Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo.

Interspecies chimera formation with human pluripotent stem cells (hPSCs) represents a necessary alternative to evaluate hPSC pluripotency in vivo and might constitute a promising strategy for various regenerative medicine applications, including the generation of organs and tissues for transplantation. Studies using mouse and pig embryos suggest that hPSCs do not robustly contribute to chimera formation in species evolutionarily distant to humans. We studied the chimeric competency of human extended pluripotent stem cells (hEPSCs) in cynomolgus monkey (Macaca fascicularis) embryos cultured ex vivo. We demonstrate that hEPSCs survived, proliferated, and generated several peri- and early post-implantation cell lineages inside monkey embryos. We also uncovered signaling events underlying interspecific crosstalk that may help shape the unique developmental trajectories of human and monkey cells within chimeric embryos. These results may help to better understand early human development and primate evolution and develop strategies to improve human chimerism in evolutionarily distant species.

Comparative Analysis of Active Ingredients and Potential Bioactivities of Essential Oils from Artemisia argyi and A. verlotorum.

Artemisia argyi H. Lév. and Vaniot is a variety of Chinese mugwort widely cultured in central China. A. verlotorum Lamotte, another variety of Chinese mugwort, has been used in the southern region of China since ancient times. Despite their similar uses in traditional medicine, little is known about the differences in their active ingredients and potential benefits. Herein, the chemical compositions of the essential oils (EOs) from both varieties were analyzed using chromatography-mass spectrometry (GC-MS). A series of databases, such as the Traditional Chinese Medicine Systems Pharmacology database (TCMSP), SuperPred database and R tool, were applied to build a networking of the EOs. Our results revealed significant differences in the chemical compositions of the two Artemisia EOs. However, we found that they shared similar ingredient-target-pathway networking with diverse bioactivities, such as neuroprotective, anti-cancer and anti-inflammatory. Furthermore, our protein connection networking analysis showed that transcription factor p65 (RELA), phosphatidylinositol 3-kinase regulatory subunit alpha (PIK3R1) and mitogen-activated protein kinase 1 (MAPK1) are crucial for the biological activity of Artemisia EOs. Our findings provided evidence for the use of A. verlotorum as Chinese mugwort in southern China.

The genome of medicinal leech (Whitmania pigra) and comparative genomic study for exploration of bioactive ingredients.

BACKGROUND: Leeches are classic annelids that have a huge diversity and are closely related to people, especially medicinal leeches. Medicinal leeches have been widely utilized in medicine based on the pharmacological activities of their bioactive ingredients. Comparative genomic study of these leeches enables us to understand the difference among medicinal leeches and other leeches and facilitates the discovery of bioactive ingredients. RESULTS: In this study, we reported the genome of Whitmania pigra and compared it with Hirudo medicinalis and Helobdella robusta. The assembled genome size of W. pigra is 177 Mbp, close to the estimated genome size. Approximately about 23% of the genome was repetitive. A total of 26,743 protein-coding genes were subsequently predicted. W. pigra have 12346 (46%) and 10295 (38%) orthologous genes with H. medicinalis and H. robusta, respectively. About 20 and 24% genes in W. pigra showed syntenic arrangement with H. medicinalis and H. robusta, respectively, revealed by gene synteny analysis. Furthermore, W. pigra, H. medicinalis and H. robusta expanded different gene families enriched in different biological processes. By inspecting genome distribution and gene structure of hirudin, we identified a new hirudin gene g17108 (hirudin_2) with different cysteine patterns. Finally, we systematically explored and compared the active substances in the genomes of three leech species. The results showed that W. pigra and H. medicinalis exceed H. robusta in both kinds and gene number of active molecules. CONCLUSIONS: This study reported the genome of W. pigra and compared it with other two leeches, which provides an important genome resource and new insight into the exploration and development of bioactive molecules of medicinal leeches.

A New World Monkey Resembles Human in Bitter Taste Receptor Evolution and Function via a Single Parallel Amino Acid Substitution.

Bitter taste receptors serve as a vital component in the defense system against toxin intake by animals, and the family of genes encoding these receptors has been demonstrated, usually by family size variance, to correlate with dietary preference. However, few systematic studies of specific Tas2R to unveil their functional evolution have been conducted. Here, we surveyed Tas2R16 across all major clades of primates and reported a rare case of a convergent change to increase sensitivity to ß-glucopyranosides in human and a New World monkey, the white-faced saki. Combining analyses at multiple levels, we demonstrate that a parallel amino acid substitution (K172N) shared by these two species is responsible for this functional convergence of Tas2R16. Considering the specialized feeding preference of the white-faced saki, the K172N change likely played an important adaptive role in its early evolution to avoid potentially toxic cyanogenic glycosides, as suggested for the human TAS2R16 gene.

In Silico Identification of Anti-SARS-CoV-2 Medicinal Plants Using Cheminformatics and Machine Learning.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen of COVID-19, is spreading rapidly and has caused hundreds of millions of infections and millions of deaths worldwide. Due to the lack of specific vaccines and effective treatments for COVID-19, there is an urgent need to identify effective drugs. Traditional Chinese medicine (TCM) is a valuable resource for identifying novel anti-SARS-CoV-2 drugs based on the important contribution of TCM and its potential benefits in COVID-19 treatment. Herein, we aimed to discover novel anti-SARS-CoV-2 compounds and medicinal plants from TCM by establishing a prediction method of anti-SARS-CoV-2 activity using machine learning methods. We first constructed a benchmark dataset from anti-SARS-CoV-2 bioactivity data collected from the ChEMBL database. Then, we established random forest (RF) and support vector machine (SVM) models that both achieved satisfactory predictive performance with AUC values of 0.90. By using this method, a total of 1011 active anti-SARS-CoV-2 compounds were predicted from the TCMSP database. Among these compounds, six compounds with highly potent activity were confirmed in the anti-SARS-CoV-2 experiments. The molecular fingerprint similarity analysis revealed that only 24 of the 1011 compounds have high similarity to the FDA-approved antiviral drugs, indicating that most of the compounds were structurally novel. Based on the predicted anti-SARS-CoV-2 compounds, we identified 74 anti-SARS-CoV-2 medicinal plants through enrichment analysis. The 74 plants are widely distributed in 68 genera and 43 families, 14 of which belong to antipyretic detoxicate plants. In summary, this study provided several medicinal plants with potential anti-SARS-CoV-2 activity, which offer an attractive starting point and a broader scope to mine for potentially novel anti-SARS-CoV-2 drugs.

Correction to: FastMM: an efficient toolbox for personalized constraint-based metabolic modeling.

An amendment to this paper has been published and can be accessed via the original article.

FastMM: an efficient toolbox for personalized constraint-based metabolic modeling.

BACKGROUND: Constraint-based metabolic modeling has been applied to understand metabolism related disease mechanisms, to predict potential new drug targets and anti-metabolites, and to identify biomarkers of complex diseases. Although the state-of-art modeling toolbox, COBRA 3.0, is powerful, it requires substantial computing time conducting flux balance analysis, knockout analysis, and Markov Chain Monte Carlo (MCMC) sampling, which may limit its application in large scale genome-wide analysis. RESULTS: Here, we rewrote the underlying code of COBRA 3.0 using C/C++, and developed a toolbox, termed FastMM, to effectively conduct constraint-based metabolic modeling. The results showed that FastMM is 2~400 times faster than COBRA 3.0 in performing flux balance analysis and knockout analysis and returns consistent outputs. When applied to MCMC sampling, FastMM is 8 times faster than COBRA 3.0. FastMM is also faster than some efficient metabolic modeling applications, such as Cobrapy and Fast-SL. In addition, we developed a Matlab/Octave interface for fast metabolic modeling. This interface was fully compatible with COBRA 3.0, enabling users to easily perform complex applications for metabolic modeling. For example, users who do not have deep constraint-based metabolic model knowledge can just type one command in Matlab/Octave to perform personalized metabolic modeling. Users can also use the advance and multiple threading parameters for complex metabolic modeling. Thus, we provided an efficient and user-friendly solution to perform large scale genome-wide metabolic modeling. For example, FastMM can be applied to the modeling of individual cancer metabolic profiles of hundreds to thousands of samples in the Cancer Genome Atlas (TCGA). CONCLUSION: FastMM is an efficient and user-friendly toolbox for large-scale personalized constraint-based metabolic modeling. It can serve as a complementary and invaluable improvement to the existing functionalities in COBRA 3.0. FastMM is under GPL license and can be freely available at GitHub site: https://github.com/GonghuaLi/FastMM.

Gain and loss events in the evolution of the apolipoprotein family in vertebrata.

BACKGROUND: Various apolipoproteins widely distributed among vertebrata play key roles in lipid metabolism and have a direct correlation with human diseases as diagnostic markers. However, the evolutionary progress of apolipoproteins in species remains unclear. Nine human apolipoproteins and well-annotated genome data of 30 species were used to identify 210 apolipoprotein family members distributed among species from fish to humans. Our study focused on the evolution of nine exchangeable apolipoproteins (ApoA-I/II/IV/V, ApoC-I~IV and ApoE) from Chondrichthyes, Holostei, Teleostei, Amphibia, Sauria (including Aves), Prototheria, Marsupialia and Eutheria. RESULTS: In this study, we reported the overall distribution and the frequent gain and loss evolutionary events of apolipoprotein family members in vertebrata. Phylogenetic trees of orthologous apolipoproteins indicated evident divergence between species evolution and apolipoprotein phylogeny. Successive gain and loss events were found by evaluating the presence and absence of apolipoproteins in the context of species evolution. For example, only ApoA-I and ApoA-IV occurred in cartilaginous fish as ancient apolipoproteins. ApoA-II, ApoE, and ApoC-I/ApoC-II were found in Holostei, Coelacanthiformes, and Teleostei, respectively, but the latter three apolipoproteins were absent from Aves. ApoC-I was also absent from Cetartiodactyla. The apolipoprotein ApoC-III emerged in terrestrial animals, and ApoC-IV first arose in Eutheria. The results indicate that the order of the emergence of apolipoproteins is most likely ApoA-I/ApoA-IV, ApoE, ApoA-II, ApoC-I/ApoC-II, ApoA-V, ApoC-III, and ApoC-IV. CONCLUSIONS: This study reveals not only the phylogeny of apolipoprotein family members in species from Chondrichthyes to Eutheria but also the occurrence and origin of new apolipoproteins. The broad perspective of gain and loss events and the evolutionary scenario of apolipoproteins across vertebrata provide a significant reference for the research of apolipoprotein function and related diseases.

Folate Deficiency and Gene Polymorphisms of MTHFR, MTR and MTRR Elevate the Hyperhomocysteinemia Risk.

BACKGROUND: Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular diseases (CVDs). We aimed to investigate the joint effect of homocysteine metabolism gene polymorphisms, as well as the folate deficiency on the risk of HHcy in a Chinese hypertensive population. METHODS: This study enrolled 480 hypertensive patients aged 28 - 75 from six hospitals in different Chinese regions from 9/2005 - 12/2005. Known genotypes of methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, methionine synthase (MTR) A2756G, and methionine synthase reductase (MTRR) A66G were detected by PCRRFLP methods. Serum Hcy was measured by high-performance liquid chromatography and serum folate was measured by chemiluminescent immunoassay. RESULTS: MTHFR C677T and MTR A2756G can independently elevate the risk of HHcy (TT vs. CC + CT, p < 0.001 and AG + GG vs. AA, p = 0.026, respectively), whereas MTHFR A1298C decreased HHcy risk (AC + CC vs. AA, p < 0.001) and showed a protective effect against HHcy risk. Importantly, the joint effect of these risk genotypes showed significantly higher odds of HHcy than non-risk genotypes, especially the patients with four risk genotypes. It is noteworthy that this deleterious effect was aggravated by folate deficiency. These findings were verified by generalized multifactor dimensionality reduction model (p = 0.001) and a cumulative effects model (p < 0.001). CONCLUSIONS: We have first demonstrated that the joint effect of homocysteine metabolism gene polymorphisms and folate deficiency lead to dramatic elevations in the HHcy risk.

Joint associations of folate, homocysteine and MTHFR, MTR and MTRR gene polymorphisms with dyslipidemia in a Chinese hypertensive population: a cross-sectional study.

BACKGROUND: Dyslipidemia is a well-established risk factor for cardiovascular disease. Serum lipids were affected by several gene polymorphisms, folate, homocysteine and other metabolite levels. We aim to investigate the effects of homocysteine metabolism enzyme polymorphisms (MTHTR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G) and their interactions with folate, homocysteine on serum lipid levels in Chinese patients with hypertension. METHODS: Participants were 480 hypertensive adults that enrolled in September to December 2005 from six different Chinese hospitals (Harbin, Shanghai, Shenyang, Beijing, Xi'an, and Nanjing). Known MTHFR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G genotypes were determined by PCR-RFLP methods. Serum folate was measured by chemiluminescent immunoassay, homocysteine was measured by high-performance liquid chromatography, serum lipids parameters were determined by an automatic biochemistry analyzer, low-density lipoprotein was calculated by Friedewald's equation. Unitary linear regression model was used to assess the associations of gene polymorphisms, folate and homocysteine on serum lipid profiles. Unconditional logistic regression model was applied to test the interactions of folate, homocysteine and gene polymorphisms on dyslipidemia. RESULTS: No correlations between gene polymorphisms and homocysteine on serum lipid profiles. Compared with normal folate patients, patients with low folate showed higher odds of hypertriglyceridemia (OR = 2.02, 95 % CI: 1.25-3.25, P = 0.004) and low levels of high-density lipoprotein cholesterol (OR = 1.88, 95 % CI: 1.07-3.28, P = 0.027). Each of four gene polymorphisms (MTHTR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G) combined with low folate showed higher odds of hypertriglyceridemia (P for trend: 0.049, 0.004, 0.007 and 0.005, respectively). MTHFR C677T and A1298C with low folate showed higher odds of low levels of high-density lipoprotein cholesterol (P for trend: 0.008 and 0.031). CONCLUSIONS: Low folate status and homocysteine metabolism gene polymorphisms (MTHTR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G) may have a synergistic effect increased the incidence of dyslipidemia in Chinese hypertensive population.

Tsaokoflavanols A1-J1: Flavanol-fatty alcohol hybrids with HPL inhibitory activity from Amomum tsao-ko.

Ten previously undescribed compounds were isolated from the fruits of Amomum tsao-ko (Zingiberaceae), including nine undescribed flavanol-fatty alcohol hybrids (1-6, 10-11, 13), and a flavanol-monoterpenoid hybrid (14), along with seven known flavanol hybrids (7-9, 12, 15-17). The structures of these compounds were determined using various analyses, such as HRESIMS, 1D/2D NMR, and ECD calculations. In terms of biological activity, compounds 1, 2, 5, and 6 exhibited inhibitions of human pancreatic lipase (HPL), with IC50 values ranging from 0.017 to 0.193 mM. Some of these values were found to be stronger than that of the positive control, orlistat (IC50, 0.067 mM). Molecular docking studies were also conducted to investigate the interactions between these compounds and HPL. The docking simulations revealed the importance of the orientation of the 3,4-dihydroxyphenyl in binding with HPL. Additionally, compound 9 demonstrated cytotoxicity against HepG2, with a CC50 value of 14.96 ± 0.62 µM as determined by the MTT assay. Flow cytometry analysis indicated that compound 9 induced apoptosis in HepG2 cells. Western blot results showed an up-regulation of apoptosis-related proteins, such as p53 protein, Bax and Caspase-3 proteins, while the expression of Bcl-2 protein was down-regulated.

Long-term in vivo chimeric cells tracking in non-human primate.

Non-human primates (NHPs) are increasingly used in preclinical trials to test the safety and efficacy of biotechnology therapies. Nonetheless, given the ethical issues and costs associated with this model, it would be highly advantageous to use NHP cellular models in clinical studies. However, developing and maintaining the naïve state of primate pluripotent stem cells (PSCs) remains difficult as does in vivo detection of PSCs, thus limiting biotechnology application in the cynomolgus monkey. Here, we report a chemically defined, xeno-free culture system for culturing and deriving monkey PSCs in vitro. The cells display global gene expression and genome-wide hypomethylation patterns distinct from monkey-primed cells. We also found expression of signaling pathways components that may increase the potential for chimera formation. Crucially for biomedical applications, we were also able to integrate bioluminescent reporter genes into monkey PSCs and track them in chimeric embryos in vivo and in vitro. The engineered cells retained embryonic and extra-embryonic developmental potential. Meanwhile, we generated a chimeric monkey carrying bioluminescent cells, which were able to track chimeric cells for more than 2 years in living animals. Our study could have broad utility in primate stem cell engineering and in utilizing chimeric monkey models for clinical studies.

Deciphering molecular heterogeneity and dynamics of human hippocampal neural stem cells at different ages and injury states.

While accumulated publications support the existence of neurogenesis in the adult human hippocampus, the homeostasis and developmental potentials of neural stem cells (NSCs) under different contexts remain unclear. Based on our generated single-nucleus atlas of the human hippocampus across neonatal, adult, aging, and injury, we dissected the molecular heterogeneity and transcriptional dynamics of human hippocampal NSCs under different contexts. We further identified new specific neurogenic lineage markers that overcome the lack of specificity found in some well-known markers. Based on developmental trajectory and molecular signatures, we found that a subset of NSCs exhibit quiescent properties after birth, and most NSCs become deep quiescence during aging. Furthermore, certain deep quiescent NSCs are reactivated following stroke injury. Together, our findings provide valuable insights into the development, aging, and reactivation of the human hippocampal NSCs, and help to explain why adult hippocampal neurogenesis is infrequently observed in humans.

New insights into the pharmacological chaperone activity of c2-substituted glucoimidazoles for the treatment of Gaucher disease.

Mutations in acid ß-glucocerebrosidase (GCase) lead to the accumulation of the sphingolipid glucosylceramide, thereby resulting in Gaucher disease (GD). Active-site-specific competitive GCase inhibitors are effective pharmacological chaperones (PCs) that act as folding agents for mutant GCase folding in the endoplasmic reticulum. In this study, we prepared a series of glucoimidazole C2-substituent derivatives, and evaluated their inhibition and PC properties with GCase. A cell-based assay with patient-derived lymphoblasts (N370S or L444P mutations) demonstrated that administration of these compounds can significantly increase GCase activity. Interestingly, the 3,3-dimethyl-N-phenyl-4-amide-1-butyl-substituted moderate inhibitor 11 had the greatest effect on activity: 2.1-fold increase in N370S lymphoblasts at 2.5 µM and 1.2-fold increase in L444P at 0.5 µM following a three-day incubation. Computer docking studies and a protease protection assay were used to elucidate the ligand-enzyme interactions responsible for the chaperone activity of 11. Western blot and immuno-fluorescence assays verified restoration of GCase trafficking to the lysosome. Together, these results indicate that 11 is a promising PC for GD treatment and provide direct evidence of the mechanism of GCase chaperoning.

Spatiotemporal proteomic atlas of multiple brain regions across early fetal to neonatal stages in cynomolgus monkey.

Fetal stages are critical periods for brain development. However, the protein molecular signature and dynamics of the human brain remain unclear due to sampling difficulty and ethical limitations. Non-human primates present similar developmental and neuropathological features to humans. This study constructed a spatiotemporal proteomic atlas of cynomolgus macaque brain development from early fetal to neonatal stages. Here we showed that (1) the variability across stages was greater than that among brain regions, and comparisons of cerebellum vs. cerebrum and cortical vs. subcortical regions revealed region-specific dynamics across early fetal to neonatal stages; (2) fluctuations in abundance of proteins associated with neural disease suggest the risk of nervous disorder at early fetal stages; (3) cross-species analysis (human, monkey, and mouse) and comparison between proteomic and transcriptomic data reveal the proteomic specificity and genes with mRNA/protein discrepancy. This study provides insight into fetal brain development in primates.

Essential oil of lemon myrtle (Backhousia citriodora) induces S-phase cell cycle arrest and apoptosis in HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Lemon myrtle (Backhousia citriodora F.Muell.) leaves, whether fresh or dried, are used traditionally in folk medicine to treat wounds, cancers, skin infections, and other infectious conditions. However, the targets and mechanisms related to anti-cancer effect of lemon myrtle are unavailable. In our study, we found that the essential oil of lemon myrtle (LMEO) showed anti-cancer activity in vitro, and we initially explored its mechanism of action. MATERIALS AND METHODS: We analyzed the chemical compositions of LMEO by GC-MS. We tested the cytotoxicity of LMEO on various cancer cell lines using the MTT assay. Network pharmacology was used also to analyze the targets of LMEO. Moreover, the mechanisms of LMEO were investigated through scratch assay, flow cytometry analysis, and western blot in the HepG2 liver cancer cell line. RESULTS: LMEO showed cytotoxicity on various cancer cell lines with values of IC50 40.90 ± 2.23 (liver cancer HepG2 cell line), 58.60 ± 6.76 (human neuroblastoma SH-SY5Y cell line), 68.91 ± 4.62 (human colon cancer HT-29 cell line) and 57.57 ± 7.61 µg/mL (human non-small cell lung cancer A549 cell line), respectively. The major cytotoxic chemical constituent in LMEO was identified as citrals, which accounted for 74.9% of the content. Network pharmacological analysis suggested that apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1), androgen receptor (AR), cyclin-dependent kinases 1 (CDK1), nuclear factor erythroid 2-related factor 2 (Nrf-2), fatty acid synthase (FASN), epithelial growth factor receptor (EGFR), estrogen receptor 1 (ERα) and cyclin-dependent kinases 4 (CDK4) are potential cytotoxic targets of LMEO. These targets are closely related to cell migration, cycle and apoptosis. Notley, the p53 protein had the highest confidence to co-associate with the eight common targets, which was further confirmed by scratch assay, flow cytometry analysis, and western blot in the HepG2 liver cancer cell line. LMEO significantly inhibited the migration of HepG2 cells in time-dependent and dose-dependent manner. Moreover, LMEO caused a S-phase blocking on HepG2 cells and promoted apoptosis in the meanwhile. Western blot results indicated that p53 protein, Cyclin A2 and Bax proteins were up-regulated, while Cyclin E1 and Bcl-2 proteins were down-regulated. CONCLUSION: LMEO showed cytotoxicity in various cancer cell lines in vitro. Pharmacological networks showed LMEO to have multi-component and multi-targeting effects that are related to inhibit migration of HepG2 cells, and affect cell cycle S-phase arrest and apoptosis through modulation of p53 protein.

A hnRNPA2B1 agonist effectively inhibits HBV and SARS-CoV-2 omicron in vivo.

The twenty-first century has already recorded more than ten major epidemics or pandemics of viral disease, including the devastating COVID-19. Novel effective antivirals with broad-spectrum coverage are urgently needed. Herein, we reported a novel broad-spectrum antiviral compound PAC5. Oral administration of PAC5 eliminated HBV cccDNA and reduced the large antigen load in distinct mouse models of HBV infection. Strikingly, oral administration of PAC5 in a hamster model of SARS-CoV-2 omicron (BA.1) infection significantly decreases viral loads and attenuates lung inflammation. Mechanistically, PAC5 binds to a pocket near Asp49 in the RNA recognition motif of hnRNPA2B1. PAC5-bound hnRNPA2B1 is extensively activated and translocated to the cytoplasm where it initiates the TBK1-IRF3 pathway, leading to the production of type I IFNs with antiviral activity. Our results indicate that PAC5 is a novel small-molecule agonist of hnRNPA2B1, which may have a role in dealing with emerging infectious diseases now and in the future.

Evolution, expansion and expression of the Kunitz/BPTI gene family associated with long-term blood feeding in Ixodes Scapularis.

BACKGROUND: Recent studies of the tick saliva transcriptome have revealed the profound role of salivary proteins in blood feeding. Kunitz/BPTI proteins are abundant in the salivary glands of ticks and perform multiple functions in blood feeding, such as inhibiting blood coagulation, regulating host blood supply and disrupting host angiogenesis. However, Kunitz/BPTI proteins in soft and hard ticks have different functions and molecular mechanisms. How these differences emerged and whether they are associated with the evolution of long-term blood feeding in hard ticks remain unknown. RESULTS: In this study, the evolution, expansion and expression of Kunitz/BPTI family in Ixodes scapularis were investigated. Single- and multi-domain Kunitz/BPTI proteins have similar gene structures. Single-domain proteins were classified into three groups (groups I, II and III) based on their cysteine patterns. Group I represents the ancestral branch of the Kunitz/BPTI family, and members of this group function as serine protease inhibitors. The group I domain was used as a module to create multi-domain proteins in hard ticks after the split between hard and soft ticks. However, groups II and III, which evolved from group I, are only present and expanded in the genus Ixodes. These lineage-specific expanded genes exhibit significantly higher expression during long-term blood feeding in Ixodes scapularis. Interestingly, functional site analysis suggested that group II proteins lost the ability to inhibit serine proteases and evolved a new function of modulating ion channels. Finally, evolutionary analyses revealed that the expansion and diversification of the Kunitz/BPTI family in the genus Ixodes were driven by positive selection. CONCLUSIONS: These results suggest that the differences in the Kunitz/BPTI family between soft and hard ticks may be linked to the evolution of long-term blood feeding in hard ticks. In Ixodes, the lineage-specific expanded genes (Group II and III) lost the ancient function of inhibiting serine proteases and evolved new functions to adapt to long-term blood feeding. Therefore, these genes may play a profound role in the long-term blood feeding of hard ticks. Based our analysis, we propose that the six genes identified in our study may be candidate target genes for tick control.

Two different aging paths in human blood revealed by integrated analysis of gene Expression, mutation and alternative splicing.

Aging is a complex life process that human organs and tissues steadily and continuously decline. Aging has huge heterogeneity, which shows different aging rates among different individuals and in different tissues of the same individual. Many studies of aging are often contradictory and show little common signature. The integrated analysis of these transcriptome datasets will provide an unbiased global view of the aging process. Here, we integrated 8 transcriptome datasets including 757 samples from healthy human blood to study aging from three aspects of gene expression, mutations, and alternative splicing. Surprisingly, we found that transcriptome changes in blood are relatively independent of the chronological age. Further pseudotime analysis revealed two different aging paths (AgingPath1 and AgingPath2) in human blood. The differentially expressed genes (DEGs) along the two paths showed a limited overlap and are enriched in different biological processes. The mutations of DEGs in AgingPath1 are significantly increased in the aging process, while the opposite trend was observed in AgingPath2. Expression quantitative trait loci (eQTL) and splicing quantitative trait loci (sQTL) analysis identified 304 important mutations that can affect both gene expression and alternative splicing during aging. Finally, by comparison between aging and Alzheimer's disease, we identified 37 common DEGs in AgingPath1, AgingPath2 and Alzheimer's disease. These genes may contribute to the shift from aging state to Alzheimer's disease. In summary, this study revealed the two aging paths and the related genes and mutations, which provides a new insight into aging and aging-related disease.