NTnC-like green fluorescent genetically encoded calcium indicators (GECIs) with two calcium ion binding sites were constructed using the insertion of truncated troponin C (TnC) from Opsanus tau into green fluorescent proteins (GFPs). These GECIs are small proteins containing the N- and C-termini of GFP; they exert a limited effect on the cellular free calcium ion concentration; and in contrast to calmodulin-based calcium indicators they lack undesired interactions with intracellular proteins in neurons. The available TnC-based NTnC or YTnC GECIs had either an inverted response and high brightness but a limited dynamic range or a positive response and fast kinetics in neurons but lower brightness and an enhanced but still limited dF/F dynamic range. Here, we solved the crystal structure of NTnC at 2.5 Å resolution. Based on this structure, we developed positive NTnC2 and inverted iNTnC2 GECIs with a large dF/F dynamic range in vitro but very slow rise and decay kinetics in neurons. To overcome their slow responsiveness, we swapped TnC from O. tau in NTnC2 with truncated troponin C proteins from the muscles of fast animals, namely, the falcon, hummingbird, cheetah, bat, rattlesnake, and ant, and then optimized the resulting constructs using directed molecular evolution. Characterization of the engineered variants using purified proteins, mammalian cells, and neuronal cultures revealed cNTnC GECI with truncated TnC from Calypte anna (hummingbird) to have the largest dF/F fluorescence response and fast dissociation kinetics in neuronal cultures. In addition, based on the insertion of truncated TnCs from fast animals into YTnC2, we developed fYTnC2 GECI with TnC from Falco peregrinus (falcon). The purified proteins cNTnC and fYTnC2 had 8- and 6-fold higher molecular brightness and 7- and 6-fold larger dF/F responses to the increase in Ca2+ ion concentration than YTnC, respectively. cNTnC GECI was also 4-fold more photostable than YTnC and fYTnC2 GECIs. Finally, we assessed the developed GECIs in primary mouse neuronal cultures stimulated with an external electric field; in these conditions, cNTnC had a 2.4-fold higher dF/F fluorescence response than YTnC and fYTnC2 and was the same or slightly slower (1.4-fold) than fYTnC2 and YTnC in the rise and decay half-times, respectively.
Matched MeSH terms: Green Fluorescent Proteins/genetics
beta-Lactamases have been identified as the major cause of antimicrobial resistance to beta-lactam antibiotics in Escherichia coli. The activities of ampicillin-sulbactam and amoxicillin-clavulanate as well as a range of beta-lactam antibiotics were studied with 87 clinical E. coli isolates from patients of the University Malaya Medical Center using the disc diffusion technique. Susceptible, intermediate and resistant categories were established based on the diameter of zones of inhibition set by the National Committee for Clinical Laboratory Standards (NCCLS). The isolates were then classified into 6 phenotypes according to the criteria stated in the methodology: S (susceptible to all beta-lactams); TL (resistant to aminopenicillins; amoxicillin-clavulanate susceptible and susceptible or intermediate to ampicillin-sulbactam); TI (resistant to aminopenicillins and ampicillin-sulbactam; susceptible to amoxicilin-clavulanate); TH-IRT (resistant to aminopenicillins; intermediate or resistant to amoxicillin-clavulanate; resistant to ampicillin-sulbactam); ESBL (resistant to aminopenicillins and oxyimino cephalosporins; positive results with the double-disc diffusion test); and CP (resistant to aminopenicillins, beta-lactam-beta-lactamase inhibitor combinations, oxyimino cephalosporins and cephamycins). Results showed that the TL phenotype was the commonest (40.2% of the isolates) followed by S (31%), TH-IRT (16.1%), ESBL and CP (3.4% each) and TI (2.3%). One isolate showed both ESBL and CP phenotypes while two isolates were classified as inconclusive. Representatives from each phenotype were further analysed for the presence of beta-lactamases which revealed a predominance of TEM and SHV enzyme producers. PCR-SSCP analysis of the SHV gene from all the ESBL and CP isolates revealed the predominance of SHV 5-type enzyme which was concurrent with our previous studies.
In a study of Malaysians of different racial groups, 1,510 sera (908 from Malays, 371 from Chinese and 231 from Indians) were identified for their protease inhibitor (Pi) types. The gene frequencies for the alleles PiM, PiS and PiX in Malays were, respectively, 0.979, 0.015, and 0.007. In Chinese, the frequencies were 0.981, 0.019 and 0.000, and in Indians they were 0.976, 0.24, and 0.000. It is interesting that the usually rare PiX type is found in appreciable frequency in the Malays. Two different types with unusual behavior and obscure origin were also found.
alpha1-Antitrypsin types were determined in 200 individual specimens of Malaysian Macaca irus. We found the pattern B in 76 samples, BC in 116, and C in 8. Assuming that these patterns are determined by codominant alleles at one locus, this distribution constitutes a significant (P less than 0.001) deviation from Hardy-Weinberg equilibrium, because of an excess of BC and low prevalence of C. We found no clear evidence for the presence of alpha1-antitrypsin deficiency comparable to the situation in man.
We conducted a prospective study to determine the role of alpha1-antitrypsin (alpha1AT) deficiency in the pathogenesis of neonatal cholestasis and other childhood liver diseases in a multi-ethnic Southeast Asian population.
Antioxidant protection provided by different doses of alpha-tocopherol was compared by determining nitric oxide synthase (NOS) activity in blood vessels of spontaneously hypertensive rats (SHR) treated with alpha-tocopherol. SHR were divided into four groups namely hypertensive control (C), treatment with 17 mg of alpha-tocopherol/kg diet (alpha1), 34 mg of alpha-tocopherol/kg diet (alpha2), and 170 mg of alpha-tocopherol/kg diet (alpha3). Wister Kyoto (WKY) rats were used as normal control (N). Blood pressure were recorded from the tail by physiography every other night for the duration of the study period of 3 months. At the end of the trial, animals were sacrificed. The NOS activity in blood vessels was measured by [3H]arginine radioactive assay and the nitrite concentration in plasma by spectrophotometry at wavelength 554 nm using Greiss reagent. Analysis of data was done using Student's t test and Pearson's correlation. The computer program Statistica was used for all analysis. Results of our study showed that for all the three alpha-tocopherol-treated groups, blood pressure was significantly (P < .001) reduced compared to the hypertensive control and maximum reduction of blood pressure was shown by the dosage of 34 mg of alpha-tocopherol/kg diet (C: 209.56 +/- 8.47 mm Hg; alpha2: 128.83 +/- 17.13 mm Hg). Also, NOS activity in blood vessels of SHR was significantly lower than WKY rats (N: 1.54 +/- 0.26 pmol/mg protein, C: 0.87 +/- 0.23 pmol/mg protein; P < .001). Although alpha-tocopherol in doses of alpha1, alpha2, and alpha3 increased the NOS activity in blood vessels, after treatment only that of alpha2 showed a statistical significance (P < .01). Plasma nitrite concentration was significantly reduced in SHR compared to normal WKY rats (N: 54.62 +/- 2.96 mol/mL, C: 26.24 +/- 2.14 mol/mL; P < .001) and accordingly all three groups showed significant improvement in their respective nitrite level (P < .001). For all groups, NOS activity and nitrite level showed negative correlation with blood pressure. It was significant for NOS activity in hypertensive control (r = -0.735, P = .038), alpha1 (r = -0.833, P = .001), and alpha2 (r = -0.899, P = .000) groups. For plasma nitrite, significant correlation was observed only in group alpha1 (r = -0.673, P = .016) and alpha2 (r = -0.643, P = .024). Only the alpha2 group showed significant positive correlation (r = 0.777, P = .003) between NOS activity and nitrite level. In conclusion it was found that compared to WKY rats, SHR have lower NOS activity in blood vessels, which upon treatment with antioxidant alpha-tocopherol increased the NOS activity and concomitantly reduced the blood pressure. There was correlation of lipid peroxide in blood vessels with NOS and nitric oxide, which implies that free radicals may be involved in the pathogenesis of hypertension.
Both zinc and the α-subunit of hypoxia-inducible factor (HIF-1α) play important roles in the remodelling of mammary gland tissues. In the present study, we examined the level and the transcriptional activity of HIF-1α in mammary cells upon zinc treatment. In MCF-7 mammary adenocarcinoma and MCF-10A mammary epithelial cell lines, the toxicity levels of zinc differ. Interestingly, both cell lines overexpress HIF-1α following zinc treatment. As it was evident from an up-regulation of its specific target gene CA9 that encodes carbonic anhydrase IX, the stabilized HIF-1α translocated to the nucleus and was transcriptionally active. Hence, we conclude that zinc causes normoxic accumulation of transcriptionally active HIF-1α by interfering with its post-translational regulation.
In most Old world monkey species, TRIM5α plays a role in combating retroviruses and restricting HIV-1. Alongside TRIM5α, the TRIMCyp fusion gene formed by the retrotransposition of a CypA pseudogene cDNA to 3' terminal or 3'-UTR of TRIM5 gene in these monkeys has become a key research area in anti HIV-1 factors. The regional differences, gene frequencies, genotypes, and retrovirus restrictive activities of TRIMCyp vary among different primate species. While the frequencies of cynomolgus TRIMCyp have been studied in several areas of Southeast Asia, the frequency and prevalence of cynomolgus TRIMCyp in China remains unclear. In this study, we screened 1, 594 cynomolgus samples from 11 monkey manufacturers located across 5 provinces in China. Our results showed that the frequencies of TRIMCyp range from 7.65% to 19.79%, markedly lower than the frequencies found in monkey species in the Philippines, Malaysia and Indonesia (ranging from 34.85% to 100%). We speculate that potentially the latter were isolated groups established since 1978. The NE haplotype frequencies of cynomolgus TRIMCyp were 4.93% in China, also significantly lower than those found in species in the Philippines, Malaysia and Indonesia (from 11.1% to 14.3%). Our research provides interesting findings that contribute towards a more firm basis of further studies of HIV-1 animal models and relevant pathogenesis.
Nipah virus (NiV), a paramyxovirus, was first discovered in Malaysia in 1998 in an outbreak of infection in pigs and humans, and incurred a high fatality rate in humans. We established a system that enabled the rescue of replicating NiVs from a cloned DNA. Using the system, we analyzed the functions of accessory proteins in infected cells and the implications in in vivo pathogenicity. Further, we have developed a recombinant measles virus (rMV) vaccine expressing NiV envelope glycoproteins, which appeared to be an appropriate to NiV vaccine candidate for use in humans.
This study was done to detect and diagnose beta-thalassemia (beta-Thal) gene quickly. We applied sequence specific Amplification (SSA) method to the analysis. 13 kinds of beta-Thal and two kinds of hemoglobin variants were able to detect under the same PCR condition. These mutations were found frequently in ten countries of Asian region (the southern part of China, Vietnam, Cambodia, Thailand, Myanmar, Malaysia, Singapore, Indonesia, Pakistan, India), and 15 kinds in total (-28CapA-->G, CD5-CT, CD8/9+-G, CD15G-->A, CD17A-->T, IVSI-1G-->T, CD41/42-4del, CD16-C, CD26G-->A(betaE), IVSI-5G-->C, CD35C-->A, CD71/72 +A, CD6A-->T (betaS), -619del, IVSII-654C-->T). More than 80% of patients are included in these mutations. To make the reagents a kit, the procedure became simple and rapid. DNA was extracted by salting out method. The PCR product was detected by polyacrylamide gel electrophoresis and silver staining. The confirmation of the variant was done by the PCR-direct sequencing method. It took approximately six hours for PCR reaction, electrophoresis and staining. This method made us to detect and diagnose beta-Thal in one day.
The molecular genetic analysis of the genomes of the virus of porcine reproductive respiratory syndrome (VPRRS) and porcine circovirus type 2 (PCV-2) circulating in the area of the Russian Federation was discussed. The results of this work showed the circulation of the strains of the European genotype VPRRS similar to those found in France and Denmark from 1998 to 2001. The homology of the fragment of one of the genes between the Russian isolates and the vaccine strain Porcilis PRRS (Intervet) was found. It requires further study. The strains representing the North American genotype VPRRS were not found. The PCV-2 genomes fall into three separate goups. One (genotype 2b) is formed by isolates in Malaysia, Brazil, Switzerland, China, Slovakia, UK, USA, isolated during the period from 2004 to the present time. The second group consists of sequences of the viruses isolated in 2000-2012 in Canada, the U.S., China, and South Korea (genotype 2a). The third group is formed by highly pathogenic isolates in 2013 from China (highly pathogenic genotype 2c). The circulation of all three known genotypes of PCV-2: 2a, 2b, and 2c in Russian Federation was demonstrated.
Matched MeSH terms: Circovirus/genetics*; Porcine respiratory and reproductive syndrome virus/genetics*; Porcine Reproductive and Respiratory Syndrome/genetics*
Objective: To confirm the laboratory diagnosis of dengue bordline cases reported in Henan Province and trace its origin from molecular level in 2017. Methods: The study samples were blood samples (3-5 ml), which came from 8 suspected cases of dengue fever reported in the 2017 direct reporting system of Henan provincial infectious disease monitoring network. Meanwhile, case investigation was conducted according to National dengue fever surveillance programme. Serum were separated from blood samples and tested for Dengue NS1 antigen, IgM & IgG antibodies, and dengue RNA. According to dengue diagnosis criteria, confirmed cases were identified by testing results. Samples carried dengue RNA performed for real-time PCR genotyping and amplification of E gene. Then, the amplicons were sequenced and homological and phylogenetic analyses were constructed. Results: 8 serum samples of suspected dengue cases were collected in Henan Province, 2017. Six of them were diagnosed as dengue confirmed cases. All the dengue confirmed cases belonged to outside imported cases, 5 of them were positive by dengue RNA testing. Genotyping results showed there were 1 DENV1 case, 2 DENV2 cases and 2 DENV3 cases. A DENV2 case and a DENV3 case of this study were traced its origin successfully. The sequence of Pakistan imported DENV2 case belongs to cosmopolitan genotype, which was the most consistent with Pakistan's DENV2 KJ010186 in 2013 (identity 99.0%). The sequence of Malaysia imported DENV3 case belongs to genotype I, which was the most consistent with Singapore's DENV3 KX224276 in 2014(identity 99.0%). Conclusion: The laboratory diagnosis and molecular traceability of dengue cases in Henan Province in 2017 confirmed that all cases were imported and did not cause local epidemics.
We report the fact that D. albomicans invaded into Shanghai suddenly in the autumn of 1991. Using 9 restriction enzymes, we analyse the RFLPs of mitochondrial DNA of 29 isofemale lines belonging to 4 populations of Shanghai, Jiading, Qinpu and Nanhui. We find that all 29 haplotypes are different from each other. Comparing with the populations of Canton, Kunming, Sanhutan (Taiwan), Sumoto (Japan), and Kuala Lumper (Malaysia), we come to the conclusion that D. albomicans caught in Shanghai and areas nearby is from a few of places in the south of China-mainland. This conclusion agrees with the viewpoint that this species is on the speciation stage of migration towards north. We also discuss the mtDNA polymorphism within the species.
To isolate and identify the pathogen of Dengue fever from Shenzhen city in 2005 - 2006, and to analyze the molecular characteristics of the isolated Dengue virus strain as well as to explore its possible origin.
To investigate the epidemiological characteristics of influenza B viruses and explore the genetic evolution characteristics of the hemagglutinin(HA) and neuraminidase(NA) genes of local isolated strains in Ningbo, Southeast China, during 2010 to 2012.
A Chicken anemia virus has been isolated from a chicken flock in Harbin of China. The genome of the ivrus was cloned through polymerase chain reaction(PCR) and sequence of the genome was analyzed. The cycle genome is made of 2298 base pairs including three overlapping open reading frames(vp1, vp2, vp3) and a regulative region. Comparing sequence of the genome through BLAST in GenBank, this sequence exhibits 96.9% identity with other genome of CA Vs and least. Multiple alignment of this genome of this virus, 26p4, strain isolated in Germany, strain isolated in Malaysia and Cux-1 found that this sequence exhibits 98.2% (42/2298), 98.2% (42/2298), 96.9% (72/2298) and 97.5% (60/2319) identify with them, respectively. A new CAV strain was isolated and it has better identify with CAV isolated in Europe countries than is Asia country Malaysia. Multiple alignment of VP1, VP2, VP3 of 26p4, strain isolated in Germany, strain isolated in Malaysia, Cux-1 and strain isolated in Harbin of China found the VP2 the most conservative.
A panel of five monoclonal antibodies (MAbs) to the HA1 molecule of the influenza B virus of the Victorian lineage with high virus-neutralizing activity was developed. For identification of the virus neutralizing epitopes in HA1 escape mutants (EM) of the influenza BIShandong/07/97 and B/Malaysia/2506/04 virus were selected using virus- neutralizing antibodies (MAbs). Three EMs had single, two--double and one--triple amino acid substitutions (AAS) in HA1 (H122N, A202E, K203T, K2031, K203N or A317V). In addition, AAS N197S was detected in three EMs. A correlation of AAS identified with peculiarities of interaction of EMs with Mabs was discussed.
In this paper, urolithiasis is remarked from the standpoint of descriptive epidemiology, which examines the frequency distribution of a given disease in a population in terms of time, place and personal characteristics with an aim of identifying risk factors or some clues to the etiology. Some descriptive epidemiological features of urolithiasis are summarized. Prevalence rate is around 4% (4-15% in males and 4-8% in females), and incidence rate varies from area to area: 53.2 per 100,000 population in 1975 in Japan, 364 in 1976 in Malaysia, and 540 in 1979 in West Germany. Prevalence and/or incidence rates have, in general, increased in the developed countries since World War II and in the developing countries as well, where upward trends are quite analogous to the trends observed in the nineteenth century in Europe. Recurrence rate, which is much higher in males than in females, ranges from 31% to 75%, depending on the follow-up periods. In the industrialized countries, upper urinary (renal and ureteral) stones account for more than 90% of total stones, which are ordinarily calcium complexes in composition. More common in the developing countries are lower urinary (bladder and urethral) stones, frequently composed of magnesium ammonium phosphate, which indicates a close association with urinary tract infections. Variations in frequency are evident by season and by region within a country. Age and sex differentials in urinary stone formers are substantial: more common in males 30-40 years old in the industrialized countries and in children under 10 years old in the developing countries. Racial differentials are also noted; blacks appear to suffer less frequently than whites. Stone formers experience more frequent episodes of stone formation in their family members, particularly father and brothers, than non-stone formers. These findings on racial differentials and family preponderance suggest the possible relevance of genetic factors in stone formation. Stone formers are more likely to be occupationally sedentary and socially affluent. This observation and differentials by age and sex suggest the probable relevance of lifestyle and environmental factors in stone formation. Epidemiological factors incriminated for stone formation will be discussed in a separate paper.
In an in vitro culture system, primary hepatocytes usually display a low proliferation capacity, accompanied with a decrease of viability and a loss of hepatocyte-specific functions. Previous studies have demonstrated that the combination introductions of certain hepatocyte-specific transcription factors are able to convert fibroblasts into functional hepatocyte-like cells. However, such combinational usage of transcription factors in primary hepatocytes culture has not yet sufficiently studied. The forkhead box protein A3 (FoxA3) and hepatocyte nuclear factor 4α (Hnf4α) are liver-enriched transcription factors that play vital roles in the differentiation, and maintenance of hepatocytes. Thus, we simultaneously overexpressed the two genes, Foxa3 and Hnf4α, in rat hepatocytes and observed that the combinational augmentation of these two transcription factors have enhanced the proliferation and stabilized the hepatocyte-specific functions of primary hepatocytes over a long-term culture period.