PaperPlayer biorxiv molecular biology

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.30.228064v1?rss=1

Authors: Zu, Z., Bani Ismail, M., Shinohara, M., Shinohara, A.

Abstract:

Homologous chromosomes pair with each other during meiosis, culminating in the formation of the synaptonemal complex (SC), which is coupled with meiotic recombination. In this study, we showed that a meiosis-specific depletion mutant of a cullin (Cdc53) of the SCF (Skp-Cullin-F-box) ubiquitin ligase, which plays a critical role in cell cycle regulation during mitosis, is deficient in SC formation, but is proficient in the formation of crossovers, indicating uncoupling of meiotic recombination with SC formation in the mutant. Furthermore, the deletion of the PCH2 gene encoding a meiosis-specific AAA+ ATPase suppresses SC-assembly defect induced by CDC53 depletion. On the other hand, the pch2 cdc53 double mutant is defective in meiotic crossover formation, suggesting the SC assembly with unrepaired DSBs. A temperature-sensitive mutant of the CDC4, which encodes a F-box protein of SCF, shows similar meiotic defects to the CDC53 depletion mutant. These suggest that SCFCdc4, probably SCFCdc4-dependnet protein ubiquitylation, regulates and collaborates with Pch2 in SC assembly and meiotic recombination.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.226845v1?rss=1

Authors: Di Muri, C., Lawson Handley, L. J., Bean, C. W., Li, J., Peirson, G., Sellers, G. S., Walsh, K., Watson, H. V., Winfield, I. J., Hänfling, B.

Abstract:

The sampling of environmental DNA (eDNA) coupled with cost-efficient and ever-advancing sequencing technology is propelling changes in biodiversity monitoring within aquatic ecosystems. Despite the growth of DNA metabarcoding approaches, the ability to quantify species biomass and abundance in natural systems remains a major challenge. Few studies have examined the association between eDNA metabarcoding data and biomass inferred by whole-organism sampling, mesocosms or mock communities, and the interpretation of sequencing reads as a measure of biomass or number of organisms is largely disputed. Here we tested whether read counts from eDNA metabarcoding provide accurate quantitative estimates of fish abundance in holding ponds with known fish biomass and number of individuals. eDNA samples were collected from two fishery ponds with high fish density and broad species diversity. In one pond, two different DNA capture strategies (on-site filtration with enclosed filters and three different preservation buffers versus lab filtration using open filters) were used to evaluate their performance in relation to fish community composition and biomass/abundance estimates. Fish species read counts were significantly correlated with both biomass and abundance, and this result, together with information on fish diversity, was repeatable when open or enclosed filters with different preservation buffers were used. This research demonstrates that eDNA metabarcoding provides accurate qualitative and quantitative information on fish communities in small ponds, and results are consistent between different methods of DNA capture. This method flexibility will be beneficial for future eDNA-based fish monitoring and their integration into fisheries management.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.30.228619v1?rss=1

Authors: Vyas, P., Trofimyuk, O., Longo, L. M., Deshmukh, F. K., Sharon, M., Tawfik, D. S.

Abstract:

The P-loop Walker A motif underlies hundreds of essential enzyme families that bind NTPs and mediate phosphoryl transfer (P-loop NTPases), including the earliest DNA/RNA helicases, translocases and recombinases. What were the primordial precursors of these enzymes? Could these large and complex proteins emerge from simple polypeptides? Previously, we showed that P-loops embedded in simple {beta}repeat proteins bind NTPs, but also, unexpectedly so, ssDNA and RNA. Here, we extend beyond the purely biophysical function of ligand binding to demonstrate rudimentary helicase-like activities. We further constructed simple 40-residue polypeptides comprising just one {beta}-(P-loop)- motif. Despite their simplicity, these P-loop prototypes confer functions such as strand separation and exchange. Foremost, these polypeptides unwind dsDNA, and upon addition of NTPs, or inorganic polyphosphates, release the bound ssDNA strands to allow reformation of dsDNA. Binding kinetics and low-resolution structural analyses indicate that activity is mediated by oligomeric forms spanning from dimers to high-order assemblies. These tantalizing resemblances to extant P-loop helicases and recombinases suggest that these engineered P-loop prototypes comprise a plausible description of the sequence, structure and function of the earliest P-loop NTPases. These prototypes also indicate that multifunctionality and dynamic assembly were key in endowing short polypeptides with elaborate, evolutionarily relevant functions.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.30.228650v1?rss=1

Authors: Moribe, F., Nishikori, M., Sasanuma, H., Akagawa, R., Arima, H., Takeda, S., Takaori-Kondo, A., Murai, J.

Abstract:

Background SLFN11 enhances cellular toxicity of genotoxic anti-cancer agents, and its important role under physiological conditions has not been appreciated yet. Somatic hypermutations and class switch recombination that can cause physiological genotoxic stress arise in germinal center B cells (GCBs). GCBs are a major origin of B-cell lymphomas that are frequently treated by cytosine arabinoside, a genotoxic anti-cancer agent. Objective To clarify the expression profile of SLFN11 in different stages of B cells and B-cell lymphomas. Methods We analyzed the expression of SLFN11 by mining publicly available databases for different stages of normal B cells and various types of B-cell lymphoma lines and also by performing immunohistochemical staining of human lymph nodes. We investigated the effects of two epigenetic modifiers, an EZH2 inhibitor, tazemetostat (EPZ6438) and a histone deacetylase inhibitor, panobinostat (LBH589) on SLFN11 expression in B-cell lymphoma lines and examined the therapeutic efficacy of these epigenetic modifiers in the combination with cytosine arabinoside. Results SLFN11 expression was specifically low in GCBs compared to non-GCBs, which was consolidated by the immunohistochemical staining for SLFN11 with human lymph nodes. SLFN11 expression levels in B-cell lymphoma lines largely correlated to those of their normal counterparts. The epigenetic modifiers upregulated SLFN11 expression in GCB-derived lymphomas and made the lymphomas further susceptible to cytosine arabinoside. Conclusions The expression of SLFN11 may be epigenetically suppressed in GCBs and GCB-derived lymphomas. GCB-derived lymphomas with low SLFN11 expression can be treated by the combination of epigenetic modifiers and cytosine arabinoside.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.30.228627v1?rss=1

Authors: Iwanicki, T., Haman, C., Liu, A., Taylor, J. S.

Abstract:

Correlations between variation in opsin expression and variation in vision are often assumed but rarely tested. We exposed starry flounder ( Platichthys stellatus ) to either broad spectrum sunlight or green-filtered light in outdoor aquaria for seven weeks and then combined digital-PCR and camouflage experiments to test two hypotheses: i) short-wavelength sensitive opsin expression decreases in a green light environment, and ii) if observed, this change in opsin expression influences colour vision as estimated using a camouflage-based behavioural assay. Of the eight visual opsins measured, Sws1 (UV sensitive) and Sws2B (blue sensitive) expression was significantly lower in fish exposed to green light. However, opsin expression in fish transferred to an arena illuminated with white LED light for three hours after the green light treatment did not differ from broad spectrum controls. Changes in opsin expression in response to artificial light environments have been reported before, but rapid changes over three hours rather than days or weeks is unprecedented. We did not observe a significant difference in a flounders camouflage response based on light environment, although broad spectrum fish increased and green-filter fish decreased the pattern contrast when on the blue-green substrate, and this difference approached significance. This pattern is intriguing considering green-filter fish expressed fewer UV and blue opsins and we recommend increased statistical power for future experiments. Together, our results show that starry flounder opsin expression changes rapidly in response to changes in light environment, however, there is no apparent effect on their visually mediated camouflage.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.30.228429v1?rss=1

Authors: Georgiadis, C., Rasaiyaah, J., Gkazi, S. A., Preece, R., Etuk, A., Christi, A., Qasim, W.

Abstract:

Targeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by "T v T" fratricide against shared antigens such as CD3 and CD7. Genome-editing can overcome such hurdles through targeted disruption of problematic shared antigens. Base editing offers the possibility of seamless disruption of gene expression through the creation of stop codons or elimination of splice donor or acceptor sites. We describe the generation of fratricide resistant, T cells by orderly removal of shared antigens such as TCR/CD3 and CD7 ahead of lentiviral mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base edited cells confirmed virtual elimination of chromosomal translocation events detected in conventional Cas9 treated cells. Interestingly, co-culture of 3CAR and 7CAR cells resulted in "self-enrichment" yielding populations that were 99.6% TCR-/CD3/-CD7-. 3CAR or 7CAR cells were able to exert specific cytotoxicity against their relevant target antigen in leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited the highest level of cytotoxicity against T-ALL targets expressing both target in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no evidence of promiscuous base conversion activity affecting CAR antigen specific binding regions which may otherwise redirect T cell specificity. Combinational infusion of fratricide resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allogeneic haematopoietic stem cell transplantation for T cell malignancies.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.30.228171v1?rss=1

Authors: Jaramillo-Madrid, A. C., Abbriano, R., Ashworth, J., Fabris, M., Ralph, P.

Abstract:

Sterols are a class of triterpenoid molecules with diverse functional roles in eukaryotic cells, including intracellular signaling and regulation of cell membrane fluidity. Diatoms are a dominant eukaryotic phytoplankton group that produce a wide diversity of sterol compounds. The enzymes 3-hydroxy-3-methyl glutaryl CoA reductase (HMGR) and squalene epoxidase (SQE) have been reported to be rate-limiting steps in sterol biosynthesis in other model eukaryotes; however, the extent to which these enzymes regulate triterpenoid production in diatoms is not known. To probe the role of these two metabolic nodes in the regulation of sterol metabolic flux in diatoms, we independently over-expressed two versions of the native HMGR and a conventional, heterologous SQE gene in the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum. Overexpression of these key enzymes resulted in significant differential accumulation of downstream sterol pathway intermediates in P. tricornutum. HMGR-mVenus overexpression resulted in the accumulation of squalene, cycloartenol, and obtusifoliol, while cycloartenol and obtusifoliol accumulated in response to heterologous NoSQE-mVenus overexpression. In addition, accumulation of the end-point sterol 24-methylenecholesta-5,24(24')-dien-3{beta}-ol was observed in all P. tricornutum overexpression lines, and campesterol increased 3-fold in P. tricornutum lines expressing NoSQE-mVenus. Minor differences in end-point sterol composition were also found in T. pseudonana, but no accumulation of sterol pathway intermediates was observed. Despite the successful manipulation of pathway intermediates and individual sterols in P. tricornutum, total sterol levels did not change significantly in transformed lines, suggesting the existence of tight pathway regulation to maintain total sterol content.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.30.229187v1?rss=1

Authors: Shi, W., Chen, M., Yang, Y., Zhou, W., Chen, S., Hu, Y., Liu, B.

Abstract:

The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is the core machinery responsible for the viral genome replication and transcription and also a major antiviral target. Here we report the cryo-electron microscopy structure of a post-translocated SARS-CoV-2 RdRp core complex, comprising one nsp12, one separate nsp8(I) monomer, one nsp7-nsp8(II) subcomplex and a replicating RNA substrate. Compared with the recently reported SARS-CoV-2 RdRp complexes, the nsp8(I)/nsp7 interface in this RdRp complex shifts away from the nsp12 polymerase. Further functional characterizations suggest that specific interactions between the nsp8(I) and nsp7, together with the rearrangement of nsp8(I)/nsp7 interface, ensure the efficient and processive RNA synthesis by the RdRp complex. Our findings provide a mechanistic insight into how nsp7 and nsp8 cofactors regulate the polymerase activity of nsp12 and suggest a potential new intervention interface, in addition to the canonical polymerase active center, in RdRp for antiviral design.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.225599v1?rss=1

Authors: Coveney, C. R., Zhu, L., Miotla-Zarebska, J., Stott, B., Parisi, I., Batchelor, V., Duarte, C., Chang, E., McSorley, E., Vincent, T. L., Wann, A. K.

Abstract:

Mechanical forces are known to drive cellular signalling programmes in cartilage development, health, and disease. Proteins of the primary cilium, implicated in mechanoregulation, control cartilage formation during skeletal development, but their role in post-natal cartilage is unknown. Ift88fl/fl and AggrecanCreERT2 mice were crossed to create a cartilage specific inducible knockout mouse AggrecanCreERT2;Ift88fl/fl. Tibial articular cartilage thickness was assessed, through adolescence and adulthood, by histomorphometry and integrity by OARSI score. In situ cell biology was investigated by immunohistochemistry (IHC) and qPCR of micro-dissected cartilage. OA was induced by destabilisation of the medial meniscus (DMM). Some mice were provided with exercise wheels in their cage.Deletion of IFT88 resulted in a reduction in medial articular cartilage thickness (atrophy) during adolescence from 102.57m, 95% CI [94.30, 119.80] in control (Ift88fl/fl) to 87.36m 95% CI [81.35, 90.97] in AggrecanCreERT2;Ift88fl/fl by 8-weeks p<0.01, and adulthood (104.00m, 95% CI [100.30, 110.50] in Ift88fl/fl to 89.42m 95% CI [84.00, 93.49] in AggrecanCreERT2;Ift88fl/fl, 34-weeks, p<0.0001) through a reduction in calcified cartilage. Thinning in adulthood was associated with spontaneous cartilage degradation. Following DMM, AggrecanCreERT2;Ift88fl/fl mice had increased OA (OARSI scores at 12 weeks Ift88fl/fl = 22.08 +/- 9.30, and AggrecanCreERT2;Ift88fl/fl = 29.83 +/- 7.69). Atrophy was not associated with aggrecanase-mediated destruction or chondrocyte hypertrophy. Ift88 expression positively correlated with Tcf7l2 and connective tissue growth factor. Cartilage thickness was restored in AggrecanCreERT2;Ift88fl/fl by voluntary wheel exercise.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.226191v1?rss=1

Authors: Owino, B. O., Mwangi, J. M., Kiplagat, S., Mwangi, H. N., Ingonga, J. M., Chebet, A., Ngumbi, P. M., Villinger, J., Masiga, D. K., Matoke-Muhia, D.

Abstract:

Background: Visceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) are of public health concern in Merti sub-County, Kenya, but epidemiological data on transmission, vector abundance, distribution, and reservoir hosts remains limited. To better understand the disease and inform control measures to reduce transmission, we investigated the abundance and distribution of sandfly species responsible for Leishmania transmission in the sub-County, and their blood-meal hosts. Methods: We conducted an entomological survey in five villages with reported cases of VL in Merti sub-County, Kenya, using CDC miniature light traps and castor oil sticky papers. Sandflies were dissected and identified to the species level using standard taxonomic keys and PCR analysis of the cytochrome c oxidase subunit 1 (COI) gene. Leishmania parasites were detected and identified by PCR and sequencing of internal transcribed spacer 1 (ITS1) genes. Bloodmeal sources of engorged females were identified by high-resolution melting analysis of vertebrate cytochrome b (cyt-b) gene PCR products. Results: We sampled 526 sandflies consisting of eight species, Phlebotomus orientalis (1.52%; n = 8) and seven Sergentomyia spp. Sergentomyia squamipleuris was the most abundant sandfly species (78.71%; n = 414) followed by Sergentomyia clydei (10.46%; n = 55). Leishmania major, Leishmania donovani, and Trypanosoma DNA were detected in S. squamipleuris specimens. Humans were the main sources of sandfly bloodmeals. However, we also detected mixed bloodmeals; one S. squamipleuris specimen had fed on both human and mouse (Mus musculus) blood, while two Ph. orientalis specimens fed on human, hyrax (Procavia capensis), and mouse (Mus musculus) blood. Conclusions: Our findings implicate the potential involvement of S. squamipleuris in the transmission of Leishmania and question the dogma that human leishmaniases in the Old World are exclusively transmitted by sandflies of the Phlebotomus genus. The presence of Trypanosoma spp. may indicate mechanical transmission, whose efficiency should be investigated. Host preference analysis revealed the possibility of zoonotic transmission of leishmaniasis and other pathogens in the sub-County. Leishmania major causes ZCL while L. donovani is responsible for VL. However, the reservoir status of the parasites is not uniform. Further studies are needed to determine the reservoir hosts of Leishmania spp. in the area. Keywords: Leishmania; leishmaniasis; sandfly; Trypanosoma; Sergentomyia; Phlebotomus; zoonotic cutaneous leishmaniasis; visceral leishmaniasis; Kenya.

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