CRISPR-Cas13-mediated RNA editing in the silkworm Bombyx mori

The CRISPR-Cas13 system,an RNA-guided editing tool,has emerged as a highly efficient and stable RNA editing technique.Although the CRISPR-Cas13 system has been developed in several insect species,its application in lepidopterans has not yet been reported.In the present study,we evaluated the RNA cleavage activity of the CRISPR-Cas13 system in the silkworm(Bombyx mori),a model lepidopteran insect,both ex vivo and in vivo.We established two stable silkworm BmE cell lines expressing PspCas13b and CasRx,respectively.Further analysis demonstrated that both PspCas13b and CasRx effectively down-regulated the transcription of exogenouslyintroduced target and endogenous genes in these cell lines.In addition,we generated two transgenic silkworm strains,one expressing CasRx and the other expressing RNA-guided CRISPR RNA targeting Sex combs reduced(Scr).Further crossing experiments showed that CasRx induced a down-regulation of Scr transcription in silkworms,which impaired systemic growth of larvae.Overall,this study demonstrated that the CRISPR-Cas13RNA editing system works efficiently in the silkworm,providing a potential alternative approach for RNA manipulation in lepidopteran insects.

麦蚜取食行为特殊刺探电位波形图(EPG)研究初报

研究了麦长管蚜(Sitobionavenae.Fab.)、禾谷缢管蚜(RhopalosiphumPadi)在不同小麦上的刺探行为,发现了一些特殊的刺探电位波形(EPG),即点G波,E0波和两种F波,并就其可能的生物学意义进行了探讨。初步认为,点G波是蚜虫在刺探途径阶段(C波阶段)的试探性主动吸食过程;E0波是蚜虫口针即将到达韧皮部时短暂的判断、休息和适应过程;一种F波是蚜虫在木质部主动吸食受阻的一种过渡波;另一种F波与蚜虫口针在植物组织中遇阻后的重复回撤与刺探行为有关。结果表明,EPG波形不仅有其稳定性,同时也有其多样性。

果树病毒RNA的二氧化硅提取法

针对果树组织含有较高的多酚和多糖物质,研究建立了适合果树病毒日常进行大量RT-PCR检测时,病毒RNA的快速、经济、高效的提取方法。该方法是基于在适宜条件下,二氧化硅对RNA的吸附和洗脱作用,避免了常规方法使用的酚、氯仿等有机溶剂。在2 h内同时可以进行数十个样品的提取,由于提取的是总RNA,1次提取,1次反转录得到的cDNA样品可以用于各种待检病毒的PCR检测。得到的总RNA和cDNA在-20℃条件下可以长期保存备用,大大提高了核酸的提取效率。

改良RNA提取法及樱桃PDV和PNRSV的RT-PCR检测(英文)

介绍了一种从木本植物组织中获得高质量RNA的快速、简单和高效的核酸提取方法。该方法是基于核酸的二氧化硅捕获,避免了使用苯酚、氯仿等有机溶剂。利用该方法从樱桃组织中提取的总RNA用RT-PCR技术检测PDV,PNRSV均获得成功。从感病植株的一年生枝的叶片、韧皮部及芽组织中扩增出了预期的目的片段,即172和449bp,而健康组织中无此扩增带。该法提取的总RNA用于RT-PCR技术检测,其敏感性至少与商业出售的QiagenRNeasy提取试剂盒相当,但简单经济。

Therapeutic proteasome inhibition in experimental acute pancreatitis

AIM： To establish the therapeutic potential of proteasome inhibition, we examined the therapeutic effects of MG132 （Z-Leu-Leu-Leu-aldehyde） in an experimental model of acute pancreatitis. METHODS： Pancreatitis was induced in rats by two hourly intraperitoneal （ip） injections of cholecystokinin octapeptide （CCK; 2×100μg/kg） and the proteasome inhibitor MG132 （10 mg/kg ip） was administered 30 min after the second CCK injection. Animals were sacrificed 4 h after the first injection of CCK. RESULTS： Administering the proteasome inhibitor MG132 （at a dose of 10 mg/kg, ip） 90 min after the onset of pancreatic inflammation induced the expression of cell-protective 72 kDa heat shock protein （HSP72） and decreased DNA-binding of nuclear factor-κB （NF-κB）. Furthermore MG132 treatment resulted in milder inflammatory response and cellular damage, as revealed by improved laboratory and histological parameters of pancreatitis and associated oxidative stress. CONCLUSION： Our findings suggest that proteasome inhibition might be beneficial not only for the prevention, but also for the therapy of acute pancreatitis.

Recent progress in preventive effect of collagen peptides on photoaging skin and action mechanism

Ultraviolet(UV)-induced photoaging skin has become an urgent issue.The functional foods and cosmetics aiming to improve skin photoaging are developing rapidly,and the demand is gradually increasing year by year.Collagen peptides have been proven to display diverse physiological activities,such as excellent moisture retention activity,hygroscopicity,tyrosinase inhibitory activity and antioxidant activity,which indicates that they have great potential in amelioration of UV-induced photoaging.The main objective of this article is to recap the main mechanisms to improve photoaging skin by collagen peptides and their physiological activities in photo-protection.Furthermore,the extraction and structural characteristics of collagen peptides are overviewed.More importantly,some clinical trials on the beneficial effect on skin of collagen peptides are also discussed.In addition,prospects and challenges of collagen peptides are emphatically elucidated in this review.This article implies that collagen peptides have great potential as an effective ingredient in food and cosmetics industry with a wide application prospect.

maturity of associating liver partition and portal vein ligation for staged hepatectomy-derived liver regeneration in a rat model

AIM To establish a rat model for evaluating the maturity of liver regeneration derived from associating liver partition and portal vein ligation for staged hepatectomy(ALPPS).METHODS In the present study, ALPPS, partial hepatecotmy(PHx), and sham rat models were established initially, which were validated by significant increase of proliferative markers including Ki-67, proliferating cell nuclear antigen, and cyclin D1. In the setting of accelerated proliferation in volume at the second and fifth day after ALPPS, the characteristics of newborn hepatocytes, as well as specific markers of progenitor hepatic cell, were identified. Afterwards, the detection of liver function followed by cluster analysis of functional gene expression were performed to evaluate the maturity.RESULTS Compared with PHx and sham groups, the proliferation of f LR was significantly higher in ALPPS group(P = 0.023 and 0.001 at second day, P = 0.034 and P < 0.001 at fifth day after stage I). Meanwhile, the increased expression of proliferative markers including Ki-67, proliferating cell nuclear antigen, and cyclin D1 verified the accelerated liver regeneration derived from ALPPS procedure. However, ALPPS-induced Sox9 positive hepatocytes significantly increased beyond the portal triad, which indicated the progenitor hepatic cell was potentially involved. And the characteristics of ALPPSinduced hepatocytes indicated the lower expression of hepatocyte nuclear factor 4 and anti-tryptase in early proliferative stage. Both suggested the immaturity of ALPPS-derived liver regeneration. Additionally, the detection of liver function and functional genes expression confirmed the immaturity of renascent hepatocytes derived in early stage of ALPPS-derived liver regeneration.CONCLUSION Our study revealed the immaturity of ALPPS-derived proliferation in early regenerative response, which indicated that the volumetric assessment overestimated the functional proliferation. This could be convincing evidence that the stage Ⅱ of ALPPS should be performed prudently in patients with marginally adequate f LR, as the ALPPS-derived proliferation in volume lags behind the functional regeneration.

Upregulation of epidermal growth factor receptor 4 in oral leukoplakia

In the present study,we investigate the expression profile of the epidermal growth factor receptor family,which comprises EGFR/ ErbBl,HER2/ErbB2,HER3/ErbB3 and HER4/ErbB4 in oral leukoplakia(LP).The expression of four epidermal growth factor receptor (EGFR) family genes and their ligands were measured in LP tissues from 14 patients and compared with levels in 10 patients with oral lichen planus(OLP) and normal oral mucosa(NOM) from 14 healthy donors by real-time polymerase chain reaction(PCR) and immunohistochemistry.Synchronous mRNA coexpression of ErbBl,ErbB2,ErbB3and ErbB4 was detected in LP lesions.Out of the receptors,only ErbB4 mRNA and protein was more highly expressed in LP compared with NOM tissues.These were strongly expressed by epithelial keratinocytes in LP lesions,as shown by immunohistochemistry.Regarding the ligands,the mRNA of Neuregulin2 and 4 were more highly expressed in OLP compared with NOM tissues.Therefore,enhanced ErbB4 on the keratinocytes and synchronous modulation of EGFR family genes may contribute to the pathogenesis and carcinogenesis of LP.

Stop Band Gap in Periodic Layers of Confined Atomic Vapor/Dielectric Medium

A stop band gap is predicted in periodic layers of a confined atomic vapor/dielectric medium.Reflection and transmission profile of the layers over the band gap can be dramatically modified by the confined atoms and the number of layer periods.These gap and line features can be ascribed to the enhanced contribution of slow atoms induced by atom-wall collision,transient behavior of atom-light interaction and Fabry–Pérot effects in a thermal confined atomic system.

Morphological and genetic differentiation in isolated populations of Mexican beech Fagus grandifolia subsp.mexicana

Mexican beech[Fagus grandifolia subsp.mexicana(Martinez)A.E.Murray]is a subspecies endemic to the Sierra Madre Oriental Mountains and considered endangered due to the low density of its populations and high degree of habitat fragmentation and environmental specificity.Because its morphological and genetic variation is associated with its ability to adapt to changes in environmental conditions,the objective of this study was to determine whether phenotypic and genotypic variation exist,and it relationships with population reduction events.In four beech populations in the states of Hidalgo and Veracruz,we analyzed 11 morphological variables for leaves and 6 micros atellite markers.The morphological variables that to discriminate between populations were related to the size of the leaf,but a robust differentiation pattern was not found,given that independent groups of leaves were identified.The populations located closest to each other,had greater genetic variation and less genetic distance;populations in the extreme north and south had the lowest genetic variation.Genetic differentiation among populations was associated with reduction in population size.In the 3 localities in Hidalgo,recent bottlenecks were identified,and in Veracruz,an old bottleneck was found.Variation in leaf morphology and genetic structure of Mexican beech populations could be the result of a combination of various geographical,climate and ecological factors.

Molecular modeling of epsilon RNA from hepatitis B virus

An initial model of the HBV epsilon RNA was built by the Biopolymer module of Insight II. While its three-dimensional structure was obtained through structure optimization based on molecular dynamics simulation, the two active sites were found. A comparison with the experimental result indicated that the active sites may be the binding sites of the epsilon RNA in the RT-epsilon interaction. The result will be helpful to further discussion about the mechanism of RT-epsilon interaction and the study of HBV genome replication.

Two Phenolic Diterpenes as Antioxidants

A DFT study was carried out to investigate the structure-activity relationship of rosmanol and carnosol. The geometry, HOMO and LUMO of parent molecules, O–H bond dissociation energy (BDE), and distribution of unpaired electron obtained by B3LYP/6-31G* were used to elucidate the antioxidant properties of the two compounds. The results proved the intramolecular hydrogen bond and delocalization of the unpaired electron to be the important factors affecting the stability of phenoxyl free radical generated after the H-abstraction.

THE ROLE OF ONCOGENES AND ONCO-SUPPRESSOR GENES IN CARCINOGENESIS

Carcinogenesis is a multi-stage process.This process may involve both genetic and epi-genetic events. Analyses of the involvement ofgenetic events (mutations in the global sensewhich include point mutations, deletions, trans-locations and rearrangements of genetic mate-rial) in carcinogenesis have implicated twomajor classes of genes, oncogenes and onco-suppressor genes. Oncogenes, a term first usedby Huebner and Todaro, are derived by muta-tion of normal cellular genes, protooncogenes.More than 40 oncogenes have now been isolatedand found to encode proteins with diverse bio-chemical functions.

Characterization of H^+ and HCO_3^- transporters in CFPAC-1 human pancreatic duct cells

AIM： To characterize H^＋ and HCO3^- transporters in polarized CFPAC-1 human pancreatic duct cells, which were derived from a cystic fibrosis patient with the AF508 CFTR mutation. METHODS： CFPAC-1 cells were seeded at high density onto permeable supports and grown to confluence. The cells were loaded with the pH-sensitive fluorescent dye BCECF, and mounted into a perfusion chamber, which allowed the simultaneous perfusion of the basolateral and apical membranes. Transmembrane base flux was calculated from the changes in intracellular pH and the buffering capacity of the cells. RESULTS： Our results showed differential permeability to HCO3^＋/CO2 at the apical and basolateral membranes of CFPAC-1 cells. Na^＋/HCO3^- co-transporters （NBCs） and Cl^-/HCO3^- exchangers （AEs） were present on the basolateral membrane, and Na＋/H＋ exchangers （NHEs） on both the apical and basolateral membranes of the cells. Basolateral HCO3 uptake was sensitive to variations of extracellular K^＋ concentration, the membrane permeable carbonic anhydrase （CA） inhibitors acetazolamide （100 μmol/L） and ethoxyzolamide （100 μmol/L）, and was partially inhibited by H2-DIDS （600 μmol/L）. The membrane-impermeable CA inhibitor 1-N-（4-sulfamoylphenylethyl）-2,4,6-trimethylpyridine perchlorate did not have any effect on HCO3^- uptake.The basolateral AE had a much higher activity than that in the apical membrane, whereas there was no such difference with the NHE under resting conditions. Also, 10 μmol/L forskolin did not significantly influence Cl^-/HCO3^- exchange on the apical and basolateral membranes. The administration of 250 μmol/L H2-DIDS significantly inhibited the basolateral AE. Amiloride （300 μmol/L） completely inhibited NHEs on both membranes of the cells. RT-PCR revealed the expression of pNBC1, AE2, and NHE1 mRNA. CONCLUSION： These data suggest that apart from the lack of CFTR and apical Cl^-/HCO3^- exchanger activity, CFPAC-1 cells express similar H^＋ and HCO3^- transporters to those observed in native animal tissue.

Haplotype-resolved genome of diploid ginger (Zingiber officinale) and its unique gingerol biosynthetic pathway

Ginger(Zingiber officinale),the type species of Zingiberaceae,is one of the most widespread medicinal plants and spices.Here,we report a high-quality,chromosome-scale reference genome of ginger‘Zhugen’,a traditionally cultivated ginger in Southwest China used as a fresh vegetable,assembled from PacBio long reads,Illumina short reads,and high-throughput chromosome conformation capture(Hi-C)reads.The ginger genome was phased into two haplotypes,haplotype 1(1.53 Gb with a contig N50 of 4.68 M)and haplotype 0(1.51 Gb with a contig N50 of 5.28 M).Homologous ginger chromosomes maintained excellent gene pair collinearity.In 17,226 pairs of allelic genes,11.9%exhibited differential expression between alleles.Based on the results of ginger genome sequencing,transcriptome analysis,and metabolomic analysis,we proposed a backbone biosynthetic pathway of gingerol analogs,which consists of 12 enzymatic gene families,PAL,C4H,4CL,CST,C3’H,C3OMT,CCOMT,CSE,PKS,AOR,DHN,and DHT.These analyses also identified the likely transcription factor networks that regulate the synthesis of gingerol analogs.Overall,this study serves as an excellent resource for further research on ginger biology and breeding,lays a foundation for a better understanding of ginger evolution,and presents an intact biosynthetic pathway for species-specific gingerol biosynthesis.

The Anatomic Pathology Evaluation of Liver with Diethylinitrosamine Treated via Intraperitoneal Injection Singly and Peros for 90 Days Carcinogenicity Study in F344 Rats

Objective：To establish the integrity experiment method of short（ medium）- term carcinogenicity test pursuant to GLP, make into relative SOP and improve the safeguard in the center. Methods：Diethylinitrosamine（DEN） is known as carcinogenic agent, whose target organ is liver. Using the two-stage carcinogenesis test method, DEN was treated to F344 rats via intraperitoneal injection singly（200 mg/kg） , and peros administrated for 90 days（10 ppm）. The liver in any group rat will be examined by light microscopy. Results：In pathologic examination, no liver cell tumor was shown in the livers of the rats that were singly treated with a carcinogenic chemical-DEN. Foci of cellular alteration were observed in the livers of these rats. The proliferation lesions of liver from slight to seveity （foci of cellular aherationepatocelluar adenoma-hepatocellular carcinoma） were observed in the livers of the rats which exposed peros to a low dose of DEN for 90 days after initiation by a single intraperitoneal injection. The incidence of hepatocelluar tumor was 35% in male animal ,which was not shown in the liver of female rat. Conclusion：For current results, it may be possible that low-dose DEN acts as a promotor of hepatocelluar tumor if it was exposed in a population for a long time. It is considered that male hormone has a synergistic effect on hepatocelluar tumor development of DEN. This two-stage carcinogenesis test might be a new model for the study of drug induced and promoted carcinogenesis, which could be used to evaluate the carcinogenesis of chemical compound fast.

Anti-inflammatory and Anti-oxidative Effects of the Ethanol Extract of Cryptocarya densiflora Blume in Lipopolysaccharide-Stimulated RAW264,7 Mouse Macrophages

CD （Cryptocarya densiflora） Blume has traditionally been used as an herbal medicine. In this study, the effects of CDEE （CD ethanol extract） on inflammation were investigated in LPS （lipopolysaccharide）-stimulated mouse RAW264.7 macrophages. We investigated the effects of CDEE on the production of NO, PGE2 interleukin （IL）-6, IL-1β, and tumor necrosis factor （TNF）-α in LPS-stimulated RAW264.7 cells. We measured the mRNA or protein expression of the pro-inflammatory mediators induced by CDEE in LPS-stimulated RAW264.7 cells. We explored the expression of Nrf-2, heme oxygenase （HO）-I and NADPH-quinone oxidoreductase （NQO）-I to elucidate the antioxidative mechanisms. CDEE significantly inhibited the production of NO, PGE2, IL-6, IL-1β and TNF-α in LPS-stimulated RAW264.7 cells. CDEE suppressed the mRNA or protein expression of iNOS, COX-2, and the MAPKs with a reduction in the translocation of NF-κB in LPS-stimulated RAW264.7 cells. In addition, CDEE significantly increased the expression of HO-I and NQO-1 with an increase in the translocation of Nrf-2 into the nucleus. These results indicate that CDEE inhibits the LPS-induced inflammatory and oxidative responses via suppression of NF-κB activation and the enhancement of Nrf2 activation. We suggest that CDEE may be therapeutic for treating inflammatory diseases.

Taxonomic revision of genus Rohanixalus(Anura:Rhacophoridae)in China with description of one new species

DEAR EDITOR,Based upon morphological and molecular evidence,the authors revised the genus Rohanixalus Biju,Garg,Gokulakrishnan,Chandrakasan,Thammachoti,Ren,Gopika,Bisht,Hamidy and Shouche,2020(Anura:Rhacophoridae)in China through describing one new species,adding one species to the fauna(R.shyamrupus)and supplementing data on one species(Rohanixalus hansenae;Supplementary Materials).

Photonic control of ligand nanospacing in self-assembly regulates stem cell fate

Extracellular matrix(ECM)undergoes dynamic inflation that dynamically changes ligand nanospacing but has not been explored.Here we utilize ECM-mimicking photocontrolled supramolecular ligand-tunable Azo^(+)self-assembly composed of azobenzene derivatives(Azo^(+))stacked via cation-πinteractions and stabilized with RGD ligand-bearing poly(acrylic acid).Near-infrared-upconverted-ultraviolet light induces cis-Azo^(+)-mediated inflation that suppresses cation-πinteractions,thereby inflating liganded self-assembly.This inflation increases nanospacing of“closely nanospaced”ligands from 1.8 nm to 2.6 nm and the surface area of liganded selfassembly that facilitate stem cell adhesion,mechanosensing,and differentiation both in vitro and in vivo,including the release of loaded molecules by destabilizing water bridges and hydrogen bonds between the Azo^(+)molecules and loaded molecules.Conversely,visible light induces trans-Azo^(+)formation that facilitates cation-πinteractions,thereby deflating self-assembly with“closely nanospaced”ligands that inhibits stem cell adhesion,mechanosensing,and differentiation.In stark contrast,when ligand nanospacing increases from 8.7 nm to 12.2 nm via the inflation of self-assembly,the surface area of“distantly nanospaced”ligands increases,thereby suppressing stem cell adhesion,mechanosensing,and differentiation.Long-term in vivo stability of self-assembly via real-time tracking and upconversion are verified.This tuning of ligand nanospacing can unravel dynamic ligand-cell interactions for stem cell-regulated tissue regeneration.

MicroRNA let-7 targets BmCDK1 to regulate cell proliferation and endomitosis of silk gland in the silkworm,Bombyx mori

MicroRNAs play critical roles in multiple developmental processes in insects.Our previous study showed that CRISPR/Cas9-mediated knock down of the microRNA let-7 in silkworms increased the size of larvae and silk glands,thereby improving the silk production capacity.In this study,we elucidate the molecular mechanism underlying of let-7 regulates growth.Identification of differentially expressed genes in response to let-7 knock down revealed enrichment of pathways associated with cell proliferation and DNA replication.let-7 dysregulation affected the cell cycle and proliferation of the Bombyx mori cell line BmN.Dual-luciferase and target site mutation assays showed that BmCDK1 is a direct target gene of let-7,with only 1 binding site on its 3′-untranslated region.RNA interference of BmCDK1 inhibited cell proliferation,but this effect was counteracted by co-transfection with let-7 antagomir.Moreover,let-7 knock down induced BmCDK1 expression and promoted cell proliferation in multiple tissues,and further induced endomitosis in the silk gland in vivo.Knock down of BmCDK1 resulted in abnormal formation of a new epidermis,and larval development was arrested at the 2nd or 3rd molt stage.Taken together,our results demonstrated that BmCDK1 is a novel target of let-7 in cell fate determination,possessing potential for improving silk yield in silkworm.