PaperPlayer biorxiv molecular biology

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.23.217315v1?rss=1

Authors: Murakami, M., Murakami, A. M., Hirota, K., Itagaki, S.

Abstract:

In this study, we introduced an efficient subcloning and expression system with two inducible prokaryotic expression promoters, arabinose and lac, in a single plasmid in Escherichia coli. This pdMAX system is a manageable size at 5811 bp. The arabinose promoter unit allows for the expression of a FLAG-tagged protein, while the isopropyl-{beta}-D-thiogalactoside (IPTG)-inducible unit allows for the expression of a Myc-tagged protein. An efficient subcloning (DNA insertion) system (iUnit) follows each promoter. The iUnit, based on a toxin that targets DNA topoisomerase of E. coli, allows for effective selection with arabinose or IPTG induction.

Interestingly, the dual induction plasmid system shows limited protein expression. To analyze the expression of inserted genes, we performed an -complementation assay, in which the -peptide of the lacZ ({beta}-galactosidase) gene is inserted in XL-10 E. coli cells. E. coli expressing the recombinant plasmid form blue colonies when plated on ampicillin/IPTG- or arabinose/X-gal-containing plates if the -peptide was correctly inserted in-frame to produce the tagged protein. This assay system assesses whether the -peptide was inserted at the restriction sites (EcoRV or SmaI), whether the inserted peptide was expressed, and whether the inserted -peptide sequence was ligated in-frame to produce a tagged protein. Frameshifts result in no -complementation and white colonies.

With the dual promoter plasmid (pdMAX) system, expressed lacZ activity was significantly decreased comparing with the solo expression (pgMAX) system. Despite this disadvantage, we believe the pdMAX system is still useful for the analysis of distinct genes in E. coli, which will enable different types of expression analysis. Overall, the novel pdMAX system allows for efficient subcloning of two different genes. Furthermore, the pdMAX system could be used to induce and analyze the expression of two distinct genes and adopted to various types of prokaryotic gene expression analyses.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.23.217141v1?rss=1

Authors: Rifkin, R. F., Vikram, S., Ramond, J. B., Cowan, D. A., Jakobsson, M., Schlebusch, C. M., Lombard, M.

Abstract:

The Stone Age record of South Africa provides some of the earliest evidence for the biological and cultural origins of Homo sapiens. While there is extensive genomic evidence for the selection of polymorphisms in response to pathogen-pressure in sub-Saharan Africa, there is insufficient evidence for ancient human-pathogen interactions in the region. Here, we analysed shotgun metagenome libraries derived from the sequencing of a Later Stone Age hunter-gatherer child who lived near Ballito Bay, South Africa, c. 2,000 years ago. This resulted in the identification of DNA sequence reads homologous to Rickettsia felis, and the reconstruction of an ancient R. felis genome, the causative agent of typhus-like flea-borne rickettsioses. The concurrent detection of DNA reads derived from Toxoplasma gondii, the causative agent of toxoplasmosis, confirms the pre-Neolithic incidence of these pathogens in southern Africa. We demonstrate that an R. felis and T. gondii co-infection, exacerbated by various additional bacterial and parasitic pathogens, contributed to the ill-health and subsequent demise of the boy from Ballito Bay.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.23.217281v1?rss=1

Authors: Bai, h., Bai, H., Bai, F., Cao, Y., Chen, Y.

Abstract:

IntroductionAngiostrogylus cantonensis (A.cantonensis) is a zoonotic parasitic nematode, with a worldwide distribution, causing eosinophilic meningitis or meningoencephalitis in human. Human infection with A.cantonensis typically occur from intentional or unintentional ingestion of infected intermediate or paratenic hosts, sometimes due to contaminated produce. Although the biology of A.cantonensis is relatively well known, little is understood about the transmission level in different zoogeographical regions, especially in Asia. Here, to evaluate the prevalence of A.cantonensis in rodent hosts in China and several Asian countries, we conducted a systematic review registered with PROSPERO (CRD42020161665).

MethodsRecords were selected systematically from 7 database (Medline via to Pubmed, Web of science, Scopus, Google Scholar, CNKI, Wangfang, CBM). Forest plots and random-effects model were used to display pooled estimates. The Freeman-Tukey double arcsine transformation of R software was used to conduct meta-analysis and statistical significance was set at 0.05.

ResultsA total of 67 studies met the inclusion criteria and were included in the systematic review and meta-analysis. The pooled prevalence estimates of A.cantonensis infection in rodents was 0.1003 (95%CI: 0.0765, 0.1268). There was significant heterogeneity in reported outcomes (p<0.0001). And we considered that there was no publication bias in the included studies.

ConclusionThe A.cantonensis infection rate among rodent hosts were still high in Asia, particularly in China, especially in Rattus norvegicus, and thus comprehensive measures should be taken for rodent hosts control to avoid an angiostrongyliasis outbreak. Due to the wide distribution and movement of rodent hosts, people in all regions of China, even in other Asia area live at risk of an infection. Hence, the development of more reliable diagnostic tests will be key for an effective identification of cases as well as improved patient care. Consequently, further studies are required to updated strategies for controlling A.cantonensis infection among human population.

Author summaryAngiostrongylus cantonensis (A.cantonensis) is a zoonotic parasitic nematode, with a worldwide distribution, causing eosinophilic meningitis or meningoencephalitis in human. Human infection with A.cantonensis typically occur from intentional or unintentional ingestion of infected intermediate or paratenic hosts, sometimes due to contaminated produce. Information about angiostrongyliasis burden, including its prevalence and incidence and geographic distribution in Asia, which is necessary to guide in planning appropriate control measures. In this study, we performed a systematic review and meta-analysis of published studies in China and several Asian countries on A.cantonensis. Our approach allowed evidence-based approximation to the estimations of the burden of A.cantonensis infection, for different rodent hosts groups, geographic regions, publication years and so on. At the same time, our review showed the A.cantonensis infection rate among rodent hosts were still high in Asia, particularly in China, especially in Rattus norvegicus. Finally, much bigger and longer-term studies are required to updated strategies for controlling A.cantonensis infection among human population.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.20.211821v1?rss=1

Authors: Kouba, T., Koval, T., Sudzinova, P., Pospisil, J., Brezovska, B., Hnilicova, J., Sanderova, H., Janouskova, M., Sikova, M., Halada, P., Sykora, M., Barvik, I., Novacek, J., Trundova, M., Duskova, J., Skalova, T., Chon, U., Murakami, K., Dohnalek, J., Krasny, L.

Abstract:

RNA synthesis is central to life, and RNA polymerase depends on accessory factors for recovery from stalled states and adaption to environmental changes. Here we investigated the mechanism by which a helicase-like factor HelD recycles RNA polymerase. We report a cryo-EM structure of an unprecedented complex between the Mycobacterium smegmatis RNA polymerase and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNA polymerase channels that are responsible for DNA binding and substrate delivery to the active site, thereby locking RNA polymerase in an inactive state. We show that HelD prevents non-specific interactions between RNA polymerase and DNA and dissociates transcription elongation complexes, but does not inhibit RNA polymerase binding to the initiation {sigma} factor. The liberated RNA polymerase can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that removes undesirable nucleic acids from RNA polymerase.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.22.215384v1?rss=1

Authors: Guo, H.-H., Jing, X.-Y., Chen, H., Xu, H.-X., Zhu, B.-M.

Abstract:

Late remote ischemia preconditioning (RIPC) and electro-acupuncture (EA) have both been suggested to reduce injury caused by myocardial ischemia/reperfusion (I/R). Our previous study has found that cardioprotection in RIPC is STAT5-dependent. Here, we aim to observe the effects of electro-acupuncture pretreatment (EAP) on I/R in the presence or absence of STAT5 in mice and investigate whether the protection of EAP is in a STAT5-dependent manner. In this study, EAP decreased myocardial infarction size (IS) /total area (TA) and rate of cardiomyocyte apoptosis. STAT5 was activated by EAP in the Stat5fl/fl mice but not in the Stat5-cKO mice, whereas, STAT3 was activated by EAP only in the Stat5-cKO but not in the Stat5fl/fl mice. Differentially expressed genes (DEGs) regulated by EAP in the Stat5fl/fl and the Stat5-cKO mice were quite distinct, indicating that EAP may activate IL-6/STAT3 signal in the absence of Stat5, and that EAP-induced cardioprotection against myocardial I/R injury was correlated with the activation of anti-apoptotic signaling and cardiomyocyte-survival signaling. Our results, for the first time, demonstrated that the protective effect of EAP was attributed to, but not dependent on, STAT5.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.21.214874v1?rss=1

Authors: Alexanian, M., Przytycki, P. F., Micheletti, R., Padmanabhan, A., Ye, L., Travers, J. G., Gonzalez Teran, B., Duan, Q., Ranade, S. S., Felix, F., Linares-Saldana, R., Huang, Y., Andreoletti, G., Yang, J., Ivey, K. N., Jain, R., McKinsey, T. A., Rosenfeld, M. G., Gifford, C., Pollard, K. S., Haldar, S. M., Srivastava, D.

Abstract:

In diseased organs, stress-activated signaling cascades alter chromatin, triggering broad shifts in transcription and cell state that exacerbate pathology. Fibroblast activation is a common stress response that worsens lung, liver, kidney and heart disease, yet its mechanistic basis remains poorly understood1,2. Pharmacologic inhibition of the BET family of transcriptional coactivators alleviates cardiac dysfunction and associated fibrosis, providing a tool to mechanistically interrogate maladaptive fibroblast states and modulate their plasticity as a potential therapeutic approach3-8. Here, we leverage dynamic single cell transcriptomic and epigenomic interrogation of heart tissue with and without BET inhibition to reveal a reversible transcriptional switch underlying stress-induced fibroblast activation. Transcriptomes of resident cardiac fibroblasts demonstrated robust and rapid toggling between the quiescent fibroblast and activated myofibroblast state in a manner that directly correlated with BET inhibitor exposure and cardiac function. Correlation of single cell chromatin accessibility with cardiac function revealed a novel set of reversibly accessible DNA elements that correlated with disease severity. Among the most dynamic elements was an enhancer regulating the transcription factor MEOX1, which was specifically expressed in activated myofibroblasts, occupied putative regulatory elements of a broad fibrotic gene program, and was required for TGF{beta}-induced myofibroblast activation. CRISPR interference of the most dynamic cis-element within the enhancer, marked by nascent transcription, prevented TGF{beta}-induced activation of Meox1. These findings identify MEOX1 as a central regulator of stress-induced myofibroblast activation associated with cardiac dysfunction. The plasticity and specificity of the BET-dependent regulation of MEOX1 in endogenous tissue fibroblasts provides new trans- and cis- targets for treating fibrotic disease.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.22.213892v1?rss=1

Authors: Stuart, R. B., Zwaanswijk, S., MacKintosh, N. D., Witikornkul, B., Prescott, M., Brophy, P. M., Morphew, R. M.

Abstract:

Fasciola hepatica (liver fluke), a significant threat to food security, causes global economic loss for the livestock production industry and is re-emerging as a food borne disease of humans. In the absence of vaccines the commonly used method of treatment control is by anthelmintics; with only Triclabendazole (TCBZ) currently effective against all stages of F. hepatica in livestock and humans. There is widespread resistance to TCBZ and detoxification by flukes might contribute to the mechanism. However, there is limited Phase I capacity in adult parasitic helminths and the major Phase II detoxification system in adults is the soluble Glutathione transferases (GST) superfamily. Previous global proteomic studies have shown that the levels of Mu class GST from pooled F. hepatica parasites respond under TCBZ-Sulphoxide (TCBZ-SO), the likely active metabolite, challenge during in vitro culture ex-host. We have extended this finding by using a sub-proteomic lead approach to measure the change in the total soluble GST profile (GST-ome) of individual TCBZ susceptible F. hepatica on TCBZ-SO-exposure in vitro culture. TCBZ-SO exposure demonstrated a FhGST-Mu29 and FhGST-Mu26 response following affinity purification using both GSH and S-hexyl GSH affinity resins. Furthermore, a low affinity Mu class GST (FhGST-Mu5) has been identified and recombinantly expressed and represents a novel low affinity mu class GST. Low affinity GST isoforms within the GST-ome was not limited to FhGST-Mu5 with second likely low affinity sigma class GST (FhGST-S2) uncovered through genome analysis. This study represents the most complete Fasciola GST-ome generated to date and has supported the sub proteomic analysis on individual adult flukes.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.21.215244v1?rss=1

Authors: Cranford-Smith, T., Wynne, M., Carter, C., Jiang, C., Jamshad, M., Milner, M. T., Djouider, Y., Hutchinson, E., Lund, P. A., Henderson, I. R., Huber, D.

Abstract:

Proteins that are translocated across the cytoplasmic membrane by Sec machinery must be in an unfolded conformation in order to pass through the protein-conducting channel during translocation. Molecular chaperones assist Sec-dependent protein translocation by holding substrate proteins in an unfolded conformation in the cytoplasm until they can be delivered to the membrane-embedded Sec machinery. For example, in Escherichia coli, SecB binds to a subset of unfolded Sec substrates and delivers them to the Sec machinery by interacting with the metal-binding domain (MBD) of SecA, an ATPase required for translocation in bacteria. Here, we describe a novel molecular chaperone involved Sec-dependent protein translocation, which we have named AscA (for accessory Sec component). AscA contains a metal-binding domain (MBD) that is nearly identical to the MBD of SecA. In vitro binding studies indicated that AscA binds to SecB and ribosomes in an MBD-dependent fashion.

Saturated transposon mutagenesis and genetics studies suggested that AscA is involved in cell-envelope biogenesis and that its function overlaps with that of SecB. In support of this idea, AscA copurified with a range of proteins and prevented the aggregation of citrate synthase in vitro. Our results suggest that AscA is molecular chaperone and that it enhances Sec-dependent protein translocation by delivering its substrate proteins to SecB.

IMPORTANCEThis research describes the discovery of a novel molecular chaperone, AscA (YecA). The function of AscA was previously unknown. However, it contains a small domain, known as the MBD, suggesting it could interact with the bacterial Sec machinery, which is responsible for transporting proteins across the cytoplasmic membrane. The work described this study indicates that the MBD allows AscA to bind to both the protein synthesis machinery and the Sec machinery. The previously function of the previously uncharacterised N-terminal domain is that of a molecular chaperone, which binds to unfolded substrate proteins. We propose that AscA binds to protein substrates as they are still be synthesised by ribosomes in order to channel them into the Sec pathway.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.21.213942v1?rss=1

Authors: Sowunmi, K., Shoga, S. M., Oriyomi, F. A., Adewumi, O. M., Sowunmi, L. A.

Abstract:

Pesticides are the substances for preventing, destroying, repelling any pest. Due to bulk handling or accidental release, they are accumulated in soil which leads to occasional entry into ecosystem that shows lethal effect on living system. An enrichment culture technique was used to isolate bacterial strains from organophosphate soil degrading high concentration of the selected pesticides. Five pure bacterial cultures were isolated. All five isolates were characterized on the basis of molecular and biochemical features like biodegradation test and substrate specificity, phosphate solubilization and screened for pesticide residue, pH, and extraction of DNA, quantity and quality check and salt tolerance. The organophosphate isolates were also tested for quantitative production. The screening of pesticide tolerance was done at for fungicides and insecticides.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.22.215491v1?rss=1

Authors: Matsumura, K., Shimomura, T., Kubo, Y., Oka, T., Kobayashi, N., Imai, S., Yanase, N., Akimoto, M., Fukuda, M., Yokogawa, M., Ikeda, K., Kurita, J.-i., Nishimura, Y., Shimada, I., Osawa, M.

Abstract:

Human ether-a-go-go-related gene potassium channel 1 (hERG) is a voltage-gated potassium channel, the voltage-sensing domain (VSD) of which is targeted by a gating-modifier toxin, APETx1. Although it is known that APETx1 inhibits hERG by stabilizing the resting state, it remains unclear where and how APETx1 interacts with the VSD in the resting state. Here, we prepared a recombinant APETx1, which is structurally and functionally equivalent to the natural product. Electrophysiological analyses using wild type and mutants of APETx1 and hERG revealed that their hydrophobic residues, in addition to a previously reported acidic hERG residue, play key roles in the inhibition of hERG by APETx1. Docking models of the APETx1-VSD complex that satisfy the results of mutational analysis suggest a molecular recognition mode between APETx1 and the resting state of hERG; this would provide a structural basis for designing ligands that control hERG function by binding to the VSD.

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