Forensic DNA evidence and the death penalty in the Philippines.

The death penalty remains a contentious issue even though it has been abolished in countries such as Australia, New Zealand, Canada, European Union member nations and some Asian countries such as Cambodia, East Timor and Nepal. Many argue that the irrevocability of the death penalty, in the face of potential erroneous convictions, can never justify its imposition. The Philippines, the first Asian country that abolished the death penalty in 1987, held the record for the most number of mandatory death offenses (30 offenses) and death eligible offenses (22 offenses) after it was re-imposed in 1994. Majority of death penalty convictions were decided based on testimonial evidence. While such cases undergo automatic review by the Supreme Court, the appellate process in the Philippines is not structured to accept post-conviction evidence, including DNA evidence. Because of the compelling nature of post-conviction DNA evidence in overturning death penalty convictions in the United States, different groups advocated its use in the Philippines. In one such case, People v Reynaldo de Villa, the defendant was charged with raping his 13-year-old niece that supposedly led to birth of a female child, a situation commonly known as 'criminal paternity'. This paper reports the results of the first post-conviction DNA test using 16 Short Tandem Repeat (STR) DNA markers in a criminal paternity case (People v Reynaldo de Villa) and discusses the implications of these results in the Philippine criminal justice system.

Evaluating DNA tests of motherless cases using a Philippine genetic database.

BACKGROUND: In 5 percent of paternity determination cases, only DNA samples from the alleged father and child pairs are tested. The absence of the mother's DNA increases the probability of false paternity inclusions, which affects laboratories that use a limited number of DNA markers. The effect of coincidental matches between unrelated individuals on DNA tests of motherless cases was determined using the Philippine population genetic database of the National Capital Region (NCR). STUDY DESIGN AND METHODS: Seven short tandem repeat (STR) markers were used, namely HUMvWA, HUMTH01, HUMCSF1PO, HUMFOLP23, D8S306, HUMFES/FPS, and HUMF13A01. Values of the probability of paternity with (W) and without a mother (W(-mother)) were determined using the equation W = cumulative likelihood ratio/cumulative likelihood ratio + 1). These values were determined for 50 volunteer families and compared with values calculated from randomly matched pairs in a reference NCR population database. RESULTS: The W and W(-mother) values of the 50 families range from 96.48 to 99.99 percent and 79.76 to 99.99 percent, respectively. In the NCR database, 195 coincidental matches in seven STR loci out of 5253 possible pairs (3.71%) were detected with W(-mother) values ranging from 12.47 to 99.83 percent. Of these, 53 and 10 random pairs have W(-mother) greater than 95.0 and 99.0 percent, respectively. CONCLUSION: W was higher than W(-mother) in the 50 families. However, the existence of unrelated individuals in the NCR database that randomly matched at seven STR loci and that has W(-mother) values greater than 99.0 percent highlights the need for greater precaution when dealing with motherless cases.

Characterization of Helicobacter felis by pulsed-field gel electrophoresis, plasmid profiling and ribotyping.

BACKGROUND: Helicobacter felis, an organism naturally infecting both canine and feline gastric mucosa, has been largely used as in animal models to study the ecology and treatment of human Helicobacter pylori infections. H. felis has not yet been studied at the genetic level. METHODS: The aims of this study were to modify an in situ DNA isolation method suitable for H. felis and, by the use of pulsed-field gel electrophoresis (PFGE), plasmid profiling, and ribotyping, to determine the degree of genetic variation among H. felis strains isolated from cats and dogs from various geographic locations, and to determine the genome size of H. felis. Furthermore, the ability of these new H. felis strains to colonize mice was tested. RESULTS: Most H. felis strains were distinguishable from each other, and 20 distinct PFGE types were detected. Four pairs of strains within a country and animal species produced identical patterns. All strains tested were found to carry several plasmids and plasmid profiling was equally discriminatory to PFGE. Ribotyping was not able to discriminate all the strains. CONCLUSIONS: The genome size of H. felis was found to be approximately 1.6 Mb.

Molecular characterization and interstrain variability of pHPS1, a plasmid isolated from the Sydney strain (SS1) of Helicobacter pylori.

The 5846-bp circular plasmid pHPS1 of Helicobacter pylori Sydney strain, SS1, was cloned, sequenced, and structurally characterized. The SS1 strain is widely used in animal studies of H. pylori infection. The sequence of pHPS1 revealed three open reading frames (ORFs), all of which are transcribed. Two ORFs encode putative plasmid replication proteins, RepA and RepB, similar to replicases resident on theta plasmids. In contrast, the function of ORF2 remains cryptic due to the absence of sequence similarity with any known protein in sequence databases. In addition, species specificity of these three coding regions was shown using DNA dot blot hybridization in 57 diverse clinical H. pylori isolates and 32 Helicobacter and Campylobacter strains. RepA appears to be the predominant plasmid replication protein of H. pylori and the deduced amino acid sequence was highly conserved (76-96%) in 8 H. pylori isolates, including SS1. RepB was detected in 3 H. pylori isolates examined in this study, 2 of which possess only the repB gene. Analysis of the protein sequences of these two replicases, together with previously characterized H. pylori plasmid replication proteins, supports the formation of a distinct class of H. pylori plasmid proteins. Moreover, comprehensive analysis of the whole genome sequence of H. pylori strain 26695, pHPS1, and other H. pylori plasmid sequences that are available revealed interesting insights as to the occurrence of plasmid-mediated recombination within H. pylori. Common regions between plasmids and chromosome sequences of H. pylori were identified in this study which could only have arisen by genetic recombination, thus providing the first line of evidence, albeit indirectly, of the contribution of H. pylori plasmids in generating an extensive genetic heterogeneity characteristic of this important gastroduodenal pathogen.

Perinatal brain iron deficiency increases the vulnerability of rat hippocampus to hypoxic ischemic insult.

Fetal brain iron deficiency occurs in human pregnancies complicated by diabetes mellitus or intrauterine growth retardation. Because neurocognitive deficits are more common in the offspring of these pregnancies, we tested the hypothesis that perinatal brain iron deficiency predisposes the neonatal hippocampus, a structure important for memory processing, to injury. Brain iron concentration was reduced by 45% in 45 neonatal rats by maternal dietary iron restriction during gestation. Right-sided neuronal injury in four hippocampal subareas was induced by hypoxic-ischemic insult (ipsilateral carotid artery ligation and subsequent hypoxia on postnatal d 7) and was quantified histochemically on d 8 by cytochrome c oxidase activity (n = 30), and on d 14 by Nissl staining (n = 15). Acute right-sided cytochrome c oxidase activity loss occurred in CA1 (P = 0.02), CA3c (P < 0.001) and dentate gyrus (P < 0.001) in the iron-deficient group, whereas only CA1 (P = 0. 003) was affected in the iron-sufficient group. Long-term right-sided Nissl substance loss occurred in CA1 (P = 0.001), CA3a,b (P < 0.001) and dentate gyrus (P = 0.008) in the iron-deficient group, but only in CA1 (P = 0.004) in the iron-sufficient group. No increase in right-sided free-iron staining was present in either group. Perinatal iron deficiency predisposes the neonatal hippocampus to a greater acute loss of neuronal metabolic activity after an hypoxic-ischemic event, suggesting compromised cellular energetics. The subsequently greater loss of hippocampal neuronal integrity suggests poorer recoverability after injury in the perinatal iron-deficient brain.

A novel method of extracting plasmid DNA from Helicobacter species.

BACKGROUND: Plasmids are extra-chromosomal DNA that may encode products that aid in virulence, pathogenesis, and the spread of antibiotic resistance among a wide spectrum of bacteria. Plasmids have been detected in Helicobacter pylori, H. felis, H. fennelliae, and H. cinaedi. However, no function has been attributed to the Helicobacter plasmids studied to date. Moreover, the characterization of plasmids in other Helicobacter species is an as yet unexplored area of research. Several laboratories have reported difficulties in the extraction and isolation of plasmid DNA from H. pylori and H. felis isolates due to the presence of large amounts of DNase, necessitating cumbersome and time-consuming purification steps. The development of a method for extracting plasmid DNA from Helicobacter species would be useful for future systematic studies of plasmids in this important group of microorganisms. MATERIALS AND METHODS: Eight H. pylori isolates, including the Sydney Strain SS1, three H. felis isolates, and one isolate each of H. hepaticus, H. bilis, H. mustelae, and H. rodentium, were screened for plasmid DNA using a novel method that includes a potassium xanthogenate-sodium dodecyl sulfate-phenol (XSP) buffer. A specific PCR targeting a highly conserved plasmid replication protein gene, repA, was used to confirm the presence of plasmids in the H. pylori isolates examined. The PCR primers used were designed based on the sequence of the H. pylori plasmid pHPM180. To demonstrate the effectiveness of this method, plasmid DNA extracted from SS1 using XSP buffer was digested using three restriction enzymes (DdeI, SpeI and MaeIII). The relative amount of DNA obtained using the protocol was also compared to the yield derived from four commercial kits commonly used in many laboratories. RESULTS: High and low molecular weight plasmids were extracted from H. pylori (n = 8) and H. felis (n = 3) isolates. The size range of these plasmids was from 3 kb to >16 kb. Attempts to isolate plasmids from H. hepaticus ATCC 51488, H. bilis ATCC 51630, H. rodentium MIT-95-2060, and H. mustelae NCTC 11574 were not successful, which was most likely due to the absence of endogenous plasmids from the strains examined. The relative amount of DNA obtained using the XSP buffer protocol was comparable to that obtained from commercial kits as assessed by direct examination of plasmid profiles on agarose gels. Plasmid DNA extracted from H. pylori SS1 using XSP buffer was successfully digested with restriction enzymes. CONCLUSION: This study reports the development of an efficient, inexpensive, and rapid method for extracting high and low molecular weight plasmids from Helicobacter species. Application of this novel method for the isolation and future characterization of plasmids from different Helicobacter species could promote a better understanding of the role of plasmids in the basic microbial physiology and ecology of this group of microorganisms.

A standardized mouse model of Helicobacter pylori infection: introducing the Sydney strain.

BACKGROUND & AIMS: Currently available Helicobacter pylori models show variable and, in some instances, poor colonization. There is a need for a strain with high colonizing ability to act as a standard for animal studies. METHODS: After screening a range of fresh clinical isolates and long-term adaptation in mice, a strain of H. pylon has been isolated with a very good colonizing ability. RESULTS: This strain, named the Sydney strain of H. pylori (strain SS1), is cagA and vacA positive. High levels of colonization (10(6)-10(7) colony-forming units/g tissue) were achieved consistently in C57BL/6 mice. Colonization levels varied depending on the mouse strain used with BALB/c, DBA/2, and C3H/He, all being colonized but in lower numbers. In all strains of mice, bacteria were clearly visible at the junctional zone between the antrum and the body. The phenotype was stable with colonizing ability remaining after 20 subcultures in vitro. The bacterium attached firmly to gastric epithelium. During 8 months, a chronic active gastritis slowly developed, progressing to severe atrophy in both C57BL/6 and BALB/c mice. CONCLUSIONS: The Sydney strain of H. pylori is available to all and will provide a standardized mouse model for vaccine development, compound screening, and studies in pathogenesis.

Immunization with glycoprotein C of equine herpesvirus-1 is associated with accelerated virus clearance in a murine model.

The glycoprotein C (gC) gene of equine herpesvirus-1 (EHV-1) was expressed in insect cells by a recombinant baculovirus as several products with apparent molecular weights of 66 kDa-80 kDa. The baculovirus EHV-1 gC products were recognised by monoclonal antibody and by EHV-1 convalescent equine sera, indicating conservation of antigenic determinants and confirming this glycoprotein as a target for the equine immune system. Mice immunized with recombinant EHV-1 gC showed accelerated clearance of EHV-1 from respiratory tissues following intranasal challenge. Virus clearance was accompanied by virus specific antibodies and by cell mediated immune responses measured by a delayed type hypersensitivity reaction and lymphocyte stimulation by killed EHV-1 as antigen.