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Intronic TNR-retained ISOPROPYLMALATE ISOMERASE LARGE SUBUNIT1 transcripts impair leaf development in Arabidopsis biorxiv.org/content/10.1101/20

Intronic TNR-retained ISOPROPYLMALATE ISOMERASE LARGE SUBUNIT1 transcripts impair leaf development in Arabidopsis

Intronic trinucleotide repeat (TNR) is widely distributed in plant genomes. In Arabidopsis accession Bur-0, abnormally expanded TTC repeat in intron-3 of the ISOPROPYLMALATE ISOMERASE LARGE SUBUNIT1 (IIL1) gene causes growth defects called the irregularly impaired leaves (iil) phenotype, triggered by DNA methylation-mediated IIL1 gene silencing at elevated temperature. However, little is known about how the reduced expression of IIL1 causes the iil phenotype. We demonstrated that the iil phenotype was resulted from the relative increase of intron-3-retained IIL1 transcripts through the experiments where the iil phenotype was reproduced by introducing the IIL1 gene harboring 100 copies of TTC repeat into Col-0. The iil phenotype appeared when the total amount of the IIL1 transcripts was decreased by co-suppression and the percentage of intron-3-retained IIL1 transcripts was increased. The IIL1 gene encodes an isopropylmalate isomerase large subunit, forming heterodimers with small subunits (AtLeuD1, AtLeuD2, or AtLeuD3). In the myb28 myb29 mutant lacking AtLeuD1 and AtLeuD2, the iil phenotype was almost completely suppressed regardless of higher percentage of intron-3-retained IIL1 transcripts. The results indicated that the iil phenotype was associated with interaction with AtLeuDs, suggesting that intronic TNR-containing transcripts were translated into abnormal proteins and perturbed the metabolic pathway supporting the leaf development.

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Morphological Attributes of Glycine max (Soybean) biorxiv.org/content/10.1101/20

Morphological Attributes of Glycine max (Soybean)

Heavy metal like lead and chromium present in environment in large quantities cause a severe environmental concern. Significant risks to the environment and agriculture are posed by heavy metal contamination of the air, water, and soil as a result of increased urbanisation and industrialization. Heavy metal pollution in soils can harm nearby ecosystems, groundwater, agricultural productivity, and human health because of its persistence and high toxicity. Heavy metals are accumulated in the tissues of plants growing in metal-contaminated soil, which can have a negative impact on the morphology of the plant. The present study was carried out to determine the effect of different concentrations (50ml, 100ml, 150ml and 200ml) of chromium and lead on morphological attributes of three varieties (JS:335, JS:80-21, JS:75-46) of soybean (Glycine max). The result of the present study showed that the exposure of Glycine max to Pb and Cr resulted in a decrease in total length, number of nodes and number of leaves of a plant. The shape of leaves in case of treated plants was also changed from ovoid to heart and oval shapes, at higher concentration of heavy metals. Among the three different varieties (JS:335, JS:80-21, JS:75-46) of Glycine max (soybean) JS:335 showed maximum reduction and the JS:80-21 showed least reduction in the growth of a plant. Lead treatment proved more toxic than chromium treatment for all the three different varieties (JS:335, JS:80-21, JS:75-46) of Glycine max. Key words: Heavy metal, Chromium, Lead, Glycine max, Morphological parameters

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High-altitude hypoxia exposure inhibits erythrophagocytosis by inducing macrophage ferroptosis in the spleen biorxiv.org/content/10.1101/20

High-altitude hypoxia exposure inhibits erythrophagocytosis by inducing macrophage ferroptosis in the spleen

High-altitude polycythemia (HAPC) occurs in high-altitude (HA) environments and involves an imbalance between erythropoiesis and eryptosis. Spleen/splenic macrophages are an important primary tissue/cell of eryptosis and iron recycling. However, the role of the spleen in the pathogenesis of HAPC and the effect of hypobaric hypoxia (HH) on the biology of the spleen and splenic macrophages are still unclear. We used a mouse hypobaric hypoxia (HH) exposure model to simulate an in vivo study of 6000 m HA exposure. For in vitro studies, we used a primary splenic macrophage model treated with 1% hypoxia. We found that the HH-treated mouse model promoted erythropoiesis and led to erythrocytosis. In addition, HH exposure resulted in marked splenic contraction followed by splenomegaly for up to 14 days. HH exposure impaired the red blood cell (RBC) handling capacity of the spleen, which was caused by a decrease in splenic macrophages in the red pulp. Moreover, HH treatment for 7 and 14 days promoted iron mobilization and ferroptosis in the spleen, as reflected by the expression of metabolism-related proteins and ferroptosis-related proteins. All of the protein expression levels were similar to the gene expression levels in human peripheral blood mononuclear cells. Single-cell sequencing of the spleen further demonstrated a significant decrease in macrophages in the spleen 7 days after HH exposure. In in vitro studies, we confirmed that primary splenic macrophages decreased and induced ferroptosis following hypoxic treatment, which was reversed by pre-treatment with the ferroptosis inhibitor ferrostatin-1. Taken together, HH exposure induces splenic ferroptosis, especially in red pulp macrophages, which further inhibits the clearance of RBCs from the spleen. As such, it promotes the retention of RBCs in the spleen and causes splenomegaly, which may further lead to the persistent production of RBCs and ultimately to the development of HAPC. ### Competing Interest Statement The authors have declared no competing interest.

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Inhibition of CD40-TRAF6 signaling protects against aneurysm development and progression biorxiv.org/content/10.1101/20

Inhibition of CD40-TRAF6 signaling protects against aneurysm development and progression

Objective: Inflammation is a critical process during the progressive development and complication of abdominal aortic aneurysm. The co-stimulatory dyad CD40-CD40L is a major driver of inflammation and modulates immune responses. This study evaluates the potential of a small molecule inhibitor, which blocks the interaction between CD40 and tumor necrosis factor (TNF) receptor-associated factor (TRAF)-6, referred to as TRAF-STOP, in the early and later phase during AAA progression. Methods and results: AAAs were induced in C57BL/6J mice by infrarenal aortic porcine pancreatic elastase infusion for 7, 14 or 28 days. Inhibition of CD40 signaling by TRAF-STOP resulted in less severe AAA formation and reduced the incidence of AAA development. TRAF-STOP treatment attenuated aortic structural remodeling, characterized by a reduced elastic fiber degradation, lowered expression of matrix metalloproteinase (MMP)-2 and MMP9, as well as preserved collagen type IV content in aneurysmal tissue. Furthermore, this is accompanied by the reduction of key pro-inflammatory genes such as TNFα. Conclusion: Pharmacological inhibition of CD40-TRAF6 signaling protects from adverse aortic structural remodeling during the early phase of AAA progression representing a translational strategy to limit progression of human AAA disease. ### Competing Interest Statement The authors have declared no competing interest.

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Reduction, removal or replacement of sodium nitrite in a model of cured and cooked meat: a joint evaluation of consequences on microbiological issues in food safety, colon ecosystem and colorectal carcinogenesis biorxiv.org/content/10.1101/20

Reduction, removal or replacement of sodium nitrite in a model of cured and cooked meat: a joint evaluation of consequences on microbiological issues in food safety, colon ecosystem and colorectal carcinogenesis

Scope: Epidemiological and experimental evidence reported that processed meat consumption is associated with colorectal cancer (CRC) risk. Several studies suggest the involvement of nitrite or nitrate additives via N-nitroso-compound formation (NOCs). Methods and results: Compared to the reference level (120 mg/kg of ham), the effects of sodium nitrite reduction (90 mg/kg of ham), removal and replacement were analysed on ham characteristics and in a CRC rat model. Sodium nitrite removal and reduction induced a similar decrease in CRC preneoplastic lesions, but only reduction led to (i) an inhibitory effect on Listeria monocytogenes growth comparable to that obtained using the reference nitrite level of 120 mg/kg and (ii) an effective control of lipid peroxidation. Among the three alternatives tested, none led to a significant gain when compared to the 120 mg/kg ham reference level: vegetable stock, due to nitrate presence, was very similar to this reference nitrite level, yeast extract induced a strong luminal peroxidation and no decrease in preneoplastic lesions despite the absence of NOCs, and polyphenol rich extract induced the clearest downward trend on preneoplastic lesions but the concomitant presence of nitrosyl iron in feces. Except vegetable stock, other alternatives were less efficient than sodium nitrite (≥ 90 mg/kg) in reducing L. monocytogenes growth. Conclusion: Nitrite reduction (90mg/kg) effectively reduced CRC risk through limiting NOC formation and lipid peroxidation, while mitigating L. monocytogenes risks from cooked hams. Going further in reduction should be possible if accompanied by antioxidants to limit lipid peroxidation and appropriate use-by dates. ### Competing Interest Statement A P Promeyrat, B F, J-L M Martin, S Jeuge and G Nassy were employed by the French Pork Institute (IFIP). This study was co-financed (50%) by IFIP. F Pierre, F Gueraud and V Sante-Lhoutellier have some research projects from their academic research teams that have been co-financed by the processed meat sector.

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Absence of Telomerase Leads to Immune Response and Tumor Regression in Zebrafish Melanoma biorxiv.org/content/10.1101/20

Absence of Telomerase Leads to Immune Response and Tumor Regression in Zebrafish Melanoma

Most cancers reactivate telomerase to maintain telomere length to acquire immortality. The importance of this process is well illustrated by the fact that telomerase promoter mutations are found at a high frequency in many cancer types, including melanoma. However, it is unclear when and if telomerase is strictly required during tumorigenesis. Here, we show that melanoma can occur in the absence of telomerase but is required to sustain later growth and to avoid tumor regression. We combined telomerase mutant zebrafish (tert-/-) with two established melanoma models and found equal melanoma incidence and invasiveness as tumors became visible. Later, however, while tert+/+ fish develop increasing larger tumors, tert-/- tumors stagnate growth and regress. tert-/- tumors showed lower cell proliferation, higher apoptosis and melanocyte differentiation. We also detected an immune response directed at tert-/- tumors. tert-/- tumors exhibited increased immune cell infiltrates and resume growth when transplanted into immunocompromised hosts. We propose that telomerase is required for melanoma in zebrafish, albeit at later stages of progression, to sustain growth while avoiding immune rejection and regression. Thus, absence of telomerase restricts melanoma through tumor-autonomous mechanisms (cell cycle arrest, apoptosis and melanocyte differentiation) and a non-tumor-autonomous mechanisms (immune rejection). ### Competing Interest Statement The authors have declared no competing interest.

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VRK3 depletion induces cell cycle arrest and metabolic reprogramming of Pontine Diffuse Midline Glioma (DMG)-K27 altered cells biorxiv.org/content/10.1101/20

VRK3 depletion induces cell cycle arrest and metabolic reprogramming of Pontine Diffuse Midline Glioma (DMG)-K27 altered cells

We previously identified VRK3 as a specific vulnerability in DMG-H3K27M cells in a synthetic lethality screen targeting the whole kinome. The aim of the present study was to elucidate the mechanisms by which VRK3 depletion impact DMG-K27M cell fitness. Gene expression studies after VRK3 knockdown emphasized the inhibition of genes involved in G1/S transition of the cell cycle resulting in growth arrest in G1. Additionally, a massive modulation of genes involved in chromosome segregation was observed, concomitantly with a reduction in the level of phosphorylation of serine 10 and serine 28 of histone H3 supporting the regulation of chromatin condensation during cell division. This last effect could be partly due to a concomitant decrease of the chromatin kinase VRK1 in DMG following VRK3 knock-down. Furthermore, a metabolic switch specific to VRK3 function was observed towards increased oxidative phosphorylation without change in mitochondria content, that we hypothesized would represent a cell rescue mechanism. This study further explored the vulnerability of DMG-H3K27M cells to VRK3 depletion suggesting potential therapeutic combinations, e.g. with the mitochondrial ClpP protease activator ONC201. ### Competing Interest Statement The authors have declared no competing interest.

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The Potential of Low Press and Hypoxia Environment in Assisting Pan-cancer Treatment biorxiv.org/content/10.1101/20

The Potential of Low Press and Hypoxia Environment in Assisting Pan-cancer Treatment

Objective: A low incidence and mortality rate of cancer has been observed in high-altitude regions, suggesting a potential positive effect of low press and hypoxia (LPH) environment on cancer. Based on this finding, our study aimed to construct a pan-cancer prognosis risk model using a series of ADME genes intervened by low oxygen, to explore the impact of LPH environment on the overall survival (OS) of various kinds of cancers, and to provide new ideas and approaches for cancer prevention and treatment. Datasets and Measures: The study used multiple sources of data to construct the pan-cancer prognosis risk model, including gene expression and survival data of 8,628 samples from the cancer genome atlas, and three gene expression omnibus databases were employed to validate the prediction efficiency of the prognostic model. The AltitudeOmics dataset was specifically used to validate the significant changes in model gene expression in LPH. To further identify the biomarkers and refine the model, various analytical approaches were employed such as single-gene prognostic analysis, weighted gene co-expression network analysis, and stepwise cox regression. And LINCS L1000, AutoDockTools, and STITCH were utilized to explore effective interacting drugs for model genes. Main Outcomes and Conclusions: The study identified eight ADME genes with significant changes in the LPH environment to describe the prognostic features of pan-cancer. Lower risk scores calculated by the model were associated with better prognosis in 25 types of tumors, with a p-value of less than 0.05. The LPH environment was found to reduce the overall expression value of model genes, which could decrease the death risk of tumor prognosis. Additionally, it is found that the low-risk group had a higher degree of T cell infiltration based on immune infiltration analysis. Finally, drug exploration led to the identification of three potential model-regulating drugs. Overall, the study provided a new approach to construct a pan-cancer survival prognosis model based on ADME genes from the perspective of LPH and offered new ideas for future tumor prognosis research. ### Competing Interest Statement The authors have declared no competing interest.

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Multiscale Computational Framework to Investigate Integrin Mechanosensing and Cell Adhesion biorxiv.org/content/10.1101/20

Multiscale Computational Framework to Investigate Integrin Mechanosensing and Cell Adhesion

Integrin mechanosensing plays an instrumental role in cell behavior, phenotype, and fate by transmitting mechanical signals that trigger downstream molecular and cellular changes. For instance, force transfer along key amino acid residues can mediate cell adhesion. Disrupting key binding sites within α5β1 integrin's binding partner, fibronectin (FN) diminishes adhesive strength. While past studies have shown the importance of these mechanosensing residues, the molecular dynamics by which they maintain adhesion locally and throughout the cell remains less explored. Here, we present a multiscale mechanical model to investigate the mechanical coupling between integrin nanoscale dynamics and whole-cell adhesion dynamics. The model's force outputs were consistent with past atomic force microscopy and fluorescence resonance energy transfer measurements from literature. The model also confirmed past studies that implicate two key sites within FN that maintain cell adhesion: the synergy site and RGD motif. Our study contributed to our understanding of molecular mechanisms by which these sites collaborate to mediate whole-cell integrin adhesion dynamics. Specifically, we showed how FN unfolding, residue binding/unbinding, and molecular structure contribute to α5β1-FN's nonlinear force-extension behavior during stretching. These dynamics could be used to understand cell differentiation via mechanosensitive sites or limit the spread of metastatic cells through targeted protein design. ### Competing Interest Statement The authors have declared no competing interest.

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Video-rate three-photon imaging in deep Drosophila brain based on a single Cr:forsterite oscillator biorxiv.org/content/10.1101/20

Video-rate three-photon imaging in deep Drosophila brain based on a single Cr:forsterite oscillator

We have demonstrated 30-Hz three-photon imaging using a single 24-MHz mode-locked Cr:forsterite oscillator with a center wavelength at 1260 nm. By managing the dispersion distribution in the resonator using double-chirped mirrors, we have produced 32-fs pulses with 22-nJ pulse energy. Using the oscillator as a driving source, we have realized multi-color three-photon images using a GFP-labeled Drosophila brain and an AF647-labeled mouse brain. To demonstrate the capability of deep-tissue imaging, we have obtained a 10-times higher SBR from the three-photon images than the two-photon results at different depths in a GFP-labeled Drosophila brain dissection. Furthermore, we have shown the impact of excitation pulse width on three-photon deep-tissue imaging. Our results indicate the superiority of using shorter pulses for deeper-tissue imaging, especially in the Drosophila brain. In addition, we have recorded the three-photon calcium imaging in vivo from the Drosophila mushroom body in response to external electric shocks. We believe our demonstration provides a robust approach for high-speed three-photon microscopy applications, especially for intravital investigations in the Drosophila brain. ### Competing Interest Statement The authors have declared no competing interest.

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Production of extracellular polymeric substances in granular sludge under selection for Accumulibacter and Competibacter biorxiv.org/content/10.1101/20

Production of extracellular polymeric substances in granular sludge under selection for Accumulibacter and Competibacter

Granular sludge intensifies the removal of nutrients from wastewater. Granules structured by extracellular polymeric substances (EPS) can be recovered as biomaterial. Links between microbial selection and EPS formation during granulation need to get uncovered. We inoculated anaerobic-aerobic sequencing batch reactors with either flocs or granules to study the relationships between microbial selection, bioaggregation, exopolymer formation, and EPS composition. Selection for slow-growing organisms like the model polyphosphate-accumulating organism "Candidatus Accumulibacter" (max. 83% vs. amplicon sequencing read counts) and glycogen-accumulating organism "Ca. Competibacter" (max. 45%) sustained granulation. Gel-forming exopolymers were produced as high as above 40% of the volatile solids of the biomass by stepwise increase of the organic loading rate (0.3 to 2.0 g CODAc d-1 LR-1). Confocal laser scanning microscopy, FT-IR spectroscopy, and HPAE-PAD chromatography revealed the complex and dynamic chemical compositions of the structural EPS in relation to microbial population shifts along reactor regimes. The analysis of 20 representative genomes of "Ca. Accumulibacter" and "Ca. Competibacter" recovered from public databases revealed their functional potential to produce EPS among other representative wastewater microorganisms. The more than 40 functional gene categories annotated highlight the complexity of EPS metabolic networks from monomers processing to assembly, export, and epimerizations. The combination of ecological engineering principles and systems microbiology will help unravel and direct the production of EPS from wastewater, valorizing residual granular sludge into beneficial biomaterials for the circular economy. ### Competing Interest Statement The authors have declared no competing interest.

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Model based concept to extract heart beat-to-beat variations beyond respiratory arrhythmia and baroreflex biorxiv.org/content/10.1101/20

Model based concept to extract heart beat-to-beat variations beyond respiratory arrhythmia and baroreflex

The heart rate (HR) and its variability (HRV) reflect modulation of the autonomous nervous system, especially sympathovagal balance. The aim of this research was to develop a personalizable HR model and an in-silico system that could identify HR regulation parameters associated with respiratory arrhythmia (RSA) and baroreflex, and subsequently capture the residual heart beat-to-beat variations from individual psychophysiological recordings in humans. Here respiration signal, blood pressure signal and time instances of R peaks of EKG are used as input for the model. The model considers traditional lower-order mechanisms of HR dynamic, extracting residual displacements of the modeled R peaks relative to real R peaks. Three components - tonic, spontaneous and 0.1 Hz changes - can be seen in these R peak displacements. These dynamic residuals can help to analyze HRV beyond RSA and baroreflex, whereas our model-based concept suggests that the residuals are not merely modeling errors. The proposed method could help to investigate the presumably additional neural regulation impulses from higher-order brain and other influences. ### Competing Interest Statement The authors have declared no competing interest.

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Pulmonary vein flow split effects in patient-specific simulations of left atrial flow biorxiv.org/content/10.1101/20

Pulmonary vein flow split effects in patient-specific simulations of left atrial flow

Disruptions to left atrial (LA) blood flow, such as those caused by atrial fibrillation (AF), can lead to thrombosis in the left atrial appendage (LAA) and an increased risk of systemic embolism. LA hemodynamics are influenced by various factors, including LA anatomy and function, and pulmonary vein (PV) inflow conditions. In particular, the PV flow split can vary significantly among and within patients depending on multiple factors. In this study, we investigated how changes in PV flow split affect LA flow transport, focusing on blood stasis in the LAA, using a high-fidelity patient-specific computational fluid dynamics (CFD) model. We analyzed LA anatomies from eight patients with varying atrial function, including three with AF and either a LAA thrombus or a history of TIAs. Using four different flow splits (60/40\% and 55/45\% through right and left PVs, even flow rate, and same velocity through each PV), we found that flow patterns are sensitive to PV flow split variations, particularly in planes parallel to the mitral valve. Changes in PV flow split also had a significant impact on blood stasis and could contribute to increased risk for thrombosis inside the LAA, particularly in patients with AF and previous LAA thrombus or a history of TIAs. Our study highlights the importance of considering patient-specific PV flow split variations when assessing LA hemodynamics and identifying patients at increased risk for thrombosis and stroke. ### Competing Interest Statement The authors have declared no competing interest.

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Normal and cancer tissues are accurately characterised by intergenic transcription at RNA polymerase 2 binding sites biorxiv.org/content/10.1101/20

Normal and cancer tissues are accurately characterised by intergenic transcription at RNA polymerase 2 binding sites

Intergenic transcription in normal and cancerous tissue is pervasive and incompletely understood. To investigate this activity at a global level, we constructed an atlas of over 180,000 consensus RNA Polymerase II (RNAP2) bound intergenic regions from more than 900 RNAP2 ChIP-seq experiments across normal and cancer samples. Using unsupervised analysis, we identified 51 RNAP2 consensus clusters, many of which map to specific biotypes and identify tissue-specific regulatory signatures. We developed a meta-clustering methodology to integrate our RNAP2 atlas with active transcription across 28,797 RNA-seq samples from TCGA, GTEx and ENCODE, which revealed strong tissue- and disease- specific interconnections between RNAP2 occupancy and transcription. We demonstrate that intergenic transcription at RNAP2 bound regions are novel per-cancer and pan-cancer biomarkers showing genomic and clinically relevant characteristics including the ability to differentiate cancer subtypes and are associated with overall survival. Our results demonstrate the effectiveness of coherent data integration to uncover and characterise intergenic transcriptional activity in both normal and cancer tissues. ### Competing Interest Statement The authors have declared no competing interest.

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Immune cell type signature discovery and random forest classification for analysis of single cell gene expression datasets biorxiv.org/content/10.1101/20

Immune cell type signature discovery and random forest classification for analysis of single cell gene expression datasets

Background: Robust immune cell gene expression signatures are central to the analysis of single cell studies. Nearly all known sets of immune cell signatures have been derived by making use of only single gene expression datasets. Utilizing the power of multiple integrated datasets could lead to high-quality immune cell signatures which could be used as superior inputs to machine learning-based cell type classification approaches. Results: We established a novel gene expression similarity-based workflow for the discovery of immune cell type signatures that leverages multiple datasets, here four single cell expression datasets from three different cancer types. We used our immune cell signatures to train random forest classifiers for immune cell type assignment of single-cell RNA-seq datasets. We obtained similar or better prediction results compared to commonly used methods for cell type assignment in two independent benchmarking datasets. Our gene signature set yields higher prediction scores than other published immune cell type gene sets in our random forest approach. Discussion and conclusion: We demonstrated the quality of our immune cell signatures and their strong performance in a random forest-based cell typing approach. We argue that classifying cells based on our comparably slim sets of genes accompanied by a random forest-based approach not only matches or outperforms widely used published approaches. It also facilitates unbiased downstream statistical analyses of differential gene expression between cell types for 90% of all genes whose expression profiles have not been used for cell type classification. ### Competing Interest Statement The authors have declared no competing interest.

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Fast centromeric repeat turnover provides a glimpse into satellite DNA evolution in Nothobranchius annual killifishes biorxiv.org/content/10.1101/20

Fast centromeric repeat turnover provides a glimpse into satellite DNA evolution in Nothobranchius annual killifishes

Satellite DNA (satDNA) is rapidly evolving class of tandem repeats with some motifs being involved in centromere organization and function. Rapid co-evolution of centromeric satDNA and associated proteins has been mostly attributed to the so-called centromere drive. To identify repeats associated with centromeric regions and test for the role of meiotic drive in their evolution, we investigated satDNA across Southern and Coastal clades of African annual killifishes of the genus Nothobranchius. C-banding showed expansion of (peri)centromeric heterochromatin regions in the Southern-clade killifishes. Molecular cytogenetic and bioinformatic analyses further revealed that two previously identified satellites, Nfu-SatA and Nfu-SatB, are associated with centromeres only in one lineage of the Southern clade. Nfu-SatB was, however, detected outside centromeres also in other members of the Coastal clade, which is consistent with the "library" hypothesis of satDNA evolution. We also identified a novel satDNA, Cl-36, associated with (peri)centromeres in N. foerschi, N. guentheri and N. rubripinnis from the Coastal clade. Our findings could be explained by centromere drive shaping karyotype change and centromeric repeat turnover in Nothobranchius species with possible reversal of spindle polarity within the Southern clade. ### Competing Interest Statement The authors have declared no competing interest.

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Rhizosphere and detritusphere habitats modulate expression of soil N-cycling genes during plant development biorxiv.org/content/10.1101/20

Rhizosphere and detritusphere habitats modulate expression of soil N-cycling genes during plant development

Interactions between plant roots and rhizosphere bacteria mediate nitrogen (N)-cycling processes and create habitats rich in low molecular weight (growing roots, rhizosphere) and complex organic molecules (decaying root litter, detritusphere) compared to bulk soil. Microbial N-cycling is regulated by a diverse suite of genes from many interconnected metabolic pathways; but most studies of soil N-cycling gene expression have focused on single pathways. Currently, we lack a comprehensive understanding of the interplay between soil N-cycling gene regulation, spatial habitat and time. Here we present an analysis of a replicated time series of soil metatranscriptomes; we followed multiple N transformations in four soil habitats (rhizosphere, detritusphere, mixed rhizo-/detriusphere, bulk soil) over a period of active root growth for the annual grass, Avena fatua. The presence of root litter and living roots significantly altered the trajectory of N-cycling gene expression. Across soil habitats, the most highly expressed N-transformation genes were related to extracellular proteases, ammonium assimilation into microbial biomass via glutamate synthase, and ammonium oxidation. Upregulation of bacterial assimilatory nitrate reduction in the rhizosphere suggests that rhizosphere bacteria were actively competing with roots for nitrate. Simultaneously, bacterial ammonium assimilatory pathways were upregulated in both rhizosphere and detritusphere soil, which could have limited N availability to plants. The detritusphere supported dissimilatory processes DNRA and denitrification. Expression of ammonium oxidation genes was almost exclusively performed by three phylotypes of Thaumarchaeota and was upregulated in unamended bulk soil. Unidirectional ammonium assimilation and its regulatory genes (glutamine synthetase/glutamate synthase, or GS/GOGAT) were upregulated in soil surrounding relatively young roots and more highly decayed root litter, suggesting N may have been limiting in these habitats (the GS/GOGAT pathway is known to be activated under low N availability). We did not detect expression of N-fixation or anammox genes. Our comprehensive metatranscriptomic time-series of organic and inorganic N-cycling in rhizosphere, detritusphere, and bulk soil, indicates that differences in C and inorganic N availability control contemporaneous transcription of N-cycling pathways in soil microhabitats that exist in close spatial proximity. ### Competing Interest Statement The authors have declared no competing interest.

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Predicting future conservation areas while avoiding competition in two alpine urodele amphibians severely threatened by climate change biorxiv.org/content/10.1101/20

Predicting future conservation areas while avoiding competition in two alpine urodele amphibians severely threatened by climate change

Climate change will cause important declines in species distributions, especially when living at high altitudes. The Critically Endangered Salamandra lanzai from SW Alps may be severely exposed to future climate change effects and its suitable climate may shrink or shift. Another Alpine salamander (S. atra) is present in the region, which in case of spatial overlap may represent a competitor for S. lanzai. It is urgent to estimate the effect of future climate change on these species and identify priority areas for conservation while accounting for competition between both species. With a Species Distribution Modelling (SDM) approach, we projected the current and future climate suitability of both salamander species. We accounted for uncertainty related to the methods (model replicates) and climate projections (data source, global circulation model and scenario) to provide a consensus map for practitioners. This map also takes into account potential competition with S. atra by penalizing the suitability scores of S. lanzai by the scores of S. atra. We predict a severe effect of climate change on both species. Most of the current habitats are projected to become largely unsuitable by 2070, regardless of the climatology and scenario. We identified important spatial disagreements between projections based on different data sources, mostly due to precipitation projections and daily temperature variation. This highlights the need to account for multiple climatologies in mountainous environments. Both species' habitats are highly fragmented, which is expected to prevent distributional shifts through natural dispersion. We suggest to explore the possibility of translocation for the most threatened populations and simultaneously develop captive breeding programs. Biotic interactions are rarely accounted for in SDMs, and we encourage the documentation of species with similar ecological requirements to improve the relevance of SDMs for future conservation planning. ### Competing Interest Statement The authors have declared no competing interest.

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Experimental heatwaves and warming cause distinctive community responses through their interactions with a novel species biorxiv.org/content/10.1101/20

Experimental heatwaves and warming cause distinctive community responses through their interactions with a novel species

As mean temperatures increase and heatwaves become more frequent, species are expanding their distributions to colonise new habitats. The resulting novel species interactions will simultaneously shape the temperature-driven reorganization of resident communities. The interactive effects of climate change and climate change-facilitated invasion have rarely been studied in multi-trophic communities, and are likely to differ depending on the nature of the climatic driver (i.e. climate extremes or constant warming). We recreated under laboratory conditions a host-parasitoid community typical of high-elevation rainforest sites in Queensland, Australia, comprising four Drosophila species and two associated parasitoid species. We subjected these communities to climate change in the form of either heatwaves or constant warming, in combination with an invasion treatment involving a novel host species from lower-elevation habitats. The two parasitoid species were sensitive to both warming and heatwaves, while the demographic responses of Drosophila species were highly idiosyncratic, reflecting the combined effects of thermal tolerance, parasitism, competition, and facilitation. After multiple generations, heatwaves (but not constant warming) promoted the establishment of low-elevation species in upland communities. The introduction of this invading species correlated negatively with the abundance of one of the parasitoid species, leading to cascading effects on its hosts and their competitors. Our study, therefore, reveals differing, sometimes contrasting, impacts of extreme temperatures and constant warming on community composition. It also highlights how the scale and direction of climate impacts could be further modified by range-expanding species within a bi-trophic community network. ### Competing Interest Statement The authors have declared no competing interest.

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