bluuebunny/crossref_metadata_2025 huggingface dataset:
HIV viral load and the efficacy of antiviral drug
Authors: Zhang Stephanie Yunfei , Taye Mesfin Asfaw
Published: November 2020

Developing antiviral drugs is an exigent task since viruses mutate to overcome the effect of antiviral drugs. As a result, the efficacy of most antiviral drugs is short-lived. To include this effect, we modify the Neumann and Dahari model. Considering the fact that the efficacy of the antiviral drug varies in time, the differential equations introduced in the previous model systems are rewritten to study the correlation between the viral load and antiviral drug. The effect of antiviral drug that either prevents infection or stops the production of a virus is explored. First, the efficacy of the drug is considered to decreases monotonously as time progresses. In this case, our result depicts that when the efficacy of the drug is low, the viral load decreases and increases back in time revealing the effect of the antiviral drugs is short-lived. On the other hand, for the antiviral drug with high efficacy, the viral load, as well as the number of infected cells, monotonously decreases while the number of uninfected cells increases. The dependence of the critical drug efficacy on time is also explored. Moreover, the correlation between viral load, the antiviral drug, and CTL response is also explored. In this case, not only the dependence for the basic reproduction ratio on the model parameters is explored but also we analyze the critical drug efficacy as a function of time. We show that the term related to the basic reproduction ratio increases when the CTL response step up. A simple analytically solvable mathematical model is also presented to analyze the correlation between viral load and antiviral drugs.
Knockdown of LC3 increases mitochondria-to-micronucleus transition
Authors: Hou Bolin , Li Erwei , Huang Haiwen , Yang Huaiyi , Xi Zhijun , Jiang Xuejun
Published: November 2020

Nuclear-localized mitochondria were discovered over sixty years ago1; however, the function of these organelles in the initiation of nuclear formation and development remains unknown. Here, we showed that mitochondria fragmented into dense particles to initiate and develop a nucleus, and multiple nuclei were separately and simultaneously formed by fragmented mitochondria in a single cell. The combination of nuclei individually constructed by the mitochondrial assembly of dense particles for nuclear transition partitioned the cytoplasm to form an intranuclear inclusion (INC), whose formation was not related to herniation or invagination of the cytoplasm. During nuclear conversion of itself and neighbouring organelles, the mitochondrion was incorporated into the nucleus to become a nuclear mitochondrion. Knockdown of microtubule-associated protein light chain 3 (LC3), a key autophagic protein, increased free micronuclei by delaying nuclear fusion and enhancing the mitochondria-to-micronuclei transition.
Reconstructing complex cancer evolutionary histories from multiple bulk DNA samples using Pairtree
Authors: Wintersinger Jeff A. , Dobson Stephanie M. , Stein Lincoln D. , Dick John E. , Morris Quaid
Published: November 2020

Cancers are composed of genetically distinct subpopulations of malignant cells. By sequencing DNA from cancer tissue samples, we can characterize the somatic mutations specific to each population and build clone trees describing the evolutionary ancestry of populations relative to one another. These trees reveal critical points in disease development and inform treatment. Pairtree constructs clone trees using DNA sequencing data from one or more bulk samples of an individual cancer. It uses Bayesian inference to compute posterior distributions over the evolutionary relationships between every pair of identified subpopulations, then uses these distributions in a Markov Chain Monte Carlo algorithm to perform efficient inference of the posterior distribution over clone trees. Pairtree also uses the pairwise relationships to detect mutations that violate the infinite sites assumption. Unlike previous methods, Pairtree can perform clone tree reconstructions using as many as 100 samples per cancer that reveal 30 or more cell subpopulations. On simulated data, Pairtree is the only method whose performance reliably improves when provided with additional bulk samples from a cancer. On 14 B-progenitor acute lymphoblastic leukemias with up to 90 samples from each cancer, Pairtree was the only method that could reproduce or improve upon expert-derived clone tree reconstructions. By scaling to more challenging problems, Pairtree supports new biomedical research applications that can improve our understanding of the natural history of cancer, as well as better illustrate the interplay between cancer, host, and therapeutic interventions. The Pairtree method, along with an interactive visual interface for exploring the clone tree posterior, is available at https://github.com/morrislab/pairtree.
Variability of Accessory Proteins Rules the SARS-CoV-2 Pathogenicity
Authors: Hassan Sk. Sarif , Choudhury Pabitra Pal , Uversky Vladimir N. , Dayhoff Guy W. , Aljabali Alaa A. A. , Uhal Bruce D. , Lundstrom Kenneth , Rezaei Nima , Seyran Murat , Pizzol Damiano , Adadi Parise , Lal Amos , Soares Antonio , Abd El-Aziz Tarek Mohamed , Kandimalla Ramesh , Tambuwala Murtaza , Azad Gajendra Kumar , Sherchan Samendra P. , Baetas-da-Cruz Wagner , Takayama Kazuo , Serrano-Aroca Ángel , Chauhan Gaurav , Palu Giorgio , Brufsky Adam M.
Published: November 2020

The coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) which is pandemic with an estimated fatality rate less than 1% is ongoing. SARS-CoV-2 accessory proteins ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF10 with putative functions to manipulate host immune mechanisms such as interferons, immune signaling receptor NLRP3 (NOD-, LRR-, and pyrin domain-containing 3) inflammasome, inflammatory cytokines such as interleukin 1β(IL-1β) are critical in COVID-19 pathology. Outspread variations of each of the six accessory proteins of all complete proteomes (available as of October 26, 2020, in the National Center for Biotechnology Information depository) of SARS-CoV-2, were observed across six continents. Across all continents, the decreasing order of percentage of unique variations in the accessory proteins was found to be ORF3a>ORF8>ORF7a>ORF6>ORF10>ORF7b. The highest and lowest unique variations of ORF3a were observed in South America and Oceania, respectively. This finding suggests that the wide variations of accessory proteins seem to govern the pathogenicity of SARS-CoV-2, and consequently, certain propositions and recommendations can be made in the public interest.
βPix sequesters IDOL and prevents LDL receptor degradation through a β2AR-regulated signaling pathway in Alport Syndrome
Authors: Chahdi Ahmed , Yousefi Keyvan , Capcha Jose Manuel Condor , Irion Camila Iansen , Lambert Guerline , Shehadeh Serene A. , Dunkley Julian C. , Lee Yee-Shuan , Khan Aisha , Ramic Melina , Andrade Nadja S. , Zeier Zane , Dykxhoorn Derek M. , Katsoufis Chryso Pefkaros , Freundlich Michael , Hare Joshua M. , Nabity Mary , Rivera Carolina , Lymperopoulos Anastasios , Shehadeh Lina A.
Published: November 2020

Alport syndrome (AS) is a rare disease of the glomerular basement membrane type IV collagen causing progressive renal failure. We reported increased accumulation of low-density lipoprotein (LDL) receptor (LDLR) and subsequent LDL cholesterol (LDL-C) uptake in renal tubular epithelial cells (TEC) in Alport mice, but the mechanisms regulating LDLR stability and function remain unknown. Here, we show that a selective β2-Adrenoceptor (β2AR) agonist, salbutamol, decreased LDLR levels and LDL-C uptake in Alport kidneys accompanied with reduced albuminuria and improved cardiac systolic and diastolic function. Similarly, salbutamol decreased LDL-C uptake in HK2 and HEK293 human renal epithelial cell lines, in smooth muscle cells from an X-linked hereditary nephropathy dog model (a large animal model of AS), and in TECs differentiated from AS patient-derived iPSCs. We show that the Rac1/Cdc42 guanine nucleotide exchange factor β1Pix blocked β2AR-induced LDLR degradation and, hence, increased LDL-C uptake. β1Pix also abrogated ubiquitination and degradation of LDLR induced by the inducible degrader of the LDLR (IDOL), an E3 ubiquitin ligase that promotes lysosomal LDLR ubiquitination and degradation. We identify a multimolecular complex comprised of βPix, IDOL, and LDLR and demonstrate that βPix counteracts β2AR-mediated LDLR degradation by sequestering IDOL. Our findings show βPix acts as a significant post-transcriptional regulator of IDOL-mediated LDLR degradation and identify β2AR activation as a potential treatment for Alport pathology.
Phosphorylation of the canonical histone H2A marks foci of damaged DNA in malaria parasites
Authors: Goyal Manish , Heinberg Adina , Mitesser Vera , Kandelis-Shalev Sofia , Singh Brajesh Kumar , Dzikowski Ron
Published: November 2020

Plasmodium falciparumparasites proliferate within circulating red blood cells and are responsible for the deadliest form of human malaria. These parasites are exposed to numerous intrinsic and external sources that could cause DNA damage, therefore, they have evolved efficient mechanisms to protect their genome integrity and allow them to proliferate in such conditions. In higher eukaryotes, double strand breaks rapidly lead to phosphorylation of the core histone variant H2A.X which marks the site of damaged DNA. We show that inP. falciparumthat lacks the H2A.X variant, the canonical PfH2A is phosphorylated on serine 121 upon exposure to sources of DNA damage in a dose dependent manner. We further demonstrate that phosphorylated PfH2A is recruited to foci of damaged chromatin shortly after exposure to sources of damage, while the non-phosphorylated PfH2A remains spread throughout the nucleoplasm. In addition, we found that PfH2A phosphorylation is dynamic and as the parasite repairs its DNA over time, this phosphorylation is removed. We also demonstrate that these phosphorylation dynamics could be used to establish a novel and direct DNA repair assay inP. falciparum.

Importance

Plasmodium falciparumis the deadliest human parasite that causes malaria when it reaches the blood stream and begins proliferating inside red blood cells where the parasites are particularly prone to DNA damage. The molecular mechanisms that allow these pathogens to maintain their genome integrity under such condition are also the driving force for acquiring genome plasticity that enable them to create antigenic variation and become resistant to essentially all available drugs. However, mechanisms of DNA damage response and repair have not been extensively studied in these parasites. The paper addresses our recent discovery, thatP. falciparumthat lacks the histone variant H2A.X, phosphorylates its canonical core histone PfH2A in response to exposure to DNA damage. The process of DNA repair in Plasmodium was mostly studied indirectly. Our findings enabled us to establish a direct DNA repair assay forP. falciparumsimilar to assays that are widely used in model organisms.
African Swine Fever Virus CD2v protein promotes β-Interferon expression and apoptosis in swine cells
Authors: Chaulagain Sabal , Delhon Gustavo , Khatiwada Sushil , Rock Daniel L.
Published: November 2020

African swine fever (ASF) is a disease of swine characterized by massive lymphocyte depletion in lymphatic tissues due to apoptosis of B and T cells, most likely triggered by proteins or factors secreted by infected adjacent macrophages. Here we describe a role for the ASF virus (ASFV) protein CD2v in apoptosis induction in lymphocytes. CD2v is a viral homolog of host CD2 that has been implicated in viral virulence and immunomodulationin vitro; however, its actual function remains unknown. We show that CD2v is secreted into culture medium of CD2v-expressing swine cells; and expression of-or treatment with CD2v led to significant induction of IFN-β/ISGs transcription and antiviral state. CD2v expression led to enhanced NF-κB-p65 nuclear translocation in these cultures and incubation with a NF-κB inhibitor reduced CD2v-induced NF-κB-p65 nuclear translocation and IFN-β transcription. We show that CD2v binds CD58, the natural CD2 ligand, and that CD58 siRNA knock-down results in significant reduction in NF-κB-p65 nuclear translocation and IFN-β transcription. Treatment of swine PBMC with purified CD2v led to enhanced NF-κB-p65 nuclear translocation and induction of IFN-β transcription. Further, induction of caspase-3 and PARP1 cleavage was observed in these swine PBMC at later times, providing a mechanism for CD2v-induced apoptosis of lymphocytes. Finally, IFN-β induction and NF-κB activation was inhibited in swine PBMC treated with purified CD2v pre-incubated with antibodies against CD2v. Overall, our results indicate that ASFV CD2v is an immunomodulatory protein that, by promoting lymphocyte apoptosis, may contribute to bystander lymphocyte depletion observed during ASFV infection in pigs.

IMPORTANCE

ASF, a severe hemorrhagic disease of domestic swine, represents a significant economic threat to swine industry worldwide. One critical pathological event observed in pigs infected with virulent isolates is an extensive destruction of lymphoid tissue and massive lymphocyte depletion. However, viral factor/s involved in this event are yet to be identified. Here we show that, by inducing NF-κB-dependent IFN signaling, ASFV CD2v is able to promote apoptosis in swine PBMC. We propose that CD2v released by ASFV-infected macrophages contributes to the massive depletion of lymphocytes observed in lymphoid tissues of infected pigs. Results here improve our understanding of ASFV pathogenesis and will encourage novel intervention approaches.
Sulfate import inSalmonellaTyphimurium impacts bacterial aggregation and the neutrophil respiratory burst
Authors: Westerman TL , Sheats MK , Elfenbein JR
Published: November 2020

During enteric salmonellosis, neutrophil generated reactive oxygen species alter the gut microenvironment favoring survival ofSalmonellaTyphimurium. While the type-3 secretion system-1 (T3SS-1) and flagellar motility are potentSalmonellaTyphimurium agonists of the neutrophil respiratory burstin vitro, neither of these pathways alone are responsible for stimulation of a maximal respiratory burst. In order to identifySalmonellaTyphimurium genes that impact the magnitude of the neutrophil respiratory burst, we performed a two-step screen of defined mutant libraries in co-culture with neutrophils. We first screenedSalmonellaTyphimurium mutants lacking defined genomic regions, followed by the individual mutants mapping to genomic regions under selection. Mutants in four genes,STM1696(sapF),STM2201(yeiE),STM2112(wcaD), andSTM2441(cysA), induced an attenuated respiratory burst. We linked the altered respiratory burst to reduced T3SS-1 expression and/or altered flagellar motility for two mutants (ΔSTM1696and ΔSTM2201). The ΔSTM2441mutant, defective for sulfate transport, formed aggregates in minimal media and adhered to surfaces in rich media, suggesting a role for sulfur homeostasis in regulation of aggregation/adherence. We linked the aggregation/adherence phenotype of the ΔSTM2441mutant to biofilm-associated protein A and flagellins and hypothesize that aggregation caused the observed reduction in the magnitude of the neutrophil respiratory burst. Our data demonstrate thatSalmonellaTyphimurium has numerous mechanisms to limit the magnitude of the neutrophil respiratory burst. These data further inform our understanding of how Salmonellamay alter neutrophil antimicrobial defenses.
Chemoreceptor Co-Expression inDrosophilaOlfactory Neurons
Authors: Task Darya , Lin Chun-Chieh , Vulpe Alina , Afify Ali , Ballou Sydney , Brbić Maria , Schlegel Philipp , Jefferis Gregory S. X. E. , Li Hongjie , Menuz Karen , Potter Christopher J.
Published: November 2020

Drosophila melanogasterolfactory neurons have long been thought to express only one chemosensory receptor gene family. There are two main olfactory receptor gene families inDrosophila, the Odorant Receptors (ORs) and the Ionotropic Receptors (IRs). The dozens of odorant binding receptors in each family require at least one co-receptor gene in order to function:Orcofor ORs, andIr25a,Ir8a, andIr76bfor IRs. Using a new genetic knock-in strategy, we targeted the four co-receptors representing the main chemosensory families inDrosophila(Orco, Ir8a, Ir76b, Ir25a). Co-receptor knock-in expression patterns were verified as accurate representations of endogenous expression. We find extensive overlap in expression among the different co-receptors. As defined by innervation into antennal lobe glomeruli,Ir25ais broadly expressed in 88% of all olfactory sensory neuron classes and is co-expressed in 82% of Orco+ neuron classes, including all neuron classes in the maxillary palp.Orco,Ir8a, andIr76bexpression patterns are also more expansive than previously assumed. Single sensillum recordings from Orco-expressingIr25amutant antennal and palpal neurons identify changes in olfactory responses. These results suggest co-expression of chemosensory receptors is common in olfactory neurons. Together, our data present the first comprehensive map of chemosensory co-receptor expression and reveal their unexpected widespread co-expression in the fly olfactory system.
Expansion of Human Papillomavirus-Specific T Cells in Periphery and Cervix in a Therapeutic Vaccine Recipient Whose Cervical High-Grade Squamous Intraepithelial Lesion Regressed
Authors: Shibata Takeo , Shah Sumit , Evans Teresa , Coleman Hannah , Lieblong Benjamin J. , Spencer Horace J. , Quick C. Matthew , Sasagawa Toshiyuki , Stephens Owen W. , Peterson Erich , Johann Donald , Lu Yong-Chen , Nakagawa Mayumi
Published: November 2020

Advances in high-throughput sequencing have revolutionized the manner with which we can study T cell responses. We describe a woman who received a human papillomavirus (HPV) therapeutic vaccine called PepCan, and experienced complete resolution of her cervical high-grade squamous intraepithelial lesion. By performing bulk T cell receptor (TCR) β deep sequencing of peripheral blood mononuclear cells before and after 4 vaccinations, 70 putatively vaccine-specific clonotypes were identified for being significantly increased using a beta-binomial model. Interferon-γ enzyme-linked immunospot assay identified significantly increased HPV-specific T cell response in the HPV 16 E6 91-115 region after 4 vaccinations (p=0.023). T cells with specificity to this region were sorted and analyzed using single-cell RNA-seq and TCR sequencing, HPV specificity in 60 of the 70 clonotypes identified to be vaccine-specific was demonstrated. TCR β bulk sequencing of the cervical liquid-based cytology samples and cervical formalin-fixed paraffin-embedded samples before and after 4 vaccinations demonstrated the presence of these HPV-specific T cells in the cervix. Combining traditional and cutting-edge immunomonitoring techniques enabled us to demonstrate expansion of HPV-antigen specific T cells in the periphery and cervix.
Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection
Authors: Brouwer Philip J. M. , Brinkkemper Mitch , Maisonnasse Pauline , Dereuddre-Bosquet Nathalie , Grobben Marloes , Claireaux Mathieu , de Gast Marlon , Marlin Romain , Chesnais Virginie , Diry Ségolène , Allen Joel D. , Watanabe Yasunori , Giezen Julia M. , Kerster Gius , Turner Hannah L. , van der Straten Karlijn , van der Linden Cynthia A. , Aldon Yoann , Naninck Thibaut , Bontjer Ilja , Burger Judith A. , Poniman Meliawati , Mykytyn Anna Z. , Okba Nisreen M. A. , Schermer Edith E. , van Breemen Marielle J. , Ravichandran Rashmi , Caniels Tom G. , van Schooten Jelle , Kahlaoui Nidhal , Contreras Vanessa , Lemaître Julien , Chapon Catherine , Ho Tsong Fang Raphaël , Villaudy Julien , Sliepen Kwinten , van der Velden Yme U. , Haagmans Bart L. , de Bree Godelieve J. , Ginoux Eric , Ward Andrew B. , Crispin Max , King Neil P. , van der Werf Sylvie , van Gils Marit J. , Grand Roger Le , Sanders Rogier W.
Published: November 2020

The SARS-CoV-2 pandemic is continuing to disrupt personal lives, global healthcare systems and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits and cynomolgus macaques. The vaccine-induced immunity protected macaques against a high dose challenge, resulting in strongly reduced viral infection and replication in upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic.
TRAF6 and TAK1 contribute to SAMHD1-mediated negative regulation of NF-κB signaling
Authors: Espada Constanza E. , Gelais Corine St. , Bonifati Serena , Maksimova Victoria V. , Cahill Michael P. , Kim Sun Hee , Wu Li
Published: November 2020

Sterile alpha motif and HD-domain–containing protein 1 (SAMHD1) restricts HIV-1 replication by limiting the intracellular dNTP pool. SAMHD1 also suppresses the activation of NF-κB in response to viral infections and inflammatory stimuli. However, the mechanisms by which SAMHD1 negatively regulates this pathway remain unclear. Here we show that SAMHD1-mediated suppression of NF-κB activation is modulated by two key mediators of NF-κB signaling, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and transforming growth factor-β-activated kinase-1 (TAK1). We compared NF-κB activation stimulated by interleukin (IL)-1β in monocytic THP-1 control and SAMHD1 knockout (KO) cells with and without partial TRAF6 knockdown (KD), or in cells treated with TAK1 inhibitors. Relative to control cells, IL-1β-treated SAMHD1 KO cells showed increased phosphorylation of the inhibitor of NF-κB (IκBα), an indication of pathway activation, and elevated levels ofTNF-αmRNA. Moreover, SAMHD1 KO combined with TRAF6 KD or pharmacological TAK1 inhibition reduced IκBα phosphorylation andTNF-αmRNA to the level of control cells. SAMHD1 KO cells infected with single-cycle HIV-1 showed elevated infection andTNF-αmRNA levels compared to control cells, and the effects were significantly reduced by TRAF6 KD or TAK1 inhibition. We further demonstrated that overexpressed SAMHD1 inhibited TRAF6-stimulated NF-κB reporter activity in HEK293T cells in a dose-dependent manner. SAMHD1 contains a nuclear localization signal (NLS), but an NLS-defective SAMHD1 exhibited a suppressive effect similar to the wild-type protein. Our data suggest that the TRAF6-TAK1 axis contributes to SAMHD1-mediated suppression of NF-κB activation and HIV-1 infection.

Importance

Cells respond to pathogen infection by activating a complex innate immune signaling pathway, which culminates in the activation of transcription factors and secretion of a family of functionally and genetically related cytokines. However, excessive immune activation may cause tissue damage and detrimental effects on the host. Therefore, in order to maintain host homeostasis, the innate immune response is tightly regulated during viral infection. We have reported SAMHD1 as a novel negative regulator of the innate immune response. Here, we provide new insights into SAMHD1-mediated negative regulation of the NF-κB pathway at the TRAF6-TAK1 checkpoint. We show that SAMHD1 inhibits TAK1 activation and TRAF6 signaling in response to proinflammatory stimuli. Interestingly, TRAF6 knockdown in SAMHD1-deficient cells significantly inhibited HIV-1 infection and activation of NF-κB induced by virus infection. Our research reveals a new negative regulatory mechanism by which SAMHD1 participates in the maintenance of cellular homeostasis during HIV-1 infection and inflammation.
Candidate Explorer: a tool for discovery, evaluation, and display of mutations causing significant immune phenotypes
Authors: Xu Darui , Lyon Stephen , Bu Chun Hui , Hildebrand Sara , Choi Jin Huk , Zhong Xue , Liu Aijie , Turer Emre E. , Zhang Zhao , Nair-Gill Evan , Shi Hexin , Wang Ying , Zhang Duanwu , Yue Tao , SoRelle Jeff , Misawa Takuma , Sun Lei , Wang Jianhui , Farokhnia Roxana , Sakla Andrew , Schneider Sara , Stewart Nathan , Coco Hannah , Coolbaugh Gabrielle , Hayse Braden , Mazal Sara , Medler Dawson , Nguyen Brandon , Rodriguez Edward , Wadley Andrew , Tang Miao , Li Xiaohong , Anderton Priscilla , Keller Katie , Scott Lindsay , Quan Jiexia , Cooper Sydney , Qin Baifang , Cardin Jennifer , Simpson Rochelle , Tadesse Meron , Sun Qihua , Santoyo John , Bronikowski Amy , Johnson Alexyss , Moresco Eva Marie Y. , Beutler Bruce
Published: November 2020

When applied to immunity, forward genetic studies use meiotic mapping to provide strong statistical evidence that a particular mutation is causative of a particular immune phenotype. Notwithstanding this, co-segregation of multiple mutations, occasional unawareness of mutations, and paucity of homozygotes may lead to erroneous declarations of cause and effect. We sought to improve the selection of authentic causative mutations using a machine learning software tool, Candidate Explorer (CE), which integrates 65 data features into a single numeric score, mathematically convertible to the likelihood of verification of any putative mutation-phenotype association. CE has identified most genes within which mutations can be causative of flow cytometric phenovariation inMus musculus. The majority of these genes were not previously known to support immune function or homeostasis. Mouse geneticists will find CE data informative in identifying causative mutations within quantitative trait loci, while clinical geneticists may use CE to help connect causative variants with rare heritable diseases of immunity, even in the absence of linkage information. CE displays integrated mutation, phenotype, and linkage data, and is freely available for query online.
Orthogonal CRISPR-Cas tools for genome editing, inhibition, and CRISPR recording in zebrafish embryos
Authors: Takasugi Paige R. , Wang Shengzhou , Truong Kimberly T. , Drage Evan P. , Kanishka Sahar N. , Higbee Marissa A. , Bamidele Nathan , Ojelabi Ogooluwa , Sontheimer Erik J. , Gagnon James A.
Published: November 2020

The CRISPR-Cas universe continues to expand. The type II CRISPR-Cas system fromStreptococcus pyogenes(SpyCas9) is the most widely used for genome editing due to its high efficiency in cells and organisms. However, concentrating on a single CRISPR-Cas system imposes limits on target selection and multiplexed genome engineering. We hypothesized that CRISPR-Cas systems originating from different bacterial species could operate simultaneously and independently due to their distinct single-guide RNAs (sgRNAs) or CRISPR-RNAs (crRNAs), and protospacer adjacent motifs (PAMs). Additionally, we hypothesized that CRISPR-Cas activity in zebrafish could be regulated through the expression of inhibitory anti-CRISPR (Acr) proteins. Here, we use a simple mutagenesis approach to demonstrate that CRISPR-Cas systems fromStreptococcus pyogenes(SpyCas9),Streptococcus aureus(SauCas9),Lachnospiraceae bacterium(LbaCas12a, previously known as LbCpf1), are orthogonal systems capable of operating simultaneously in zebrafish. CRISPR systems fromAcidaminococcussp. (AspCas12a, previously known as AsCpf1) andNeisseria meningitidis(Nme2Cas9) were also active in embryos. We implemented multichannel CRISPR recording using three CRISPR systems and show that LbaCas12a may provide superior information density compared to previous methods. We also demonstrate that type II Acrs (anti-CRISPRs) are effective inhibitors of SpyCas9 in zebrafish. Our results indicate that at least five CRISPR-Cas systems and two anti-CRISPR proteins are functional in zebrafish embryos. These orthogonal CRISPR-Cas systems and Acr proteins will enable combinatorial and intersectional strategies for spatiotemporal control of genome editing and genetic recording in animals.
Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype
Authors: Lee Young-Ho , Lee Hyeong Ji , Kim Hyung Cheol , Lee Yujean , Nam Su Kyung , Hupperetz Cedric , Ma Jennifer SY , Wang Xinxin , Singer Oded , Kim Won Seog , Kim Seok Jin , Koh Youngil , Jung Inkyung , Kim Chan Hyuk
Published: November 2020

CD19-targeting chimeric antigen receptor (CAR) T cells have become an important therapeutic option for patients with relapsed and refractory B cell malignancies. However, recent clinical data indicate that a significant portion of patients still do not benefit from the therapy owing to various resistance mechanisms, including high expression of multiple inhibitory immune checkpoint receptors on activated CAR T cells. Here, we report a lentiviral two-in-one CAR T approach in which two checkpoint receptors are downregulated simultaneously by a dual short-hairpin RNA (shRNA) cassette integrated into a CAR vector. Using this system, we evaluated CD19-targeting CAR T cells in the context of four different checkpoint combinations—PD-1/TIM-3, PD-1/LAG-3, PD-1/CTLA-4 and PD-1/TIGIT—and found that CAR T cells with PD-1/TIGIT downregulation uniquely exerted synergistic antitumor effects in mouse xenograft models compared with PD-1 single downregulation, and maintained cytolytic and proliferative capacity upon repeated antigen exposure. Importantly, functional and phenotypic analyses of CAR T cells as well as analyses of transcriptomic profiles suggested that downregulation of PD-1 enhances short-term effector function, whereas downregulation of TIGIT is primarily responsible for maintaining a less-differentiated/exhausted state, providing a potential mechanism for the observed synergy. The PD-1/TIGIT–downregulated CAR T cells generated from diffuse large B-cell lymphoma patient-derived T cells using a clinically applicable manufacturing process also showed robust antitumor activity and significantly improved persistencein vivocompared with conventional CD19-targeting CAR T cells. Overall, our results demonstrate that the cell-intrinsic PD-1/TIGIT dual downregulation strategy may prove effective in overcoming immune checkpoint-mediated resistance in CAR T therapy.
A marine bacterial community that degrades poly(ethylene terephthalate) and polyethylene
Authors: Gao Rongrong , Sun Chaomin
Published: November 2020

Plastic wastes have become the most common form of marine debris and present a growing global pollution problem. Recently, microorganisms-mediated degradation has become a most promising way to accomplish the eventual bioremediation of plastic wastes due to their prominent degradation potentials. Here, a marine bacterial community which could efficiently colonize and degrade both poly (ethylene terephthalate) (PET) and polyethylene (PE) was discovered through a screening with hundreds of plastic waste associated samples. Using absolute quantitative 16S rRNA sequencing and cultivation methods, we obtained the abundances and pure cultures of three bacteria mediating plastic degradation. We further reconstituted a tailored bacterial community containing above three bacteria and demonstrated its efficient degradation of PET and PE through various techniques. The released products from PET and PE degraded by the reconstituted bacterial community were determined by the liquid chromatography-mass spectrometry. Finally, the plastic degradation process and potential mechanisms mediated by the reconstituted bacterial community were elucidated through transcriptomic methods. Overall, this study establishes a stable and effective marine bacterial community for PET and PE degradation and sheds light on the degradation pathways and associated mechanistic processes, which paves a way to develop a microbial inoculant against plastic wastes.
Bacteroidetes contribute to the carbon and nutrient cycling of deep sea through breaking down diverse glycans
Authors: Zheng Rikuan , Cai Ruining , Liu Rui , Liu Ge , Sun Chaomin
Published: November 2020

Bacteroidetes are thought to be specialized for the degradation of algae-derived ocean polysaccharides and are a major contributor to the marine carbon and nutrient cycling. Here, we first show Bacteroidetes are the second most abundant phylum bacteria in deep-sea cold seep and possess more genes associated with polysaccharides degradation than other bacteria through metagenomics methods. We further isolate a novel Bacteroidetes species,Maribellus comscasisWC007T, which can efficiently degrade numerous different polysaccharides including: cellulose, pectin, fucoidan, mannan, xylan and starch. These results are verified by transcriptomic analyses and growth assays. Notably, we find cellulose promotes abundant bacterial growth, and using transcriptomics and metabolomics we finally report on the underlying mechanisms of cellulose degradation and utilization, as well as potential contributions to the carbon cycling. Overall, our results suggest Bacteroidetes play key roles in the deep-sea carbon and nutrient cycling, likely due to their high abundance and prominent polysaccharide degradation capabilities.

One Sentence Summary

Bacteroidetes contribute to ocean carbon and nutrient cycle.
Enhancing the prediction of protein coding regions in biological sequence via a deep learning framework with hybrid encoding★,★★
Authors: Wei Chao , Zhang Junying , Yuan Xiguo
Published: November 2020

Protein coding regions prediction is a very important but overlooked subtask for tasks such as prediction of complete gene structure, coding/noncoding RNA. Many machine learning methods have been proposed for this problem, they first encode a biological sequence into numerical values and then feed them into a classifier for final prediction. However, encoding schemes directly influence the classifier’s capability to capture coding features and how to choose a proper encoding scheme remains uncertain. Recently, we proposed a protein coding region prediction method in transcript sequences based on a bidirectional recurrent neural network with non-overlapping 3-mer feature, and achieved considerable improvement over existing methods, but there is still much room to improve the performance. First, 3-mer feature that counts the occurrence frequency of trinucleotides in a biological sequence only reflect local sequence order information between the most contiguous nucleotides, which loses almost all the global sequence order information. Second, kmer features of lengthklarger than three (e.g., hexamer) may also contain useful information. Based on the two points, we here present a deep learning framework with hybrid encoding for protein coding regions prediction in biological sequences, which effectively exploit global sequence order information, non-overlapping gapped kmer (gkm) features and statistical dependencies among coding labels. 3-fold cross-validation tests on human and mouse biological sequences demonstrate that our proposed method significantly outperforms existing state-of-the-art methods.
Genetic composition and evolution of the prevalentMycobacteriumtuberculosis lineages 2 and 4 in the Chinese and Zhejiang Province populations
Authors: Wu Beibei , Zhu Wenlong , Wang Yue , Wang Qi , Zhou Lin , Liu Zhengwei , Bi Lijun , Barun Mathema , Kreiswirth Barry N. , Chen Liang , Chen Songhua , Wang Xiaomeng , Wang Weibing
Published: November 2020

The causative agent of tuberculosis (TB) comprises seven human-adapted lineages. Human movements and host genetics are crucial to TB dissemination. We analyzed whole-genome sequencing data for a countrywide collection of 1154 isolates and a provincial collection of 1296 isolates, constructed the best-scoring maximum likelihood phylogenetic tree, conducted Bayesian evolutionary analysis to compute the most recent common ancestors of lineages 2 and 4, and assessed the antigenic diversity in human T cell epitopes by calculating pairwise dN/dS ratios. Of the 1296 Zhejiang isolates, 964 (74.38%) belonged to lineage 2 and 332 (25.62%) belonged to lineage 4. L2.2 is the most ancient sub-lineage in Zhejiang, first appearing approximately 6897 years ago (95% HDI: 6513-7298). L4.4 is the most modern sub-lineage, first appearing approximately 2217 years ago (95% HDI: 1864-2581). The dN/dS ratios revealed that the epitope and non-epitope regions of lineage 2 strains were significantly (P<0.001) more conserved than those of lineage 4. An increase in the frequency of lineage 4 may reflect its successful transmission over the last 20 years. The recent common ancestors and transmission routes of the sub-lineages are related to the entry of humans into China and Zhejiang Province.
Epilepsy-associated Mutations in the Calcium-sensing Receptor Disrupt the Regulation of NALCN Sodium-leak Channel by Extracellular Calcium in Neurons
Authors: Cang Chunlei , Lu Boxun , Ren Dejian
Published: November 2020

Most mammalian neurons have a resting membrane potential (RMP) of ~ −50 mV to −70 mV, significantly above the equilibrium potential of K+(EK) of ~ −90 mV. The resting Na+-leak conductance is a major mechanism by which neurons maintain their RMPs above EK. In the hippocampal neurons, the TTX-insensitive, voltage-independent Na+leak is mediated by the NALCN cation channel. Extracellular Ca2+(Ca2+e) also controls the sizes of NALCN current (INALCN) in a G-protein-dependent fashion. The molecular identities of the basal Na+conductances and their regulation in other regions in the central nervous system and in the peripheral nervous system are less established. Here we show that neurons cultured from mouse cortices, ventral tegmental area, spinal cord and dorsal root ganglia all have a NALCN-dependent basal Na+-leak conductance that is absent in NALCN knockout mice. Like in hippocampal neurons, a decrease in [Ca2+]eincreases INALCN. Using shRNA knockdown, we show that the regulation of INALCNby Ca2+ein neurons requires the Ca2+-sensing G-protein-coupled receptor CaSR. Surprisingly, the functional coupling from [Ca2+]eto NALCN requires CaSR’s distal C-terminal domain that is dispensable for the receptor’s ability to couple [Ca2+]eto its canonical signaling targets such as PLC and MAPK. In addition, several epilepsy-associated human CaSR mutations, though sparing the receptor’s ability to sense Ca2+eto maintain systemic [Ca2+], disrupt the ability of CaSR to regulate NALCN. These findings uncover a unique mechanism by which CaSR regulates neuronal excitability via NALCN in the central and peripheral nervous system.
Interactions between axon-like projections extended byDrosophilaFollicle Stem Cells dictate cell fate decisions
Authors: Lee Eric H. , Zinshteyn Daniel , Wang Melissa , Reinach Jessica , Chau Cindy , Costa Kelly , Vargas Alberto , Johnson Aminah , Alexander Jennifer I. , O’Reilly Alana M.
Published: November 2020

Stem cells cycle between periods of quiescence and proliferation to promote healthy tissue aging. Once proliferation is initiated, mechanisms that control the balance between self-renewal and differentiation must be engaged to ensure maintenance of stem cell pools until the next quiescent cycle occurs. Here, we demonstrate that dynamic axon-like projections extended by Follicle Stem Cells (FSCs) in theDrosophilaovary control the self-renewal-differentiation balance. Known axon growth regulatorsstill lifeandsickieare necessary and sufficient for FSC projection growth, mediating organization of germline cyst architecture during follicle formation, controlling targeting of projections to FSCs or germ cells, and regulating expression of the cell fate determinants Eyes Absent (Eya) and Castor (Cas). Our results support a model in which FSC projections function similarly to axons, providing structural organization to a dynamic organ while mediating communication between distinct cell types to effect the key cell fate decision to self-renew or differentiate.
InMycobacterium abscessus, the stringent factor Rel regulates metabolism, but is not the only (p)ppGpp synthase
Authors: Hunt-Serracín Augusto César , Kazi Misha I. , Boll Joseph M. , Boutte Cara C.
Published: November 2020

The stringent response is a broadly conserved stress response system that exhibits functional variability across bacterial clades. Here, we characterize the role of the stringent factor Rel in the non-tuberculous mycobacterial pathogen,Mycobacterium abscessus(Mab). We found that deletion ofreldoes not ablate (p)ppGpp synthesis, and thatreldoes not provide a survival advantage in several stress conditions, or in antibiotic treatment. Transcriptional data show that RelMabis involved in regulating expression of anabolism and growth genes in stationary phase. However, it does not activate transcription of stress response or antibiotic resistance genes, and actually represses transcription of many antibiotic resistance genes. This work shows that there is an unannotated (p)ppGpp synthetase inMab.

Importance

In this study, we examined the functional roles of the stringent factor Rel inMycobacterium abscessus(Mab). In most species, stringent factors synthesize the alarmone (p)ppGpp, which globally alters transcription to promote growth arrest and survival under stress and in antibiotic treatment. Our work shows that inMab, an emerging pathogen which is resistant to many antibiotics, the stringent factor Rel is not solely responsible for synthesizing (p)ppGpp. We find that RelMabdownregulates many metabolic genes under stress, but does not upregulate stress response genes and does not promote antibiotic tolerance. This study implies that there is another critical but unannotated (p)ppGpp synthetase inMab, and suggests that RelMabinhibitors are unlikely to sensitizeMabinfections to antibiotic treatment.
K+efflux through postsynaptic NMDA receptors suppresses local astrocytic glutamate uptake
Authors: Tyurikova Olga , Shih Pei‐Yu , Dembitskaya Yulia , Savtchenko Leonid P. , McHugh Thomas J. , Rusakov Dmitri A. , Semyanov Alexey
Published: November 2020

Glutamatergic transmission prompts K+efflux through postsynaptic NMDA receptors. K+depolarizes local presynaptic terminals, boosting glutamate release, but whether it also affecting glutamate uptake remains unknown. Here, we find that the pharmacological blockade, or conditional knockout, of NMDA receptors suppresses the progressive use‐ dependent increase in the amplitude and decay time of the astrocytic glutamate transporter current (IGluT), whereas blocking the astrocytic inward‐rectifying K+channels prevents the decay time increase only. Glutamate spot‐uncaging reveals that local astrocyte depolarization, rather than extracellular K+rises on their own, reduces the amplitude and prolong the decay of IGluT. Biophysical simulations confirm that local transient elevations of extracellular K+can inhibit local glutamate uptake in fine astrocytic processes. Optical glutamate sensor imaging and a two‐pathway test relate postsynaptic K+efflux to enhanced extrasynaptic glutamate signaling. Thus, postsynaptic K+efflux facilitates presynaptic release while reducing local glutamate uptake.
Cognitive insights from evolutionarily new brain structures in prefrontal cortex
Authors: Voorhies Willa I. , Miller Jacob A. , Yao Jewelia K. , Bunge Silvia A. , Weiner Kevin S.
Published: November 2020

While the disproportionate expansion of lateral prefrontal cortex (LPFC) in humans compared to non-human primates is accepted, the relationship between evolutionarily new LPFC brain structures and uniquely human cognitive skills is largely unknown. Here, we tested the relationship between variability in evolutionarily new LPFC tertiary sulci and reasoning skills in a pediatric cohort. A novel data-driven approach in independentdiscoveryandreplicationsamples revealed that the depth of specific LPFC tertiary sulci predicts individual differences in reasoning skills beyond age. These findings support a classic, yet untested, theory linking the protracted development of tertiary sulci to late-developing cognitive processes. We suggest that deeper LPFC tertiary sulci reflect reduced short-range connections in white matter, which in turn, improve the efficiency of local neural signals underlying cognitive skills such as reasoning that are central to human cognitive development. To expedite discoveries in future neuroanatomical-behavioural studies, we share sulcal definitions with the field.
Downstream Effects: Impact of Antibiotic Pollution on an Aquatic Host-Parasite Interaction
Authors: Melchiorre Hannah G. , Gutierrez Stephanie O. , Minchella Dennis J. , Vannatta J. Trevor
Published: November 2020

The global increase in antibiotic use has led to contamination of freshwater environments occupied by parasites and their hosts. Despite the identified impacts of antibiotics on humans and wildlife, the effect of antibiotics on host-parasite life cycles is relatively unexplored. We utilize the trematode parasiteSchistosoma mansoni, and its snail intermediate hostBiomphalaria glabratato investigate the influence of an ecologically relevant antibiotic concentration on the life history characteristics of both parasite and host. Our results demonstrate that antibiotics not only accelerate parasite development time, but also increase host reproduction and delay parasite-induced host castration. We propose that antibiotic exposure alters host microbiome composition, leading to increased host susceptibility and higher parasite production. Using a mathematical model, we suggest that life history alterations associated with antibiotics are likely to increase parasite transmission and disease burden. Our study suggests that antibiotic pollution could impact freshwater ecosystems by influencing host-parasite dynamics and potentially increase the burden of schistosomiasis in endemic regions.
A human iPSC-astroglia neurodevelopmental model reveals divergent transcriptomic patterns in schizophrenia
Authors: Szabo Attila , Akkouh Ibrahim A. , Vandenberghe Matthieu , Osete Jordi Requena , Hughes Timothy , Heine Vivi , Smeland Olav B. , Glover Joel C. , Andreassen Ole A. , Djurovic Srdjan
Published: November 2020

While neurodevelopmental abnormalities have been associated with schizophrenia (SCZ), the role of astroglia in disease pathophysiology remains poorly understood. In this study we used a human induced pluripotent stem cell (iPSC)-derived astrocyte model to investigate the temporal patterns of astroglia differentiation during developmental stages critical for SCZ using RNA-sequencing. The model generated astrocyte-specific patterns of gene expression during differentiation, and demonstrated that these patterns correspond well to astroglia-specific expression signatures ofin vivocortical fetal development. Applying this model, we were able to identify SCZ-specific expression dynamics in human astrocytes, and found that SCZ-associated differentially expressed genes were significantly enriched in the medial prefrontal cortex, striatum, and temporal lobe, targetingVWA5AandADAMTS19. In addition, SCZ astrocytes displayed alterations in calcium signaling, and significantly decreased glutamate uptake and metalloproteinase activity relative to controls. These results provide strong support for the validity of our astrocyte model, and implicate novel transcriptional dynamics in astrocyte differentiation in SCZ together with functional changes that are potentially important biological components of SCZ pathology.