Title: SERS-ELISA determination of human carboxylesterase 1 using metal-organic framework doped with gold nanoparticles as SERS substrate Feng J, Lu H, Yang Y, Huang W, Cheng H, Kong H, Li L Ref: Mikrochim Acta, 188:280, 2021 : PubMed
By in situ synthesis of gold nanoparticles (AuNPs) within the acid-etched (AE) MIL-101 (Cr) framework, AE-MIL-101 (Cr) nanocomposites embedded with AuNPs (AuNP/AE-MIL-101 (Cr)) were prepared as surface-enhanced Raman scattering (SERS) substrate. AuNPs are uniformly distributed and stabilized inside the metal-organic framework (MOF), thus forming more SERS hotspots. The SERS performance of AuNP/AE-MIL-101 (Cr) was evaluated using 4-mercaptophenylboronic acid (4-MPBA), 4-mercaptobenzoic acid (4-MBA), benzidine, and rhodamine 6G (R6G). The SERS substrate displays satisfying stability with very low background signal. When benzidine is used as the Raman reporter, the limit of detection (LOD) can reach 6.7 x 10(-13) mol.L(-1), and the relative standard deviation (RSD) of the intra- and inter-batch repetitive tests is less than 5.2%. On this basis, we developed a method for the detection of human carboxylesterase 1 (hCE 1) in human serum using AuNP/AE-MIL-101 (Cr) nanocomposite as SERS substrate and enzyme-linked immunosorbent assay (ELISA) colorimetric substrate as SERS marker. This method was used to determine hCE 1 in clinical serum samples without complicated sample pretreatment, and the detection results were consistent with the data determined by ELISA. In the concentration range 0.1-120 ng.mL(-1), the SERS signal intensity of benzidine at 1609 cm(-1) gradually decreases with the increase of hCE 1 concentration (R(2) = 0.9948). The average recoveries of hCE 1 in human serum are in the range 84 to 108%, with RSDs lower than 7.7%. By using AuNP/acid etching-MIL-101(Cr) metal organic framework (MOF) as SERS substrate and enzyme-linked immunosorbent assay (ELISA) colorimetric substrate as the SERS marker, a rapid and sensitive method for the determination of human carboxylesterase 1 (hCE1) in human serum samples has been developed.
Title: Portable electrochemical biosensor based on laser-induced graphene and MnO(2) switch-bridged DNA signal amplification for sensitive detection of pesticide Liu X, Cheng H, Zhao Y, Wang Y, Li F Ref: Biosensors & Bioelectronics, 199:113906, 2021 : PubMed
Developing portable, quantitative, and user-friendly analytical tools for sensitive pesticide assay is of significant importance for guaranteeing food safety. Herein, a novel electrochemical biosensor was constructed by integrating laser-induced graphene (LIG) electrode on polyimide (PI) foil and MnO(2) nanosheets loaded on the paper for point-of-care test (POCT) of organophosphorus (OPs) residues. The principle of this biosensor relied on acetylcholinesterase (AChE)-catalyzed hydrolytic product-triggered disintegration of MnO(2) nanosheets for releasing assistant DNA to initiate nicking enzyme-aided recycling amplification. In the presence of OPs, the activity of AChE was inhibited and could not initiate the cleavage of the electroactive molecules-labeled hairpin probe on the electrode, resulting in the maintenance of the electrochemical response to realize a "sign-on" determination of OPs. The proposed biosensor exhibited satisfactory analytical performance for OPs assay with a linear range from 3 to 4000 ng/mL and a limit of detection down to 1.2 ng/mL. Moreover, the biosensor was useful for evaluating the residual level of pesticides in the vegetables. Therefore, this novel biosensor holds great promise for OPs assay and opens a new avenue on the development of higher-performance POCT device for sensing applications in the environment and food safety fields.
The gene encoding collagen like tail subunit of asymmetric acetylcholinesterase (COLQ) is responsible for the transcription of three strands of collagen of acetylcholinesterase, which is attached to the endplate of neuromuscular junctions. Mutations in the COLQ gene are inherited in an autosomal-recessive manner and can lead to type V congenital myasthenia syndrome (CMS), which manifests as decreased muscle strength at birth or shortly after birth, respiratory failure, restricted eye movements, drooping of eyelids, and difficulty swallowing. Here we reported three variants within COLQ in two unrelated children with CMS. An intronic variant (c.393+1G>A) and a novel missense variant (p.Q381P) were identified as compound heterozygous in a 13-month-old boy, with the parents being carriers of each. An intragenic deletion including exons 14 and 15 was found in a homozygous state in a 12-year-old boy. We studied the relative expression of the COLQ and AChE gene in the probands' families, performed three-dimensional protein structural analysis, and analyzed the conservation of the missense mutation c.1142A>C (p.Q381P). The splicing mutation c.393+1G>A was found to affect the normal splicing of COLQ exon 5, resulting in a 27-bp deletion. The missense mutation c.1142A>C (p.Q381P) was located in a conserved position in different species. We found that homozygous deletion of COLQ exons 14-15 resulted in a 241-bp deletion, which decreased the number of amino acids and caused a frameshift translation. COLQ expression was significantly lower in the probands than in the probands' parents and siblings, while AChE expression was significantly higher. Moreover, the mutations were found to cause significant differences in the predicted three-dimensional structure of the protein. The splicing mutation c.393+1G>A, missense mutation c.1A>C (p.Q381P), and COLQ exon 14-15 deletion could cause CMS.
Title: Identification of NDRG Family Member 4 (NDRG4) and CDC28 Protein Kinase Regulatory Subunit 2 (CKS2) as Key Prognostic Genes in Adrenocortical Carcinoma by Transcriptomic Analysis Yang Z, Cheng H, Zhang Y, Zhou Y Ref: Med Sci Monit, 27:e928523, 2021 : PubMed
BACKGROUND Adrenocortical carcinoma (ACC) is an aggressive cancer with heterogeneous outcomes. In this study, we aimed to investigate genomic and prognostic features of ACC. MATERIAL AND METHODS Clinical, pathologic, and transcriptomic data from 2 independent datasets derived from ACC samples (TCGA-ACC dataset, GEO-GSE76021 dataset) were collected. Weighted gene co-expression network analysis (WGCNA) and survival analyses were performed to identify prognostic genes. Pathway analysis was performed for mechanistic analysis. xCell deconvolution was performed for tumor microenvironment analysis. RESULTS In the TCGA-ACC cohort, WGCNA identified a prognostic module of 5408 genes. Differential expression analysis identified 1969 genes that differed in expression level between long-term and short-term survivors. Univariate Cox regression model analysis identified 8393 genes with prognostic value. The intersection of these gene sets included 820 prognostic genes. Similar protocols were performed for the GSE76021 dataset, and 5 candidate genes were identified. Further intersection of these genes finally identified NDRG4 and CKS2 as key prognostic genes. Multivariate Cox regression model analysis validated the prognostic value of NDRG4 (HR=0.61, 95% CI 0.46-0.80) and CKS2 (HR=2.52, 95% CI 1.38-4.60). Moreover, NDRG4 and CKS2 expression predicted survival in patients treated with mitotane (P<0.001). Further mechanism exploration found an association between CKS2 and DNA mismatch repair pathways. Moreover, NDRG4 positively correlated with CD8 T cell infiltration, while CKS2 negatively correlated with it. CONCLUSIONS We identified NDRG4 and CKS2 expression as key prognostic genes in ACC, which may help in risk stratification of ACC. Moreover, a close relationship was found between CKS2 and mismatch repair pathways. Moreover, immune cell infiltration differed according to NDRG4 and CKS2 expression.
Title: A magnetic SERS immunosensor for highly sensitive and selective detection of human carboxylesterase 1 in human serum samples Feng J, Xu Y, Huang W, Kong H, Li Y, Cheng H, Li L Ref: Anal Chim Acta, 1097:176, 2020 : PubMed
Hepatocellular carcinoma (HCC) is a common and lethal cancer. New serum markers for detecting HCC are urgently needed. Human carboxylesterase 1 (hCE1) is an important member of the serine hydrolase superfamily and is closely related to the occurrence of HCC. It can be used as a good serum marker for early diagnosis of HCC. Here, we developed a surface enhanced Raman scattering (SERS)- based magnetic immunosensor that specifically recognizes and detects trace amounts of hCE1 in human serum via a sandwich structure consisting of a SERS tags, magnetic supporting substrates, and target antigen (hCE1). The SERS tags are 4-mercaptobenzoic acid (4-MBA)-labeled AgNPs, and the SERS supporting substrates are composed of a raspberry-like morphology of Fe3O4@SiO2@AgNPs magnetic nanocomposites surface-functionalized with a hCE1 antibody. The prepared SERS magnetic immunosensor exhibits excellent selectivity and extremely high sensitivity for hCE1 detection. The SERS signal and logarithm of hCE1 concentration presented a wide linear response range of 0.1ngmL(-1) to 1.0mgmL(-1), and the detection limit of hCE1 was 0.1ngmL(-1). The results indicate that the immunosensor can be used for the rapid determination of hCE1 in human serum without a complicated sample pre-treatment. Furthermore, the immunosensor has good reproducibility and stability, and has a promising prospect for the quantitative detection of other tumor markers in early clinical diagnosis.
Title: Antioxidant and pancreatic lipase inhibitory effects of flavonoids from different citrus peel extracts: An in vitro study Huang R, Zhang Y, Shen S, Zhi Z, Cheng H, Chen S, Ye X Ref: Food Chem, 326:126785, 2020 : PubMed
Obesity and oxidative damage are two important risk factors associated closely with metabolic syndrome. Utilization of functional food ingredients is considered as a feasible way to tackle these challenges. In the present study, eight representative species of citrus peel extracts (CPEs) were evaluated and compared for their flavonoid profiles, antioxidant activities, and pancreatic lipase (PL) inhibitory capacities and mechanisms. Results indicated that hesperidin, naringin, neohesperidin, narirutin and eriocitrin were the five major flavonoids in CPEs, among which hesperidin was the main active PL inhibitor. Moreover, hesperidin could interact with PL by hydrogen bonds and van der Waals forces, and the interaction would not obviously change the secondary structure of PL. Overall, ponkan peel extract, having the strongest overall antioxidant activity, the highest content of hesperidin and total phenolic compounds among all tested CPEs, is a promising natural ingredient to scavenge free radicals and manage obesity.
The ability to directly measure acetylcholine (ACh) release is an essential step toward understanding its physiological function. Here we optimized the GRAB(ACh) (GPCR-activation-based ACh) sensor to achieve substantially improved sensitivity in ACh detection, as well as reduced downstream coupling to intracellular pathways. The improved version of the ACh sensor retains the subsecond response kinetics, physiologically relevant affinity and precise molecular specificity for ACh of its predecessor. Using this sensor, we revealed compartmental ACh signals in the olfactory center of transgenic flies in response to external stimuli including odor and body shock. Using fiber photometry recording and two-photon imaging, our ACh sensor also enabled sensitive detection of single-trial ACh dynamics in multiple brain regions in mice performing a variety of behaviors.
Cellular studies indicate that endocannabinoid type-1 retrograde signaling plays a major role in synaptic plasticity. Disruption of these processes by delta-9-tetrahydrocannabinol (THC) could produce alterations either in structural and functional brain connectivity or in their association in cannabis (CB) users. Graph theoretic structural and functional networks were generated with diffusion tensor imaging and resting-state functional imaging in 37 current CB users and 31 healthy non-users. The primary outcome measures were coupling between structural and functional connectivity, global network characteristics, association between the coupling and network properties, and measures of rich-club organization. Structural-functional (SC-FC) coupling was globally preserved showing a positive association in current CB users. However, the users had disrupted associations between SC-FC coupling and network topological characteristics, most perturbed for shorter connections implying region-specific disruption by CB use. Rich-club analysis revealed impaired SC-FC coupling in the hippocampus and caudate of users. This study provides evidence of the abnormal SC-FC association in CB users. The effect was predominant in shorter connections of the brain network, suggesting that the impact of CB use or predispositional factors may be most apparent in local interconnections. Notably, the hippocampus and caudate specifically showed aberrant structural and functional coupling. These structures have high CB1 receptor density and may also be associated with changes in learning and habit formation that occur with chronic cannabis use.
Alzheimer's disease (AD) is a multifaceted and progressive neurodegenerative disease characterized by accumulation of amyloid-beta (Abeta) and deficits of acetylcholine. Accordingly, the intra-/extra-cerebral level of high density lipoprotein (HDL) is crucial on the pathogenesis of AD; and most of all, various HDL-protein subtypes play a double-edged role in AD pathology, of which apolipoprotein A-I (apoA-I) gives protective outcomes. Inspired from "HDL bionics", we proposed biologically reassembled nanodrugs, donepezil-loaded apolipoprotein A-I-reconstituted HDL (rHDL/Do) that concurrently executed dual-missions of Abeta-targeting clearance and acetylcholinesterase (AChE) inhibition in AD therapy. Once prepared, rHDL/Do nanodrug achieved high drug encapsulation efficiency of 90.47%, and mimicked the configurations and properties of natural lipoproteins aiming to significantly enhance BBB penetration and modulate Abeta-induced neuronal damage both in vitro and in vivo. Surface plasmon resonance (SPR) analysis confirmed that rHDL/Do facilitated microglial-mediated Abeta intake and degradation, demonstrating low KD value with Abeta affinity (2.45x10(-8) of Abeta monomer and 2.78x10(-8) of Abeta oligomer). In AD animal models, daily treatment of rHDL/Do efficiently inhibited AChE activity, ameliorated neurologic variation, promoted Abeta clearance, and rescued memory loss at a safe level. The collective findings indicated that the biological nanodrug was provided with the capacities of BBB penetration, Abeta capture and degradation via microglial cells, and cholinergic dysfunction amelioration after controlled donepezil release. In summary, rHDL/Do nanodrugs could offer a promising strategy to synergize both symptom control and disease modification in AD therapy.
Perfluorododecanoic acid (PFDoA), an artificial perfluorochemical, has been widely distributed in different ambient media and has been reported to have the potential to cause developmental neurotoxicity. However, the specific mechanism is largely unknown. In the current study, zebrafish embryos were treated with 0, 0.24, 1.2, and 6mg/L PFDoA for 120h. Exposure to PFDoA causes serious decreases in hatching delay, body length, as well as decreased locomotor speed in zebrafish larvae. Additionally, the acetylcholine (ACh) content as well as acetylcholinesterase (AChE) activity were determined to be significantly downregulated in PFDoA treatment groups. The level of dopamine was upregulated significantly after treating with 1.2 and 6mg/L of PFDoA. Gene expressions related to the nervous system development were also analyzed, with the exception of the gene mesencephalic astrocyte-derived neurotrophic factor (manf), which is upregulated in the 6mg/L treatment group. All other genes were significantly downregulated in larvae in the PFDoA group in different degrees. In general, the results demonstrated that PFDoA exposure could result in the disruption of the cholinergic system, dopaminergic signaling, and the central nervous system.
Title: Two novel deep-sea sediment metagenome-derived esterases: residue 199 is the determinant of substrate specificity and preference Huo YY, Cheng H, Rong Z, Cui HL, Xu XW Ref: Microbial Cell Factories, 17:16, 2018 : PubMed
Background
The deep-sea environment harbors a vast pool of novel enzymes. Owing to the limitations of cultivation, cultivation-independent has become an effective method for mining novel enzymes from the environment. Based on a deep-sea sediment metagenomics library, lipolytic-positive clones were obtained by activity-based screening methods.
Results
Two novel esterases, DMWf18-543 and DMWf18-558, were obtained from a deep-sea metagenomic library through activity-based screening and high-throughput sequencing methods. These esterases shared 80.7% amino acid identity with each other and were determined to be new members of bacterial lipolytic enzyme family IV. The two enzymes showed the highest activities toward p-nitrophenyl (p-NP) butyrate at pH 7.0 and 35-40 C and were found to be resistant to some metal ions (Ba2+, Mg2+, and Sr2+) and detergents (Triton X-100, Tween 20, and Tween 80). DMWf18-543 and DMWf18-558 exhibited distinct substrate specificities and preferences. DMWf18-543 showed a catalytic range for substrates of C2-C8, whereas DMWf18-558 presented a wider range of C2-C14. Additionally, DMWf18-543 preferred p-NP butyrate, whereas DMWf18-558 preferred both p-NP butyrate and p-NP hexanoate. To investigate the mechanism underlying the phenotypic differences between the esterases, their three-dimensional structures were compared by using homology modeling. The results suggested that residue Leu199 of DMWf18-543 shortens and blocks the substrate-binding pocket. This hypothesis was confirmed by the finding that the DMWf18-558-A199L mutant showed a similar substrate specificity profile to that of DMWf18-543.
Conclusions
This study characterized two novel homologous esterases obtained from a deep-sea sediment metagenomic library. The structural modeling and mutagenesis analysis provided insight into the determinants of their substrate specificity and preference. The characterization and mechanistic analyses of these two novel enzymes should provide a basis for further exploration of their potential biotechnological applications.
Title: Parental transfer of microcystin-LR induced transgenerational effects of developmental neurotoxicity in zebrafish offspring Wu Q, Yan W, Cheng H, Liu C, Hung TC, Guo X, Li G Ref: Environ Pollut, 231:471, 2017 : PubMed
Microcystin-LR (MCLR) has been reported to cause developmental neurotoxicity in zebrafish, but there are few studies on the mechanisms of MCLR-induced transgenerational effects of developmental neurotoxicity. In this study, zebrafish were exposed to 0, 1, 5, and 25 mug/L MCLR for 60 days. The F1 zebrafish embryos from the above-mentioned parents were collected and incubated in clean water for 120 h for hatching. After examining the parental zebrafish and F1 embryos, MCLR was detected in the gonad of adults and F1 embryos, indicating MCLR could potentially be transferred from parents to offspring. The larvae also showed a serious hypoactivity. The contents of dopamine, dihydroxyphenylacetic acid (DOPAC), serotonin, gamma-aminobutyric acid (GABA) and acetylcholine (ACh) were further detected, but only the first three neurotransmitters showed significant reduction in the 5 and 25 mug/L MCLR parental exposure groups. In addition, the acetylcholinesterase (AChE) activity was remarkably decreased in MCLR parental exposure groups, while the expression levels of manf, bdnf, ache, htr1ab, htr1b, htr2a, htr1aa, htr5a, DAT, TH1 and TH2 genes coincided with the decreased content of neurotransmitters (dopamine, DOPAC and serotonin) and the activity of AChE. Neuronal development related genes, alpha1-tubulin, syn2a, mbp, gfap, elavl3, shha and gap43 were also measured, but gap43 was the gene only up-regulated. Our results demonstrated MCLR could be transferred to offspring, and subsequently induce developmental neurotoxicity in F1 zebrafish larvae by disturbing the neurotransmitter systems and neuronal development.
Recent efforts have attempted to convert non-blood cells into hematopoietic stem cells (HSCs) with the goal of generating blood lineages de novo. Here we show that hematopoietic transcription factors Scl, Lmo2, Runx1 and Bmi1 can convert a developmentally distant lineage (fibroblasts) into 'induced hematopoietic progenitors' (iHPs). Functionally, iHPs generate acetylcholinesterase+ megakaryocytes and phagocytic myeloid cells in vitro and can also engraft immunodeficient mice, generating myeloerythoid and B-lymphoid cells for up to 4 months in vivo. Molecularly, iHPs transcriptionally resemble native Kit+ hematopoietic progenitors. Mechanistically, reprogramming factor Lmo2 implements a hematopoietic programme in fibroblasts by rapidly binding to and upregulating the Hhex and Gfi1 genes within days. Moreover the reprogramming transcription factors also require extracellular BMP and MEK signalling to cooperatively effectuate reprogramming. Thus, the transcription factors that orchestrate embryonic hematopoiesis can artificially reconstitute this programme in developmentally distant fibroblasts, converting them into engraftable blood progenitors.
Post-stroke cognitive impairment (PSCI), commonly seen in the clinical practice, is a major factor impeding patient rehabilitation. Enriched environment (EE) intervention is a simple and effective way to improve cognitive impairment, partially due to the rebalancing of the basal forebrain-hippocampus cholinergic signaling pathway. Epigenetic changes have been identified in many cognitive disorders. However, studies on the effects of EE on epigenetic regulation of cholinergic circuits in PSCI animal models have not yet been reported. In this study, we established a photothrombotic mouse PSCI model and showed that after EE intervention, mice with PSCI had significantly improved water maze performance, better induction of hippocampal long-term potentiation (LTP), enhanced function of the basal forebrain-hippocampus cholinergic circuits of contralateral side of stroke and relatively balanced acetylation homeostasis compared to those of PSCI mice in standard environments (SE). In addition, PSCI mice in EE expressed much higher levels of p-CREB and CBP than in SE, and the chromatins bound to M-type promoter of ChAT gene were more acetylated. These results demonstrate that EE plays an important role in the improvement of PSCI and the underlying mechanism may involve in the acetylation of histones bound to the ChAT gene promoter in cholinergic circuits.
Title: Complete genome sequencing and comparative analysis of the linezolid-resistant Enterococcus faecalis strain DENG1 Yu Z, Chen Z, Cheng H, Zheng J, Li D, Deng X, Pan W, Yang W, Deng Q Ref: Arch Microbiol, 196:513, 2014 : PubMed
Genome level analysis of bacterial strains provides information on genetic composition and resistance mechanisms to clinically relevant antibiotics. To date, whole genome characterization of linezolid-resistant Enterococcus faecalis isolated in the clinic is lacking. In this study, we report the entire genome sequence, genomic characteristics and virulence factors of a pathogenic E. faecalis strain, DENG1. Our results showed considerable differences in genomic characteristics and virulence factors compared with other E. faecalis strains (V583 and OG1RF). The genome of this LZD-resistant E. faecalis strain can be used as a reference to study the mechanism of LZD resistance and the phylogenetic relationship of E. faecalis strains worldwide.
Title: Role of paraoxonase-1 in bone anabolic effects of parathyroid hormone in hyperlipidemic mice Lu J, Cheng H, Atti E, Shih DM, Demer LL, Tintut Y Ref: Biochemical & Biophysical Research Communications, 431:19, 2013 : PubMed
Hyperlipidemia blunts anabolic effects of intermittent parathyroid hormone (PTH) on cortical bone, and the responsiveness to PTH are restored in part by oral administration of the antioxidant ApoA-I mimetic peptide, D-4F. To evaluate the mechanism of this rescue, hyperlipidemic mice overexpressing the high-density lipoprotein-associated antioxidant enzyme, paraoxonase 1 (Ldlr(-/-)PON1(tg)) were generated, and daily PTH injections were administered to Ldlr(-/-)PON1(tg) and to littermate Ldlr(-/-) mice. Expression of bone regulatory genes was determined by realtime RT-qPCR, and cortical bone parameters of the femoral bones by micro-computed tomographic analyses. PTH-treated Ldlr(-/-)PON1(tg) mice had significantly greater expression of PTH receptor (PTH1R), activating transcription factor-4 (ATF4), and osteoprotegerin (OPG) in femoral cortical bone, as well as significantly greater cortical bone mineral content, thickness, and area in femoral diaphyses compared with untreated Ldlr(-/-)PON1(tg) mice. In contrast, in control mice (Ldlr(-/-)) without PON1 overexpression, PTH treatment did not induce these markers. Calvarial bone of PTH-treated Ldlr(-/-)PON1(tg) mice also had significantly greater expression of osteoblastic differentiation marker genes as well as BMP-2-target and Wnt-target genes. Untreated Ldlr(-/-)PON1(tg) mice had significantly greater expression of PTHR1 than untreated Ldlr(-/-) mice, whereas sclerostin expression was reduced. In femoral cortical bones, expression levels of transcription factors, FoxO1 and ATF4, were also elevated in the untreated, control Ldlr(-/-)PON1(tg) mice, suggesting enhancement of cellular protection against oxidants. These findings suggest that PON1 restores responsiveness to PTH through effects on oxidant stress, PTH receptor expression, and/or Wnt signaling.
Streptonigrin (STN, 1) is a highly functionalized aminoquinone alkaloid with broad and potent antitumor activity. Here, we reported the biosynthetic gene cluster of STN identified by genome scanning of a STN producer Streptomyces flocculus CGMCC4.1223. This cluster consists of 48 genes determined by a series of gene inactivations. On the basis of the structures of intermediates and shunt products accumulated from five specific gene inactivation mutants and feeding experiments, the biosynthetic pathway was proposed, and the sequence of tailoring steps was preliminarily determined. In this pathway, a cryptic methylation of lavendamycin was genetically and biochemically characterized to be catalyzed by a leucine carboxyl methyltransferase StnF2. A [2Fe-2S](2+) cluster-containing aromatic ring dioxygenase StnB1/B2 system was biochemically characterized to catalyze a regiospecific cleavage of the N-C8' bond of the indole ring of the methyl ester of lavendamycin. This work provides opportunities to illuminate the enzymology of novel reactions involved in this pathway and to create, using genetic and chemo-enzymatic methods, new streptonigrinoid analogues as potential therapeutic agents.
Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen that causes a wide range of both hospital- and community-acquired infections. The high prevalence of MRSA and the extensive use of vancomycin in Mainland China may lead to the emergence of vancomycin-intermediate S. aureus (VISA) isolates. In this case, we report a VISA isolate from a 34-year-old male patient with steam burn. The isolate was determined to be sequence type 239 staphylococcal cassette chromosome mec type III, the most prevalent MRSA clone in Mainland China.
Pelagibacterium halotolerans B2(T) is a marine halotolerant bacterium that was isolated from a seawater sample collected from the East China Sea. Here, we present the complete genome sequence of the type strain P. halotolerans B2(T), which consists of one chromosome (3,944,837 bp; 61.4% G+C content) and one plasmid (4,050 bp; 56.1% G+C content). This is the first complete genome of a member of the Pelagibacterium genus.
Title: Cloning, expression and characterization of a halotolerant esterase from a marine bacterium Pelagibacterium halotolerans B2T Jiang X, Huo Y, Cheng H, Zhang X, Zhu X, Wu M Ref: Extremophiles, 16:427, 2012 : PubMed
An esterase PE10 (279 aa) from Pelagibacterium halotolerans B2(T) was cloned and overexpressed in Escherichia coli Rosetta in a soluble form. The deduced protein was 29.91 kDa and the phylogenetic analysis of the deduced amino acids sequence showed it represented a new family of lipolytic enzymes. The recombinant protein was purified by Ni-NTA affinity chromatography column and the characterization showed its optimal temperature and pH were 45 degrees C and pH 7.5, respectively. Substrate specificity study showed PE10 preferred short chain p-nitrophenyl esters and exhibited maximum activity toward p-nitrophenyl acetate. In addition, PE10 was a halotolerant esterase as it was still active under 4 M NaCl. Three-dimensional modeling of PE10 suggested that the high negative electrostatic potential on the surface may relevant to its tolerance to high salt environment. With this halotolerance property, PE10 could be a candidate for industrial use.
Childhood obesity is increasingly prevalent in the community and is related to many adult diseases. Lipoprotein lipase (LPL) plays a central role in dyslipidemia, and polymorphisms of the LPL gene may result in the disturbance in the lipid's metabolism. The aim of this study is to test the hypothesis that genetic variants of LPL and serum lipid levels are associated with the risk of childhood obesity. We genotyped +495T > G and PvuII T > C in an LPL gene and measured the serum lipid levels in a case-control study of 124 obese children and 346 frequency-matched normal controls in preschool Chinese children. The variant genotypes of LPL + 495GG and PvuII CC were associated with a significantly increased risk of childhood obesity [adjusted odds ratio (OR) = 2.39, 95% CI = 1.09-5.23 for +495 GG; adjusted OR = 2.00, 95% CI = 1.04-3.83 for PvuII CC], compared with their wild-type genotypes, respectively. In addition, compared with the lower serum level cut off by the control median, the higher level of serum triglyceride (TG) (>0.59 mmol/L) was associated with a 1.32-fold increased risk of childhood obesity, and the higher level of high density lipoprotein cholesterol (HDLC) (>1.14 mmol/L) was associated with a 36% decrease in risk of childhood obesity. Furthermore, the median levels of TG were higher in obese children carrying LPL +495TT/TG and PvuII TT/CT genotypes than those in controls, the HDLC levels were lower in obese children carrying LPL +495TG and PvuII CT/CC genotypes than those in controls. In conclusion, the LPL gene +495T > G and PvuII T > C polymorphisms may modulate the magnitude of dyslipidemia in Chinese early-onset obesity.
Ketogulonicigenium vulgare is characterized by the efficient production of 2KGA from L-sorbose. Ketogulonicigenium vulgare Y25 is known as a 2-keto-L-gulonic acid-producing strain in the vitamin C industry. Here we report the finished, annotated genome sequence of Ketogulonicigenium vulgare Y25.
Proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases generates beta-amyloid (Abeta) peptides, which accumulate in the brains of individuals affected by Alzheimer disease. Detergent-resistant membrane microdomains (DRM) rich in cholesterol and sphingolipid, termed lipid rafts, have been implicated in Abeta production. Previously, we and others reported that the four integral subunits of the gamma-secretase associate with DRM. In this study we investigated the mechanisms underlying DRM association of gamma-secretase subunits. We report that in cultured cells and in brain the gamma-secretase subunits nicastrin and APH-1 undergo S-palmitoylation, the post-translational covalent attachment of the long chain fatty acid palmitate common in lipid raft-associated proteins. By mutagenesis we show that nicastrin is S-palmitoylated at Cys(689), and APH-1 is S-palmitoylated at Cys(182) and Cys(245). S-Palmitoylation-defective nicastrin and APH-1 form stable gamma-secretase complexes when expressed in knock-out fibroblasts lacking wild type subunits, suggesting that S-palmitoylation is not essential for gamma-secretase assembly. Nevertheless, fractionation studies show that S-palmitoylation contributes to DRM association of nicastrin and APH-1. Moreover, pulse-chase analyses reveal that S-palmitoylation is important for nascent polypeptide stability of both proteins. Co-expression of S-palmitoylation-deficient nicastrin and APH-1 in cultured cells neither affects Abeta40, Abeta42, and AICD production, nor intramembrane processing of Notch and N-cadherin. Our findings suggest that S-palmitoylation plays a role in stability and raft localization of nicastrin and APH-1, but does not directly modulate gamma-secretase processing of APP and other substrates.
We have sequenced the genome of Shigella flexneri serotype 2a, the most prevalent species and serotype that causes bacillary dysentery or shigellosis in man. The whole genome is composed of a 4 607 203 bp chromosome and a 221 618 bp virulence plasmid, designated pCP301. While the plasmid shows minor divergence from that sequenced in serotype 5a, striking characteristics of the chromosome have been revealed. The S.flexneri chromosome has, astonishingly, 314 IS elements, more than 7-fold over those possessed by its close relatives, the non-pathogenic K12 strain and enterohemorrhagic O157:H7 strain of Escherichia coli. There are 13 translocations and inversions compared with the E.coli sequences, all involve a segment larger than 5 kb, and most are associated with deletions or acquired DNA sequences, of which several are likely to be bacteriophage-transmitted pathogenicity islands. Furthermore, S.flexneri, resembling another human-restricted enteric pathogen, Salmonella typhi, also has hundreds of pseudogenes compared with the E.coli strains. All of these could be subjected to investigations towards novel preventative and treatment strategies against shigellosis.
The microbiological transformation of N-heptyl physostigmine (L-693,487) (1), a semisynthetic physostigmine cholinesterase inhibitor, was investigated using Verticillium lecanii MF 5713 (ATCC 74148), Acremonium sp MF 5723 (ATCC 74164) and Actinoplanes sp MA 6559 (ATCC 53771). Nine microbial metabolites (2-10) of 1 were isolated and purified using reversed-phase HPLC. The structures of the metabolites were established using spectroscopic techniques including MS and NMR. Some of the microbial metabolites were identical to metabolites present in urine of a dog treated with 1.
