The big bang of genome editing technology: development and application of the CRISPR/Cas9 system in disease animal models

Targeted genome editing technology has been widely used in biomedical studies. The CRISPR- associated RNA-guided endonuclease Cas9 has become a versatile genome editing tool. The CRISPR/Cas9 system is useful for studying gene function through efficient knock-out, knock-in or chromatin modification of the targeted gene loci in various cell types and organisms. It can be applied in a number of fields, such as genetic breeding, disease treatment and gene functional investigation In this review, we introduce the most recent developments and applications, the challenges, and future directions of Cas9 in generating disease animal model. Derived from the CRISPR adaptive immune system of bacteria, the development trend of Cas9 will inevitably fuel the vital applications from basic research to biotechnology and bio- medicine.

Experimental primates and non-human primate(NHP) models of human diseases in China: current status and progress

Non-human primates （NHPs） are phylogenetically close to humans, with many similarities in terms of physiology, anatomy, immunology, as well as neurology, all of which make them excellent experimental models for biomedical research. Compared with developed countries in America and Europe, China has relatively rich primate resources and has continually aimed to develop NHPs resources. Currently, China is a leading producer and a major supplier of NHPs on the international market. However, there are some deficiencies in feeding and management that have hampered China＇s growth in NHP research and materials. Nonetheless, China has recently established a number of primate animal models for human diseases and achieved marked scientific progress on infectious diseases, cardiovascular diseases, endocrine diseases, reproductive diseases, neurological diseases, and ophthalmic diseases, etc. Advances in these fields via NHP models will undoubtedly further promote the development of China＇s life sciences and pharmaceutical industry, and enhance China＇s position as a leader in NHP research. This review covers the current status of NHPs in China and other areas, highlighting the latest developments in disease models using NHPs, as well as outlining basic problems and proposing effective to better utilize NHP resources and further foster NHP research in China.

Isolation and characterization of glacier VMY22, a novel lytic cold-active bacteriophage of Bacillus cereus

As a unique ecological system with low temperature and low nutrient levels, glaciers are considered a "living fossil" for the research of evolution. In this work, a lytic cold-active bacteriophage designated VMY22 against Bacillus cereus MYB41-22 was isolated from Mingyong Glacier in China, and its characteristics were studied. Electron microscopy revealed that VMY22 has an icosahedral head(59.2 nm in length, 31.9 nm in width) and a tail(43.2 nm in length). Bacteriophage VMY22 was classified as a Podoviridae with an approximate genome size of 18 to 20 kb. A one-step growth curve revealed that the latent and the burst periods were 70 and 70 min, respectively, with an average burst size of 78 bacteriophage particles per infected cell. The pH and thermal stability of bacteriophage VMY22 were also investigated. The maximum stability of the bacteriophage was observed to be at pH 8.0 and it was comparatively stable at p H 5.0–9.0. As VMY22 is a cold-active bacteriophage with low production temperature, its characterization and the relationship between MYB41-22 and Bacillus cereus bacteriophage deserve further study.

Analysis of immunoglobulin, complements and CRP levels in serum of captive northern pig-tailed macaques （Macaca leonina）

The northem pig-tailed macaque （NPM, Macaca leonina） has become a widely used animal model in biomedical research. In this study, we measured serum immunoglobulin IgG, IgM, IgA, complement C3, C4 and CRP levels in 3-11 year old captive northem pig-tailed macaques using HITACHI 7600-20 automated chemistry analyzer in order to determine the influences of age and gender on these items. The results showed that serum IgA, IgM, C3 and C4 levels were not correlated with age （P〉0.05）, while serum IgG levels increased progressively with age （r=0.202; P=0.045）. Serum IgG, IgA, IgM and C3 levels were higher in females than in males （P〈0.05）. Moreover, serum C3 concentration was both positively and strongly correlated with that of C4 （r=0.700; P〈0.0001）. This study provides basic serum immunoglobulin and complement data of captive northem pig-tailed macaques, which may prove useful for future breeding efforts and biomedical research.

Expression patterns and action analysis of genes associated with blood coagulation responses during rat liver regeneration

AIM: To study the blood coagulation response after partial hepatectomy (PH) at transcriptional level. METHODS: After PH of rats, the associated genes with blood coagulation were obtained through reference to the databases, and the gene expression changes in rat regenerating liver were analyzed by the Rat Genome 230 2.0 array. RESULTS: It was found that 107 genes were associated with liver regeneration. The initially and totally expressing gene numbers occurring in initiation phase of liver regeneration (0.5-4 h after PH), G0/G1 transition (4-6 h after PH), cell proliferation (6-66 h after PH), cell differentiation and structure-function reconstruction (66-168 h after PH) were 44, 11, 58, 7 and 44, 33, 100, 71 respectively, showing that the associated genes were mainly triggered in the forepart and prophase, and worked at different phases. According to their expression similarity, these genes were classified into 5 groups: only up-, predominantly up-, only down-, predominantly down-, up- and down-regulation, involving 44, 8, 36, 13 and 6 genes, respectively, and the total times of their up- and down-regulation expression were 342 and 253, respectively, demonstrating that the number of the up-regulated genes was more than that of the down- regulated genes. Their time relevance was classified into 15 groups, showing that the cellular physiological and biochemical activities were staggered during liver regeneration. According to gene expression patterns, they were classified into 29 types, suggesting that their protein activities were diverse and complex during liver regeneration.CONCLUSION: The blood coagulation response is enhanced mainly in the forepart, prophase and anaphase of liver regeneration, in which the response in the forepart, prophase of liver regeneration can prevent the bleeding caused by partial hepatectomy, whereas that in the anaphase contributes to the structure-function reorganization of regenerating liver. In the process, 107 genes associated with liver regeneration play an important role.

Expression patterns and action analysis of genes associated with hepatitis virus infection during rat liver regeneration

AIM: To study the action of hepatitis virus infection-associated genes at transcription level during liver regeneration (LR).METHODS: Hepatitis virus infection-associated genes were obtained by collecting the data from databases and retrieving the correlated articles, and their expression changes in the regenerating rat liver were detected with the rat genome 230 2.0 array. RESULTS: Eighty-eight genes were found to be associated with liver regeneration. The number of genes initially and totally expressed during initial LR [0.5-4 h after partial hepatectomy (PH)], transition from G0 to G1 (4-6 h after PH), cell proliferation (6-66 h after PH), cell differentiation and reorganization of structure-function (66-168 h after PH) was 37, 8, 48, 3 and 37, 26, 80, 57, respectively, indicating that the genes were mainly triggered at the early stage of LR (0.5-4 h after PH), and worked at different phases. These genes were classified into 5 types according to their expression similarity, namely 37 up-regulated, 9 predominantly up-regulated, 34 down-regulated, 6 predominantly down-regulated and 2 up/down-regulated genes. Their total up- and down-regulation frequencies were 359 and 149 during LR, indicating that the expression of most genes was enhanced, while the expression of a small number of genes was attenuated during LR. According to time relevance, they were classified into 12 groups (0.5 and 1 h, 2 and 4 h, 6 h, 8 and 12 h, 16 and 96 h, 18 and 24 h, 30 and 42 h, 36 and 48 h, 54 and 60 h, 66 and 72 h, 120 and 144 h, 168 h), demonstrating that the cellular physiological and biochemical activities duringLR were fluctuated. According to expression changes of the genes, their expression patterns were classified into 23 types, suggesting that the cellular physiological and biochemical activities during LR were diverse and complicated. CONCLUSION: The anti-virus infection capacity of regenerating liver can be enhanced and 88 genes play an important role in LR.

Advances and perspectives in the application of CRISPR/Cas9 in insects

Insects compose more than half of all living organisms on earth, playing essential roles in global ecosystems and forming complex relationships with humans. Insect research has significant biological and practical importance. However, the application of genetic manipulation technology has long been restricted to several model insects only, such as gene knockout in Drosophila, which has severely restrained the development of insect biology research. Recently, with the increase in the release of insect genome data and the introduction of the CRISPR/Cas9 system for efficient genetic modification, it has been possible to conduct meaningful functional studies in a broad array of insect species. Here, we summarize the advances in CRISPR/Cas9 in different insect species, discuss methods for its promotion, and consider its application in future insect studies. This review provides detailed information about the application of the CRISPR/Cas9 system in insect research and presents possible ways to improve its use in functional studies and insect pest control.

Purification and characterization of cholecystokinin from the skin of salamander Tylototriton verrucosus

As a group of intestinal hormones and neurotransmitters, cholecystokinins（CCKs） regulate and affect pancreatic enzyme secretion, gastrointestinal motility, pain hypersensitivity, digestion and satiety, and generally contain a DYMGWMDFG sequence at the C-terminus. Many CCKs have been reported in mammals. However, only a few have been reported in amphibians, such as Hyla nigrovittata, Xenopus laevis, and Rana catesbeiana, with none reported in urodele amphibians like newts and salamanders. Here, a CCK called CCK-TV was identified and characterized from the skin of the salamander Tylototriton verrucosus. This CCK contained an amino acid sequence of DYMGWMDF-NH2 as seen in other CCKs. A c DNA encoding the CCK precursor containing 129 amino acid residues was cloned from the c DNA library of T. verrucosus skin. The CCK-TV had the potential to induce the contraction of smooth muscle strips isolated from porcine gallbladder, eliciting contraction at a concentration of 5.0x10-11 mol/L and inducing maximal contraction at a concentration of 2.0x10-6 mol/L. The EC50 was 13.6 nmol/L. To the best of our knowledge, this is the first report to identify the presence of a CCK in an urodele amphibian.

Molecular cloning and anti-HIV-1 activities of APOBEC3s from northern pig-tailed macaques(Macaca leonina)

Northern pig-tailed macaques （NPMs, Macaca leonina） are susceptible to HIV-1 infection largely due to the loss of HIV-l-restricting factor TRIM5a. However, great impediments still exist in the persistent replication of HIV-1 in vivo, suggesting some viral restriction factors are reserved in this host The APOBEC3 proteins have demonstrated a capacity to restrict HIV-1 replication, but their inhibitory effects in NPMs remain elusive. In this study, we cloned the NPM A3A-A3H genes, and determined by BLAST searching that their coding sequences （CDSs） showed 99% identity to the corresponding counterparts from rhesus and southern pig-tailed macaques. We further analyzed the anti-HIV-1 activities of the A3A-A3H genes, and found that A3G and A3F had the greatest anti-HIV-1 activity compared with that of other members. The results of this study indicate that A3G and A3F might play critical roles in limiting HIV-1 replication in NPMs in vivo. Furthermore, this research provides valuable information for the optimization of monkey models of HIV-1 infection.

Generation of a tree shrew breast cancer model using lentivirus expressing PIK3CA-H1047R

DEAR EDITOR,Breast cancer is the most common malignant tumor in women,posing a serious threat to health. Tree shrews(Tupaia belangeri chinensis) are evolutionarily closer to humans than rodents and are emerging as an attractive experimental animal model for breast cancer. The PIK3CA gene is frequently mutated in both human and tree shrew breast tumors. Herein, we effectively overexpressed PIK3CA-H1047R in the mammary epithelial cells of tree shrews using a lentivirus to induce breast tumors.

Plant functional trait diversity and structural diversity co-underpin ecosystem multifunctionality in subtropical forests

Tree species diversity is assumed to be an important component in managing forest ecosystems because of effects on multiple functions or ecosystem multifunctionality.However,the importance of tree diversity in determining multifunctionality in structurally complex subtropical forests relative to other regulators(e.g.,soil microbial diversity,stand structure,and environmental conditions)remains uncertain.In this study,effects of aboveground(species richness and functional and structural diversity)and belowground(bacterial and fungal diversity)biodiversity,functional composition(community-weighted means of species traits),stand structure(diameter at breast height and stand density),and soil factors(pH and bulk density)on multifunctionality(including biomass production,carbon stock,and nutrient cycling)were examined along a tree diversity gradient in subtropical forests.The community-weighted mean of tree maximum height was the best predictor of ecosystem multifunctionality.Functional diversity explained a higher proportion of the variation in multifunctionality than that of species richness and fungal diversity.Stand structure-played an important role in modulating the effects of tree diversity on multifunctionality.The work highlights that species composition and maximizing forest structural complexity are effective strategies to increase forest multifunctionality while also conserving biodiversity in the management of multifunctional forests under global environmental changes.

Isozyme expression of Chinese and Japanese populations of Chlamys farreri and their reciprocal hybrids

Chinese and Japanese population of Chlamys farreri and their reciprocal hybrids were surveyed in isozyme variability at 13 loci by polyacrytamide gel electrophoresis (PAGE). Isozyme banding patterns indicated these hybrids were diploid. Loci that were observed as being monomorphic in inbred populations of C. farreri were also found to be monomorphic in filial progeny; loci that observed to be polymorphic in parental type gen- erations were also polymorphic in hybrid generations. Differences existed among allelic frequency of the four types of cross. Within the reciprocal hybrids the expression of malic enzyme (ME) isozyme was sufficient to distinguishing individual hybrids because of the band, Rf=0.38. However, there were no noticeable variations among all the samples to differentiate one from another. Inbreeding was likely to be the main problem in aqua- culture. The introduction of new broodstock can improve the genetic diversity. Hybrid vigor has manifested to a certain extent in the present study.

Synthesis and characterization of A1 doped δ-laminar crystal sodium silicate

An modified laminar crystal sodium silicate （A1-SKS-6） was synthesized by spray drying and high temperature crystallization using sodium silicate as silicon source, A1 atom as inorganic source and hexadecyltrimethy ammonium bromide （CTMAB） as the structure template. The hydrothermal stability of product was at least 1 h. The synthesized material was characterized by XRD and SEM. The results indicated that shape and intensity of XRD diffraction peak at 20 took changes, but raw material 8 phase crystal form kept unchanged, and assured high Ca^2＋ and Mg^2＋ binding capacity of the products. The Ca^2＋ and Mg^2＋ binding capacity of product is up to 380 mg Ca/g and 410mg Mg/g respectively. It was found that the hole of product became smaller and appeared rod structure based on SEM. The hydrothermal stability was increased due to the pillaring of AI atom.

Mitochondrial phylogenomics provides insights into the phylogeny and evolution of spiders(Arthropoda:Araneae)

Spiders are among the most varied terrestrial predators,with highly diverse morphology,ecology,and behavior.Morphological and molecular data have greatly contributed to advances in the phylogeny and evolutionary dynamics of spiders.Here,we performed comprehensive mitochondrial phylogenomics analysis on 78 mitochondrial genomes(mitogenomes)representing 29 families;of these,23 species from eight families were newly generated.Mesothelae retained the same gene arrangement as the arthropod ancestor(Limulus polyphemus),while Opisthothelae showed extensive rearrangement,with 12 rearrangement types in transfer RNAs(tRNAs)and control region.Most spider tRNAs were extremely truncated and lacked typical dihydrouridine or TΨC arms,showing high tRNA structural diversity;in particular,trnS1 exhibited anticodon diversity across the phylogeny.The evolutionary rates of mitochondrial genes were potentially associated with gene rearrangement or truncated tRNAs.Both mitogenomic sequences and rearrangements possessed phylogenetic characteristics,providing a robust backbone for spider phylogeny,as previously reported.The monophyly of suborder,infraorder,retrolateral tibial apophysis clade,and families(except for Pisauridae)was separately supported,and high-level relationships were resolved as(Mesothelae,(Mygalomorphae,(Entelegynae,(Synspermiata,Hypochilidae)))).The phylogenetic positions of several families were also resolved(e.g.,Eresidae,Oecobiidae and Titanoecidae).Two reconstructions of ancestral web type obtained almost identical results,indicating that the common ancestor of spiders likely foraged using a silk-lined burrow.This study,the largest mitochondrial phylogenomics analysis of spiders to date,highlights the usefulness of mitogenomic data not only for providing efficient phylogenetic signals for spider phylogeny,but also for characterizing trait diversification in spider evolution.

Linkage of microbial living communities and residues to soil organic carbon accumulation along a forest restoration gradient in southern China

Background:Forest restoration has been considered an effective method to increase soil organic carbon(SOC),whereas it remains unclear whether long-term forest restoration will continuously increase SOC.Such large uncertainties may be mainly due to the limited knowledge on how soil microorganisms will contribute to SOC accumulation over time.Methods:We simultaneously documented SOC,total phospholipid fatty acids(PLFAs),and amino sugars(AS)content across a forest restoration gradient with average stand ages of 14,49,70,and>90 years in southern China.Results:The SOC and AS continuously increased with stand age.The ratio of fungal PLFAs to bacterial PLFAs showed no change with stand age,while the ratio of fungal AS to bacterial AS significantly increased.The total microbial residue-carbon(AS-C)accounted for 0.95-1.66% in SOC across all forest restoration stages,with significantly higher in fungal residue-C(0.68-1.19%)than bacterial residue-C(0.05-0.11%).Furthermore,the contribution of total AS-C to SOC was positively correlated with clay content at 0-10 cm soil layer but negatively related to clay content at 10-20 cm soil layer.Conclusions:These findings highlight the significant contribution of AS-C to SOC accumulation along forest restoration stages,with divergent contributions from fungal residues and bacterial residues.Soil clay content with stand age significantly affects the divergent contributions of AS-C to SOC at two different soil layers.

Psychological impact of the COVID-19 pandemic on Chinese population:An online survey

BACKGROUND The ongoing coronavirus disease 2019(COVID-19)pandemic infiltrates every aspect of our life,including the psychological impact.China has experienced the first wave of this epidemic,and it is now affecting the global population.AIM This study aimed to investigate the prevalence and associated factors of posttraumatic stress disorder(PTSD)among the general Chinese population.METHODS A detailed questionnaire,comprising of 38 questions designed in both English and Chinese,was developed.The survey was conducted via WeChat,a multipurpose messaging,social media,and mobile payment app,which is widely used by the Chinese population.RESULTS In total,1082 individuals from 31 provinces,autonomous regions,and municipalities participated in the survey by filling the questionnaires through the WeChat app.97.8%of the participants had an Impact of Event-Scale-Revised(IES-R)total score above 20,which is an indicator of PTSD.The IES-R total and all the three subscales,including intrusion,avoidance,and hyperarousal,are significantly correlated with age.In addition,age,profession,marital status,and level of education are significantly correlated with the degree of PTSD symptoms.CONCLUSION The COVID-19 epidemic has widely caused PTSD among the general Chinese population.These results bear important implications for regions struggling with the pandemic to implement effective interventions to cope with these mental health problems.

WRKY11 up-regulated dirigent expression to enhance lignin/lignans accumulation in Lilium regale Wilson during response to Fusarium wilt

Lilium are highly economically valuable ornamental plants that are susceptible to Fusarium wilt caused by Fusarium oxysporum.Lilium regale Wilson,a wild lily native to China,is highly resistant to F.oxysporum.In this study,a WRKY transcription factor,WRKY11,was isolated from L.regale,and its function during the interaction between L.regale and F.oxysporum was characterized.The ectopic expression of LrWRKY11 in tobacco increased the resistance to F oxysporum,moreover,the transcriptome sequencing and UHPLC-MS/MS analysis indicated that the methyl salicylate and methyl jasmonate levels rose in LrWRKY11 transgenic tobacco,meanwhile,the expression of lignin/lignans biosynthesis-related genes including a dirigent(DiR)was up-regulated.The lignin/lignans contents in LrWRKY11-transgenic tobacco also significantly increased compared with the wild-type tobacco.In addition,the resistance of L.regale scales in which LrWRKY11 expression was silenced by RNAi evidently decreased,and additionally,the expression of lignin/lignans biosynthesis-related genes including LrDIR1 was significantly suppressed.Therefore,LrDIR1 and its promoter(PLrDIR1)sequence containing the W-box element were isolated from L.regale.The interaction assay indicated that LrWRKY11 specifically bound to the W-box element in PLrDIR1 and activated LrDIR1 expression.Additionally,β-glucuronidase activity in the transgenic tobacco co-expressing LrWRKY11/PLrDIR1-β-glucuronidase was higher than that in transgenic tobacco expressing PLrDIR1-β-glucuronidase alone.Furthermore,the ectopic expression of LrDIR1 in tobacco enhanced the resistance to F.oxysporum and increased the lignin/lignans accumulation.In brief,this study revealed that LrWRKY11 positively regulated L.regale resistance to F.oxysporum through interaction with salicylic acid/jasmonic acid signaling pathways and modulating LrDIR1 expression to accumulate lignin/lignans.

Influence of Thinning on Soil CO_2 Efflux in Chinese Fir Plantations

Forest management is expected to influence soil CO2 efflux (FCO2) as a result of changes in microenvironmental conditions, soil microclimate, and root dynamics. Soil FCO2 rate was measured during the growing season of 2006 in both thinning and non-thinning locations within stands ranging from 0 to 8 years after the most recent thinning in Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) plantations in Huitong Ecosystem Research Station, Hunan, China. Soil temperature and moisture were also measured to examine relationships between FCO2 and soil properties. Forest thinning resulted in huge changes in FCO2 that varied with time since cutting. Immediately following harvest (year 0) FCO2 in thinning area increased by about 30%, declined to 20%–27% below pre-cutting levels during years 4–6, and recovered to pre-cutting levels at 8 years post-cutting. A similar temporal pattern, but with smaller changes, was found in non-thinning locations. The initial increase in FCO2 could be attributed to a combination of root decay, soil disturbance, and increased soil temperature in gaps, while the subsequent decrease and recovery to the death and gradual regrowth of active roots. Strong effects of soil temperature and soil water content on FCO2 were found. Forest thinning mainly influenced FCO2 through changes in tree root respiration, and the net result was a decrease in integrated FCO2 flux through the entire felling cycle.

MiR-182 is up-regulated and targeting Cebpa in hepatocellular carcinoma

MicroRNAs （miRNAs） are endogenous small non-coding RNAs that repress their targets at post transcriptional level.Existing studies have shown that miRNAs are important regulatory genes in hepatocellular carcinoma （HCC）,as either tumor suppressors or oncogenes.MiR-122 is normally downregulated in HCC and regarded as a tumor suppressor.Recently miR-122 has been reported to be regulated by CEBPA,which is then involved in a novel pathway to influence proliferation of tumor cells.However it is unknown whether CEBPA is regulated by miRNAs in HCC.In this study,we find that miR182 is upregulated in HCC model rat,and represses CEBPA in both rat and human.This further improves the current CEBPA/miR-122 pathway that controls the proliferation of tumor cells.These results suggest that miR-182 is a potential oncogene in HCC and could be used as a diagnostic marker and drug target of HCC.