Biology subjects

Fan, X.

Publications and source records attributed to Fan, X..

At least 145 records · Page 8Linked to original sources

bioRxiv2022

Differential expression patterns and potential regulatory function of piRNAs in western honey bee larval response to Ascosphaera apis infestation

piRNAs are a class of small non-coding RNAs that play an essential part in genomic defense, as well as in modulation of gene expression and diverse biological processes such as host-pathogen interaction. However, little is known about the expression pattern of the regulatory function of piRNAs in interactions between honey bees and pathogens. In this work, on the basis of our previously obtained high-quality small RNA-seq datasets from western honey bee (Apis mellifera) larval guts, the differential expression profile of piRNAs in A. mellifera larval guts after Ascosphaera apis infestation was analyzed, followed by structural characterization, target prediction, and regulatory network investigation. The potential roles of differentially expressed piRNAs (DEpiRNAs) in regulating host response, especially immune response, were further discussed. In this study, 504, 657, and 587 piRNAs were respectively identified in the 4-, 5-, and 6-day-old larval guts infected by A. apis, with 411 (53.24%) piRNAs shared. The length distribution of these piRNAs ranged from 24 nt to 33 nt and their first base had a "C" bias, similar to piRNAs discovered in other mammals and insects. Additionally, 96, 103, and 143 DEpiRNAs were detected in the 4-, 5-, and 6-day-old comparison groups; among these, piR-ame-149736, piR-ame-1066173, and piR-ame-1125190 were the most up-regulated, while piR-ame-1202932 was the most down-regulated. There were 68 DEpiRNAs shared between these three comparison groups. The targets of the DEpiRNAs in the three comparison groups were engaged in a suite of crucial functions associated with biological processes, molecular function, and cellular components, including molecular transducer activity, biological regulation, and membrane part. These targets were also relevant to diverse vital pathways such as the phosphatidylinositol signaling system, inositol phosphate metabolism, and Wnt signaling pathway. Further investigation demonstrated that targets of DEpiRNAs were involved in three energy metabolism-related pathways, seven development-associated signaling pathways, and seven immune-relevant pathways, including lysosome and endocytosis, as well as the MAPK and Jak-STAT signaling pathways. The expression trends of five randomly selected DEpiRNAs were verified using a combination of RT-PCR and RT-qPCR. Moreover, the expression levels of six genes targeted by piR-ame-945760 were detected by RT-qPCR, with the results showing that their expression trends were the same as the expression trend of piR-ame-945760, indicative of the positive correlation between piR-ame-945760 and these targets. These results suggest that A.apis infestation increased the overall expression level of piRNAs and altered the expression pattern of piRNAs in A. mellifera larval guts. DEpiRNAs potentially participate in the A. apis response of the host by modulating the expression of target genes associated with energy metabolism and development, as well as cellular and humoral immune response. Our findings not only offer novel insights into A. mellifera larva-A. apis interaction, but also lay the groundwork for clarifying the mechanisms underlying DEpiRNA-regulated larval response.

molecular biology↗

bioRxiv2022

Effect of Ascosphaera apis infestation on the activities of four antioxidant enzymes in Asian honey bee larval guts

Ascosphaera apis exclusively infects bee larvae and causes chalkbrood, a lethal fungal disease that results in the sharp reduction in adult bees and colony productivity. However, little is known about the effect of A. apis infestation on the activities of antioxidant enzymes in bee larvae. Here, A. apis spores were purified and used to inoculate Asian honey bee (Apis cerana) larvae, followed by detection of the host survival rate and evaluation of the activities of four major antioxidant enzymes. At 6 days post inoculation (dpi) with A. apis spores, white mycelia penetrated the posterior end of the larva, extended to the anterior end, and eventually covered the entire larval body surface, presenting an obvious symptom of chalkbrood disease similar to that occurs in Apis mellifera larvae. Additionally, PCR identification showed that the expected fragment was amplified from the A. apis-inoculated larval guts and the A. apis spores, verifying the A. apis infection of A. cerana larvae. The survival rate of larvae inoculated with A. apis was high at 1-2 dpi, sharply decreased to 4.16% at 4 dpi, and reached 0% at 5 dpi; whereas that of un-inoculated larvae was always high at 1~8 dpi, with an average survival rate of 95.37%, indicating the negative impact of A. apis infection on larval survival. Furthermore, in comparison with those in the corresponding un-inoculated groups, the superoxide dismutase (SOD) and catalase (CAT) activities in the 4-day-old larval gut in the A. apis-inoculated groups were reduced (p > 0.05), while those in the 5- and 6-day-old larval guts were significantly decreased (p < 0.05); the glutathione S-transferase (GST) activity in the 4- and 5-day-old larval guts was significantly increased (p < 0.05), while that in the 6-day-old larval gut was reduced (p > 0.05); the polyphenol oxidase (PPO) activity in 4-day-old larval gut was increased (p > 0.05) and that in the 5-day-old larval gut was significantly increased (p < 0.05), whereas that in the 6-day-old larval gut was significantly reduced (p < 0.01). These results together suggested that the activities of SOD and CAT in the larval guts were suppressed during the process of A. apis infestation, while the GST activity was induced to activation, and the PPO activity was first enhanced and then inhibited. Our findings not only unravel the response of A. cerana larvae to A. apis infestation from a biochemical perspective, but also offer a valuable insight into the interaction between Asian honey bee larvae and A. apis.

molecular biology↗

bioRxiv2022

ame-miR-34 modulates the larval body weight and immune response of Apis mellifera workers to Ascosphara apis invasion

MiRNAs are critical regulators of numerous physiological and pathological processes. Ascosphaera apis exclusively infects bee larvae and causes chalkbrood disease. However, the function and mechanism of miRNAs in the bee larval response to A. apis infection is poorly understood. Here, ame-miR-34, a previously predicted miRNA involved in the response of Apis mellifera ligustica larvae to A. apis invasion, was subjected to molecular validation, and overexpression and knockdown were then conducted to explore the regulatory functions of ame-miR-34 in larval body weight and immune response. Stem-loop RT-PCR and Sanger sequencing confirmed the authenticity of ame-miR-34 in the larval gut of A. m. ligustica. RT-qPCR results demonstrated that compared with that in the uninfected larval guts, the expression level of ame-miR-34 was significantly downregulated (P < 0.001) in the guts of A. apis-infected 4-, 5-, and 6-day-old larvae, indicative of the remarkable suppression of host ame-miR-34 due to A. apis infection. In comparison with the corresponding negative control (NC) groups, the expression level of ame-miR-34 in the larval guts in the mimic-miR-34 group was significantly upregulated (P < 0.001), while that in the inhibi- tor-miR-34 group was significantly downregulated (P < 0.01). Similarly, effective overexpression and knockdown of ame-miR-34 were achieved. In addition, the body weights of 5- and 6-day-old larvae were significantly increased compared with those in the mimic-NC group; the weights of 5-day-old larvae in the inhibitor-miR-34 group were significantly decreased in comparison with those in the inhibitor-NC group, while the weights of 4- and 6-day-old larvae in the inhibi- tor-miR-34 group were significantly increased, indicating the involvement of ame-miR-34 in modulating larval body weight. Furthermore, the expression levels of both hsp and abct in the guts of A. apis-infected 4-, 5- and 6-day-old larvae were significantly upregulated after ame-miR-34 overexpression. In contrast, after ame-miR-34 knockdown, the expression levels of the aforementioned two key genes in the A. apis-infected 4-, 5- and 6-day-old larval guts were significantly downregu- lated. Together, the results demonstrated that effective overexpression and knockdown of ame-miR-34 in both noninfected and A. apis-infected A. m. ligustica larval guts could be achieved by the feeding method, and ame-miR-34 exerted a regulatory function in the host immune response to A. apis invasion through positive regulation of the expression of hsp and abct. Our findings not only provide a valuable reference for the functional investigation of bee larval miRNAs but also reveal the regulatory role of ame-miR-34 in A. mellifera larval weight and immune response. Additionally, the results of this study may provide a promising molecular target for the treatment of chalkbrood disease.

molecular biology↗

bioRxiv2022

Integrated transcriptome and lineage analyses reveal novel catecholaminergic cardiomyocytes contributing to the cardiac conduction system in murine heart

Cardiac conduction system (CCS) morphogenesis is essential for correct heart function yet is incompletely understood. Here we established the transcriptional landscape of cell types populating the developing heart by integrating single-cell RNA sequencing and spatial enhanced resolution omics-sequencing (Stereo-seq). Stereo-seq provided a spatiotemporal transcriptomic cell fate map of the murine heart with a panoramic field of view and in situ cellular resolution of the CCS. This led to the identification of a previously unrecognized cardiomyocyte population expressing dopamine beta-hydroxylase (Dbh+-CMs), which is closely associated with the CCS in transcriptomic analyses. To confirm this finding, genetic fate mapping by using DbhCre/Rosa26-tdTomato reporter mouse line was performed with Stereo-seq, RNAscope, and immunohistology. We revealed that Dbh+-derived CMs first emerged in the sinus venosus at E12.5, then populated the atrial and ventricular CCS components at E14.5, with increasing abundance towards perinatal stages. Further tracing by using DbhCFP reporter and DbhCreERT/Rosa26-tdTomato inducible reporter, we confirmed that Dbh+-CMs are mostly abundant in the AVN and ventricular CCS and this persists in the adult heart. By using DbhCre/Rosa26-tdTomato/Cx40-eGFP compound reporter line, we validated a clear co-localization of tdTomato and eGFP signals in both left and right ventricular Purkinje fibre networks. Finally, electrophysiological optogenetic study using cell-type specific Channelrhodopsin2 (ChR2) expression further elucidated that Dbh+-derived CMs form a functional part of the ventricular CCS and display similar photostimulation-induced electrophysiological characteristics to Cx40CreERT/ChR2-tdTomato CCS components. Thus, by utilizing advanced transcriptomic, mouse genetic, and optogenetic functional analyses, our study provides new insights into mammalian CCS development and heterogeneity by revealing novel Dbh+-CMs. HighlightsO_LIStereo-seq provided a spatiotemporal transcriptomic cell fate map of the murine heart with a panoramic field of view and in situ cellular resolution of the CCS. C_LIO_LIEstablished the transcriptional landscape of cell types populating the developing murine heart. C_LIO_LIRevealed previously unreported catecholaminergic cardiomyocyte populations contributing to the developing and mature murine cardiac conduction system. C_LI

cell biology↗

bioRxiv2022

First identification and investigation of piRNAs in the larval guts of Asian honey bee, Apis cerana

Piwi-interacting RNAs (piRNAs), a kind of small non-coding RNAs (ncRNAs), play pivotal parts in maintaining the genomic stability and modulating biological processes such as growth and development via regulation of gene expression. However, piRNAs in Asian honey bee (Apis cerana) is still largely unknown at present. In this current work, on basis of previously gained high-quality small RNA-seq datasets, piRNAs in the larval guts of Apis cerana cerana, the nominate species of A. cerana, was for the first time identified, followed by in-depth investigation of the regulatory roles of differentially expressed piRNAs (DEpiRNAs) in the developmental process of the A. c. cerana. Here, a total of 621 piRNAs were identified in the A. c. cerana larval guts, among which 499 piRNAs were shared by 4- (Ac4 group), 5- (Ac5 group), and 6-day-old (Ac6 group) larval guts, while the numbers of unique ones were 79, 37, and 11, respectively. piRNAs each group were ranged from 24 nt to 33 nt in length, and the first base of piRNAs had a cytosine (C) bias. Additionally, five up-regulated and five down-regulated piRNAs were identified in the Ac4 vs. Ac5 comparison group, 9 of which could target 9, 011 mRNAs; these targets were involved in 41 GO terms and 137 pathways. Comparatively, 22 up-regulated piRNAs were detected in the Ac5 vs. Ac6 comparison group, 21 of which could target 28, 969 mRNAs; these targets were engaged in 46 functional terms and 164 pathways. The results suggested the overall alteration of expression pattern of piRNAs during the developmental process of A. c. cerana larvae. Regulatory network analysis showed that piR-bmo-748815 and piR-bmo-512574 in the Ac4 vs. Ac5 comparison group as well as piR-bmo-716466 and piR-bmo-828146 in the Ac5 vs. Ac6 comparison group linked to the highest number of targets. Further investigation indicated that targets of DEpiRNAs in the above-mentioned two comparison groups could be annotated to several growth and development-associated pathways, such as Jak/STAT, TGF-{beta}, and Wnt signaling pathways, indicating the involvement of DEpiRNAs in modulating larval gut development via these crucial pathways. Moreover, the expression trends of six randomly selected DEpiRNAs were verified using a combination of stem-loop RT-PCR and RT-qPCR. These results not only provide a novel insight into the development of the A. c. cerana larval guts, but also lay a foundation for uncovering the epigenetic mechanism underlying the larval gut development.

molecular biology↗

bioRxiv2022

Cuproptosis is correlated with clinical status, tumor immune microenvironment and immunotherapy in colorectal cancer: a multi-omic analysis

Copper, like double-edged sword, either too little or too much can lead to cell death. Cuproptosis, a novel identified cell death form induced by copper, is characterized by aggregation of lipoylated mitochondrial enzymes and the destabilization of Fe-S cluster proteins. However, the function and potential clinical value of cuproptosis in colorectal cancer remains largely unknown. In this study, 16 cuproptosis-related molecules (CPRMs) were identified and analyzed by transcriptomics, genomics, and single-cell transcriptome profiling from multiple databases. We established two cuproptosis-related molecular phenotypes (CMP1 and 2) to distinguish CRC individuals, in which there were significantly differences in prognosis, biological function, somatic mutation frequency, immune cell infiltration in CRC individuals. A novel cuproptosis-related scoring system (CuproScore) was also constructed to predict the prognosis of CRC individuals, TME and the response to immunotherapy. Of notion, the value of CuproScore was also confirmed in our transcriptome cohort, demonstrating that CRC individuals in the high CuproScore group tended to have higher immune cell infiltrations and higher immune checkpoint expression. We also checked and analyzed the expression and clinical significance of 16 CPRMs in CRC cell lines and CRC tissues. This study indicated that cuproptosis and CPRMs played significant role in CRC progression and in modeling the TME. Inducing cuproptosis may be a useful tool for tumor therapy in the future.

cancer biology↗

bioRxiv2022

Transcriptome-wide characterization of piRNAs during the developmental process of European honey bee larval guts

piRNAs play pivotal roles in suppressing transposons, maintaining genome stability, regulating gene expression, and modulating development and immunity. However, there are few piRNA-associated studies on honey bee, and the regulatory role of piRNAs in the development of bee guts is largely unknown. In this current work, the differential expression pattern of piRNAs during the developmental process of the European honey bee (Apis mellifera) was analyzed, and target prediction of differentially expressed piRNAs (DEpiRNAs) was then conducted, followed by investigation of regulatory networks and the potential function of DEpiRNAs in regulating larval gut development. Here, a total of 843 piRNAs were identified in the larval guts of A. mellifera; among these, 764 piRNAs were shared by 4- (Am4 group), 5- (Am5 group), and 6-day-old (Am6 group) larval guts, while 11, 67, and 1, respectively, were unique. The first base of piRNAs in each group had a cytosine (C) bias. Additionally, 61 up-regulated and 17 down-regulated piR-NAs were identified in the Am4 vs. Am5 comparison group, further targeting 9, 983 genes, which were involved in 50 GO terms and 142 pathways, while two up-regulated and five down-regulated piRNAs were detected in the Am5 vs. Am6 comparison group, further targeting 1, 936 genes, which were engaged in 41 functional terms and 101 pathways. piR-ame-742536 and piR-ame-856650 in the Am4 vs. Am5 comparison group as well as piR-ame-592661 and piR-ame-31653 in the Am5 vs. Am6 comparison group were found to link to the highest number of targets. Further analysis indicated that targets of DEpiRNAs in these two comparison groups were seven development-associated signaling pathways such as Hippo and Notch signaling pathways, seven immune-associated pathways including endocytosis and the Jak/STAT signaling pathway, and three energy metabolism pathways, namely sulfur metabolism, nitrogen metabolism, and oxidative phosphorylation. Moreover, the expression trends of five randomly selected DEpiRNAs were verified based on stem-loop RT-PCR and RT-qPCR. These results were suggestive of the overall alteration of piRNAs during the larval developmental process and demonstrated that DEpiRNAs potentially modulate development-, immune-, and energy metabolism-associated pathways by regulating expression of corresponding genes via target binding, further affecting the development of A. mellifera larval guts. Our data offer novel insights into the development of bee guts and lay a basis for clarifying the developmental mechanism underlying the larval guts of European honey bee.

molecular biology↗

bioRxiv2022

SHINE: Protein Language Model based Pathogenicity Prediction for Inframe Insertion and Deletion Variants

Inframe insertion and deletion variants (indels) alter protein sequence and length. Accurate pathogenicity predictions are important in genetic studies of human diseases. Indel Interpretation is challenging due to limitations in the available number of known pathogenic variants for training. Existing methods largely use manually encoded features including conservation, protein structure and function, and allele frequency. Recent advances in deep learning modeling of protein sequences and structures provide an opportunity to improve the representation of salient features based on large numbers of protein sequences. We developed a new pathogenicity predictor for SHort Inframe iNsertion and dEletion (SHINE). SHINE uses pre-trained protein language models to construct a latent representation of an indel and its protein context from protein sequences and multiple protein sequence alignments, and feeds the latent representation into supervised machine learning models for pathogenicity prediction. We curated training data from ClinVar and gnomAD, and created two test datasets from different sources. SHINE achieved better prediction performance than existing methods for both deletion and insertion variants in these two test datasets. Our work suggests that unsupervised protein language models can provide valuable information about proteins, and new methods based on these models can improve variant interpretation in genetic analyses.

bioinformatics↗

bioRxiv2022

Control of Motor Landing and Processivity by the CAP-Gly Domain in the KIF13B Tail

Microtubules are a major component of the eukaryotic cytoskeleton that play crucial roles in diverse cellular process. Posttranslational modifications (PTMs) of tubulin dimers regulate the dynamics and organization of microtubules, as well as the interactions between microtubules and microtubule-associated proteins (MAPs). One unique PTM that occurs on microtubules is the cyclical removal and re-addition of the C-terminal tyrosine of -tubulin. CAP-Gly (cytoskeleton-associated protein glycine-rich) domain containing proteins specifically recognize tyrosinated microtubules, a property exploited to regulate and spatially localize diverse microtubule effectors. KIF13B is a member of the long-distance transport kinesin-3 family, and the only kinesin motor that contains a conserved C-terminal CAP-Gly domain. What role the CAP-Gly domain plays in KIF13Bs motility along microtubules is unknown. Here, we investigated the interaction between KIF13Bs CAP-Gly domain, and the tyrosinated C-terminal tail domain of -tubulin. We found that KIF13Bs CAP-Gly domain strongly influences the initial motor-microtubule interaction, as well as the processive motility of KIF13B along microtubules. The effect of the CAP-Gly domain on kinesin-microtubule binding is specific to the nucleotide state of the motor domain, suggesting an interplay between the N-terminal motor domain and C-terminal CAP-Gly domain underlies the KIF13B-microtubule interaction. These results reveal that specialized kinesin tail domains play active roles in the initiation and continuation of motor movement.

biophysics↗

bioRxiv2022

Enhanced Single RNA Imaging Reveals Dynamic Gene Expression in Live Animals

Imaging endogenous mRNAs in live animals is technically challenging. Here we describe an MS2 based signal Amplification with Suntag System that enables live-cell RNA imaging of high temporal resolution and with 8xMS2 stem-loops, which overcomes the obstacle of inserting a 1,300 nt 24xMS2 into the genome for the imaging of endogenous mRNAs. Using this tool we were able to image the activation of gene expression and the dynamics of endogenous mRNAs in the epidermis of live C. elegans.

cell biology↗

bioRxiv2022

Generation of cardiomyocytes from human induced pluripotent stem cells resembling atrial cells with ability to respond to adrenoceptor agonists

Cardiovascular disease is the leading cause of global mortality and morbidity. Cardiac dysrhythmias contribute significantly to this disease burden. Atrial fibrillation (AF) is the most common chronic dysrhythmia. Human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-AMs) present an exciting new model for AF but currently fail to reach maturity and so are limited in translational potential currently. We report a new approach using a combination of Gremlin 2 and retinoic acid treatment of human iPSCs for generating cardiomyocytes resembling atrial cells. More than 40% of myocytes generated by this approach showed rod-shaped morphology, expression of cardiomyocyte proteins (including RyR2 receptors, a-actinin-2, F-actin) and typically a striated appearance, all of which were broadly similar to the characteristics of adult atrial myocytes. Isolated myocytes were electrically quiescent until stimulated to fire action potentials with an atrial myocyte profile and an amplitude of approximately 100 mV, arising from a resting potential of approximately -70 mV. Single-cell RNA sequence (scRNASeq) analysis showed a high level of expression of several atrial specific transcripts including NPPA, MYL7, HOXA3, SLN, KCNJ4, KCNJ5 and KCNA5. Amplitudes of calcium transients recorded from spontaneously beating cultures were increased by the stimulation of -adrenoceptors (activated by phenylephrine and blocked by prazosin) or {beta}-adrenoceptors (activated by isoproterenol and blocked by CGP20712A). Thus, our new method provides an efficient approach for differentiating human atrial myocytes with mature characteristics from hiPSCs. This preparation will be very useful for studying signalling pathways in human atrial myocytes, and provides a valuable model for investigating atrial fibrillation and drug discovery.

bioengineering↗

bioRxiv2022

Characteristics of circadian rhythm-related genes and establishment of a prognostic scoring system (CRscore) for lung adenocarcinoma with experimental verification

BackgroundNon-small-cell lung cancer (NSCLC) is one of the most common malignant tumors worldwide. Lung adenocarcinoma (LUAD), which is the m ain subtype of NSCLC, has a poor prognosis. In recent years, circadian rhythm (CR)-related genes (CRRGs) have demonstrated associations with tumor occurrence and development, but the relationship between CRRGs and LUAD is not clear. MethodsBased on data from The Cancer Genome Atlas and Gene Expression Omnibus databases, we explored the biological function and immune cell infiltration of LUAD in different CR clusters and quantified CR genes using principal component analysis. Then, we built a CR scoring system (CRscore) to explore the relationship between CRRGs and LUAD prognosis. ResultsPatients were divided into three clusters (A, B, and C). Biological characteristics, such as survival, immune cell infiltration, and gene enrichment, were significantly different among the three clusters. We then established the usefulness of the CR score, which could effectively predict the prognosis of LUAD. Specifically, patients with a high CR score had a better prognosis and were more sensitive to chemotherapy than patients with a low CR score. ConclusionCRRGs can be used to assess the prognosis of patients with LUAD. Quantification of CR using the CRscore tool in patients with LUAD could help to guide personalized cancer immunotherapy strategies in the future. Thus, the CRscore may be a powerful prognostic tool for LUAD.

genomics↗

bioRxiv2022

In-depth investigation of microRNA-mediated cross-kingdom regulation between Asian honey bee and microsporidian

Asian honey bee Apis cerana is the original host for Nosema ceranae, a unicellular fungal parasite that causes bee nosemosis throughout the world. Currently, interaction between A. cerana and N. ceranae is largely unknown. Here, based on our previously gained high-quality RNA-seq and small RNA-seq data from N. ceranae-infected A. c. cerana workers midguts and clean spores, differentially expressed mRNAs (DEmiRNAs) in N. ceranae targeted by DEmiRNAs in host midguts and A. c. cerana DEmRNAs targeted by microsporidian DEmiRNAs were predicted using bioinformatics, and then target DEmRNAs in microsporidian and host were annotated and investigated, with a focus on targets involved in N. ceranae glycolysis/glyconeogenesis and virulence factors as well as A. c. cerana energy mechanism and immune response. Its found that 97 down-regulated (60 up-regulated) mRNAs in NcCKM vs NcTM1 were potentially targeted by eight up-regulated (six down-regulated) miRNAs in AcCKMI1 vs AcTMI1, 44 down-regulated (15 up-regulated) mRNAs in NcCKM vs NcTM2 were putative targets of seven up-regulated (two down-regulated) miRNAs in AcCKMI2 vs AcTMI2. Additionally, miR-60-y and miR-676-y were found to up-regulate in AcCKMI1 vs AcTMI1 and target genes engaged in spore wall protein and glycolysis/gluconeogenesis, while miR-60-y in AcCKMI1 vs AcTMI1 was up-regulated and potentially targeted a glycolysis/gluconeogenesis-associated gene. Comparatively, 343 down-regulated (138 up-regulated) mRNAs in AcCKM1 vs AcTM1 were putative targets of 121 up-regulated (112 down-regulated) miRNAs in NcCKMI vs NcTMI1, 247 down-regulated (110 up-regulated) mRNAs were putatively targeted by 110 up-regulated (104 down-regulated) miRNAs in NcCKMI vs NcTMI2. Further analysis showed that 31 up-regulated miRNAs in NcCKMI vs NcTMI1 potentially targeted 12 down-regulated mRNAs in AcCKM1 vs AcTM1, which were involved in five immune-related pathways such as phagasome and Jak-STAT signaling pathway, whereas nine up-regulated miRNAs in NcCKMI vs NcTMI2 putatively targeted five down-regulated mRNAs in AcCKM2 vs AcTM2, which were engaged in three immune-related pathways including endocytosis, lysosomes, and regulation of autophagy. In addition, miR-21-x was observed to up-regulate in NcCKMI vs NcTMI1 and target a oxidative phosphorylation-related gene. Finally, potential targeting relationship between two host DEmiRNAs-microsporidian DEmRNAs pairs and two microsporidian DEmiRNAs-host DEmRNAs pairs were verified on basis of RT-qPCR. Our findings not only lay a foundation for exploring the molecular mechanism underlying cross-kingdom regulation between A. c. cerana workers and N. ceranae, but also offer valuable insights into Asian honey bee-microsporidian interaction.

pathology↗

bioRxiv2022

CircRNA-regulated immune response of Asian honey bee workers to microsporidian infection

Nosema ceranae is a widespread fungal parasite for honey bees, causing bee nosemosis. Based on deep sequencing and bioinformatics, identification of circular RNAs (circRNAs) in Apis cerana cerana workers midguts and circRNA-regulated immune response of host to N. ceranae invasion were conducted in this current work, followed by molecular verification of back-splicing sites and expression trends of circRNAs. Here, 10185 and 7405 circRNAs were identified in the midguts of workers at 7 d (AcT1) and 10 d (AcT2) post inoculation (dpi) with N. ceranae. PCR amplification result verified the back-splicing sites in three specific circRNAs (novel_circ_005123, novel_circ_007177, and novel_circ_015140) expressed in N. ceranae-inoculated midgut. In combination with transcriptome data from corresponding un-inoculated midguts (AcCK1 and AcCK2), 2266 circRNAs were found to be shared by the aforementioned four groups, whereas the numbers of specific ones were 2618, 1917, 5691 and 3723 respectively. Further, 83 (52) differentially expressed circRNAs (DEcircRNAs) were identified in AcCK1 vs AcT1 (AcCK2 vs AcT2) comparison group. Source genes of DEcircRNAs in workers midgut at 7 dpi were involved in two cellular immune-related pathways such as endocytosis and ubiquitin mediated proteolysis. Additionally, competing endogenous RNA network analysis showed that 23 (13) DEcircRNAs in AcCK1 vs AcT1 (AcCK2 vs AcT2) can target 18 (14) miRNAs and further link to 1111 (1093) mRNAs. These target mRNAs were annotated to six cellular immunity pathways including endocytosis, lysosome, phagosome, ubiquitin mediated proteolysis, metabolism of xenobiotics by cytochrome P450, and insect hormone biosynthesis. Moreover, 284 (164) IRES and 54 (26) ORF were identified from DEcircRNAs in AcCK1 vs AcT1 (AcCK2 vs AcT2) comparison group; additionally, ORFs in DEcircRNAs in midgut at 7 dpi with N. ceranae were associated with several crucial pathways including endocytosis and ubiquitin-mediated proteolysis. Finally, RT-qPCR results showed that the expression trends of six DEcircRNAs were consistent with those in transcriptome data. These results demonstrated that N. ceranae altered the expression pattern of circRNAs in A. c. cerana workers midguts, and DEcircRNAs were likely to regulate host cellular and humoral immune response to microsporidian infection. Our findings lay a foundation for clarifying the mechanism underlying host immune response to N. ceranae infection and provide a new insight into interaction between Asian honey bee and microsporidian.

pathology↗

bioRxiv2022

A scalable sparse neural network framework for rare cell type annotation of single-cell transcriptome data

Cell type annotation is critical to understand the cell population heterogeneity in the single-cell RNA sequencing (scRNA-seq) analysis. Due to their fast, precise, and user-friendly advantages, automatic annotation methods are gradually replacing traditional unsupervised clustering approaches in cell type identification practice. However, current supervised annotation tools are easily overfitting, thus favoring large cell populations but failing to learn the information of smaller populations. This drawback will significantly mislead biological analysis, especially when the rare cell types are important. Here, we present scBalance, an integrated sparse neural network framework that leverages the adaptive weight sampling and dropout techniques for the auto-annotation task. Using 20 scRNA-seq datasets with different scales and different imbalance degrees, we systematically validate the strong performance of scBalance for both intra-dataset and inter-dataset annotation tasks. Furthermore, we also demonstrate the scalability of scBalance on identifying rare cell types in million-level datasets by uncovering the immune landscape in bronchoalveolar cells. Up to now, scBalance is the first and only auto-annotation tool that expands scalability to 1.5 million cells dataset. In addition, scBalance also shows a fast and stable speed outperforming commonly used tools across all scales of datasets. We implemented scBalance in a user-friendly manner that can easily interact with Scanpy, which makes scBalance a superior tool in the increasingly important Python-based platform.

bioinformatics↗

bioRxiv2022

Circular RNA ROR B; regulates TGFBR1 by decoying miR-140 in alcohol-exposed lungs and fibroblasts

Alcohol ingestion exaggerates transforming growth factor-beta 1 (TGF{beta}1) expression and signaling leading to fibroproliferation. Inhibition of TGF{beta} receptor type 1 (TGF{beta}R1) mitigates the effect of TGF{beta}1 signaling. We showed that alcohol can modulate microRNA (miRNA) expressions. The mechanism by which alcohol modulates microRNA and how it ties to TGF{beta}1 signaling has not been well elucidated. Circular RNA (circRNAs or circ) emerges as a potential therapeutic target based on its stability, tissue specificity, and its ability to modify miRNAs. In this study, we showed that alcohol upregulates TGF{beta}R1 and circRNA form of retinoic acid receptor-related orphan receptor beta (circ-ROR{beta}) in lung fibroblasts (LF) and the lung. We identified miR-140 to have binding sites for both TGF{beta}R1 3 UTR and circ-ROR{beta} and alcohol attenuated miR-140 expression in LF and the lung. We demonstrated that inhibition of circ-ROR{beta} upregulated miR-140 and completely abrogated alcohol-induced miR-140 suppression. We further showed that inhibition of circ-ROR{beta} attenuated alcohol-induced TGF{beta}R1, fibronectin (FN1), and -smooth muscle actin (SMA) expressions and myofibroblast development as seen by an attenuation of SMA stress fiber formation in LF. Taken together, these findings identify circ-ROR{beta}-miR-140-TGF{beta}R1 axis as a novel mechanism by which alcohol induces TGF{beta}1 signaling and promotes FMD. HighlightsAlcohol induces circ-ROR{beta} expression in lung fibroblasts Circ-ROR{beta} regulates TGF{beta}R1 by decoying miR-140 in lung fibroblasts Inhibition of Circ-ROR{beta} restores miR-140 expression Inhibition of Circ-ROR{beta} mitigates alcohol-mediated myofibroblast differentiation This is the first description of circ-ROR{beta} functional significance in lung fibroblast

physiology↗

bioRxiv2022

Reversal of lactate and PD-1-mediated macrophage immunosuppression controls growth of PTEN/p53-deficient prostate cancer

PTEN loss-of-function occurs in approximately 50% of mCRPC patients, and is associated with a poor prognosis, therapeutic outcomes and resistance to immune-checkpoint inhibitors. Recent clinical studies demonstrated that dual PI3K/AKT pathway inhibition and androgen axis blockade led to a modest improvement in progression-free survival of PTEN-deficient mCRPC patients, but the mechanistic basis for this limited efficacy is unknown. To elucidate potential resistance mechanism(s), we performed co-clinical trials in a prostate-specific PTEN/p53-deficient genetically-engineered mouse model, and discovered that the recruitment of PD-1-expressing tumor-associated macrophages (TAM) thwarts the phagocytosis-mediated anti-tumor efficacy of androgen deprivation therapy (ADT)/PI3K inhibitor (PI3Ki) combination. Strikingly, we observed a TAM-dependent [~]3-fold enhancement in the overall response rate with the addition of PD-1 antibody (aPD-1) to ADT/PI3Ki combination therapy. Mechanistically, decreased lactate production from PI3Ki-treated tumor cells suppressed histone lactylation (H3K18lac) within TAM, resulting in their phagocytic activation, which was augmented by concurrent ADT/aPD-1 treatment. Consistent with our murine observations, single cell RNA-sequencing analysis of human metastatic PC samples revealed a direct correlation between high glycolytic activity and phagocytosis suppression. Critically, feedback activation of Wnt/{beta}-catenin signaling observed in non-responder mice following ADT/PI3Ki/aPD-1 combination treatment, restored lactate-mediated H3K18lac and suppressed phagocytosis within macrophages. Altogether, these data suggest that reversal of lactate and PD-1-mediated TAM immunosuppression by PI3Ki and aPD-1, respectively, controls tumor growth in combination with ADT, and warrants further clinical investigation in PTEN/p53-deficient mCRPC patients. One Sentence SummaryInhibition of tumor-cell intrinsic lactate production suppresses PTEN/p53-deficient prostate cancer growth via macrophage activation/phagocytosis

cancer biology↗

bioRxiv2022

Reconstruction of the cell pseudo-space from single-cell RNA sequencing data with scSpace

Tissues are highly complicated with spatial heterogeneity in gene expression. However, the cutting-edge single-cell RNA-seq technology eliminates the spatial information of individual cells, which contributes to the characterization of cell identities. Herein, we propose single-cell spatial position associated co-embeddings (scSpace), an integrative algorithm to distinguish spatially variable cell subclusters by reconstructing cells onto a pseudo-space with spatial transcriptome references (Visium, STARmap, Slide-seq, etc.). We demonstrated that scSpace can define biologically meaningful cell subpopulations neglected by single-cell RNA-seq or spatially resolved transcriptomics. The use of scSpace to uncover the spatial association within single-cell data, reproduced, the hierarchical distribution of cells in the brain cortex and liver lobules, and the regional variation of cells in heart ventricles and the intestinal villus. scSpace identified cell subclusters in intratelencephalic neurons, which were confirmed by their biomarkers. The application of scSpace in melanoma and Covid-19 exhibited a broad prospect in the discovery of spatial therapeutic markers.

bioinformatics↗