Analysis of the electron transfer pathway in small laccase by EPR and UV-vis spectroscopy coupled with redox titration

Bacterial small laccases(SLAC) are promising industrial biocatalysts due to their ability to oxidize a broad range of substrates with exceptional thermostability and tolerance for alkaline p H. Electron transfer between substrate, copper centers, and O2is one of the key steps in the catalytic turnover of SLAC. However, limited research has been conducted on the electron transfer pathway of SLAC and SLAC-catalyzed reactions, hindering further engineering of SLAC to produce tunable biocatalysts for novel applications. Herein, the combinational use of electron paramagnetic resonance(EPR) and ultraviolet-visible(UV-vis) spectroscopic methods coupled with redox titration were employed to monitor the electron transfer processes and obtain further insights into the electron transfer pathway in SLAC. The reduction potentials for type 1 copper(T1Cu), type 2 copper(T2Cu) and type 3copper(T3Cu) were determined to be 367 ± 2 mV, 378 ± 5 m V and 403 ± 2 mV,respectively. Moreover, the reduction potential of a selected substrate of SLAC, hydroquinone(HQ), was determined to be 288 mV using cyclic voltammetry(CV). In this way, an electron transfer pathway was identified based on the reduction potentials. Specifically,electrons are transferred from HQ to T1Cu, then to T2Cu and T3Cu, and finally to O2.Furthermore, superhyperfine splitting observed via EPR during redox titration indicated a modification in the covalency of T2Cu upon electron uptake, suggesting a conformational alteration in the protein environment surrounding the copper sites, which could potentially influence the reduction potential of the copper sites during catalytic processes. The results presented here not only provide a comprehensive method for analyzing the electron transfer pathway in metalloenzymes through reduction potential measurements, but also offer valuable insights for further engineering and directed evolution studies of SLAC in the aim for biotechnological and industrial applications.

Origin of Traditional Chinese Medicine in Cuba in the 19th Century from Its Main Exponents and Some Notable Medical Descendants in the 20th Century

The Cuban people are made up of three major migratory currents, the Chinese are one of them. They brought their culture, the methods, and procedures of traditional Chinese medicine(TCM) in the 19th century. Few were able to return and so they created families in Cuba;some of their descendants dedicated themselves to medicine. In order to investigate the practices that were predecessors of TCM in Cuba in the 19th century, a qualitative phenomenological research was carried out, reviewing what was published by various sources, applying documentary analysis, logical historical analysis, abstraction, synthesis, and systematization of the results on the regularities of the work and human behavior of Chinese doctors in the Cuban 19th and 20th centuries. This made it possible to identify six Chinese doctors in the 19th century in Cuba who gave rise to the beginning of some practices of TCM in Cuba, and five from the 20th century, descendants of coolies who dedicated themselves to other specialties of medicine. It was found that despite their geographical and time disperse, they were all notorious for their outstanding professional and human behavior, with a trail of accumulated successes in achieving “almost the impossible” with the patient. They have left their mark on Cuban culture.

Identification of an LDLR variant in a Chinese familial hypercholesterolemia and its relation to ROS/NLRP3-Mediated pyroptosis in hepatic cells

BACKGROUND Familial hypercholesterolemia(FH)is a common autosomal dominant hereditary disease.Its early diagnosis and intervention significantly improve the patient’s quality of life.However,there are few types of research on the FH pathogenic genes in China.METHODS In this study,we recruited a family diagnosed with FH and used whole exome sequencing(WES)to analyze the proband variants.Intracellular cholesterol level,reactive oxygen species(ROS)level,and the expression of pyroptosis-related genes were detected after overexpression of wild-type or variant LDLR in L02 cells.RESULTS A heterozygous missense variant predicted to be deleterious to LDLR(c.1879G>A,p.Ala627Thr)was identified in the proband.Mechanistically,intracellular cholesterol level,ROS level,and the expression of pyroptosis-related genes,nucleotidebinding oligomerization domain-like receptor family protein 3(NLRP3)inflammasome and components(caspase 1,apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC)and NLRP3),gasdermin D(GSDMD),interleukin(IL)-18,IL-1βwas elevated in the variant LDLR group,which was attenuated by inhibition of ROS.CONCLUSIONS FH is associated with a variant(c.1879G>A,p.Ala627Thr)in the LDLR gene.Regarding the mechanism,the ROS/NLRP3-mediated pyroptosis in hepatic cells may contribute to the pathogenesis of the LDLR variant.

Evaluation of novel decellularizing corneal stroma for cornea tissue engineering applications

AIM:To develop a new decellularization method depended upon the natural corneal structure and to harvest an ideal scaffold with good biocompatibilities for corneal reconstruction.METHODS:The acellular cornea matrix (ACM) were prepared from de-epithelium fresh porcine corneas (DFPCs) by incubation with 100% fresh human sera and additional electrophoresis at 4℃. Human corneal epithelial cells (HCEs) were used for the cytotoxicity tests of ACM. ACM were implanted into the Enhanced Green Fluorecence Protein (eGFP) transgenic mouse anterior chamber for evaluation of histocompatibility.RESULTS:HE and GSIB4 results showed fresh porcine cornea matrix with 100% human sera and electrophoresis could entirely decellularize stromal cell without reducing its transparency. ACM has no cytotoxic effect ex vivo. Animal test showed there was no rejection for one month after surgery.CONCLUSION:These results provide a decellularizing approach for the study of corneal tissue engineering and had the broader implications for the field of biological tissue engineering in other engineered organ or tissue matrix.

Human fetal mesenchymal stem cells differentiate into brown and white adipocytes： a role for ERRα in human UCP1 expression

We investigated the ability of fetal mesenchymal stem cells （fMSCs） to differentiate into brown and white adi- pocytes and compared the expression of a number of marker genes and key regulatory factors. We showed that the expression of key adipocyte regulators and markers during differentiation is similar to that in other human and mu- rine adipocyte models, including induction of PPARy2 and FABP4. Notably, we found that the preadipocyte marker, Pref-1, is induced early in differentiation and then declines markedly as the process continues, suggesting that fMSCs first acquire preadipocyte characteristics as they commit to the adipogenic lineage, prior to their differentiation into mature adipocytes. After adipogenic induction, some stem cell isolates differentiated into cells resembling brown adi- pocytes and others into white adipocytes. Detailed investigation of one isolate showed that the novel brown fat-deter- mining factor PRDM16 is expressed both before and after differentiation. Importantly, these cells exhibited elevated basal UCP-1 expression, which was dependent on the activity of the orphan nuclear receptor ERRa, highlighting a novel role for ERRa in human brown fat. Thus fMSCs represent a useful in vitro model for human adipogenesis, and provide opportunities to study the stages prior to commitment to the adipocyte lineage. They also offer invaluable in- sights into the characteristics of human brown fat.

Blockage of PPARδ increases the expression of inflammatory factors in 3T3-L1 cells stimulated with TNFα

Objective： To investigate the role of peroxisome proliferator-activated receptors δ （PPARδ） in inflammatory reaction and its possible mechanism in adipocyte. Methods：Lentivirus-mediated RNA interference （RNAi） was used to block the expression of PPARδ in 3T3-L1 cells. In order to induce inflammation in 3T3-L1, cells were stimulated with tumor necrosis factor-α（TNFα, 20 ng/ml） for 4 h. The expression of PPARδ, nuclear factor κB （NFκB） and C reactive protein （CRP） were determined by Western blot analysis. Results：The expression of PPARδ was reduced by 80% after RNAi. Blockage of PPARδ promoted the expression of CRP and NFκB in cells stimulated with TNFα but had no effect on normal cells. Conclusion： PPARδ is involved in inflammatory reaction in adipocyte. Blockage of PPARδ can promote the inflammation mediated by inflammatory factors and increase the expression of NFκB and CRP in 3T3-L1 cells stimulated with TNFα.

Unilateral neuropathic arthropathy of the shoulder secondary to syringomyelia: Diagnostic challenges

Neuropathic arthropathy of the shoulder is a rare disorder characterized by joint degeneration, and is associated with loss of sensory innervation. Syringomyelia is a disease in which fluid-containing cavities(syrinxes) form within the spinal cord. Here, we report a case of neuropathic arthropathy of the shoulder secondary to syringomyelia in a 40-year-old woman. X-rays of the left shoulder revealed damage to bone and joint architecture. Blood tests indicated vitamin D deficiency and secondary hyperparathyroidism. Magnetic resonance imaging of the cervical spine showed a large syrinx from the second cervical spine to the second dorsal spine. Although neuropathic arthropathy is uncommon, it should be considered in cases of unexplained pain, discomfort, or limited range of motion of the affected joint. Symptoms related to the affected joint may precede or overshadow neurological deficits. Appropriate radiological examinations and diagnoses are imperative to prevent misdiagnosis or undetected bone and joint disorders.

Critical review of bone health,fracture risk and management of bone fragility in diabetes mellitus

The risk of fracture is increased in both type 1 diabetes mellitus(T1DM)and type 2 diabetes mellitus(T2DM).However,in contrast to the former,patients with T2DM usually possess higher bone mineral density.Thus,there is a considerable difference in the pathophysiological basis of poor bone health between the two types of diabetes.Impaired bone strength due to poor bone microarchitecture and low bone turnover along with increased risk of fall are among the major factors behind elevated fracture risk.Moreover,some antidiabetic medications further enhance the fragility of the bone.On the other hand,antiosteoporosis medications can affect the glucose homeostasis in these patients.It is also difficult to predict the fracture risk in these patients because conventional tools such as bone mineral density and Fracture Risk Assessment Tool score assessment can underestimate the risk.Evidence-based recommendations for risk evaluation and management of poor bone health in diabetes are sparse in the literature.With the advancement in imaging technology,newer modalities are available to evaluate the bone quality and risk assessment in patients with diabetes.The purpose of this review is to explore the patho-physiology behind poor bone health in diabetic patients.Approach to the fracture risk evaluation in both T1DM and T2DM as well as the pragmatic use and efficacy of the available treatment options have been discussed in depth.

Regulated upon activation,normal T cell expressed and secreted(RANTES)levels in the peripheral blood of patients with Alzheimer’s disease

Alzheimer’s disease(AD)is the most common type of dementia,but it is very difficult to diagnose with certainty,so many AD studies have attempted to find early and relevant diagnostic markers.Regulated upon activation,normal T cell expressed and secreted(RANTES,also known as C-C chemokine ligand)is a chemokine involved in the migration of T cells and other lymphoid cells.Changes in RANTES levels and its expression in blood or in cerebrospinal fluid have been reported in some neurodegenerative diseases,such as Parkinson’s disease and multiple sclerosis,but also in metabolic diseases in which inflammation plays a role.The aim of this observational study was to assess RANTES levels in peripheral blood as clinical indicators of AD.Plasma levels of RANTES were investigated in 85 AD patients in a relatively early phase of AD(median 8.5 months after diagnosis;39 men and 46 women;average age 75.7 years),and in 78 control subjects(24 men and 54 women;average age 66 years).We found much higher plasma levels of RANTES in AD patients compared to controls.A negative correlation of RANTES levels with age,disease duration,Fazekas scale score,and the medial temporal lobe atrophy(MTA)score(Scheltens’s scale)was found in AD patients,i.e.,the higher levels corresponded to earlier stages of the disease.Plasma RANTES levels were not correlated with cognitive scores.In AD patients,RANTES levels were positively correlated with the levels of pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-α,which is consistent with the wellknown fact that AD is associated with inflammatory processes.RANTES levels were also positively correlated with insulin levels in AD patients,with insulin resistance(HOMA-R)and pancreatic beta cell function(HOMA-F).This study evaluated several clinical and metabolic factors that may affect plasma levels of RANTES,but these factors could not explain the increases in RANTES levels observed in AD patients.Plasma levels of RANTES appear to be an interesting peripheral marker for early stages of AD.The study was approved by the Ethics Committee of Institute of Endocrinology,Prague,Czech Republic on July 22,2011.

A panel of monoclonal antibodies against the prion protein proves that there is no prion protein in human pancreatic ductal epithelial cells

Prion diseases are a group of neurodegenerative diseases that are fatal. The study of these unique diseases in China is hampered by a lack of resources. Amongst the most important resources for biological study are monoclonal antibodies. Here, we characterize a panel of monoclonal antibodies specific for cellular prion protein by enzyme-linked immunosorbent assay(ELISA), immunofluorescent staining, flow cytometry, and western blotting. We identify several antibodies that can be used for specific applications and we demonstrate that there is no prion protein expression in human pancreatic ductal epithelial cells(HPDC).

Effects of Rhodiola rosea supplementation on mental performance,physical capacity,and oxidative stress biomarkers in healthy men

Purpose： The objective of this study was to investigate the effects of chronic Rhodiola rosea （R. rosea） supplementation on mental and physical performance, as well as hormonal and oxidative stress biomarkers. Methods： Twenty-six healthy male students received either R. rosea extract （600 mg/day; RR） or placebo （PL） in a randomized double-blind trial. Prior to supplementation （Term I） and following 4 weeks of supplementation （Term II）, the students underwent psychomotor tests for simple and choice reaction time, included in the Vienna Test System. Also, the subjects performed VO2peak test. Blood samples were obtained before and after the test to measure the hormonal profile （cortisol, testosterone, and growth hormone）, as well as the biomarkers of oxidative stress （lipid hydroperoxides, total antioxidant capacity, and superoxide dismutase） and muscle damage （creatine kinase）. Results： R. rosea ingestion shortened reaction time and total response time. Moreover, a greater relative increase in the number of correct responses was observed in RR group as compared to the PL group. No changes in endurance exercise capacity and hormonal profile were observed after R. rosea ingestion. R. rosea ingestion raised plasma total antioxidant capacity. It did not, however, affect other measured parameters. Conclusion： Chronic R. rosea ingestion does not affect physical performance, but can improve the results of some psychomotor tests （simple and choice reaction time） in young, healthy, and physically active men. The improvements in mental performance, however, at least in our study, seem not to be related to changes in cortisol release or antioxidant activity of R. rosea extract. Thus, the specific mechanisms responsible for these effects still need to be elucidated.2018 Published by Elsevier B.V. on behalf of Shanghai University of Sport. This is an open access article under the CC BY-NC-ND license. （http：//creativecommons.org/licenses/by-nc-nd/4.0/）.

Familial primary pigmented nodular adrenocortical disease without Carney complex (CNC): A case report and review of literature

Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of familial Cushing's syndrome. It is characterized by bilateral adrenocortical hyperplasia with small to normal-sized adrenal glands containing multiple small adrenal cortical pigmented nodules [1,2]. PPNAD may occur in an isolated form or as familial PPNAD. Familial cases of PPNAD are usually associated with Carney complex in which Cushing’s syndrome is the most common endocrine manifestation [3]. Familial cases of PPNAD without associated Carney complex are very rare. Only a few cases of familial isolated PPNAD have been reported in the literature, mostly in females [4]. Isolated familial PPNAD has got a better prognosis than familial PPNAD associated with Carney Complex. This observation has important consequences for clinical management, follow-up and genetic counselling of such patients. Familial cases of PPNAD are rare and mostly present in females with associated Carney complex. We herein report a case of familial Cushing’s syndrome in male siblings due to PPNAD without associated Carney complex.

Flutamide-induced alterations in transcriptional profiling of neonatal porcine ovaries

Background: Androgens are involved in the regulation of ovarian development during fetal/neonatal life.Environmental chemicals displaying anti-androgenic activities may affect multiple signal transduction pathways by blocking endogenous androgen action.The aim of the current study was to examine effects of the anti-androgen flutamide on the expression of coding transcripts and long non-coding RNAs(lncRNAs) in neonatal porcine ovaries.By employing RNA-Seq technology we aimed to extend our understanding of the role of androgens in neonatal folliculogenesis and examine the impact of the anti-androgen flutamide on ovarian function.Method: Piglets were subcutaneously injected with flutamide(50 mg/kg BW) or corn oil(controls) between postnatal days 1 and 10(n = 3/group).Ovaries were excised from the 11-day-old piglets and total cellular RNAs were isolated and sequenced.Results: Flutamide-treated piglet ovaries showed 280 differentially expressed genes(DEGs;P-adjusted < 0.05 and log2 fold change ≥1.0) and 98 differentially expressed lncRNAs(DELs;P-adjusted < 0.05 and log2 FC ≥ 1.0).The DEGs were assigned to GO term,covering biological processes,molecular functions and cellular components,which linked the DEGs to functions associated with cellular transport,cell divisions and cytoskeleton.In addition,STRING software demonstrated strongest interactions between genes related to cell proliferation.Correlations between DEGs and DELs were also found,revealing that a majority of the genes targeted by the flutamide-affected lncRNAs were associated with intracellular transport and cell division.Conclusions: Our results suggest that neonatal exposure of pigs to flutamide alters the expression of genes involved in ovarian cell proliferation,ovarian steroidogenesis and oocyte fertilization,which in turn may affect female reproduction in adult life.

两个家族中的慢性皮肤黏膜念珠菌病和原发性甲状腺功能减退

笔者对2例患有慢性皮肤粘膜念珠菌病(CMC)和原发性甲状腺功能减退的2个家庭的临床和免疫学特征进行了描述。A家庭有3名兄妹同时患有念珠菌病和甲低,还有4名成员仅有甲低。B家庭有4名成员患有念珠菌病,其中1例男孩还患有甲低。所有CMC患者均从婴儿期即患有口腔念珠菌病和甲癣。面部溢脂性皮炎、全身性毛囊炎及剥脱性眼睑炎是其主要表现。于幼儿期甲低变得明显。在A家庭患病的兄妹身上没有发现甲状腺抗体,但B家庭那名患有甲低的男孩,在诊断时即查出有抗甲状腺过氧化物酶的抗体。免疫学检查显示血清免疫球蛋白浓度、淋巴细胞分类和增殖反应上存在个体差异,但并未观察到相同的异常。

同时患家族性甲状腺髓样癌及先天性巨结肠的一个罕见的捷克家族

Purpose: The RET proto-oncogene is involved in neural crest disorders. Activa ting germline mutations in the RET protooncogene cause the development of famili al medullary thyroid carcinoma (FMTC) or medullary thyroid carcinoma (MTC) as a part of multiple endocrine neoplasia type 2 (MEN2) syndrome. Inactivating germli nemutations in the RET proto-oncogene are detected in Hirschsprung’s disease ( HSCR). Only in a very small number of families are these 2 diseases expressed to gether. Methods: This study presents a novel Czech kindred with FMTC-HSCR pheno type. Two family members (mother and daughter) were tested for RET germline muta tions in exons 10, 11, 13, 14, 15, and 16. Results: Direct fluorescent sequencin g of genomic DNA revealed a heterozygous mutation in the RET proto-oncogene in exon 10 at codon C609Y in both persons tested. This family was reclassified, tha nks to genetic screening from the apparently sporadic MTC-HSCR to FMTC-HSCR. C onclusion: The germline mutation was detected because of the systematic genetic screening of the RET proto-oncogene, which is useful for genetic counseling of potential risk of HSCR and MTC in other family members. This family could be add ed to the small worldwide cohort of families with MEN2A/FMTC-HSCR.

Two Different Forms of Gonadotropin-releasing Hormone in Amphioxus

Since a GnRH was isolated from mammalian hypothalamus and purified in 1971 and 1972, severalvariants have been identified in various forms of lower vertebrates. However, the presence of GnRHin amphioxus is still an open question. Chang et al. (1984) observed the presence of immunopositivegranules for GnRH in Hatschek's pit of amphioxus. In this paper, HPLC was used for the isolationand purification of GnRH-like peptide from amphioxus tissues, while radioimmunoassay was appliedto determine the immunoreactivity of the peptide. Based on the immunological and chromatographiccharacteristics, two kinds of GnRH (mGnRH and sGchH) were identified in amphioxus and theseGnRH-like peptide were found to be present in the 'head', 'middle' and 'tail' regions ofamphioxus.

Effect of Gossypol on Bovine Blastocyst Attachment and Trophoblastic outgrowth in Vitro

The presont study was carried out to investigate the effect of different amounts of gossypol on bovineblastocyst attachment and trophoblastic outgrowth in vitro. Bovine oocyte were collected from theovaries of slaughtered cows and were matured and fertilized in vitro. Cleaved oocyte were culturedin CRlaa + BOEC and TC-199 + 10% FCS combined in an 1:1 ratio. After 8 days of co-culture,the hatched blastocysts were randomly allotted to different treatment groups. All were cultured ona fetal fibroblast monolaycr (prepared from bovine fetuses) in TC-199 culture medium supplementedwith 10% fetal calf serum (TCFCS). But the groups differed from one another in the dose ofgossypol given: 0.01 μg, 0.1 μg, 1 μg, 10 μs/ml, and no gossypol as control. All cultures wereperformed in 24-well culture plates at 39℃ with 5% CO_2 in air. The results indicate that the ratesof attached and outgrowing blastocysts in the medium containing 1 μg/ml gossypol were significantlylowcr than the control group (p<0.01) and outgrowth were inhibited by gossypol in a dose-dependent manner.

Large-scale manufacturing of functional single-atom ink for convenient glucose sensing

Printing techniques hold great potential in the manufacture of electronics such as sensors,micro-supercapacitors,and flexible electronics.However,developing large-scale functional conductive inks with appropriate rheological properties and active components still remains a challenge.Herein,through optimizing the formulations of ink,iron single sites supported N-doped carbon black(Fe_(1)-NC)inks can serve as both conductive electrodes and high-reactive catalysts to realize convenient glucose detection,which pronouncedly reduces the dosage of enzyme and simplifies the sensors preparation.In detail,utilizing in-situ pyrolysis method,Fe_(1)-NC single-atom catalysts(SACs)are prepared in bulk(dekagram-level).The batched Fe_(1)-NC SACs materials can be uniformly mixed with modulated ink to realize the screen printing with high resolution and uniformity.Also,the whole scalable preparation and ink-functional process can be extended to various metals(including Co,Ni,Cu,and Mn).The introduction of highly active Fe_(1)-NC sites reduces the amount of enzyme used in glucose detection by at least 50%,contributing to the cost reduction of sensors.The strategy in harnessing the SACs onto the carbon inks thus provides a broad prospect for the low-cost and large-scale printing of sensitive sensing devices.

肝细胞NLRP3与PKCε互作驱动肝脏胰岛素抵抗和脂肪变性

肝脏胰岛素抵抗(IR)是非酒精性脂肪性肝病(NAFLD)的重要特征之一,与肝脏脂肪变性密切相关.肝脏IR发病机制仍不清楚,本研究通过转录组学筛选鉴定到肝细胞NLRP3是肝脏IR的重要诱导因子,进一步证实其在人和小鼠NAFLD模型肝脏组织中表达增加.功能方面,肝细胞特异性NLRP3功能丧失或获得分别改善或加重高脂饮食(HFD)诱导的肝脏IR及脂肪变性.机制方面,NLRP3直接结合并促进蛋白激酶C蛋白(PKCs)的激活,从而损害胰岛素信号通路和增加肝脏脂肪变性;相反,抑制PKCe明显阻断HFD诱导的NLRP3缺陷小鼠的有益作用.此外,研究筛选发现转录因子阴阳1(YY1)调控NLRP3启动子.治疗方面,腺相关病毒血清型8介导的肝脏NLRP3敲低减轻瘦素受体缺陷(db/db)小鼠的肝脏IR和脂肪变性.同时,药理抑制NLRP3显著减轻饮食诱导的代谢紊乱.综上,本研究揭示了肝细胞NLRP3是肝脏IR和脂肪变性的直接驱动因素,并提示YY1-NLRP3-PKCε轴为NAFLD的潜在治疗靶点.

ASGR1:an emerging therapeutic target in hypercholesterolemia

In a recent study published in Nature,Wang et al.1 discovered that inhibition of asialoglycoprotein receptor 1(ASGR1)increased cholesterol efflux and thus lowered blood cholesterol and reduced atherosclerosis.This study offers an emerging new therapeutic target in hypercholesterolemia and its comorbidities and complications(such as fatty liver and atherosclerosis),which are major threats to public health.