin , Kim Heung-Chul , Yang Yu , Li Tao , Pollett Simon D. , Jarman Richard G. , Berry Irina Maljkovic , Klein Terry A. , Hang Jun
Published: March 2021

Recent outbreaks of emerging and re-emerging viruses such as Zika, West Nile and Japanese encephalitis (JEV) viruses have shown that timely detection of novel arboviruses with epidemic potential is essential to mitigate human health risks. There have been rising concerns that an emergent JEV genotype (genotype V, GV) is circulating in Asia, against which the current US-FDA-approved JEV vaccine may not be efficacious. To ascertain if JEV GV and other arboviruses are circulating in East Asia, we conducted next-generation sequencing on 260 pools ofCulex tritaeniorhynchusandCulex bitaeniorhynchusmosquitoes (6,540 specimens) collected at Camp Humphreys, Republic of Korea (ROK), from mid-May - October 2018. Metagenomic analysis demonstrated a highly abundant and diverse virome with correlates of health and ecological relevance. Additionally, two complete JEV GV genome sequences were obtained from separate mosquito pools, indicating that JEV GV is circulating in the Pyeongtaek area near Seoul, ROK. Retrospective sample and sequence analyses showed that JEV GV was also present in 2016 mosquito pools collected in Seoul, ROK. Sequence-based analysis of JEV GV indicates a divergent genotype that is the most distant from the GIII derived live attenuated SA14-14-2 vaccine strain. A GV E protein investigation and 3D modeling in context to SA14-14-2 indicated likely regions responsible for reduced antibody affinity, including clusters of significant amino acid changes at externally exposed domains. These data highlight the critical need for continued mosquito surveillance as a means of detecting and identifying emerging and re-emerging arboviruses of public health relevance. Importantly, our results emphasize recent concerns that there may be a possible shift in the circulating JEV genotype in East Asia and highlights the critical need for a vaccine proven to be efficacious against this re-emergent virus.
CNPY4 inhibits the Hedgehog pathway by modulating membrane sterol lipids
Authors: Lo Megan , Sharir Amnon , Paul Michael D. , Torosyan Hayarpi , Agnew Christopher , Raleigh David R. , Jura Natalia , Klein Ophir D.
Published: March 2021

Introductory paragraph

The Hedgehog (HH) pathway is critical for development and adult tissue homeostasis1. Aberrant HH signaling can cause congenital malformations, such as digit anomalies and holoprosencephaly2, and other diseases, including cancer3. Signal transduction is initiated by HH ligand binding to the Patched 1 (PTCH1) receptor on primary cilia, thereby releasing inhibition of Smoothened (SMO), a HH pathway activator4. Although cholesterol and several oxysterol lipids, which are enriched in the ciliary membrane, play a crucial role in HH activation4,5, the molecular mechanisms governing the regulation of these lipid molecules remain unresolved. Here, we identify Canopy 4 (CNPY4), a Saposin-like protein, as a regulator of the HH pathway that controls membrane sterol lipid levels.Cnpy4−/−embryos exhibit multiple defects consistent with HH signaling perturbations, most notably changes in digit number. Knockdown ofCnpy4hyperactivates the HH pathway at the level of SMOin vitro, and elevates membrane levels of accessible sterol lipids such as cholesterol, an endogenous ligand involved in SMO activation6. Thus, our data demonstrate that CNPY4 is a negative regulator that fine-tunes the initial steps of HH signal transduction, revealing a previously undescribed facet of HH pathway regulation that operates through control of membrane composition.
Eukaryotic initiation factor EIF-3.G augments mRNA translation efficiency to regulate neuronal activity
Authors: Blazie Stephen M , Takayanagi-Kiya Seika , McCulloch Katherine A , Jin Yishi
Published: March 2021

The translation initiation complex eIF3 imparts specialized functions to regulate protein expression. However, understanding of eIF3 activities in neurons remains limited despite widespread dysregulation of eIF3 subunits in neurological disorders. Here, we report a selective role of theC. elegansRNA-binding subunit EIF-3.G in shaping the neuronal protein landscape. We identify a missense mutation in the conserved Zinc-Finger (ZF) of EIF-3.G that acts in a gain-of-function manner to dampen neuronal hyperexcitation. Using neuron type-specific seCLIP, we systematically mapped EIF-3.G-mRNA interactions and identified EIF-3.G occupancy on GC-rich 5′UTRs of a select set of mRNAs enriched in activity-dependent functions. We demonstrate that the ZF mutation in EIF-3.G alters translation in a 5′UTR dependent manner. Our study reveals anin vivomechanism for eIF3 in governing neuronal protein levels to control activity states and offers insights into how eIF3 dysregulation contributes to neuronal disorders.
Automated Modeling of Protein Accumulation at DNA Damage Sites using qFADD.py
Authors: Bowerman Samuel , Mahadevan Jyothi , Benson Philip , Rudolph Johannes , Luger Karolin
Published: March 2021

Cells are exposed to a plethora of influences that can cause damage to DNA and alter the genome, often with detrimental consequences for health. Cells mitigate this damage through a variety of repair protein pathways, and accurate measurement of the accumulation, action, and dissipation timescales of these repair proteins is required to fully understand the DNA damage response. Recently, we described the Q-FADD (Quantitation ofFluorescenceAccumulation afterDNADamage) method, which enhances the analytical power of the widely used laser microirradiation technique. In that study, Q-FADD and its preprocessing operations required licensed software and a significant amount of user overhead to find the model of best fit. Here, we present “qFADD.py”, an open-source implementation of the Q-FADD algorithm that is available as both a stand-alone software package and on a publicly accessible webserver (https://qfadd.colorado.edu/). Furthermore, we describe significant improvements to the fitting and preprocessing methods that include corrections for nuclear drift and an automated grid-search for the model of best fit. To improve statistical rigor, the grid-search algorithm also includes automated simulation of replicates. As an example, we discuss the recruitment dynamics of the signaling protein PARP1 to DNA damage sites, and we show how to compare different populations of qFADD.py models.

Statement of Significance

Cells are constantly bombarded by factors that can alter or damage their genome, and they have evolved a variety of proteins that can identify and fix this damage. To fully understand how these proteins interact in repair pathways, we need robust methods to quantify the timescales between the initial identification of the DNA damage event and the subsequent protein-protein interactions that lead to repair. Laser microirradiation is a popular method for studying these repair protein cascades in vivo, and methods for quantifying the timescales of recruitment in these experiments have historically been simple to implement but lacking in physical interpretation. Here, we present qFADD.py, the next iteration of the Q-FADD method, which uses Monte Carlo diffusion models to interpret repair protein recruitment timescales to sites of DNA damage. By moving towards automated fitting procedures with minimal bias from the user, qFADD.py provides a statistically robust but low-effort means to analyze laser microirradiation experiments through a biophysical framework.
Visuomotor information drives interference between the hands more than dynamic motor information during bimanual reaching
Authors: Desrochers Phillip C. , Brunfeldt Alexander T. , Kagerer Florian A.
Published: March 2021

During complex bimanual movements, interference can occur in the form of one hand influencing the action of the contralateral hand. Interference likely results from conflicting sensorimotor information shared between brain regions controlling hand movements via neural crosstalk. However, how visual and force-related feedback processes interact with each other during bimanual reaching is not well understood. In this study, four groups experienced either a visuomotor perturbation, dynamic perturbation, combined visuomotor and dynamic perturbation, or no perturbation in their right hand during bimanual reaches, with each hand controlling its own cursor. The left hand was examined for interference as a consequence of the right-hand perturbation. The results indicated that the visuomotor and combined perturbations showed greater interference in the left hand than the dynamic perturbation, but that the combined and visuomotor perturbations were equivalent. This suggests that dynamic sensorimotor and visuomotor processes do not interact between hemisphere-hand systems, and that primarily visuomotor processes lead to interference between the hands.
Global effects ofade8deletion on budding yeast metabolism
Authors: Kokina Agnese , Tanilas Kristel , Ozolina Zane , Pleiko Karlis , Svirksts Karlis , Vamza Ilze , Liepins Janis
Published: March 2021

Purine auxotrophy is a typical marker for many laboratory yeast strains. Supplementation of additional purine source (like adenine) is necessary to cultivate these strains. If not supplied in adequate amounts, purine starvation sets in. We tested purine starvation effects in budding yeastSaccharomyces cerevisiae ade8knockout. We explored effects brought by purine starvation in cellular, central carbon metabolism and in the global transcriptome level.

We observed that cells cultivated in purine depleted media became significantly more tolerant to severe thermal, oxidative and desiccation stresses when compared to the cells cultivated in media with all necessary supplements. When starved for purine, cells stop their cell cycle in G1 or G0 state; intracellular concentration of ATP, ADP and AMP decreases, but adenylate charge remains stable. Intracellular RNA concentration decreases and massive downregulation of ribosomal RNA occurs.

We think that purine auxotrophic starvation in a way mimics “natural” nitrogen or carbon starvations and therefore initiates elements of a transcriptional program typical for stationary phase cells (cell cycle arrest, increased stress resistance). Therefore our results demonstrate that organised metabolic response is initiated not only via “natural starvations”, but also when starving for metabolic intermediates, like purines.
BIONIC: Biological Network Integration using Convolutions
Authors: Forster Duncan T. , Li Sheena C. , Yashiroda Yoko , Yoshimura Mami , Li Zhijian , Isuhuaylas Luis Alberto Vega , Itto-Nakama Kaori , Yamanaka Daisuke , Ohya Yoshikazu , Osada Hiroyuki , Wang Bo , Bader Gary D. , Boone Charles
Published: March 2021

Biological networks constructed from varied data, including protein-protein interactions, gene expression data, and genetic interactions can be used to map cellular function, but each data type has individual limitations such as bias and incompleteness. Network integration promises to address these limitations by combining and automatically weighting input information to obtain a more accurate and comprehensive representation of the underlying biology. However, existing network integration methods may fail to adequately scale to the number of nodes and networks present in genome-scale data, may perform poorly, and may not handle partial network overlap. To address these issues, we developed a deep learning-based network integration algorithm that incorporates a graph convolutional network (GCN) framework to effectively learn dependencies between any input network. Our method, BIONIC (BiologicalNetworkIntegration usingConvolutions), learns features which contain substantially more functional information compared to existing approaches, linking genes that share diverse functional relationships, including co-complex and shared bioprocess annotation. BIONIC can integrate networks in a fully unsupervised manner if functional gene annotations are not available, and it can also leverage available annotations in a semi-supervised manner. BIONIC is scalable in both size and quantity of the input networks, making it feasible to integrate numerous networks on the scale of the human genome. To demonstrate the utility of BIONIC in identifying novel biology, we predicted essential gene chemical-genetic interactions from a small set of diagnostic non-essential gene profiles in yeast, and experimentally validated these predictions. BIONIC correctly predicted many chemical-genetic interactions, and it correctly predicted genes that are required for proper β-1,6-glucan synthesis as significant interactions with the bioactive compound pseudojervine.
Assessing the Role of Calmodulin’s Linker Flexibility in Target Binding
Authors: Sun Bin , Kekenes-Huskey Peter M.
Published: March 2021

1

Calmodulin (CaM) is a universal Ca2+binding protein known to bind at least 300 targets. The selectivity and specificity towards these targets are partially attributed to the protein’s flexible alpha-helical linker that connects its N- and C-domains. How this flexible linker mediates the driving forces guiding CaM’s binding to regulatory targets is not well-established. Therefore, we utilized the Martini coarse-grained (CG) molecular dynamics simulations to probe interrelationships between CaM/target assembly and the role of its linker region. As a model system, we simulated the binding of CaM to the CaM binding region (CaMBR) of calcineurin (CaN). The simulations were conducted assuming a ‘wild-type’ calmodulin with normal flexibility of its linker, as well as a labile, highly flexible linker variant. For the wild-type model, 98% of the 600 simulations across three ionic strengths adopted a bound complex within 2µs of simulation time; of these, 1.7% sampled the fully-bound state observed in experimentally-determined crystallographic structure. By calculating the mean-first-passage-time for these simulations, we estimated the association rate to beka= 5.9 × 108M−1s−1, which is similar to the experimentally-determined rate of 2.2 × 108M−1s−1[1]. Further, our simulations recapitulated the inverse relationship between the association rate and solution ionic strength reported in the literature. In contrast, although over 97% of the labile linker simulations formed tightly-bound complexes, only 0.3% achieved the fully-bound configuration. This effect appears to stem from a difference in the ensembles of extended and collapsed states controlled by the linker properties. Specifically, the labile linker variant samples fewer extended states compatible with target peptide binding. Therefore, our simulations suggest that variations in the CaM linker’s propensity for alpha-helical secondary structure can modulate the kinetics of target binding. This finding is important, as the linker region houses several CaM variants sites for post-translational modifications, that may alter the protein’s normal regulatory functions.
Inflammatory signals are sufficient to elicit TOX expression in mouse and human CD8 T cells
Authors: Maurice Nicholas J , Berner Jacqueline , Taber Alexis K , Zehn Dietmar , Prlic Martin
Published: March 2021

T cell receptor (TCR) stimulation leads to expression of the transcription factor TOX. Prolonged TCR signaling, such as encountered during chronic infections or in tumors, leads to sustained TOX expression, which induces a state of exhaustion or dysfunction. While CD8 memory T cells (Tmem) in specific pathogen-free laboratory mice typically do not express TOX, functional human Tmemshow heterogeneous TOX expression levels. Whether TCR-independent mechanisms can alter TOX expression in human and murine Tmemhas not been defined. We report that human and mouse Tmemincrease TOX expression following stimulation with inflammatory cytokines IL-12, IL-15, and IL-18. TOX and PD-1 expression patterns often appear to be directly correlated, however, we found that TOX is not necessary for cytokine-driven expression of PD-1. Together, these observations highlight that inflammation is sufficient to alter TOX and PD-1 expression and that the signals regulating TOX expression appear well conserved in human and murine Tmem.
Thermal preconditioning in a reef-building coral alleviates oxidative damage through a BI-1 mediated antioxidant response
Authors: Majerová Eva , Drury Crawford
Published: March 2021

Global coral reef decline is largely driven by the breakdown of the coral-algal symbiosis during temperature stress. Corals can acclimatize to higher temperatures, but the cellular processes underlying this ability are poorly understood. We show that preconditioning-based improvements in thermal tolerance inPocillopora acutaare accompanied by increases in host glutathione reductase (GR) activity and expression, which prevents DNA damage. A strong correlation betweenGRandBI-1expression in heat-stressed preconditioned corals and the presence of an antioxidant response element (ARE) in theGRpromoter suggest BI-1 could regulateGRexpression through Nrf2/ARE pathway. To fortify this link, we developed an siRNA- mediated gene knockdown protocol and targeted the coral BI-1 gene. BI-1 knock-down specifically decreased GR expression and activity and increased oxidative DNA damage in heat- stressed preconditioned corals, showing that BI-1-mediated enhanced antioxidant response during acute heat stress is a key mechanism that prevents oxidative DNA damage after preconditioning.

Teaser

Preconditioning improves redox homeostasis and prevents oxidative stress in a thermally stressed reef-building coral
Methylation studies in Peromyscus: aging, altitude adaptation, and monogamy
Authors: Horvath Steve , Haghani Amin , Zoller Joseph A. , Naderi Asieh , Soltanmohammadi Elham , Farmaki Elena , Kaza Vimala , Chatzistamou Ioulia , Kiaris Hippokratis
Published: March 2021

DNA methylation-based biomarkers of aging have been developed for humans and many other mammals and could be used to assess how stress factors impact aging. Deer mice (Peromyscus) are long living rodents that have emerged as an informative model to study aging, adaptation at extreme environments, and monogamous behavior. In the present study we have undertaken an exhaustive, genome-wide analysis of DNA methylation in Peromyscus, spanning different species, stocks, sexes, tissues and age cohorts. We describe DNA methylation-based estimators of age for different species of deer mice based on novel DNA methylation data generated on highly conserved mammalian CpGs measured with a custom array. The multi-tissue epigenetic clock for deer mice was trained on 3 tissue sources (tail, liver, brain). Two dual species human-peromyscus clocks accurately measure age and relative age defined as the ratio of chronological age to maximum age. These analyses also allowed us to accurately manifest the increasing impact of age, sex, genetic relatedness, and ultimately tissue identity, in that order, in the acquisition of specific methylation patterns in the genome. Genes that were differentially methylated across different biological variables were determined and their potential impact is discussed. This study describes highly accurate DNA methylation-based estimators of age in deer mice and illustrates how differential methylation may be linked to adaptation at different conditions.
A gut-restricted lithocholic acid analog as an inhibitor of gut bacterial bile salt hydrolases
Authors: Adhikari Arijit A. , Ramachandran Deepti , Chaudhari Snehal N. , Powell Chelsea E. , McCurry Megan D. , Banks Alexander S. , Devlin A. Sloan
Published: March 2021

Bile acids play crucial roles in host physiology by acting as both detergents that aid in digestion and as signaling molecules that bind to host receptors. Gut bacterial bile salt hydrolase (BSH) enzymes perform the gateway reaction leading to the conversion of host-produced primary bile acids into bacterially modified secondary bile acids. Small molecule probes that target BSHs will help elucidate the causal roles of these metabolites in host physiology. We previously reported the development of a covalent BSH inhibitor with low gut permeability. Here, we build on our previous findings and describe the development of a second-generation gut-restricted BSH inhibitor with enhanced potency, reduced off-target effects, and durable in vivo efficacy. SAR studies focused on the bile acid core identified a compound,AAA-10, containing a C3-sulfonated lithocholic acid scaffold and an alpha-fluoromethyl ketone warhead as a potent pan-BSH inhibitor. This compound inhibits BSH activity in conventional mouse fecal slurries, bacterial cultures, and purified BSH proteins and displays reduced toxicity against mammalian cells compared to first generation compounds. Oral administration ofAAA-10to wild-type mice for 5 days resulted in a decrease in the abundance of the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) in the mouse GI tract with low systemic exposure ofAAA-10, demonstrating thatAAA-10is an effective tool for inhibiting BSH activity and modulating bile acid pool composition in vivo.
Natural Occurring Non-Synonymous Single Nucleotide Polymorphisms in Integrase and RNase H Regulate Assembly and Autoprocessing of HIV-1
Authors: Imamichi Tomozumi , Bernbaum John G. , Laverdure Sylvain , Yang Jun , Chen Qian , Highbarger Helene , Hao Ming , Sui Hongyan , Dewar Robin , Chang Weizhong , Lane H. Clifford
Published: March 2021

Recently, a genome-wide association study using plasma HIV RNA reported that 14 naturally occurring non-synonymous single nucleotide polymorphisms (SNPs) in HIV derived from anti-retrovirus naïve patients were associated with virus load (VL). However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP using site-directed mutagenesis and examined their replicative abilities and biological properties. An HIV variant containing Met-to-Ile change at codon 50 in integrase (HIV(IN:M50I)) was found an impaired virus. Despite the mutation being in integrase, a quantification assay demonstrated that the virus release was significantly suppressed (P<0.001). Transmission electron microscopy analyses revealed that the accumulation of abnormal shapes of buds on the plasma membrane and the released virus particles retained immature forms. Western blot analysis demonstrated a defect in autoprocessing of GagPol and Gag polyproteins in the HIV(IN:M50I) particles. Förster Resonance Energy Transfer (FRET) assay displayed that GagPol containing IN:M50I (GagPol(IN:M50I)) significantly increased the efficiency of homodimerization (P<0.05) and heterodimerization with Gag (P<0.001), compared to GagPol(WT). HIV replication assay using a series of variants of HIV(IN:M50I) elucidated that the C-terminus residues, Asn at codon 288, plays a key role in the defect and the impaired maturation and replication capability was rescued by two other VL-associated SNPs, Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser change at codon 79 in RNase H. These data demonstrate that Gag and GagPol assembly, virus release and autoprocessing are not only regulated by integrase but also RNase H.

Importance

A nascent HIV-1 is noninfectious. To become an infectious virus, Gag and GagPol polyproteins in the particles need to be cleaved by mature HIV protease (PR). PR is initially translated as an inactive embedded enzyme in a GagPol polyprotein. The embedded PR in homodimerized GagPol polyproteins catalyzes a proteolytic reaction to release the mature PR. This excision step by a self-cleavage is called autoprocessing. Here, during the evaluation of roles of naturally emerging non-synonymous SNPs in HIV RNA, we found that autoprocessing is inhibited by Met-to-Ile change at codon 50 in integrase in GagPol which increases the efficiency of heterodimerization with Gag. This defect was recovered by co-existing of other SNPs: Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser mutation at codon 79 in RNase H, suggesting that autoprocessing is regulated by not only integrase but also RNase H in GagPol polyprotein.
Amotosalen is a bacterial multidrug efflux pump substrate potentially affecting its pathogen inactivation activity
Authors: Green Alex B. , Zulauf Katelyn E. , Truelson Katherine A. , Chiaraviglio Lucius , Cui Meng , Zhang Zhemin , Ware Matthew P. , Flegel Willy A. , Haspel Richard L. , Yu Ed , Kirby James E
Published: March 2021

Pathogen inactivation is a strategy to improve the safety of transfusion products. The Cerus Intercept technology makes use of a psoralen compound called amotosalen in combination with UVA light to inactivate bacteria, viruses and protozoa. Psoralens have structural similarity to bacterial multidrug-efflux pump substrates. As these efflux pumps are often overexpressed in multidrug-resistant pathogens and with recent reported outbreaks of transfusion-associated sepsis withAcinetobacter, we tested whether contemporary drug-resistant pathogens might show resistance to amotosalen and other psoralens based on multidrug efflux mechanisms through microbiological, biophysical and molecular modeling analysis. The main efflux systems inEnterobacteralesandAcinetobacter baumannii, tripartite RND (resistance-nodulation-cell division) systems which span the inner and outer membranes of Gram-negative pathogens and expel antibiotics from the bacterial cytoplasm into the extracellular space, were specifically examined. We found that amotosalen was an efflux substrate for the TolC-dependent RND efflux pumps inE. coliand the AdeABC efflux pump fromAcinetobacter baumannii, and that minimal inhibitory concentrations for contemporary bacterial isolatesin vitroapproached and exceeded the concentration of amotosalen used in the approved platelet and plasma inactivation procedures. These findings suggest that otherwise safe and effective inactivation methods should be further studied to exclude possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens.

Importance

Pathogen inactivation is a strategy to enhance the safety of transfused blood products. We identify the compound, amotosalen, widely used for pathogen inactivation, as a bacterial multidrug efflux substrate. Specifically, experiments suggest that amotosalen is pumped out of bacteria by the major TolC-dependent RND efflux pumps inE. coliand the AdeABC efflux pump inAcinetobacter baumannii. Such efflux pumps are often overexpressed in multidrug-resistant pathogens. Importantly, the minimal inhibitory concentrations for contemporary multidrug-resistantEnterobacterales,Acinetobacter baumannii,Pseudomonas aeruginosa,Burkholderiaspp.,and Stenotrophomonas maltophiliaisolates approached or exceeded the amotosalen concentration used in approved platelet and plasma inactivation procedures, potentially as a result of efflux pump activity. Although there are important differences in methodology between our experiments and blood product pathogen inactivation, these findings suggest that otherwise safe and effective inactivation methods should be further studied to exclude possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens.
Finger representation in the cortex of the congenitally blind
Authors: Wesselink D.B. , Kikkert S. , Bridge H. , Makin T.R.
Published: March 2021

Hand representation in the primary somatosensory cortex (S1) is thought to be shaped by experience. Individuals with congenital blindness rely on their sense of touch for completing daily tasks that in sighted people would be informed by vision, and possess superior tactile acuity. It has therefore been proposed that their S1 hand representation should differ from that of sighted individuals. Alternatively, it has been proposed that the improved tactile acuity in blind individuals is due to cross-modal plasticity, when regions in the occipital and temporal cortex are typically used for processing vision become activated by touch. We probed finger representation using psychophysics and 7T fMRI (1 mm3resolution) in three individuals with bilateral anophthalmia, a rare condition in which both eyes fail to develop, as well as sighted controls. Despite anophthalmic individuals’ increased reliance on touch and superior tactile acuity, we found no evidence that they had more pronounced hand representation in S1. This is in line with recent research highlighting the stability of early sensory cortex, despite altered sensorimotor experience in adulthood. Unlike sighted controls, anophthalmic individuals activated the left human middle temporal complex (hMT+) during finger movement. This area did not express any hallmark of typical sensorimotor organisation, suggesting this and previously reported activity does not indicate low-level sensorimotor hand representation. However, left hMT+ contained some single finger information, beyond that found in sighted controls. This latter finding suggests that when the developmentally flexible area hMT+ is unaffected by retinal input, it can acquire novel cross-modal processes, which are potentially unrelated to the area’s function in sighted people. As such, our findings highlight the opportunity for other organising principles, beyond domain specific plasticity, in shaping cross-modal reorganisation.
Cannabidiol attenuates cognitive deficits and neuroinflammation induced by early alcohol exposure in a mice model
Authors: García-Baos Alba , Puig-Reyne Xavi , García-Algar Óscar , Valverde Olga
Published: March 2021

Foetal alcohol spectrum disorder (FASD) is the umbrella term used to describe the physical and mental disabilities induced by alcohol exposure during development. Early alcohol exposure induces cognitive impairments resulting from damage to the central nervous system (CNS). The neuroinflammatory response accompanied by neurodegenerative mechanisms contribute to those detrimental alterations. Cannabidiol (CBD) has recently emerged as an anti-inflammatory drug that might be useful to treat several neuropsychiatric disorders. In our study, we assessed the effects of CBD on long-lasting cognitive deficits induced by early alcohol exposure. Furthermore, we analysed long-term pro-inflammatory and apoptotic markers within the prefrontal cortex and hippocampus. To model alcohol binge drinking during gestational and lactation periods, we used pregnant C57BL/6 female mice with time-limited access to 20% v/v alcohol solution. Following the prenatal and lactation alcohol exposure (PLAE), we treated the male and female offspring with CBD from post-natal day (PD) 25 until PD34, and we evaluated their cognitive performance at PD60. Our results showed that CBD treatment during peri-adolescence period ameliorates cognitive deficits observed in our FASD-like mouse model, without sex differences. Moreover, CBD restores the PLAE-induced increased levels of TNFα and IL-6 in the hippocampus. Thus, our study provides new insights for CBD as a therapeutic agent to counteract cognitive impairments and neuroinflammation caused by early alcohol exposure.
Targeting Glioblastoma Signaling and Metabolism with A Re-Purposed Brain-Penetrant Drug
Authors: Bi Junfeng , Khan Atif , Tang Jun , Wu Sihan , Zhang Wei , Gimple Ryan C. , Koga Tomoyuki , Armando Aaron M. , Miki Shunichiro , Yang Huijun , Prager Briana , Curtis Ellis J. , Wainwright Derek A. , Furnari Frank B. , Rich Jeremy N. , Cloughesy Timothy F. , Quehenberger Oswald , Kornblum Harley I. , Rzhetsky Andrey , Cravatt Benjamin F. , Mischel Paul S.
Published: March 2021

The highly lethal brain cancer glioblastoma (GBM) poses a daunting challenge because the blood-brain barrier renders potentially druggable amplified or mutated oncoproteins relatively inaccessible. Here, we identify SMPD1, an enzyme that regulates the conversion of sphingomyelin to ceramide and a critical regulator of plasma membrane structure and organization, as an actionable drug target in glioblastoma. We show that the safe and highly brain-penetrant antidepressant fluoxetine, potently inhibits SMPD1 activity, killing GBMs, in vitro and in patient-derived xenografts, through inhibition of EGFR signaling and via activation of lysosomal stress. Combining fluoxetine with the chemotherapeutic agent temozolomide, a standard of care for GBM patients, causes massive increases in GBM cell death, and complete and long-lived tumor regression in mice. Incorporation of real-world evidence from electronic medical records from insurance databases, reveals significantly increased survival in glioblastoma patients treated with fluoxetine, which was not seen in patients treated with other SSRI anti-depressants. These results nominate the repurposing of fluoxetine as a potentially safe and promising therapy for GBM patients and suggest prospective randomized clinical trials.