Detection of p16 Hypermethylation in Gastric Carcinomas Using a Seminested Methylation-Specific PCR

Aberrant DNA methylation of a CpG site is among the earliest and most frequent alterations in various tumors including gastric carcinoma. The aim of this study is to detect tumor-associated aberrant hypermethylation of the p16 gene from 60 gastric tumor and corresponding normal tissues using a seminested methylation-specific PCR (MSP). The results indicated that hypermethylation of the p16 gene could be detected in 80% (48/60) of the gastric tumor samples from the first PCR. However, the frequency increased significantly to 86.7% (52/60) of the gastric tumor samples after the second PCR. These results show that this technique increases the sensitivity of detecting p16 hypermethylation from tumor samples. Furthermore, the aberrant methylation of p16 was observed in all of the stages, confirming that this epigenetic alteration is an early event during gastric carcinogenesis. Clinicopathologic parameters such as age, sex, and histological differentiation of GC were not significantly associated with the methylation status.     

A simple and improved PCR-based technique for white-tailed deer (<Emphasis Type="Italic">Odocoileus virginianus</Emphasis>) sex identification

We describe a simple single-reaction technique for identifying the sex of white-tailed deer (Odocoileus virginianus) based on the PCR amplification of a zinc-finger intron using one pair of primers. Although Sry-coamplification confirmed sex identities, use of the Sry marker was unnecessary due to dimorphic alleles on the X and Y chromosomes at the zinc-finger locus. Insertions in intron 7 of the Y-linked allele (417 bp) make it nearly twice as long as the X-linked allele (236 bp) and thus the amplification products are easily discernable by simple agarose gel electrophoresis. The relatively short size of these products makes them useful for DNA-based sex identification from potentially low-yield tissue samples (e.g., hair, feces). This technique will provide ecologists, conservation geneticists and wildlife managers with a mechanism to readily and reliably identify the sex of unknown white-tailed deer tissue samples, and likely similar samples from other cervid species.     

Molecular identification of crocodile species using novel primers for forensic analysis

All crocodilians are under varying degrees of threat due to over exploitation and these species have been listed in Appendix I or II of CITES. The lack of molecular techniques for the identification of confiscated samples makes it difficult to enforce the law. Conclusive forensic identification of species requires a complete gene sequence which is difficult in case of degraded samples. We have developed two novel sets of primers to amplify two partial cytochrome b gene sequences of six crocodile species i.e. Crocodylus palustris, Crocodylus porosus, Crocodylus siamensis, Crocodylus niloticus, Gavialis gangeticus and Caiman crocodilus. These partial sequences were edited to give a complete cyt b gene sequence, which can be used as an effective tool for forensic authentication of crocodile species. A phylogeny of crocodile species was reconstructed using these sequences. The described primers hold great promise in forensic identification of crocodile species, which can aid in the effective enforcement of law and conservation of these ancient species.     

Sex determination of human skeletal populations using latent profile analysis

Accurately estimating biological sex from the human skeleton can be especially difficult for fragmentary or incomplete remains often encountered in bioarchaeological contexts. Where typical anatomically dimorphic skeletal regions are incomplete or absent, observers often take their best guess to classify biological sex. Latent profile analysis (LPA) is a mixture modeling technique which uses observed continuous data to estimate unobserved categorical group membership using posterior probabilities. In this study, sex is the latent variable (male and female are the two latent classes), and the indicator variables used here were eight standard linear measurements (long bone lengths, diaphyseal and articular breadths, and circumferences). Mplus (Muthén and Muthén: Mplus user's guide, 6th ed. Los Angeles: Muthén & Muthén, 2010) was used to obtain maximum likelihood estimates for latent class membership from a known sample of individuals from the forensic data bank (FDB) (Jantz and Moore‐Jansen: Database for forensic anthropology in the United States 1962–1991, Ann Arbor, MI: Interuniversity Consortium for Political and Social Research, 2000) (n = 1,831), yielding 87% of correct classification for sex. Then, a simulation extracted 5,000 different random samples of 206 complete cases each from the FDB (these cases also had known sex). We then artificially imposed patterns of missing data similar to that observed in a poorly preserved bioarchaeological sample from Medieval Asturias, Spain (n = 206), and ran LPA on each sample. This tested the efficacy of LPA under extreme conditions of poor preservation (missing data, 42%). The simulation yielded an average of 82% accuracy, indicating that LPA is robust to large amounts of missing data when analyzing incomplete skeletons. Am J Phys Anthropol 151:538–543, 2013. © 2013 Wiley Periodicals, Inc.     

Sex identification of Eurasian otter (<Emphasis Type="Italic">Lutra lutra</Emphasis>) non-invasive DNA samples using ZFX/ZFY sequences

We developed a new PCR/RFLP system targeted to amplify and cut a segment of the ZFX/ZFY gene in non-invasive otter (Lutra lutra) samples. This assay produced one sex-specific fragment in females (XX genotypes) and two fragments in males (XY genotypes), and is intrinsically more reliable than alternative systems (e.g., SRY genes) that are based on the amplification of a single Y-linked fragment. The new primer pair correctly identified the sex of 23 DNA extracted from sexed otter tissues. This procedure was used to sex unknown DNA extracted from otter scats. A multi-tube approach led to identify the sex of 72/91 (79%) samples, using a minimum of two PCR replicates. This procedure is currently used in non-invasive genetic monitoring of Italian otter populations.     

Mandibular ramus flexure and gonial eversion as morphologic indicators of sex

Recently, two mandibular traits--ramus flexure and gonial eversion--have come under close scrutiny (Loth & Henneberg 1996, 2000). The present study investigates the reliability of these two traits when each is applied as a single and independent indicator of sex, including the question of repeatability. The investigation was designed to give insights into possible confounding factors such as age and remodeling after tooth loss. Two samples, one of forensic (N = 153) and one of archaeological provenance (N = 80), were examined. The forensic sample was evaluated by a single observer while the archaeological sample was independently scored by three different observers. The results document that age and localized tooth loss seriously reduce the accuracy of these traits. For ramus flexure, male accuracy was only 66%, while female accuracy was even lower (32%). Overall accuracy was 59%. It is believed that the original scoring system devised by Loth and Henneberg (1996) creates an inherent bias in favor of males. For gonial eversion, a similar picture emerged (75.4% for males, 45.2% for females and 69.3% overall accuracy). Furthermore, both indicators are prone to intra- as well as inter-observer bias. While both possess some merit as sex indicators, they show marked functional and adaptive responses and may not be suitable for all samples.     

DNA extraction from processed wood: A case study for the identification of an endangered timber species (Gonystylus bancanus)

We applied human forensic techniques to the extraction of whole genomic DNA from processed wood samples to explore the possibility of identifying an endangered tropical timber species by using DNA sequencing technology. High-yield and high-quality DNA samples were obtained from 2 commercial wood and 3 herbarium samples. Large PCR fragments ranging from 500–800 bp were successfully amplified from 2 chloroplast and 1 mitochondrial regions in all 5 samples, indicating limited degradation of the cytoplasmic genomes. DNA extraction from stem wood taken from herbarium specimens appeared superior to that from stem wood with bark intact or from leaf samples. DNA sequences from thetrn regions allowed for easy identification of the focal species based on GenBank Blast search. Little sequence variation was observed in the 3 regions, with the mitochondrialcox3 region completely conserved. Extraction of high-quality and large intact DNA fragments makes dry wood materials amenable to various DNA marker-based applications, including fingerprinting and historical approaches. By sampling stemwood, the wealth of historical information housed in international herbaria can be explored with minimal damage to taxonomically important features.     

Application of Short Tandem Repeat markers in diagnosis of chromosomal aneuploidies and forensic DNA investigation in Pakistan

Purpose

Short Tandem Repeat (STR) genetic markers hold great potential in forensic investigations, molecular diagnostics and molecular genetics research. AmpFlSTR® Identifiler™ PCR amplification kit is a multiplex system for co-amplification of 15 STR markers used worldwide in forensic investigations. This study attempts to assess forensic validity of these STRs in Pakistani population and to investigate its applicability in quick and simultaneous diagnosis and tracing parental source of common chromosomal aneuploidies.

Methodology

Samples from 554 healthy Pakistani individuals from 5 different ethnicities were analyzed for forensic parameters using Identifiler STRs and 74 patients' samples with different aneuploidies were evaluated for diagnostic strengths of these markers.

Results

All STRs hold sufficient forensic applicability in Pakistani population with paternity index between 1.5 and 3.5, polymorphic information content from 0.63 to 0.87 and discrimination power ≥ 0.9 (except TPOX locus). Variation from Hardy–Weinberg equilibrium was observed at some loci reflecting selective breeding and intermarriages trend in Pakistan. Among aneuploidic samples, all trisomies were precisely detectable while aneuploidies involving sex chromosomes or missing chromosomes were not clearly detectable using Identifiler STRs. Parental origin of aneuploidy was traceable in 92.54% patients.

Conclusion

The studied STR markers are valuable tools for forensic application in Pakistan and utilizable for quick and simultaneous identification of some common trisomic conditions. Adding more sex chromosome specific STR markers can immensely increase the diagnostic and forensic potential of this system.     

Isolation of microbial pathogens of subclinical mastitis from raw sheep's milk of Epirus (Greece) and their role in its hygiene

The natural raw milk microflora is a factor that expresses its sensorial characteristics. The microbial charge into the mammary gland of healthy animal is low and the application of right and healthy conditions during milking and cheese making procedure, prevents from contaminating as well as maintains the natural microflora in order to lend the particular characteristics of milk.The purpose of the present project was the study of the Total Viable Count (T.V.C.) and the count of total psychrotropic bacteria of raw sheep milk from Boutsiko and Karamaniko breeds, collected from healthy animals, as well as the isolation, identification and enumeration of pathogenic bacteria related with the hygiene and the quality of raw sheep milk (with a particular interest in bacteria that may cause human infection).During the experiment we examined two hundred forty (240) samples of raw sheep milk. In these samples a) Staphylococcus aureus, Salmonella sp., Escherichia coli, Clostridium perfringens (vegetative cells and spores) and Bacillus sp. were isolated and identified b) the Total Viable Count and the total number of psychrotropic bacteria were also specified. The sampling, the preparation of samples and decimal dilutions were based on international methods. The Total viable count was determined using the standard methods of the American Public Health Association, 2002. The total number of psychrotropic bacteria was determined using APHA 1976, 1978 rules. The identification of the bacteria was carried out according to the Bergey’s manual. Microscopic examination of Gram stained cells, catalase, oxidase and biochemical tests were performed when necessary to further identify.From the 240 milk samples tested, only 5% were E. coli positive, with mean counts ranged from 2 × 10³ to 2.4 × 10⁴ cfu/ml. S. aureus was isolated from 24% of the samples and the mean count per ml was ranged from <10 to 3.4 × 10². Meanwhile, Bacillus spp. was also detected in 29% samples. Vegetative forms and spores of C. perfringens were detected in 13% and 63% of the samples respectively. However, microbiological analyses revealed the presence of a small number of selected pathogens in milk samples such as Salmonella, which was only detected in 5% of the samples. Listeria sp., Pseudomonas sp. and Vibrio cholerae were never found.From the experimental results, the Total Viable Count from raw sheep milk samples, fulfils the microbiological criteria of EU Legislation in a percentage of approximately 97%.     

Gender variation inSilene acaulis (Caryophyllaceae)

Silene acaulis (Caryophyllaceae) is an alpine-arctic plant with a gynodioecious breeding system, but significant variation in sex expression has been reported. In addition, population sex ratio may be modified by the anther-smutMicrobotryum violaceum (Pers.)Deml & Oberwinkler, which sterilizes individuals of both sexes. A survey was undertaken at several sites on Baffin Island, Canada, to determine sex ratio and assess variation in female function among morphologically hermaphroditic individuals. The degree of anthersmut infection was also measured. Six sites had high female frequencies ranging from 72–80% and < 2% smut infection. High female frequencies may indicate cytoplasmic control of male-sterility. A seventh site from a mesic habitat had only 50% females and a higher rate of smut infection (22%). Of the three sites studied in detail, 84% of females set at least one capsule compared to only 25% of the hermaphrodites, indicating reduced female function. Hermaphrodites displayed significant variability in female function. Flowers with short styles (< 4.0mm) had degenerated ovules, and field estimates confirmed that only 5% of these individuals set capsules, but comprised the majority (> 55%) of hermaphrodites. Although hermaphrodites with short-styled flowers functioned solely as males, there was no increase in pollen production compared to long-styled hermaphrodites. Long-styled hermaphrodites produced the same number of ovules as females, and all set at least one capsule but these plants were uncommon (< 11%) at all sites.     

Linkage Arrangement of RFLP loci in progenies from crosses between doubled haploid Asparagus officinalis L. clones

A preliminary genetic map of the dioecious species Asparagus officinalis L. (2n = 20) has been constructed on the basis of restriction fragment length polymorphism (RFLP) and isozyme marker data. With DNA samples digested with either EcoRI or HindIII 61 out of 148 probes (41%) identified RFLPs in six families of doubled haploid lines obtained through anther culture. A higher level of polymorphism (65%) was observed when a single family was screened for RFLPs using six distinct restriction enzymes. Segregation analysis of the BC progenies (40–80 individuals) resulted in a 418-cM extended map comprising 43 markers: 39 RFLPs, three isozymes and one morphological (sex). These markers are clustered in 12 linkage groups and four of them exhibited significant deviations from the expected 11 ratio. One isozyme and three RFLP markers were assigned to the sex chromosome.     

Molecular identification of dolphinfish species (genus Coryphaena) using multiplex haplotype-specific PCR of mitochondrial DNA

A rapid and reliable method of dolphinfish species identification was designed based on PCR amplification of diagnostic DNA fragments from the mitochondrial cytochrome b gene. It consisted in a tetraplex reaction producing a positive control amplicon and species-specific fragments in Coryphaena hippurus and C. equiselis. It was successfully tested in specimens of known identity and in nominal C. hippurus samples among which two C. equiselis were discovered. This approach has significant advantages over other molecular species identification methods and may help in determining species composition of mixed catches, and in forensic and food control applications of dolphinfish specimens or products.     

Dispersal and mating structure of a parasitoid with a female-biased sex ratio: Implications for theory

Summary Species of parasitic Hymenoptera that manifest female-biased sex ratios and whose offspring mate only with the offspring of the natal patch are assumed to have evolved biased sex ratios because of Local Mate Competition (LMC). Off-patch matings, i.e. outcrossing, are inconsistent with the conditions favouring biased sex ratios because they foster a mating structure approaching panmixia. Such a mating structure favours parents who invest equally in daughters and sons, assuming the production of each sex is of equal cost.Pachycrepoideus vindemiae (Rondani) is a solitary pupal parasitoid of patchily distributed frugivorousDrosophila, whose offspring manifest a female-biased sex ratio. Thus this species appears to manifest a population structure and progeny sex ratio consistent with LMC. However, preliminary observations and subsequent greenhouse experiments suggest that the males participate in off-patch matings and that this propensity is unlikely to be an experimental artefact. FemaleP. vindemiae dispersed from patches in which either the males were lacking (12% of the emigrant females), both resident (sibling) and immigrant males were present (23% of the females), only immigrant males were present (14% of the females), or their opportunity to mate could not be determined (14% of the females). Of the 12% that emigrated from a patch lacking males, an estimated 7% mated at an oviposition site and 5% remained unmated, presumably because they arrived at an oviposition site that lacked males before they were dissected to determine whether they were inseminated. Thus the degree of bias in the sex ratios of the progeny (18% males), coupled with the suggested outcrossing potential from the experiments (26–37%), is inconsistent with the assumptions of LMC or variants of it, i.e. asynchronous brood maturation. Thus the explanation for a biased sex ratio in the offspring ofP. vindemiae remains a conundrum. More importantly,P. vindemiae does not appear to be an isolated example.     

Pooling hair samples to increase DNA yield for PCR

Hairs are useful non-invasive sources of DNA, but the DNA yield can be very small, thus promoting genotyping errors. Using multiple hairs can counter this problem, but may introduce multiple contributors to a sample if collected remotely. With microsatellite genotyping, samples representing multiple animals are obvious if three or more alleles are detected at any locus: these samples can then be removed from any analyses. However, some multiple-individual samples may have only one or two alleles at each of the loci examined. We investigated the probability of failing to identify mixed pooled samples by simulating pooled samples (10 000 replicates) from microsatellite data from the northern and southern hairy-nosed wombats (NHNW, Lasiorhinus krefftii; SHNW, L. latifrons), species with low and high genetic diversity respectively. The majority (81.7%) of the 40 000 simulated samples had three or four alleles, so were readily identified as mixed. In the remaining 1-or-2-allele SHNW samples, forensic science software (DNAMIX) correctly identified mixed versus single-individual samples for all cases when the probability of locus failure was low (P _(LF) = 0.1), and 99% of samples when locus failure was high (P _(LF) = 0.5). For NHNW however, the probability of failing to identify a mixed sample was too high for population size estimation (0.05), even when the probability of locus failure was low. In cases such as this, pooled samples may be adequate for less demanding tasks, such as estimation of allele proportions. However, for animal populations with at least average levels of genetic variation, pooling of samples could safely be utilised for most applications. This revised version was published online in July 2006 with corrections to the Cover Date.     

Parasitism rates and sex ratios of a parasitoid wasp: effects of herbivore and plant quality

Summary We studied interactions among collards, Brassica oleracea var. acephala, the diamondback moth (DBM), Plutella xylostella (Lepidoptera: Yponomeutidae) and its parasitoid Diadegma insulare (Hymenoptera: Ichneumonidae) by manipulating plant nitrogen (N) concentrations in field and laboratory experiments. Parasitoid abundance strongly reflected DBM abundance and was related to total leaf N. Parasitism rates were high (70.7%) and density-independent. Wasp sex ratios varied markedly (3–93% female) in response to the herbivores, the plants, or both. Higher proportions of female wasps emerged from DBM larvae on plants with high leaf N than on unfertilized plants. More female wasps also emerged from larvae parasitized as larger instars. We suggest that wasps have the potential to control DBM populations through long-term numerical responses mediated by variable sex ratios.     

Interactions between Chlamydia pneumoniae and trace elements: a possible link to aortic valve sclerosis

An association between Chlamydia pneumoniae and atherosclerotic cardiovascular diseases has been suggested. However, other factors may interact in the pathogenesis of valve sclerosis. Therefore, trace elements important for C. pneumoniae growth and host defense and markers of C. pneumoniae infection were studied in sclerotic valves and serum. Forty-six patients undergoing surgical valve replacement due to advanced aortic sclerosis were prospectively studied. Valves from 15 forensic cases with no heart valve disease and plasma from 46 healthy volunteers served as controls. C. pneumoniae was detected in 16/46 (34.8 %) sclerotic valves and in 0/15 forensic controls. IgG and IgA antibodies to C. pneumoniae were present in 54.3% and 26.1 % patients, respectively. In the patients’ valves, iron, magnesium, and zinc each correlated to calcium, a marker of the histopathological severity of disease. Patients showed 10- to 70-fold increases of these trace elements in valves and an increased copper/zinc ratio in serum. In a majority of aortic sclerosis patients, one of several markers of C. pneumoniae infection were detected and all patients had a disturbed trace element balance in valves and serum suggestive of active immune process and infection. The pattern of trace element changes was essentially similar regardless of positive makers of C. pneumoniae, suggesting a similar etiopathogenesis in both subgroups. The 20-fold increase in iron, essential for C. pneumoniae growth, in sclerotic valves suggests a new possible link to this infection in aortic sclerosis.     

Sex-biases in the hatching sequence of cooperatively breeding apostlebirds <Emphasis Type="Italic">Struthidea cinerea</Emphasis>

In the cooperatively breeding apostlebird (Struthidea cinerea, Corcoracidae) both sexes are philopatric and help to raise offspring. However, male helpers provision nestlings more often than females, an activity associated with reduced nestling starvation and enhanced fledgling production. Presuming that males are the more helpful sex, we examined the helper repayment hypothesis by testing the predictions that offspring sex ratio should be skewed toward the production of males (a) among breeding groups with relatively few helpers, and (b) in the population as a whole. The relationship between sex and hatching order was examined as a potential mechanism of biasing sex allocation. The sex ratio of all sexed offspring was male biased (57.9%; n = 171) as was the mean brood sex ratio (0.579; n = 70 broods). These biases were less pronounced in the subset of clutches/broods in which all offspring were sexed. This overall bias appeared to result from two distinct patterns of skew in the hatching order. First, mothers in small breeding groups produced significantly more males among the first-hatching pair. This is consistent with the helper repayment hypothesis given that later hatching chicks were less likely to survive, particularly in small groups. Second, almost all fourth-hatching chicks, usually the last in the brood, were male (91.7%, n = 12). This bias is difficult to interpret but demonstrates the value of examining hatching sequences when evaluating specific predictions of sex allocation theory in birds.     

<Emphasis Type="Italic">Sarcocystis</Emphasis> sp. in red foxes (<Emphasis Type="Italic">Vulpes vulpes</Emphasis>) from northern Croatia

The prevalence of Sarcocystis sp. was assessed in foxes from northern Croatia (city of Varadin). The intestine of 50 (29 male and 21 female) foxes aged 1–5 years, killed during the rabies control campaign, were examined. Sarcocystis sp. was identified in 62% of the fox intestinal samples examined. There was no sex difference in the rate of invasion. However, the rate of invasion differed significantly between the animals aged 1 year (47%), and those aged 2 (71%) and 3–5 years (71%).