An efficient strategy for generation of transgenic mice by lentiviral transduction of male germline stem cells in vivo

Background： Male germline stem cells（MGSCs） are a subpopulation of germ cells in the testis tissue. MGSCs are capable of differentiation into spermatozoa and thus are perfect targets for genomic manipulation to generate transgenic animals.Method： The present study was to optimize a protocol of production of transgenic mice through transduction of MGSCs in vivo using lentiviral-based vectors. The recombinant lentiviral vectors with either EF-1 or CMV promoter to drive the expression of enhanced green fluorescent protein（e GFP） transgene were injected into seminiferous tubules or inter-tubular space of 7-day-old and 28-day-old mouse testes. At 5 or 6 wk post-surgery, these pre-founders were mated with wild-type C57BL/6J female mice（1.5 to 2.0-month-old）.Results： Sixty-seven percent of F1 generation and 55.56 % of F2 offspring were positive for eG FP transgene under the control of EF-1 promoter via PCR analysis. The transgenic pups were generated in an injection site-and age-independent manner. The expression of transgene was displayed in the progeny derived from lentiviral vector containing CMV promoter to drive transgene, but it was silenced or undetectable in the offspring derived from lentiviral vector with transgene under EF-1 promoter. The methylation level of g DNA in the promoter region of transgene was much higher in the samples derived lentiviral vectors with EF-1 promoter than that with CMV promoter,suggesting e GFP transgene was suppressed by DNA methylation in vivo.Conclusion： This research reported here an effective strategy for generation of transgenic mice through transduction of MGSCs in vivo using lentivirus vectors with specific promoters, and the transgenic offspring were obtained in an injection site-and age-independent manner. This protocol could be applied to other animal species, leading to advancement of animal transgenesis in agricultural and biomedical fields.

Deletion of heterogeneous nuclear ribonucleoprotein K in satellite cells leads to inhibited skeletal muscle regeneration in mice

Heterogeneous nuclear ribonucleoprotein K(hnRNPK)is a predominantly nuclear RNA-binding protein that can bind to DNA or RNA through three KH domains and interact with multiple proteins by interactive region.These binding activities enable hnRNPK to link the function in a wide array of diverse cellular processes,such as chromatin remodeling,gene transcription,RNA metabolism,protein translation,DNA repair,and cell signal transduction,thereby playing crucial roles in many biological processes,including development,axonal regeneration,spermatogenesis,cell cycle,apoptosis,differentiation,and carcinogenesis.

Mitochondrial aldehyde dehydrogenase rescues against diabetic cardiomyopathy through GSK3β-mediated preservation of mitochondrial integrity and Parkin-mediated mitophagy

Mitochondrial aldehyde dehydrogenase(ALDH2)offers proven cardiovascular benefit,although its impact on diabetes remains elusive.This study examined the effects of ALDH2 overexpression and knockout on diabetic cardiomyopathy and the mechanism involved with a focus on mitochondrial integrity.Mice challenged with streptozotocin(STZ,200 mg/kg,via intraperitoneal injection)exhibited pathological alterations,including reduced respiratory exchange ratio,dampened fractional shortening and ejection fraction,increased left ventricular end-systolic and diastolic diameters,cardiac remodeling,cardiomyocyte contractile anomalies,intracellular Ca2+defects,myocardial ultrastructural injury,oxidative stress,apoptosis,and mitochondrial damage,which were overtly attenuated or accentuated by ALDH2 overexpression or knockout,respectively.Diabetic patients also exhibited reduced plasma ALDH2 activity,cardiac remodeling,and diastolic dysfunction.In addition,STZ challenge altered expression levels of mitochondrial proteins(PGC-1αand UCP2)and Ca2+regulatory proteins(SERCA,Na+–Ca2+exchanger,and phospholamban),dampened autophagy and mitophagy(LC3B ratio,TOM20,Parkin,FUNDC1,and BNIP3),disrupted phosphorylation of Akt,GSK3β,and Foxo3a,and elevated PTEN phosphorylation,most of which were reversed or worsened by ALDH2 overexpression or knockout,respectively.Furthermore,the novel ALDH2 activator torezolid,as well as the classical ALDH2 activator Alda-1,protected against STZ-or high glucose-induced in vivo or in vitro cardiac anomalies,which was nullified by inhibition of Akt,GSK3β,Parkin,or mitochondrial coupling.Our data discerned a vital role for ALDH2 in diabetic cardiomyopathy possibly through regulation of Akt and GSK3βactivation,Parkin mitophagy,and mitochondrial function.

TBC1D15 deficiency protects against doxorubicin cardiotoxicity via inhibiting DNAPKcs cytosolic retention and DNA damage

Clinical application of doxorubicin(DOX)is heavily hindered by DOX cardiotoxicity.Several theories were postulated for DOX cardiotoxicity including DNA damage and DNA damage response(DDR),although the mechanism(s)involved remains to be elucidated.This study evaluated the potential role of TBC domain family member 15(TBC1D15)in DOX cardiotoxicity.Tamoxifen-induced cardiac-specific Tbcldi5 knockout(Tbcldi5^(CKO))or Tbcldi5 knockin(Tbcldi5^(CKI))male mice were challenged with a single dose of DOx prior to cardiac assessment 1 week or 4 weeks following DOX challenge.Adenoviruses encoding TBC1D15 or containing shRNA targeting Tbcld15 were used for Tbcld15 overexpression or knockdown in isolated primary mouse cardiomyocytes.Our results re-vealed that DOX evoked upregulation of TBC1D15 with compromised myocardial function and overt mortality,the effects of which were ameliorated and accentuated by Tbcldi5 deletion and Tbcld15 overexpression,respectively.DOX overtly evoked apoptotic cell death,the effect of which was alleviated and exacerbated by Tbcld15 knockout and overexpression,respectively.Meanwhile,DOX provoked mitochondrial membrane potential collapse,oxidative stress and DNA damage,the effects of which were mitigated and exacerbated by Tbcld15 knockdown and overexpression,respectively.Further scrutiny revealed that TBC1D15 fostered cytosolic accumulation of the cardinal DDR element DNA-dependent protein kinase catalytic subunit(DNA-PKcs).Liquid chromatography-tandem mass spectrometry and coimmunoprecipitation denoted an interaction between TBCID15 and DNA-PKcs at the segment 594-624 of TBC1D15.Moreover,overexpression of TBC1D15 mutant(A594-624,deletion of segment 594-624)failed to elicit accentuation of DOX-induced cytosolic retention of DNA-PKcs,DNA damage and cardiomyocyte apoptosis by TBC1D15 wild type.However,Tbcld15 deletion ameliorated DOXinduced cardiomyocyte contractile anomalies,apoptosis,mitochondrial anomalies,DNA damage and cytosolic DNA-PKcs accumulation,which were canceled off by DNA-PKcs inhibition or ATM activation.Taken together,our findings denoted a pivotal role for TBCID15 in DOX-induced DNA damage,mitochondrial injury,and apoptosis possibly through binding with DNA-PKcs and thus gate-keeping its cytosolic retention,a route to accentuation of cardiac contractile dysfunction in DOX-induced cardiotoxicity.

Author correction to‘Ablation of Akt2 and AMPKα2 rescues high fat diet-induced obesity and hepatic steatosis through Parkin-mediated mitophagy’[Acta Pharmaceutica Sinica B 11(2021)3508-3526]

The authors of the above-mentioned paper noticed an error in Fig.5a as reported.H&E staining was shown in wrong color during figure processing and DKO-LF group was accidently copied and pasted twice.Original H&E images have been replaced with the correct color with the accurate grouping information.No text content in figure legend or conclusion is affected by this error.The authors sincerely apologize for any inconvenience caused to the journal and readers.

镉胁迫下地钱转录组的性别特异性响应机制

地钱(Marchantiapolymorpha)作为雌雄异株的苔纲植物,具有对重金属胁迫敏感的特性,且种群中雌雄比例差异较大,是研究重金属环境下植物性二态的理想对象之一。为探究雌雄地钱转录组对重金属镉的响应机制差异,利用高通量测序和加权基因共表达网络分析(WGCNA),鉴定了在镉胁迫条件下与不同性别地钱有关的模块和基因。结果表明,镉胁迫响应转录因子雌雄差异主要集中于bHLH、ERF和MYB家族。关键差异响应基因包括MARPO_0147s0038、MARPO_1326s0001和MARPO_0015s0058。差异响应通路雌性主要集中在信号转导通路,雄性则集中于类黄酮生物合成。研究结果有助于揭示雌雄异株地钱对镉胁迫的反应过程和响应机制,可为理解雌雄异株植物对重金属胁迫的性别特异性响应机制提供参考。

Ablation of Akt2 and AMPKα2 rescues high fat diet-induced obesity and hepatic steatosis through Parkin-mediated mitophagy

Given the opposing effects of Akt and AMP-activated protein kinase(AMPK)on metabolic homeostasis,this study examined the effects of deletion of Akt2 and AMPKα2 on fat diet-induced hepatic steatosis.Akt2–Ampkα2 double knockout(DKO)mice were placed on high fat diet for 5 months.Glucose metabolism,energy homeostasis,cardiac function,lipid accumulation,and hepatic steatosis were examined.DKO mice were lean without anthropometric defects.High fat intake led to adiposity and decreased respiratory exchange ratio(RER)in wild-type(WT)mice,which were ablated in DKO but not Akt2^(-/-) and Ampkα2^(-/-) mice.High fat intake increased blood and hepatic triglycerides and cholesterol,promoted hepatic steatosis and injury in WT mice.These effects were eliminated in DKO but not Akt2^(-/-) and Ampkα2^(-/-) mice.Fat diet promoted fat accumulation,and enlarged adipocyte size,the effect was negated in DKO mice.Fat intake elevated fatty acid synthase(FAS),carbohydrate-responsive element-binding protein(CHREBP),sterol regulatory element-binding protein 1(SREBP1),peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGC-1α),peroxisome proliferator-activated receptor-α(PPARα),PPARγ,stearoyl-CoA desaturase 1(SCD-1),phosphoenolpyruvate carboxykinase(PEPCK),glucose 6-phosphatase(G6Pase),and diglyceride O-acyltransferase 1(DGAT1),the effect was absent in DKO but not Akt2^(-/-) and Ampkα2^(-/-) mice.Fat diet dampened mitophagy,promoted inflammation and phosphorylation of forkhead box protein O1(FoxO1)and AMPKα1(Ser^(485)),the effects were eradicated by DKO.Deletion of Parkin effectively nullified DKO-induced metabolic benefits against high fat intake.Liver samples from obese humans displayed lowered microtubule-associated proteins 1A/1B light chain 3B(LC3B),Pink1,Parkin,as well as enhanced phosphorylation of Akt,AMPK(Ser^(485)),and FoxO1,which were consolidated by RNA sequencing(RNAseq)and mass spectrometry analyses from rodent and human livers.These data suggest that concurrent deletion of Akt2 and AMPKα2 offers resilience to fat diet-induced obesity and hepatic steatosis,possibly through preservation of Parkin-mediated mitophagy and lipid metabolism.

TAX1BP1 protects against myocardial infarction-associated cardiac anomalies through inhibition of inflammasomes in a RNF34/MAVS/NLRP3-dependent manner

Acute myocardial infarction(MI),one of the most common cardiovascular emergencies,is a leading cause of morbidity and mortality.Ample evidence has revealed an essential role for inflammasome activation and autophagy in the pathogenesis of acute MI.Tax1-binding protein 1(TAX1BP1),an adaptor molecule involved in termination of proinflammatory signaling,serves as an important selective autophagy adaptor,but its role in cardiac ischemia remains elusive.This study examined the role of TAX1BP1 in myocardial ischemic stress and the underlying mechanisms involved.Levels of TAX1BP1 were significantly downregulated in heart tissues of patients with ischemic heart disease and in a left anterior descending(LAD)ligation-induced model of acute MI.Adenovirus carrying TAX1BP1 was delivered into the myocardium.The acute MI induced procedure elicited an infarct and cardiac dysfunction,the effect of which was mitigated by TAX1BP1 overexpression with little effect from viral vector alone.TAX1BP1 nullified acute MI-induced activation of the NLRP3 inflammasome and associated mitochondrial dysfunction.TAX1BP1 overexpression suppressed NLRP3 mitochondrial localization by inhibiting the interaction of NLRP3 with mitochondrial antiviral signaling protein(MAVS).Further investigation revealed that ring finger protein 34(RNF34)was recruited to interact with TAX1BP1 thereby facilitating autophagic degradation of MAVS through K27-linked polyubiquitination of MAVS.Knockdown of RNF34 using siRNA nullified TAX1BP1 yielded protection against hypoxia-induced MAVS mitochondrial accumulation,NLRP3 inflammasome activation and associated loss of mitochondrial membrane potential.Taken together,our results favor a cardioprotective role for TAX1BP1 in acute MI through repression of inflammasome activation in a RNF34/MAVS-dependent manner.

Enhancement of precursor amino acid supplies for improving bacitracin production by activation of branched chain amino acid transporter BrnQ and deletion of its regulator gene lrp in Bacillus licheniformis

Bacitracin,a new type of cyclic peptide antibiotic,is widely used as the feed additive in feed industry.Branched chain amino acids(BCAAs)are the key precursors for bacitracin synthesis.In this research,soybean meal was served as the raw material to supply precursor amino acids for bacitracin synthesis,and enhanced production of bacitracin was attempted by engineering BCAA transporter BrnQ and its regulator Lrp in the bacitracin industrial production strain Bacillus licheniformis DW2.Firstly,our results confirmed that Lrp negatively affected bacitracin synthesis in DW2,and deletion of lrp improved intracellular BCAA accumulations,as well as the expression level of BCAA transporter BrnQ,which further led to a 14.71%increase of bacitracin yield,compared with that of DW2.On the contrary,overexpression of Lrp decreased bacitracin yield by 12.28%.Secondly,it was suggested that BrnQ acted as a BCAA importer in DW2,and overexpression of BrnQ enhanced the intracellular BCAA accumulations and 10.43%of bacitracin yield.While,the bacitracin yield decreased by 18.27%in the brnQ deletion strain DW2△brnQ.Finally,BrnQ was further overexpressed in lrp deletion strain DW2△lrp,and bacitracin yield produced by the final strain DW2△lrp::BrnQ was 965.34 U/mL,increased by 22.42%compared with that of DW2(788.48 U/mL).Collectively,this research confirmed that Lrp affected bacitracin synthesis via regulating the expression of BCAA transporter BrnQ and BCAA distributions,and provided a promising strain for industrial production of bacitracin.

Primary malignant tumours and malignant transformation of cysts in the retrorectal space:MRI diagnosis and treatment outcomes

Background:There are no clear guidelines for the diagnosis and treatment of malignant retrorectal tumours.The purpose of this study was to increase preoperative diagnostic knowledge and to describe the outcomes of treatment for these patients.Methods:This retrospective study was conducted on patients who underwent complete retrorectal tumour resection between May 2006 and July 2018,and had confirmed post-operative pathology reports.Demographic and clinical data(including imaging,perioperative,pathological,and prognostic data)were collected and analysed.Results:Malignant lesions were identified in 15(9[60%],female)patients.The median age of the patients was 59 years(range,34–72 years).Primary malignant tumours were identified in seven patients with solid tumours,in which gastrointestinal stromal tumours accounted for 71.4%(five of seven)and the remainder were chordoma or mucinous adenocarcinoma.Malignant transformation of cysts occurred in another eight patients with heterogeneous tumours,while histopathological features were present in 75%(six of eight)of patients with mucinous adenocarcinoma,and the remainder were squamouscell carcinoma or neuroendocrine tumour(Grade 2).The malignant characteristics of the solid portions observed using magnetic resonance imaging(MRI)were as follows:the cyst wall of the tumour was irregularly thickened;the surface was convex or lobed;the solid tumour had no capsule,or the capsule was destroyed;and the surface had a gyrus-like morphology.At a median follow-up time of 52 months(range,13–100 months),the overall recurrence-free survival rate was 40.0%and the survival rate was 46.7%.Conclusion:Some MRI features can be used to distinguish malignant retrorectal tumours from benign retrorectal tumours.The survival rate of patients with malignant retrorectal tumours is poor.