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Background and Purpose: Invasive candidiasis is one of the most common systemicmycoses, and studies have shown mixed yeast infections. Malaysia lacks mixed yeastculture data.Materials and Methods: Yeast isolates were collected in Sultan Abdul Halim Hospital,North Malaysia, from October 2020 to October 2021. Chromogenic Candida differentialagar media and PCR-RFLP were used to identify yeast species.Results: A total of 206 yeast isolates were collected from different body sites of patients.The majority of the yeast isolates (n=104) were obtained from the urine. Other isolates were extracted from blood (n=52), vaginal swabs (n=45), ear discharge (n=2), tracheal aspirate (n=2), tissue (n=2), skin (n=1), nail (n=1), sputum (n=1), and cerebrospinal fluid (n=1). In total, 200 yeast samples were identified as single species, and six isolates were a mixture of Candida species.Conclusion: Malaysia lacks accurate epidemiological data on mixed yeast infections.We identified all samples to the species level, including mixed yeast cultures, using theMspI enzyme and PCR-RFLP.

CYP1A1 polymorphisms are known to play a crucial role in the development and metastasis of malignant diseases including nasopharyngeal cancer (NPC). Individual susceptibility to cancer from environmental agents may be influenced by polymorphic metabolic gene (CYP1A1). The present study was carried out to analyze the relationship between CYP1A1 genetic polymorphism and the risk of developing nasopharyngeal cancer (NPC). The study involved 70 patients with nasopharyngeal carcinoma, compared with 100 healthy controls. Genomic DNA samples were assayed for restriction fragment length polymorphisms in the CYP1A1 by PCR amplification followed by digestion with MspI and BsrDI. The frequency comparison between the two groups was determined by the χ(2) test. Showed that the distribution of CYP1A1 mt/mt allele frequency was found to be significantly different between controls (6%) and cancer cases (11%) along with twice increase of the NPC risk in Algeria (OR=2.5, CI 95% [1.72-2.30]) Our results are in line with those found for other populations and subsequent they emphasize the importance of the involvement of this gene in the development of the NPC detoxification process, which exhibited more and more strong association with environmental factors.

The study aimed to investigate the CAST gene's genetic polymorphism, known as a candidate gene for meat quality and quantities, using the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method in Karayaka sheep. The blood samples were collected from 105 animals belong to four subpopulations (Samsun, Ordu, Giresun, and Tokat) in Turkey’s Black Sea region provinces. The genomic DNA was isolated by using an extraction kit. A fragment of 622 bp on CAST gene was amplified by PCR and then genotypes of the CAST gene for all individuals were determined with the restriction endonuclease MspI. At the end of the work, MM and MN’s genotypic frequencies were found to be 84 and 16% for the Karayaka breed. While the MN genotype was not observed only in the Ordu subpopulation, the NN genotype was not observed in any subpopulations studied. As a result, the obtained findings provided information about the CAST gene polymorphism for Karayaka breed at the population level. This result could be considered for genomic selection works to improve the meat quality traits of Karayaka in the future. [ABSTRACT FROM AUTHOR]

A label-free electrochemical method was developed for sensitive determination of ten-eleven translocation protein 1 (TET1) which can mediate the demethylation of DNA. This strategy is mainly based on MspI-mediated restriction endonuclease reaction. Current response difference of the biosensor before and after cleavage by MspI was dependent on the activity and concentration of TET1. With the aid of Au nanoparticles, this method shows a good linear range from 0.0042 μg μL−1 to 0.0210 μg μL −1 with a correlation coefficient of 0.9350 and a low limit of detection 0.00098 μg μL −1. Finally, this method was used to investigate the effects of n-oxalylglycine (NOG) and taxol on activity of TET1. The results indicated that NOG could inhibit TET1 activity but taxol could not. So this electrochemical biosensor could be applied to TET activity evaluation and inhibitor screening in field of biomedicine and clinical diagnosis. Image 1 • A novel electrochemical biosensor was developed for TET1 detection and its inhibitor screening. • This biosensor shows a wide linear range from 0.0042 to 0.0210 μg μL -1 with a low detection limit of 0.00098 μg μL -1. • This method is an attractive candidate for construction platform for monitoring DNA methylation and demethylation.. [ABSTRACT FROM AUTHOR]

Two strains of Spodoptera frugiperda (J.E. Smith) were first described in the United States; in Brazil, in the states of Rio Grande do Sul, Mato Grosso, and western Paraná they have also been reported. This study was aimed at identifying these strains in Norte Pioneiro and Campos Gerais, in the states of Paraná and southwestern São Paulo. Larvae of S. frugiperda were collected in the cities of Ponta Grossa, Tibagi, Arapoti, and Wenceslau Braz in Paraná, and in the city of Itaberá, in São Paulo. PCR-RFLP genotyping of the COI gene was carried out using sixty-six specimens. Based on their electrophoretic pattern, 51 individuals were identified as corn strain, five as rice strain, and 10 as hybrids (Rice in MspI and Maize in SacI). Our findings indicate that both S. frugiperda strains are present the study areas.

The goal of this study was investigation of the calpastatin gene polymorphism and its effect on early body weight traits in Zandi sheep by polymerase chain reaction restriction fragment length polymorphism (PCRRFLP) method. Calpastatin is the main inhibitors of calpain-calpastatin enzyme system that play an important role in regulating the protein regeneration process. Genomic DNA was extracted by whole blood samples from 124 randomly selected Zandi breed sheep using salting out method. Birth weight, weight on 150 days old and daily weight gain from birth to 150 days old were recorded. The exon and intron 1 of ovine calpastatin gene were amplified to produce a 622 bp fragment. The PCR products were digested with MspI restriction enzyme and electrophoresised on agarose gel. Results showed 80.6% and 19.4% allele frequencies for A and B alleles and 65.32%, 4.04% and 30.64% genotype frequencies for AA, BB and AB genotypes, respectively. X2 and G2 tests showed the Hardy-Weinberg equilibrium for studied locus. The effect of calpastatin genotype on growth traits was insignificant, but genotype BB had the best performance among all the three traits, which indicates the better performance of normal allele (B) than mutant allele (A) for these traits. [ABSTRACT FROM AUTHOR]

Context: Candida spp. is an emerging cause of bloodstream infections worldwide. Delay in speciation of Candida isolates by conventional methods and resistance to antifungal drugs in various Candida species are responsible for the increase in morbidity and mortality due to candidemia. Hence, the rapid identification of Candida isolates is very important for the proper management of patients with candidemia. Aims: The aim was to re-evaluate the identification of various Candida spp. by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and to evaluate the accuracy, speed, and cost of phenotypic methodology versus PCR-RFLP. Settings and Design: Hospital-based cross-sectional study. Materials and Methods: Ninety consecutive clinical isolates of seven Candida species, isolated from blood of neonates and identified by routine phenotypic methods, were re-evaluated using universal primers internal transcribed spacer 1 (ITS1) and ITS4 for PCR amplification and Msp I restriction enzyme for RFLP. Statistical Analysis Used: Kappa test for agreement. Results: The results of PCR-RFLP were 100% in agreement with those obtained using conventional phenotypic methods. Identification could be achieved within 3 work days by both the methods. Our routine methods proved to be cost effective than PCR-RFLP. Conclusions: We can continue with our routine phenotypic methods and PCR-RFLP can be used for periodic quality control or when conventional methods fail to identify a species.

• New label-free electrochemical method with high sensitivity was designed for TET1 detection. • Hairpin DNA was used for the formation of peroxidase-mimicking DNAzymes to amplify the signal. • Low detection limit of 0.027 ng/μL and a wide linear range of 0.7–10.5 ng/μL were obtained. • This method has been proven to be feasible for inhibitor screening of TET1. Ten-eleven translocation 1 (TET1) is a member of the TET enzyme family of dioxygenases, which plays an important role in active DNA demethylation. Therefore, the sensitive TET1 detection could help us better understand DNA methylation-demethylation in epigenetics. Here we report a detection method that consists of electrode fabrication, TET1 modification, DNA digestion, signal-on oxidoreduction, and current peak monitoring. An exquisitely designed 5′end-G-rich oligodeoxynucleotide was synthesized bearing a methylated cytosine (5-mC), which formed into hairpin dsDNA with the MspI recognition sequence (Cm C GG/GGCC). Then hairpin dsDNA was fabricated onto gold nanoparticles modified glassy carbon electrode (DNA/AuNPs/GCE) via Au-S bond. The combination uses of restriction enzyme MspI and hemin converted fabricated-dsDNA into peroxidase-mimicking DNAzyme, thereby promoting the reduction of H 2 O 2 with a current peak. However, the current peak was extremely decreased once TET1 and T4 β-GT were used in advance. We confirmed a delicately linear relationship matching between the current difference and TET1 activity from 0.7 to 10.5 ng μL−1 with a detection limit of 0.027 ng μL−1, which outcompeted the former methods at least one order of magnitudes. The TET1 activity evaluation in the existence of Bobcat339 was also tested as the proof of concept of inhibitors screening. Our strategy provides a novel, label-free, and sensitive electrochemical approach that enables us to complete both TET1 activity evaluation and potential TET1 inhibitors screening. [ABSTRACT FROM AUTHOR]

Background: In recent years, the incidence of opportunistic fungal infections such as candidiasis, especially in immunocompromised patients, has considerably increased. Rapid and accurate identification of candida isolates is necessary for effective antifungal therapy and hospital infections management. In this study, polymerase chain reaction (PCR)-based technique using a one-enzyme restriction fragment length polymorphism (RFLP) was used for discrimination of candida species. Methods: 98 yeast strains were obtained from 90 patients with candidiasis during one-year period, from December 2013 to December 2014, in Birjand city, Iran. Clinical samples were cultured on Sabouraud dextrose agar with choramphenicol at 32°C and CHROMagar™ at 35°C for 48 hours to produce species-specific colors. In next stage, identification of Candida species was performed using PCR-RFLP method with the MspI restriction enzyme. Findings: Totally, 98 candida isolates successfully were isolated from different clinical samples and identified via PCR-RFLP method using MspI. The age group of infancy to 10-year-old had the highest prevalence of candidiasis. In clinical samples, most of the Candida isolates were isolated from urine (83.86%). The most commonly identified species were Candida albicans in 41 cases (41.84%), Candida glabrata in 16 case (16.32%), Candida tropicalis in 12 cases (12.24%), Candida krusei in 10 cases (10.2%), Candida parapsilosis in 8 cases (8.08%), Candida Lusitania in 7 cases (7.14%), and Candida kefyr and Candida guilliermondii each one in 2 cases (2.04%). Conclusion: PCR-RFLP assay with restriction enzyme MspI is an easy, rapid, and reliable method for identification of Candida species.

[b]Introduction and objective[/b]. Lead has a negative effect on the central nervous system (CNS), [i]inter alia [/i]indicated by disturbance of posture. However, knowledge of the CNS effects at low exposure is insufficient. Individuals differ in vulnerability, possibly because of genetic predisposition. Polymorphisms in the δ-aminolevulinic acid dehydratase (ALAD) and vitamin D receptor (VDR) genes may modify lead metabolism and neurotoxicity. The objective of the study was to determine whether very low lead exposure affects posture in children, and whether ALAD and VDR genotypes modify the effect. Materials and methods. In 1996–2001, 108 (age 5–13, mean 6.9), and in 2008–2010 231 (age 5–11, mean 7.9) children from Poland were tested by posturography and blood-lead concentration (B-Pb; medians 50 and 36 µg/L, respectively). The children were genotyped for ALAD ([i]RsaI, MspI[/i]) and VDR ([i]FokI, BsmI, TaqI[/i]). [b]Results[/b]. There were correlations between posture and B-Pb (sway area with closed eyes: rS=0.24, P

Background and Purpose: The increasing rate of opportunistic infections caused by Candida and other yeasts is becoming a major health concern worldwide. However, systematic data on the epidemiology and the yeast species infections in Malaysia is still limited. In this regard, the present research aimed to identify pathogenic yeasts utilizing an economically practical and easily available molecular technique and evaluate the prevalence of pathogenic yeasts in a Malaysian tertiary care hospital., Materials and Methods: Yeast isolates were collected from Sultan Abdul Halim Hospital, Kedah, Malaysia, from October 2020 to October 2021. Molecular identification of the isolates was performed by one enzyme-based polymerase chain reaction-restriction fragment length polymorphism method., Results: Candida albicans was the most prevalent species, accounting for 120 isolates (59%) in total. The most prevalent non- albicans Candida species were C. tropicalis (n=33, 16%), C. krusei (Pichia kudriavzevii) (n=12, 5.8%), C. glabrata (n=12, 5.8%), and C. parapsilosis (n=6, 3%). Other unusual Candida species were C. guilliermondii (2), C. metapsilosis (2), C. orthopsilosis (1), C. lusitaniae (1), C. rugosa (1), C. haemulonii (1), C. bracarensis (1), and C. dubliniensis (1). Moreover, Talaromyces marneffei (1), Kodamaea ohmeri (1), Cryptococcus neoformans (3), and Cryptococcus laurentii (1) were among the other yeasts identified., Conclusion: The Molecular technique used in this study identified 96% of isolates, including mixed species. According to the findings, the most prevalent species are C. albicans , C. tropicalis , C. krusei , and C. glabrata ., Competing Interests: There is no conflict of interest to declare., (Copyright: © 2021, Published by Mazandaran University of Medical Sciences on behalf of Iranian Society of Medical Mycology and Invasive Fungi Research Center.)

The Lactobacillus species by 16S Amplified Ribosomal DNA Restriction Analysis (16S-ARDRA) was identified. Lactobacilli are bacteria often found in foodstuffs of both animal and vegetable origins. On one hand, they play an important role in the food spoilage and, on the other hand, they are used as starter cultures in food fermentation processes. The species-specific identification by traditional biochemical methods is time consuming and not always fully effective. Therefore, more efficient techniques are searched for. We focused on rapid identification of Lactobacillus isolates from different habitats. Forty-nine collection strains and isolates belonging to the genus Lactobacillus were discriminated. ARDRA was carried out with two restriction endonucleases. For the comparison of similarity, the Jaccard coefficient and clustering by the unweighted pair group method with arithmetic averages (UPGMA) were used. The percentages of similarity between profiles varied from 22% to 100% (AluI) and from 27% to 100% (MspI). This method proved applicable to the differentiation of 10 species.

Reduced representation bisulfite sequencing (RRBS) enriches CpG-rich genomic regions using the MspI restriction enzyme-which cuts DNA at all CCGG sites, regardless of their DNA methylation status at the CG site-and enables the measurement of DNA methylation levels at 5% ~ 10% of all CpG sites in the mammalian genome. RRBS has been utilized in a large number of studies as a cost-effective method to investigate DNA methylation patterns, mainly at gene promoters and CpG islands. Here, we describe protocols for gel-free preparation of RRBS libraries, quality control, sequencing, and data analysis. Our protocols typically require nine cycles of polymerase chain reaction (PCR) amplification to obtain a sufficient amount of library for sequencing when 100 ng of genomic DNA is used as a starting material; moreover, it takes 3 days to complete library preparation and quality control procedures for up to eight samples., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The calpastatin (CAST) gene has a major effect on muscle growth and meat tenderness after slaughter; it is located on the fifth chromosome in sheep. Blood samples were collected from 720 animals in total from Kıvırcık (KIV), Sakız (SZ), Karacabey Merino (KM), and Gökçeada (GA) sheep populations raised in West Anatolia. The PCR products were digested by the restriction endonuclease MspI. Allele frequencies for M and N alleles of the gene were found to be 0.85 and 0.15 in KIV, 0.80 and 0.20 in KM, 0.99 and 0.01 in GA, and 0.34 and 0.66 in SZ sheep, respectively. It was determined that NN genotype frequency was quite lower in KIV (0.04) and KM (0.07) populations than in the SZ breed (0.40). On the other hand, the NN genotype was not observed in the GA population. Populations other than KM were found to be in Hardy-Weinberg equilibrium. In general, allele and genotype frequencies of CAST were similar to those in other studies on different sheep populations. This research is a beginning step for finding candidate genes' effects on meat quantity and quality. [ABSTRACT FROM AUTHOR]

Background DNA methylation plays a key role in development, contributes to genome stability, and may also respond to external stimuli supporting adaptation and evolution. To connect different types of stimuli with particular biological processes, identifying genome regions with altered 5-methylcytosine distribution at a genome-wide scale is important. Many researchers are using the simple, reliable, and relatively inexpensive Methylation Sensitive Amplified Polymorphism (MSAP) method that is particularly useful in studies of epigenetic variation. However, electrophoretic patterns produced by the method are rather difficult to interpret, particularly when MspI and HpaII isoschizomers are used because these enzymes are methylation-sensitive, and any C within the CCGG recognition motif can be methylated in plant DNA. Results Here, we evaluate MSAP patterns with respect to current knowledge of the enzyme activities and the level and distribution of 5-methylcytosine in plant and vertebrate genomes. We discuss potential caveats related to complex MSAP patterns and provide clues regarding how to interpret them. We further show that addition of combined HpaII + MspI digestion would assist in the interpretation of the most controversial MSAP pattern represented by the signal in the HpaII but not in the MspI profile. Conclusions We recommend modification of the MSAP protocol that definitely discerns between putative hemimethylated mCCGG and internal CmCGG sites. We believe that our view and the simple improvement will assist in correct MSAP data interpretation. [ABSTRACT FROM AUTHOR]

Background and Purpose: Invasive candidiasis is one of the most common systemic mycoses, and studies have shown mixed yeast infections. Malaysia lacks mixed yeast culture data., Materials and Methods: Yeast isolates were collected in Sultan Abdul Halim Hospital, North Malaysia, from October 2020 to October 2021. Chromogenic Candida differential agar media and PCR-RFLP were used to identify yeast species., Results: A total of 206 yeast isolates were collected from different body sites of patients. The majority of the yeast isolates (n=104) were obtained from the urine. Other isolates were extracted from blood (n=52), vaginal swabs (n=45), ear discharge (n=2), tracheal aspirate (n=2), tissue (n=2), skin (n=1), nail (n=1), sputum (n=1), and cerebrospinal fluid (n=1). In total, 200 yeast samples were identified as single species, and six isolates were a mixture of Candida species., Conclusion: Malaysia lacks accurate epidemiological data on mixed yeast infections. We identified all samples to the species level, including mixed yeast cultures, using the MspI enzyme and PCR-RFLP., Competing Interests: There is no conflict of interest., (Copyright: © 2021, Published by Mazandaran University of Medical Sciences on behalf of Iranian Society of Medical Mycology and Invasive Fungi Research Center.)

The Lactobacillus species by 16S Amplified Ribosomal DNA Restriction Analysis (16S-ARDRA) was identified. Lacto-bacilli are bacteria often found in foodstuffs of both animal and vegetable origins. On one hand, they play an important role in the food spoilage and, on the other hand, they are used as starter cultures in food fermentation processes. The species-specific identification by traditional biochemical methods is time consuming and not always fully effective. Therefore, more efficient techniques are searched for. We focused on rapid identification of Lactobacillus isolates from different habitats. Forty-nine collection strains and isolates belonging to the genus Lactobacillus were discriminated. ARDRA was carried out with two restriction endonucleases. For the comparison of similarity, the Jaccard coefficient and clustering by the unweighted pair group method with arithmetic averages (UPGMA) were used. The percentages of similarity between profiles varied from 22% to 100% (aluI) and from 27% to 100% (MspI). This method proved applicable to the differentiation of 10 species. [ABSTRACT FROM AUTHOR]

Abstract: Genetic engineering of Clostridium cellulolyticum has been developed slowly compared with that of other clostridial species, and one of the major reasons might be the restriction and modification (RM) system which degrades foreign DNA. Here, a putative MspI endonuclease gene, ccel2866, was inactivated by a ClosTron-based gene disruption method. The resulting C. cellulolyticum mutant H10ΔmspI lost the MspI endonuclease activity and can accept unmethylated DNA efficiently. Following that, an oxygen-independent green fluorescence protein gene was introduced into H10ΔmspI without methylation, generating a convenient reporter system to evaluate the expression of heterologous protein in C. cellulolyticum by green fluorescence. To further demonstrate the efficiency of the H10ΔmspI, double mutants H10ΔmspIΔldh and H10ΔmspIΔack were constructed by disrupting lactate dehydrogenase gene ccel2485 and acetate kinase gene ccel2136 in H10ΔmspI, respectively, without DNA methylation, and the stability of the double mutation was confirmed after the 100th generation. The mutant H10ΔmspI constructed here can be used as a platform for further targeted gene manipulation conveniently and efficiently. It will greatly facilitate the metabolic engineering of C. cellulolyticum aiming at faster cellulose degradation and higher biofuel production at the molecular level. [Copyright &y& Elsevier]

To identify a predictor to forecast superovulation response on the basis of associations between superovulation performance and gene polymorphism, the PCR-RFLP method was applied to detect an A>G transition determining an MspI polymorphism at position 192 in the exon I of the bovine inhibin alpha ( INHA) gene and evaluate its associations with superovulatory response in 118 Chinese Holstein cows treated for superovulation. Association analysis showed that cows with the GG genotype resulted in a significant increase in the number of ova (TNO) than AG and AA genotypes in the first ( P = 0.023), second ( P = 0.004) and third ( P = 0.002) superovulation treatments and produced more transferable embryos (NTE) than that of AG and AA genotypes in the third ( P = 0.045) superovulation treatment. Moreover, individuals with GG genotype produced more transferable embryos than AA ( P < 0.05) genotype in the second superovulation treatment and all cows without superovulation response were mutations with genotypes of AA and AG. These results indicate that INHA gene can be used as a predictor for superovulation in Chinese Holstein cows, and imply that cows with AA genotype should be excluded for superovulation practices. [ABSTRACT FROM AUTHOR]

A fragment of 570 bp corresponding to exon 5 and 6 of integrin beta 2 (ITGB2) gene was amplified for screening D128G mutation in one hundred and fifty two buffaloes (Bubalus bubalis) which causes bovine leukocyte adhesion deficiency syndrome (BLAD) in cattle, as well as to ascertain polymorphism. TaqI PCR-RFLP revealed no such mutation thus indicating the absence of bubaline leukocyte adhesion deficiency (BuLAD) allele in animals under study. However, the polymorphism studies using MspI restriction enzyme revealed two genotypic patterns viz. AA pattern (bands of 293, 141, 105, and 31 bp) and BB pattern (bands of 293, 105, 77, 64, and 31 bp). The sequences of A and B alleles were submitted to the GenBank (EU853307 and AY821799). [ABSTRACT FROM AUTHOR]

Four typical coastal sites (rocky shore, sandy shore, mud flat shore, and artificial harbor) at the Yellow Sea were chosen to investigate the spatial and seasonal variations in bacterial communities. This was accomplished by using terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR amplified 16S rDNA fragments. Two kinds of tetrameric restriction enzymes, HhaI and MspI, were used in the experiment to depict the bacterial community diversity in different marine environments. It was found that the community compositions digested by the two enzymes separately were different. However, the results of bacterial community diversity derived from them were similar. The MDA analysis results of T-RFLP profiles coming from HhaI and MspI both exhibited a significant seasonal community shift for bacteria and a relatively low spatial variation among the four locations. With HhaI as the sample, the pair wise T-tests also revealed that variations were minor between each pair of marine environments, with R ranging from 0.198 to 0.349. However, the bacterial community structure in the mud flat site depicted a larger difference than each of the other three sites ( R ranging from 0.282 to 0.349). [ABSTRACT FROM AUTHOR]

Abstract: Medical schools can assist students by providing them with quality career counseling to help them choose a medical specialty. Many schools use interest inventories to help identify students’ specialty interests. This study examined the predictive validity of one such inventory, the Medical Specialty Preference Inventory (MSPI). In a longitudinal design, we used discriminant function analysis to examine how well students’ scores on the MSPI fit their chosen medical specialty one year later. The MSPI correctly predicted students’ future medical specialty choice 58.1% of the time. These results can help career advisors interpret MSPI scores, and identify students’ most likely medical specialty choice, as well as their second most likely choice. [Copyright &y& Elsevier]

Abstract: The Medical Specialty Preference Inventory (MSPI; Zimny, G.H. (1979). Manual for the Medical Specialty Preference Inventory. St. Louis, MO: St. Louis University School of Medicine), a measure of medical students’ interests, was substantively and empirically examined to identify an underlying factor structure. A factor model for the original MSPI based on 38 factors in five general areas was evaluated on a national sample of 1014 medical students and yielded poor fit to the data. Exploratory factor analyses at the item level utilizing the full pool of MSPI items produced an 11 factor solution with 88 items. Sub-scales were identified within this model and an 11–18 higher-order model and an 18 sub-scale model also were proposed. The relative fits of the three models were evaluated by confirmatory factor analysis with the 18 sub-scale model shown to be superior. This model was cross-validated on a separate sample of 1016 medical students and fit the data well. All sub-scales exhibited adequate internal consistency across samples. These findings support the need for a revised MSPI based on 18 scales. Implications of these findings for MSPI scoring practices are discussed along with future directions. [Copyright &y& Elsevier]

Abstract: We explored the effects of particulate matter <10μm (PM10) exposure along with CYP1A1 polymorphisms of MspI (T6235C) and NcoI (Ile462Val) on reduced birth weight (BW). A prospective cohort study was done with women who delivered from 2001 to 2004 at Ewha Womans University Hospital, Seoul, Korea. We compared the estimated least squares means of BW in the generalized linear model, after adjusting for controlling factors. High PM10 exposure at the 90th percentile level and above during the 1st trimester conferred a significant risk for reduced BW, compared with low PM10 exposure below the 90th percentile level. The effect of high PM10 exposure during the 1st trimester of pregnancy compared with low PM10 exposure was greater for women with MspI TC/CC and NcoI IleVal/ValVal genotypes than for those with MspI TT and NcoI IleIle genotypes. In conclusion, high PM10 exposure during the 1st trimester increased the risk for reduced BW in concert with MspI TC/CC and NcoI IleVal/ValVal genotypes in Korean women. [Copyright &y& Elsevier]

Abstract: Changes in genomic DNA methylation are recognized as important events in normal and pathological cellular processes, contributing both to normal development and differentiation as well as cancer and other diseases. Here, we report a novel method to estimate genome-wide DNA methylation, referred to as LUminometric Methylation Assay (LUMA). The method is based on combined DNA cleavage by methylation-sensitive restriction enzymes and polymerase extension assay by Pyrosequencing™. The method is quantitative, highly reproducible and easy to scale up. Since no primary modification of genomic DNA, such as bisulfite treatment, is needed, the total assay time is only 6 h. In addition, the assay requires only 200–500 ng of genomic DNA and incorporates an internal control to eliminate the problem of varying amounts of starting DNA. The accuracy and linearity of LUMA were verified by in vitro methylated lambda DNA. In addition, DNA methylation levels were assessed by LUMA in DNA methyltransferase knock-out cell lines and after treatment with the DNA methyltransferase inhibitor (5-AzaCytidine). The LUMA assay may provide a useful method to analyze genome-wide DNA methylation for a variety of physiological and pathological conditions including etiologic, diagnostic and prognostic aspects of cancer. [Copyright &y& Elsevier]

The crystal structure of the Type IIP restriction endonuclease MspI bound to DNA containing its cognate recognition sequence has been determined in both monoclinic and orthorhombic space groups. Significantly, these two independent crystal forms present an identical structure of a novel monomer-DNA complex, suggesting a functional role for this novel enzyme-DNA complex. In both crystals, MspI interacts with the CCGG DNA recognition sequence as a monomer, using an asymmetric mode of recognition by two different structural motifs in a single polypeptide. In the crystallographic asymmetric unit, the two DNA molecules in the two MspI-DNA complexes appear to stack with each other forming an end-to-end pseudo-continuous 19-mer duplex. They are primarily B-form and no major bends or kinks are observed. For DNA recognition, most of the specific contacts between the enzyme and the DNA are preserved in the orthorhombic structure compared with the monoclinic structure. A cation is observed near the catalytic center in the monoclinic structure at a position homologous to the 74/45 metal site of EcoRV, and the orthorhombic structure also shows signs of this same cation. However, the coordination ligands of the metal are somewhat different from those of the 74/45 metal site of EcoRV. Combined with structural information from other solved structures of Type II restriction enzymes, the possible relationship between the structures of the enzymes and their cleavage behaviors is discussed. [ABSTRACT FROM AUTHOR]

In 180 unrelated Japanese individuals 18 examples of allele 27 were detected at the locus D1S80 (MCT118). On 6% polylacrylamide gels 5 out of these 18 alleles were found to migrate between allele 26 and allele 27, but closer to allele 27, and thus were labelled variants of allele 27. All 18 examples of allele 27 were sequenced and the results were compared. Although all had the same number of base pairs (578 bp) the five variants could be subdivided into three types V1, V2 and V3. The variants and the standard allele were composed of the same kinds of repeat units, but the order of arrangement was different. We investigated whether it was possible to distinguish the standard allele 27, and the variants V1, V2, and V3 by PCR-RFLP. EcoRII and MspI which have restriction sites within the repeat units were adopted as restriction enzymes. The variants could be discriminated from each other after treatment of the PCR fragments with EcoRII or MspI, followed by PAG electrophoresis. [ABSTRACT FROM AUTHOR]

The article presents a study that characterized and ascertained polymorphism in the exon 5 insulin like growth factor -1 (IGF-1) gene of mithun. The study showed a monomorphic pattern between the IGF system and birth weight. Mithun was used to amplify a 396 bp fragment of IGF-1 gene, which include complete exon 5.

• Potential of 3 bio-inspired algorithms to improve ANFIS's ability in drought prediction. • The algorithms of ACO, GA and PSO are implemented. • Three drought indexes PDSI, SPI and MSPI are considered. • ACO and GA had best performance, which could improve ANFIS 45.9% and 43.2% in order. Monitoring agricultural drought utilizing the least variables can be useful at the areas with just rain gauge stations, especially in Iran, which is located in arid belt of the world. In this study, all of the stations having long duration data in Iran (31 stations with more than 59 years' data), have been used to calculate three drought indices: (1) Palmer Drought Severity Index (PDSI); (2) Standardized Precipitation Index (SPI); (3) Multivariate Standardized Precipitation Index (MSPI). According to the recommendation of world meteorological organization (WMO), Palmer index has been defined as the reference index and SPI 9 (9-month time window of SPI), SPI 10 , SPI 11 & SPI 12 , and also MSPI 9–12 (9–12 months' time window), was evaluated by Palmer index. The results showed significant relation between SPI 11 or SPI 12 and PDSI in Iran's climates. MSPI 9 – 12 has a significant relation with PDSI too, and the correlation of MSPI with Palmer Index was found to be stronger than the SPI, in all stations. The relation between MSPI and PDSI is also stronger in extra-arid climates, and weaker in humid and per-humid areas. So, MSPI can be used for agricultural drought monitoring in the places where there are restrictions in meteorological dataset (when precipitation data are only available). In the second part, prediction of MSPI 9 – 12 has been done using Adaptive Neuro-Fuzzy Inference System (ANFIS) merged with bio-inspired optimization algorithms. The meta-heuristic models implemented are ANFIS-ACO (ANFIS merged with Ant Colony Optimization), ANFIS-GA (ANFIS merged with Genetic Algorithm) and ANFIS-PSO (ANFIS merged with Particle Swarm Optimization). The models had their best predictions in arid, semi-arid and Mediterranean climates while humid climate provided the weakest predictions. Among the mentioned algorithms, the ACO and GA had the best performances to optimize ANFIS; they improved the ANFIS's accuracy by 45.9% and 43.2%, respectively. [ABSTRACT FROM AUTHOR]

Overwhelming evidence suggests that in addition to the genetic changes of DNA mutations, epigenetic changes of DNA methylation and histone modifications play important role in regulation of gene expression. DNA methylation is the most frequent epigenetic alteration observed in mammalian genomes, and generally it is negatively correlated with gene expression. Various methods are available for the detection of DNA methylation changes. Although the recent high-throughput methods for DNA methylation analysis have various advantages, they require high levels of technical expertise, costly equipment, and reagents. Because of these reasons, many of the global DNA methylation analysis methods are mainly performed at core facility, and laboratories with limited resources and expertise are not able to use these methods. Methylation-Sensitive-Random Amplified Polymorphic DNA-Polymerase Chain Reaction (MS-RAPD-PCR) is a restriction enzyme digestion and PCR-based method for the analysis of DNA methylation changes. This method is cost-effective, requires simple and basic instrumentation, and therefore can easily be performed in any laboratory with basic setup having a regular DNA thermal cycler and DNA gel electrophoresis system. Additional advantages of this method over other methods for DNA methylation analysis are that it requires very less amount of DNA and can screen DNA methylation changes globally at genome-wide level with high sensitivity. This method has been successfully used to detect changes in DNA methylation either occurring naturally or induced by various toxicants and environmental factors. A detail experimental protocol for MS-RAPD-PCR is described in this chapter.

Here we describe a method for genome wide investigation of methylation and hydroxymethylation status of cytosines. This protocol is an improvement of the HELP-tagging protocol previously described by Suzuki et al. It involves the glucosylation of 5-hydroxymethylcytosines (5-hmC) with β-glucosyl transferase (β-GT), thus rendering them resistant to digestion by MspI. Parallel digestion of β-GT treated samples with MspI, untreated sample with MspI and another untreated sample with HpaII, followed by adapter ligation, parallel sequencing and bioinformatics processing results in a differential display of MspI digestion sites that allows the determination of the distribution of 5-methylcytosines (5-mC) and 5-hmC at these sites.