The leakage of sewer systems and the impact on the 'black and odorous water bodies' and WWTPs in China.

China has achieved significant progress on wastewater treatment and aquatic environmental protection. However, leakage (in- and exfiltration) of sewer systems is still an issue. By using the statistical data of water and wastewater in 2016 in China, and the person loads (PLs) of water and wastewater in Singapore, the leakage fractions of hydraulic flow, organic carbon (COD), nitrogen (N) and phosphorus (P) mass loading, and in-sewer COD biological removal in the sewer systems of China (except Hong Kong, Macau and Taiwan), Shanghai, Guangzhou and Beijing were reported for the first time. The fractions of hydraulic flow infiltration (13%, Shanghai and Guangzhou) and exfiltration (39%, China) were calculated. Except Beijing, whose sewer networks are under appropriate management with small leakage fractions, the exfiltration fractions of COD (including in-sewer biological COD removal) ranged from 41% (Shanghai) to 66% (China) and averaged 55%; N ranged from 18% (Shanghai) to 48% (China) and averaged 33%; and P ranged from 23% (Shanghai and Guangzhou) to 44% (China) and averaged 30%. The exfiltrated sewage, COD, N and P not only wastes resources, but also contaminates the aquatic environment (especially groundwater) and contributes to 'black and odorous water bodies'. In- and exfiltration in the sewer network leads to low influent COD concentration, C/N ratio and high inorganic solids and inert particulate COD concentrations of many municipal wastewater treatment plants (WWTPs) causing high cost for nutrient removal, poor resource recovery, additional reactor/settler volume requirement and other operational problems. Therefore, tackling sewer leakage is of primary importance to today's environment in China. Recommendations for the inspection of sewer systems and the rehabilitation of damaged sewers as well as the development of design and operation guidelines of municipal WWTPs tailored to the specific local sewage characteristics and other conditions are proposed.

MicroRNAs as novel biomarkers for the differentiation of malignant versus benign thyroid lesions: a meta-analysis.

The aim of this meta-analysis was to systematically evaluate the diagnostic accuracy of microRNAs (miRNAs) in distinguishing malignant thyroid lesions from benign ones and to determine the potential of miRNAs as diagnostic biomarkers for thyroid cancer. The random-effect model was used to summarize the pooled estimates of diagnostic accuracy, including sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR). The summary receiver-operating characteristic curve (SROC) and area under the SROC curve (AUC) were used to further evaluate the overall diagnostic value. Overall, 20 studies from 7 articles, including 266 thyroid cancer patients and 277 controls with benign thyroid disease, were available for analysis. The pooled sensitivity, specificity, PLR, NLR, and DOR were: 0.78 (95%CI = 0.74-0.81), 0.73 (95%CI = 0.69-0.77), 3.17 (95%CI = 2.28-4.40), 0.30 (95%CI = 0.23-0.39), and 12.6 (95%CI = 8.26-19.4), respectively, and the AUC value was 0.85. The multiple miRNA assay yielded better diagnostic performance than the single miRNA assay, with sensitivity of 0.90 versus 0.75, specificity of 0.86 versus 0.71, PLR of 6.14 versus 2.71, NLR of 0.13 versus 0.36, DOR of 44.5 versus 8.81, and AUC of 0.95 versus 0.82, suggesting that the multiple miRNA assay is a more credible method for thyroid cancer detection. In summary, miRNA assays, especially multiple miRNA assays, may play an important role as a second-line diagnostic tool to improve the diagnostic accuracy of fine needle aspiration biopsy in indeterminate lesions. However, further studies are warranted to confirm our findings.

Gene therapy for streptozotocin-induced diabetic mice by electroporational transfer of naked human insulin precursor DNA into skeletal muscle in vivo.

Transfer of naked plasmid with insulin precursor DNA into skeletal muscle of streptozotocin (STZ)-induced diabetic mice through electroporation and detection of gene expression is described. Four different human insulin precursor DNA fragments were inserted into pcDNA3.1(-), downstream of a CMV promoter. Three of them, with a secretion signal sequence, succeeded in lowering blood glucose at a range of 30-50% in STZ diabetic mice. The other, with a synthetic DNA fragment encoding human proinsulin, failed. The mortality rate of very seriously STZ diabetic mice was reduced significantly by the treatment. The circulating insulin-like protein (mouse insulin, human proinsulin, or intermediates during conversion of proinsulin to insulin) level in the blood of less seriously STZ diabetic mice treated with the human preproinsulin gene with an intron was about 15-23 microU/ml, while that of STZ diabetic mice treated with empty vector was only about 6 microU/ml and that of normal mice was about 18 microU/ml. Transcription of the three human insulin precursor DNAs in mouse skeletal muscle was also detected by RT-PCR. The human preproinsulin gene with the intron showed a slightly higher potency in reducing blood glucose of mildly diabetic mice. These studies indicate that the skeletal muscle transferred with appropriate preproinsulin DNA by electroporation in vivo can secrete insulin-like protein resulting in reduction of blood glucose, and a basal blood insulin level can be achieved for at least 1 month.

Inhibition of platelet aggregation of a mutant proinsulin molecule engineered by introduction of a native Arg-Gly-Asp sequence.

A 13 amino acid sequence, CRVARGDWNDNYC, originated from disintegrin eristostatin, was introduced into an inactive human proinsulin molecule between the B29 and A2 sites to replace proinsulin C-peptide by molecular cloning techniques. The constructed Arg-Gly-Asp (RGD)-proinsulin gene was cloned into a temperature-inducible vector pBV220 and expressed in Escherichia coli. The expressed RGD-proinsulin was refolded and purified by Sephadex G50 and DEAE-Sephadex A25 separations. The chemical identity was confirmed by both amino acid composition and mass spectrometry analyses. This RGD-proinsulin showed an inhibitory activity of adenosine 5'-diphosphate-induced human platelet aggregation with an IC50 value of 200 nM. Its insulin receptor binding activity remained as low as 0.03% with native insulin as a control, and its insulin immune activity retained 27.6% compared with proinsulin.

Structural and functional studies of CCAAT/enhancer-binding protein epsilon.

CCAAT/enhancer-binding protein (C/EBP) epsilon is a critical transcription factor for differentiation of myeloid cells. Structural and functional relationships of C/EBPepsilon were explored by recombinant protein studies, gene mutation, and transactivation assays. Evidence strongly suggested that C/EBPepsilon does not have disulfide bonds. Transactivation analysis of C/EBPepsilon having mutations of each of three conserved cysteines (C345, C148S, and C280S) indicated that the three mutant proteins had almost the same activity as the wild type. Dimer formation of C/EBPepsilon was not detected using both reducing and non-reducing SDS-polyacrylamide gel electrophoresis with Western blot analysis from either bacterial or mammalian expressed C/EBPepsilon. Furthermore, C/EBPepsilon mutant C280S gave a gel band similar to that for wild type, suggesting that this C-terminal, conserved cysteine is not involved in disulfide bond formation in vivo, even though previous data for C/EBPbeta suggested that dimers may form in vitro utilizing this conserved cysteine residue. Mutational studies of conserved residues in the activating domain 1 (ADM1) and ADM2 of the amino region of the gene indicated that negative charge is critical for transactivational activity of C/EBPepsilon. Mutational analyses of hydrophobic amino acids in ADM1 suggested that these residues do not play a key role in transactivational activity. Further mutational studies indicated that, although the N-terminal 32-amino acid peptide of C/EBPepsilon isoform p32 did not greatly influence the transactivation activity compared with p30 isoform, this peptide does modulate transactivation activity. Domain swapping experiments substituting the ADM1 domain of various C/EBPs for C/EBPepsilon showed that the C/EBPalpha and -delta but not -beta ADM1 markedly enhanced the chimeric C/EBPepsilon transcriptional activity. Based on mutational data and possible mRNA structure, we hypothesized about the effect of mRNA structure on translation of the two major C/EBPepsilon isoforms: p32 and p30. The data suggested a very stable 8-base pair double helical structure with one strand sequence including the initial codon for p32 and complementary strand with the initial codon for p30.

Intra-A chain disulphide bond forms first during insulin precursor folding.

In this study, we investigated the folding pathway of insulin precursor and compared it with that of insulin-like growth factor I (IGF-I). The intra-A chain disulphide bond was found to form early in insulin precursor folding, whereas the corresponding disulphide bond in IGF-I formed late. Intra-A chain disulphide-bond deleted [A6, A11-Ser] proteins, including proinsulin, insulin, and A chain, were employed for this investigation. Under the same conditions the recombination yield of insulin from S-sulphonates of native A and B chains was 22%, while the yield of [A6, A11-Ser] insulin from S-sulphonates of [A6, A11-Ser] A chain and native B chains was only approx. 7%. This indicated that the intra-A chain disulphide bond may serve to stabilize the A chain folding intermediate so as to facilitate the correct recognition and pairing with the B chain. Time courses of oxidation of reduced insulin A chains, reduced A and B chains, and reduced proinsulins showed that the intra-A chain disulphide bond formed first during insulin precursor folding. The formation of intra-A chain disulphide bond further accelerated the formation of the other two inter-chain disulphide bonds. The time course of helix structure formation of insulin A chains also indicated that the intra-A chain disulphide bond formed first, and could stabilize partially folded A chain helix structure. The rate of intra-A chain disulphide bond formation was almost the same as that for both helix structure formation and insulin molecule formation, indicating that the formation of the intra-A chain disulphide bond was the rate limiting step for the folding of insulin precursor.

Separation and characterization of trypsin and carboxypeptidase B-digested products of Met-Lys-human proinsulin.

Met-Lys-human proinsulin could be converted into insulin in vitro with the treatment of trypsin and carboxypeptidase B (CPB). Under less effective conditions, the enzymatic reaction does not proceed perfectly, and two main bands have been identified by native-polyacrylamide gel electrophoresis (PAGE) analysis. These two main products were thus separated and purified by DEAE-Sephadex A25 chromatography in a Tris-isopropanol system with an NaCl gradient. The isopropanol and NaCl were removed by a second DEAE-Sephadex column. Native-PAGE, mass spectrometric, and amino acid composition analyses indicate that one fraction of these two major products contains human insulin and desB30-insulin and that the other fraction is a mixture of human insulin analogs, which have one more basic amino acid than human insulin owing to the unsuitable amount of proteases, especially the lack of CPB. Furthermore, both receptor binding assay and radioimmunoassay have been utilized for the activity determination, and both fractions display almost full biological activity with porcine insulin as the standard. Present results provide further evidence for the quality control of recombinant human insulin production.

RGD-containing trypsin with both platelet aggregation inhibitory activity and proteolytic activity.

Arg-Gly-Asp (RGD) motif mediates cell adhesion as a major determinant in interactions of disintegrins with cell surface receptors. In order to obtain a mutant trypsin with both high affinity to integrins and retained proteolytic activity, RGDS and RGD were inserted into a rigid turn region and a flexible loop of trypsin, respectively. Wild type trypsin and substituted mutant trypsins, 37RGDS and 77RGD, were expressed in E. coli and purified. Kinetic properties, autolytic stability as well as platelet aggregation inhibitory activity of both 37RGDS and 77RGD were determined. 37RGDS and 77RGD give retained proteolytic activities of 34% and 87%, respectively, and both become less stable to autolysis. 37RGDS shows an obvious inhibition rate of 29% for platelet aggregation and 77RGD gives a rather weak rate of 14% at the same protein concentration of 3.5 microM, while the wild type trypsin shows no inhibitory activity.

Anti-autolysis of trypsin by modification of autolytic site Arg117.

In order to improve the stability of trypsin, an approach to knock out the autolytic site has been carried out in this investigation. Compared with trypsins from other species, the autolytic site Arg117-Val118 of rat trypsin is the most interesting candidate to work on. The Arg117 residue was designed to be deleted or replaced by other amino acid residues to destroy the autolytic site. With DNA site-directed mutagenesis method, one deletion mutant and several replacement mutants were selected. After expression and purification, the kinetic and anti-autolytic properties of mutant trypsins were studied. No net charge difference of the trypsin molecules was observed by native PAGE analysis. Kinetic studies show that the activities of mutants vary from one another. R117L gives 32 times the activity of wild type trypsin while R117C has no detective activity. Among 8 selected mutants with characteristic properties, 7 of them give prolonged half life during anti-autolytic assay with the exception of R117M which is more sensitive to autolysis.

Three-dimensional structure of the major autoantigen in primary biliary cirrhosis.

BACKGROUND & AIMS: Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the presence of antimitochondrial autoantibodies in patients' serum. The major autoantigen, recognized by antibodies from > 95% of patients with PBC, has been identified as the E2 component (E2p) of the pyruvate dehydrogenase multienzyme complex. Immunodominant sites on E2p have been localized to the inner of the two lipoyl domains, where the essential cofactor lipoic acid is attached covalently. The aim of this study was to determine the three-dimensional structure of the inner lipoyl domain of human E2p. METHODS: The domain was expressed in Escherichia coli; after purification, its structure was analyzed using nuclear magnetic resonance spectroscopy. RESULTS: The structure of the lipoyl domain from human E2p was determined, and the implications of the structure for autoimmune recognition were assessed. CONCLUSIONS: Knowledge of the structure further defines the major epitope and may help in the design of antigen-specific immunotherapy for treatment of PBC.

Hydroxyl group of insulin A19Tyr is essential for receptor binding: studies on (A19Phe)insulin.

One mutant proinsulin gene was constructed through PCR with the code of A19Tyr changed into that of Phe. The mutant proinsulin, (A19Phe)-lysproinsulin (F19KPI) was expressed in E. coli and purified. After trypsin and carboxypeptidase B cleavage and Resource Q separation, (A19Phe)-human insulin (F19HI) was obtained. With native insulin as standard, activity assay and structural analysis were carried out. It was found that the conformation of F19HI is very similar to that of native insulin according to the data from native PAGE, reverse-phase FPLC and circular dichroism spectrum, which are also in agreement with the result of radioimmune assay. F19HI retains almost full immune activity, but displays only 13.3% of receptor binding activity. These results suggest that the hydroxyl group of insulin A19Tyr is essential for receptor binding.

Effects of deleting A19 tyrosine from insulin.

A mutant proinsulin gene was constructed through PCR mediated mutagenesis. The code of A19Tyr was deleted. The mutant proinsulin, (deltaY)19-lys-proinsulin [(deltaY)19KPI], was expressed in E. coli and purified. After treatment with trypsin and carboxypeptidase B, and Resource Q separation, (deltaY)19-human insulin [(deltaY)19HI] was obtained. It retains 63.6% of receptor binding activity but only 2.2% of immune activity, and shows a longer retaining time on reverse-phase FPLC and a slower mobility by native PAGE analysis. These results suggest that the deletion of A19Tyr causes some conformational changes on insulin, which plays a minor role on the affinity of insulin to its receptor, and a major role on immunogenicity of the hormone.

Autolytic site mutant R105C of rat trypsin.

The autolytic site Arg105 of rat trypsin was replaced with Cys by DNA site-directed mutagenesis method. Comparison of expression and purification of R105C trypsin along with the wild type and some other Arg105 mutants indicates that R105C trypsin could be expressed as well but with a lower expression level. It is unexpected that R105C trypsin has no detectable activity toward trypsin substrate TAME, quite different from the wild type and other Arg105 mutants. Native gel electrophoresis analysis indicates that R105C trypsin has a similar mobility rate to that of wild type trypsin. FPLC also gives similar retaining time. The loss of activity of R105C trypsin may result from the conformational change around active site, but not the dimer formation.

Epidemiological survey of oral submucous fibrosis in Xiangtan City, Hunan Province, China.

Oral submucous fibrosis is a high risk precancerous condition and is suggested to be caused by areca nut chewing. Areca nut chewing is popular in Hunan Province of China, and is more concentrated in Xiangtan City. Two and nine cases of oral submucous fibrosis (OSF) were first noticed in 1984 and 1985 respectively, and an epidemiologic survey was subsequently performed in 1986. The epidermiologic method of cluster sampling was used. The Yuhu District, one of the five urban districts of the Xiangtan City with a population of 100,000 was selected as a whole body in the survey, 57 independent units of various professions were randomly selected as group samples and more than 70% of subjects in each unit were examined. Definite fibrous band on palpation was used as a main diagnostic criterion for OSF. A total of 11046 individuals were examined; among them were 3907 areca nut chewers (35.37%) and 7139 non-chewers (64.63%). 335 cases of OSF were found, comprising a prevalence rate of 3.03%. The disease involved mainly the middle third of the oral cavity. All of the OSF cases were areca nut chewers. No case had been found in non-chewers. Four cases of oral carcinoma were found on the basis of OSF, the malignant transformation rate was 1.19%. The high prevalence of OSF may be due to areca nut chewing plus extensive and heavy use of hot pepper in Xiangtan people. The result supports the role of the areca nut as an etiologic factor in the development of OSF. The low malignant transformation rate of 1.19% compared with the 7.6% in an Indian report may be because Xiangtan people chew areca nut without tobacco.

Characteristic, activity and conformational studies of [A6-Ser, A11-Ser]-insulin.

Met-[A6-Ser, A11-Ser]-human proinsulin (Mut-HPI) was prepared in the same way as described previously for Met-human proinsulin (Met-HPI). After trypsin and carboxypeptidase B cleavage and DEAE-Sephadex A25 separation, Met-[A6-Ser, A11-Ser]-human insulin (Mut-HI) and Met-human insulin (Met-HI) were obtained. Their amino-acid compositions are in a good agreement with those expected. Mut-HI keeps full immuno activity, but loses almost all of its receptor binding activity with Met-HI as the control. Some physico-chemical behaviors of Mut-HI have changed to a certain extent. CD studies of mutant insulin demonstrates that the conformation of Met-HI is very similar to that of HI, while that of Mut-HI has altered slightly. The most important observation is that the binding ability of Mut-HI to the receptor has decreased abruptly. These results suggest that though the intra-A chain disulfide bond is deleted, the other two inter-chain disulfide bonds are still correctly paired, and that the intra-A chain disulfide bond is essential for insulin displaying its biological activity.

Formation of inclusion bodies may be the key factor for the stability of expressed products in E. coli.

Des-B30 single-chain insulin gene was constructed from chemically synthesized DNA encoding human proinsulin by oligo-nucleotide induced deletion, cloned into the expression plasmid pBV220, and expressed in E. coli with a level of around 5%. The expressed product was in the form of inclusion bodies. After downstream processing, 45 mg of des-B30 single-chain insulin with a purity of up to 90% was obtained from 1 liter of high density fermentation medium. It is suggested that the fusion protein model to increase the stability of small proteins seems inadequate to some extent, the formation of inclusion bodies may be the key factor for the stability of some expressed products, large or small, in the cytoplasm of E. coli cells.

Production of human insulin in an E. coli system with Met-Lys-human proinsulin as the expressed precursor.

The construction of a gene encoding Lys-human proinsulin, its direct expression in E. coli, and the simple purification procedure are described here. The temperature inducible promotor was employed for induction in a very short time. The expression level could reach 20-30%. After simple downstream processing and only one step of Sephadex G50 purification, 150 mg recombinant Lys-human proinsulin with a purity of up to 90% could be obtained easily from 1 L of high density fermentation medium. The obtained product is in the form of Met-Lys-human proinsulin because of the failure of the bacterial host to remove the initiator methionine residue. The Lys-human proinsulin could be changed into human insulin by trypsin and carboxypeptidase B treatment in later steps. After separation with DEAE-Sephadex A25, human insulin with expected amino acid composition and full native biological activity could be obtained with a yield of 50 mg/L of fermentation medium.

Double-C-peptide human proinsulin.

A fusion gene encoding double-C-peptide human proinsulin was constructed by insertion of a DNA fragment encoding human C-peptide into the 5'-terminal C-peptide coding sequence of a synthetic human proinsulin gene with correct reading frame and over-expressed in E. coli. The purified double-C-peptide human proinsulin shows decreased activity in receptor binding and insulin immune assays as compared with human proinsulin. Disulphide bond reconstitution studies demonstrate that there is not much more influence of the protein concentration on the yield of refolded double-C-peptide human proinsulin. The double-C-peptide human proinsulin shows a 1.86-fold human C-peptide immune activity as compared with that of human proinsulin and gives a good yield of the molecule with correct disulphide bonds in reconstitution studies strongly suggesting the existence of very flexible conformation of the C-peptide.

Intra-A chain disulfide bond (A6-11) of insulin is essential for displaying its activity.

The mutant proinsulin gene was constructed with the codons for A6 and A11 Cys changed to Ser to delete intra-A chain disulfide bond. After expression and purification, the mutations in the protein were further confirmed by amino acid composition. Electrophoretic mobility of the mutant proinsulin is similar to that of human proinsulin, so are the products of tryptic digestions. The mutant proinsulin, which retains its full radioimmuno activity, shows only 5.4% of receptor binding activity of human proinsulin. This suggests that though the intra-A chain disulfide bond disappears, the other two inter-chain disulfide bonds are still correctly paired, and hence the three dimensional structure has not been altered significantly. This intra-chain disulfide bond is essential for insulin displaying its activity.

Plasma cells producing lambda light chains are predominant in human gut and tonsils. An immunohistomorphometric study.

An immunohistomorphometric study was performed on human samples of duodenum (10) and tonsil (25) to assess the numbers of plasma cells producing kappa or lambda chains. Different reagents were used and carefully assayed for specificity and absence of cross-reactivity. Kappa chains were found predominantly with these antibodies in 46 bone marrow-derived B-cell proliferations used as reagents' control samples. In contrast, plasma cells producing lambda chains were found to be more numerous in the samples of mucosal tissues. The kappa/lambda ratio observed was 0.53. This finding could be another feature reflecting the autonomy of the immune system of mucosae (MALT) in humans.