Erratum to “Optical Detection of Distal Lung Enzyme Activity in Human Inflammatory Lung Disease”

In the research article“Optical Detection of Distal Lung Enzyme Activity in Human Inflammatory Lung Disease”[1],the data availability statement was inadvertently omitted by the publisher.This has now been corrected in the PDF and HTML(full text).

香烟烟雾提取物对鼠成肌细胞分化的影响的研究

目的:为了探讨COPD患者周围骨骼肌萎缩的可能的分子机制,用香烟烟雾的提取物(CSE)对鼠成肌细胞系(C2C12)进行了体外实验研究。方法:把C2C12细胞按150个/ml的浓度接种在12孔的平底塑料盘里,分为对照组和实验组,24小时后,在实验组的细胞中加入不同浓度(1%,2.5%,3.5%,5%)的CSE.我们把加入CSE的当天记录为第0天(day 0),第二天记录为第1天(day1),依次类推。CSE作用24小时后,用温的杜氏磷酸缓冲液(PBS)洗涤两次后换为10%马血清细胞分化生长液使细胞分化,而对照组更换新鲜的细胞生长液即可。并观察细胞的变化,并在加入CSE后的第0、3、6天,分别检测各组的肌酸激酶(CK)活性,并用激光扫描共焦荧光显微镜技术,观察各组的肌球蛋白重链(MHC)的生成情况。结果:重复相同实验4次后,对各组数值进行单因素方差分析的统计学处理,结果发现1%,2.5%组与对照组比较无显著差异(P>0.05);3.5%实验组与对照组比较day3有显著差异(P<0.05),day6无显著差异(P>0.05);5%实验组与对照组比较,day3,day6均有显著差异(P<0.05);并且激光扫描共焦荧光显徽镜技术观察对照组和实验组(3.5%)的MHC的照片显示CSE对MHC的形成有影响。结论:体外研究显示,CSE对C2C12的分化有影响。

Heme oxygenase system in hepatic ischemia-reperfusion injury

Hepatic ischemia-reperfusion injury (IRI) limits access to transplantation. Heme oxygenase-1 (HO-1) is a powerful antioxidant enzyme which degrades free heme into biliverdin,free iron and carbon monoxide. HO-1 and its metabolites have the ability to modulate a wide variety of inflammatory disorders including hepatic IRI. Mechanisms of this protective effect include reduction of oxygen free radicals,alteration of macrophage and T cell phenotype. Further work is required to understand the physiological importance of the many actions of HO-1 identified experimentally,and to harness the protective effect of HO-1 for therapeutic potential.

肌萎缩性侧索硬化蛋白激活小胶质细胞NLRP3炎性小体

小胶质细胞NLRP3炎性小体激活正在成为神经退行性变过程中神经炎症的关键因素。诸如β-淀粉样蛋白和α-突触核蛋白之类的致病性蛋白质聚集体触发小胶质NLRP3激活,从而导致半胱天冬酶-1激活和IL-1β的分泌。在小鼠肌萎缩性侧索硬化症(ALS)的SOD1G93A模型中,半胱天冬酶-1和IL-1β均促进疾病进展,提示小胶质NLRP3在该进程中发挥作用。然而先前的研究表明,SOD1G93A小鼠小胶质细胞不表达NLRP3,SOD1G93A蛋白在小胶质细胞中产生独立于NLRP3的IL-1β。本研究论证了使用Nlrp3-GFP基因敲入小鼠,在SOD1G93A小鼠中小胶质细胞表达NLRP3。本研究显示聚集和可溶性SOD1G93A均可激活小鼠原代小胶质细胞中的炎性小体,导致半胱天冬酶-1和IL-1β裂解,ASC斑点形成以及呈剂量和时间依赖性的IL-1β分泌。重要的是,SOD1G93A无法从缺乏Nlrp3的小胶质细胞或者用特异性NLRP3抑制剂MCC950预处理的小胶质细胞中诱导IL-1β分泌,从而证实NLRP3是介导SOD1诱导的小胶质细胞IL-1β分泌的关键炎症小体复合物。在TDP-43Q331K ALS小鼠模型中也观察到小胶质NLRP3上调,TDP-43野生型和突变蛋白亦可以NLRP3依赖性的方式激活小胶质炎性小体。从机制上讲,本研究确定了活性氧簇和ATP的生成是SOD1G93A介导的NLRP3激活所需的关键事件。总之,本研究的数据表明ALS小胶质细胞表达NLRP3,而病理ALS蛋白激活小胶质NLRP3炎性小体。因此,NLRP3抑制可能是阻止小胶质细胞神经炎症和ALS疾病进展的潜在治疗方法。

Quantitative multiparametric magnetic resonance imaging can aid non-alcoholic steatohepatitis diagnosis in a Japanese cohort

BACKGROUND Non-invasive assessment of non-alcoholic steatohepatitis(NASH)is increasing in desirability due to the invasive nature and costs associated with the current form of assessment;liver biopsy.Quantitative multiparametric magnetic resonance imaging(mpMRI)to measure liver fat(proton density fat fraction)and fibroinflammatory disease[iron-corrected T1(cT1)],as well as elastography techniques[vibration-controlled transient elastography(VCTE)liver stiffness measure],magnetic resonance elastography(MRE)and 2D Shear-Wave elastography(SWE)to measure stiffness and fat(controlled attenuated parameter,CAP)are emerging alternatives which could be utilised as safe surrogates to liver biopsy.AIM To evaluate the agreement of non-invasive imaging modalities with liver biopsy,and their subsequent diagnostic accuracy for identifying NASH patients.METHODS From January 2019 to February 2020,Japanese patients suspected of NASH were recruited onto a prospective,observational study and were screened using noninvasive imaging techniques;mpMRI with LiverMultiScan®,VCTE,MRE and 2DSWE.Patients were subsequently biopsied,and samples were scored by three independent pathologists.The diagnostic performances of the non-invasive imaging modalities were assessed using area under receiver operating characteristic curve(AUC)with the median of the histology scores as the gold standard diagnoses.Concordance between all three independent pathologists was further explored using Krippendorff’s alpha(a)from weighted kappa statistics.RESULTS N=145 patients with mean age of 60(SD:13 years.),39%females,and 40%with body mass index≥30 kg/m2 were included in the analysis.For identifying patients with NASH,MR liver fat and cT1 were the strongest performing individual measures(AUC:0.80 and 0.75 respectively),and the mpMRI metrics combined(cT1 and MR liver fat)were the overall best non-invasive test(AUC:0.83).For identifying fibrosis≥1,MRE performed best(AUC:0.97),compared to VCTE-liver stiffness measure(AUC:0.94)and 2D-SWE(AUC:0.94).For assessment of steatosis≥1,MR liver fat was the best performing non-invasive test(AUC:0.92),compared to controlled attenuated parameter(AUC:0.75).Assessment of the agreement between pathologists showed that concordance was best for steatosis(a=0.58),moderate for ballooning(a=0.40)and fibrosis(a=0.40),and worst for lobular inflammation(a=0.11).CONCLUSION Quantitative mpMRI is an effective alternative to liver biopsy for diagnosing NASH and non-alcoholic fatty liver,and thus may offer clinical utility in patient management.

c-MET immunohistochemical expression in sporadic and inflammatory bowel disease associated lesions

BACKGROUND Post-colonoscopy colorectal cancer(CRC)rates for patients with inflammatory bowel disease(IBD)are unacceptably high.During colonoscopy,an intravenous fluorescent anti-c-MET probe may improve endoscopic detection of lesions.However,c-MET expression in IBD lesions is poorly defined,limiting translational studies.AIM To comprehensively define c-MET expression in sporadic and IBD-associated colorectal carcinogenesis.METHODS c-MET expression was immunohistochemically assessed in 319 formalin-fixed paraffin-embedded tissue specimens,colonoscopically or surgically retrieved between 1994-2017.Tissue included:30 normal colorectal biopsies,30 hyperplastic polyps(HP),31 sessile serrated lesions(SSL),55 tubular/tubulovillous adenomas with low(TA-LGD,n=32)or high grade dysplasia(TA-HGD,n=23),26 sporadic(s)-CRCs,16 quiescent IBD biopsies,11 active/inflamed IBD biopsies,18 IBDassociated dysplastic lesions(IBD-dys),and 102 IBD-CRCs.Expression was scored by two independent observers as:0=absent,1=weak,2=moderate or 3=strong.Mann-Whitney U and Kruskal-Wallis tests were used to assess significance.RESULTS Positive epithelial cytoplasmic and membranous c-MET expression was observed in all tissues,indicating there is ubiquitous expression in the colorectum.c-MET expression was weak in normal colonic epithelium compared with each of the sporadic colonic lesions,including TA-LGD(P<0.001),TA-HGD(P=0.004),HP(P<0.001),SSL(P<0.001),and s-CRC(P<0.001).Specifically,in sporadic(non-IBD)lesions,expression was stronger in TA-LGD compared with normal mucosa(P<0.001),and stronger in s-CRC compared with TA-HGD(P=0.004).However,there was no significant difference between TA-LGD and TA-HGD(P=0.852).Further,there was no difference in c-MET expression between HP and SSL(P=0.065).In IBD,expression was weaker in quiescent colonic mucosa compared with inflamed colonic mucosa(P<0.001).There was no difference between inflamed colonic mucosa and IBD-dys(P=0.512)or IBD-CRC(P=0.296).However,expression was stronger in IBD-dys(P<0.001)and IBD-CRC(P<0.001)compared with quiescent IBD colonic mucosa.CONCLUSION The characterisation of c-MET expression suggest that an intravenous probe may improve the endoscopic detection of lesions in both non-IBD patients and IBD patients with quiescent disease.

小胶质细胞缺乏加速朊病毒病但不增强朊病毒在大脑中的积累

朊病毒病是与朊病毒蛋白错误折叠相关的传染性神经退行性疾病。先前的研究表明,减少小胶质细胞会加速中枢神经系统(CNS)朊病毒病并增加朊病毒在大脑中的积累,这表明小胶质细胞通过吞噬和破坏朊病毒以提供神经保护作用。在Csf1rΔFIRE小鼠中,删除Csf1r中的增强子会特异性地阻止小胶质细胞的发育。然而该小鼠大脑发育正常,并且未显示出如其他小胶质细胞缺陷模型中报道的缺陷。用Csf1rΔFIRE小鼠作为研究小胶质细胞缺乏对中枢神经系统朊病病的影响的改良模型。尽管Csf1rΔFIRE小鼠比野生型小鼠更早地死于CNS朊病毒病,但它们大脑中朊病毒的积累却减少了。而星形胶质细胞则表现出更早的非极化反应性激活,增强了对神经元内容物的吞噬作用和非折叠蛋白质反应。我们的数据表明,小胶质细胞在中枢神经系统朊病毒病中的作用并非简单地吞噬和破坏朊病毒,而是提供宿主保护并限制反应性星形胶质细胞的有害活动。

肌萎缩侧索硬化蛋白激活小胶质细胞NLRP3炎性体

小胶质细胞NLRP3炎症小体激活正在成为神经退行性变期间神经炎症的关键因素。β-淀粉样蛋白和α-突触核蛋白等致病蛋白的聚集体可触发小胶质细胞NLRP3激活,并导致半胱氨酸天冬氨酸酶激活和白介素-1β分泌。半胱氨酸天冬氨酸酶和白介素-1β均促进肌萎缩侧索硬化(ALS)小鼠SOD1G93A模型的疾病进展,这提示小胶质细胞NLRP3在该进程中起作用。然而,先前的研究表明SOD1G93A模型小鼠的小胶质细胞并不表达NLRP3,并且SOD1G93A蛋白在小胶质细胞中产生白介素-1β不依赖于NLRP3。本研究展示了使用Nlrp3-GFP基因敲入小鼠,在SOD1G93A小鼠中小胶质细胞表达NLRP3。结果表明,聚集和可溶性SOD1G93A均以剂量和时间依赖性方式激活小鼠原代小胶质细胞中的炎性体,导致半胱氨酸天冬氨酸酶和白介素-1β裂解、ASC斑点形成和白介素-1β分泌。重要的是,SOD1G93A不能诱导缺乏Nlrp3的小胶质细胞或用特异性NLRP3抑制剂MCC950预处理的小胶质细胞分泌白介素-1β,这证实NLRP3是介导SOD1诱导的小胶质细胞白介素-1β分泌的关键炎性体复合物。在TDP-43Q331K ALS小鼠模型中也观察到小胶质细胞NLRP3上调,并且TDP-43野生型和突变蛋白也能以NLRP3依赖性方式激活小胶质细胞炎症小体。从机制上,本研究确定活性氧簇和ATP的产生是SOD1G93A介导的NLRP3激活所需的关键事件。总之,本研究的数据表明ALS小胶质细胞表达NLRP3,病理性ALS蛋白激活了小胶质细胞NLRP3炎性体。因此,抑制NLRP3可能是阻止小胶质细胞神经炎症和ALS疾病进展的潜在治疗方法。

Optical Detection of Distal Lung Enzyme Activity in Human Inflammatory Lung Disease

Objective and Impact Statement.There is a need to develop platforms delineating inflammatory biology of the distal human lung.We describe a platform technology approach to detect in situ enzyme activity and observe drug inhibition in the distal human lung using a combination of matrix metalloproteinase(MMP)optical reporters,fibered confocal fluorescence microscopy(FCFM),and a bespoke delivery device.Introduction.The development of new therapeutic agents is hindered by the lack of in vivo in situ experimental methodologies that can rapidly evaluate the biological activity or drug-target engagement in patients.Methods.We optimised a novel highly quenched optical molecular reporter of enzyme activity(FIB One)and developed a translational pathway for in-human assessment.Results.We demonstrate the specificity for matrix metalloproteases(MMPs)2,9,and 13 and probe dequenching within physiological levels of MMPs and feasibility of imaging within whole lung models in preclinical settings.Subsequently,in a first-in-human exploratory experimental medicine study of patients with fibroproliferative lung disease,we demonstrate,through FCFM,the MMP activity in the alveolar space measured through FIB One fluorescence increase(with pharmacological inhibition).Conclusion.This translational in situ approach enables a new methodology to demonstrate active drug target effects of the distal lung and consequently may inform therapeutic drug development pathways.

Targeting PKM2 signaling cascade with salvianic acid A normalizes tumor blood vessels to facilitate chemotherapeutic drug delivery

Aberrant tumor blood vessels are prone to propel the malignant progression of tumors,and targeting abnormal metabolism of tumor endothelial cells emerges as a promising option to achieve vascular normalization and antagonize tumor progression.Herein,we demonstrated that salvianic acid A(SAA)played a pivotal role in contributing to vascular normalization in the tumor-bearing mice,thereby improving delivery and effectiveness of the chemotherapeutic agent.SAA was capable of inhibiting glycolysis and strengthening endothelial junctions in the human umbilical vein endothelial cells(HUVECs)exposed to hypoxia.Mechanistically,SAA was inclined to directly bind to the glycolytic enzyme PKM2,leading to a dramatic decrease in endothelial glycolysis.More importantly,SAA improved the endothelial integrity via activating theβ-Catenin/Claudin-5 signaling axis in a PKM2-dependent manner.Our findings suggest that SAA may serve as a potent agent for inducing tumor vascular normalization.

An inducible model for specific neutrophil depletion by diphtheria toxin in mice

Neutrophils are crucial for immunity and play important roles in inflammatory diseases;however,mouse models selectively deficient in neutrophils are limited,and neutrophil-specific diphtheria toxin(DT)-based depletion system has not yet been established.In this study,we generated a novel knock-in mouse model expressing diphtheria toxin receptor(DTR)under control of the endogenous Ly6G promoter.We showed that DTR expression was restricted to Ly6G+neutrophils and complete depletion of neutrophils could be achieved by DT treatment at 24-48 h intervals.We characterized the effects of specific neutrophil depletion in mice at steady-state,with acute inflammation and during tumor growth.Our study presents a valuable new tool to study the roles of neutrophils in the immune system and during tumor progression.

Repeated photoporation with graphene quantum dots enables homogeneous labeling of live cells with extrinsic markers for fluorescence microscopy

In the replacement of genetic probes,there is increasing interest in labeling living cells with high-quality extrinsic labels,which avoid over-expression artifacts and are available in a wide spectral range.This calls for a broadly applicable technology that can deliver such labels unambiguously to the cytosol of living cells.Here,we demonstrate that nanoparticle-sensitized photoporation can be used to this end as an emerging intracellular delivery technique.We replace the traditionally used gold nanoparticles with graphene nanoparticles as photothermal sensitizers to permeabilize the cell membrane upon laser irradiation.We demonstrate that the enhanced thermal stability of graphene quantum dots allows the formation of multiple vapor nanobubbles upon irradiation with short laser pulses,allowing the delivery of a variety of extrinsic cell labels efficiently and homogeneously into live cells.We demonstrate high-quality time-lapse imaging with confocal,total internal reflection fluorescence(TIRF),and Airyscan superresolution microscopy.As the entire procedure is readily compatible with fluorescence(super resolution)microscopy,photoporation with graphene quantum dots has the potential to become the long-awaited generic platform for controlled intracellular delivery of fluorescent labels for live-cell imaging.

Immunology of a unique biological structure:the Echinococcus laminated layer

The larval stages of the cestode parasites belonging to the genus Echinococcus grow within internal organs of humans and a range of animal species.The resulting diseases,collectively termed echinococcoses,include major neglected tropical diseases of humans and livestock.Echinococcus larvae are outwardly protected by the laminated layer(LL),an acellular structure that is unique to this genus.The LL is based on a fibrillar meshwork made up of mucins,which are decorated by galactose-rich O-glycans.In addition,in the species cluster termed E.granulosus sensu lato,the LL features nano-deposits of the calcium salt of myo-inositol hexakisphosphate(Insp_(6)).The main purpose of our article is to update the immunobiology of the LL.Major recent advances in this area are(i)the demonstration of LL“debris”at the infection site and draining lymph nodes,(ii)the characterization of the decoy activity of calcium Inp6 with respect to complement,(iii)the evidence that the LL mucin carbohydrates interact specifically with a lectin receptor expressed in Kupffer cells(Clec4F),and(iv)the characterization of what appear to be receptor-independent effects of LL particles on dendritic cells and macrophages.Much information is missing on the immunology of this intriguing structure:we discuss gaps in knowledge and propose possible avenues for research.

Sterile signals generate weaker and delayed macrophage NLRP3 inflammasome responses relative to microbial signals

Inflammation is the host response to microbial infection or sterile injury that aims to eliminate the insult, repair the tissue and restore homeostasis. Macrophages and the NLRP3 inflammasome are key sentinels for both types of insult. Although it is well established that the NLRP3 inflammasome is activated by microbial products and molecules released during sterile injury, it is unclear whether the responses elicited by these different types of signals are distinct. In this study, we used lipopolysaccharide and tumor necrosis factor as prototypical microbial and sterile signal 1 stimuli, respectively, to prime the NLRP3 inflammasome. We then used the bacterial toxin nigericin and a common product released from necrotic cells, ATP, as prototypical microbial and sterile signal 2 stimuli, respectively, to trigger the assembly of the NLRP3 inflammasome complex in mouse and human macrophages. We found that NLRP3 inflammasome responses were weakest when both signal 1 and signal 2 were sterile, but responses were faster and stronger when at least one of the two signals was microbial. Ultimately, the most rapid and potent responses were elicited when both signals were microbial. Together, these data suggest that microbial versus sterile signals are distinct, both kinetically and in magnitude, in their ability to generate inflammasome-dependent responses. This hierarchy of NLRP3 responses to sterile versus microbial stimuli likely reflects the urgent need for the immune system to respond rapidly to the presence of infection to halt pathogen dissemination.

Near-infrared benzodiazoles as small molecule environmentally-sensitive fluorophores

The development of fluorophores emitting in the near-infrared spectral window has gained increased attention given their suitable features for biological imaging.In this work,we have optimised a general and straightforward synthetic approach to prepare a small library of near-infrared-emitting C-bridged nitrobenzodiazoles using commercial precursors.C-bridged benzodiazoles have low molecular weight and neutral character as important features that are not common in most nearinfrared dyes.We have investigated their fluorescence response in the presence of a wide array of 60 different biomolecules and identified compound 3i as a potential chemosensor to discriminate between Fe2+and Fe^(3+)ions in aqueous media.

Expression, secretion and functional characterization of three laccases in E. coli

Endocrine Disrupting Chemicals(EDCs)are a group of molecules that can influence hormonal balance,causing disturbance of the reproductive system and other health problems.Despite the efforts to eliminate EDC in the environment,all current approaches are inefficient and expensive.In previous research,studies revealed that laccase-producing microorganisms may be a potential candidate for EDC degradation,as laccases have been found to be able to degrade many kinds of EDCs effectively and steadily.Here,we created two recombinant laccases,each fused with secretion peptide,Novel Signal Peptide 4(NSP4),and expressed them in Escherichia coli(E.coli,BL21),together with one laccase without secretion peptide.We first optimized the culture condition of expressing these laccases.Then,we test the activity of the recombinant laccases of decolorizing of a synthetic dye,indigo carmine.Finally,we confirmed the secreted can degrade one of the EDCs,β-estradiol,showing the potential of using the laccase secretion system to degrade toxic compounds.

Using recombinant adhesive proteins as durable and green flame-retardant coatings

Current fire retardants are known to be toxic to humans and our environment.As environmental-friendly flame retardants(FRs),protein-based flame retardants have been studied extensively recently,even though they are not durable.In this study,we designed,synthesized and tested a durable protein-based FR through the fusion of the adhesion domain from either mussel foot protein-5(mfp-5)or cellulose-binding domain(CBD)with flame retardant protein(SR protein and alpha casein).We first verified the expression of the recombinant proteins in Escherichia coli using Western blot.Then,we coated the fusion protein(carrying cell lysates)to cotton fabrics and wood and verified with Infrared(IR)spectroscopy.Using a vertical burning test and wood flammability test,we confirmed the flame retardancy of the materials after the protein coating.In the vertical burning test,the SR protein and alpha casein flame retardant proteins with the CBD adhesion domain showed a 50.0%and 43.3%increase in flame retardancy.The data is also consistent in the wood flame retardancy test.Confocal imaging experiments also suggested these new fire retardants can be preserved on the materials well even after washing.Overall,our results showed that flame-retardant proteins with adhesion domains are high potential candidates of green alternative flame retardants.

敲除附睾β-防御素对小鼠精子表型的影响

β-防御素是抗菌肽大家族中的一员。尽管它们在体外能杀灭微牛物，与免疫细胞相互作用，但在体内的确切功能还不清楚。虽然它们经诱导后常在粘膜表面出现，主要表达部位是附睾。最新的研究证实这一多肽在精子的成熟过程中起到重要作用。我们也己证实9个β-防御素基因簇纯合缺失会导致雄性小鼠不育。这螳突变型小鼠的精子运动能力下降，且其脆性增加。与野生型小鼠相比，突变型小鼠附睾尾部的精子表现为过早的获能以及自发顶体反应的增强。除此之外，这些突变型精子与卵母细胞透明带的结合能力降低。附睾尾部的突变型精子出现微管结构破坏，而在头部的精子中未观察到这一现象。突变型精子的胞内钙离子浓度显著增加，这也与过早的顶体反应和运动增加相一致。本研究证实β-防御素对精于的成熟起到重要的作用。敲除β-防御素会增加细胞内钙离子水下，从而导致过早的获能及自发的顶体反应，以及鞭毛丝微管结构的破坏。确定这九个β-防御素基因中哪些与精子的表型和功能相关，对未来男性不育的研究有重要意义。