Temporal Temperature Gradient Electrophoresis Research Articles

Estructura de la comunidad bacteriana en diferentes tejidos de Lobatus gigas silvestres (Linnaeus, 1758) de la Reserva de Biosfera Seaflower del Caribe

The microbial diversity of Lobatus gigas has not been thoroughly studied despite of them is a specie endangered. Knowledge of microbiota may help to improve the conservation and cultivation of this species. The objective of this study was to evaluate the bacterial populationsassociated with the gonad and the gut compartments of the wild endangered L. gigas from the Caribbean Seaflower Biosphere Reserve, using microbiological methods and culture-independent molecular tools. The genetic profiles of the bacterial populations were generated and Temporal Temperature Gradient Electrophoresis (TTGE) was used to compare them with total DNA. A genetic and statistical analysis of the bacterial communities revealed a low level of diversity in gonad tissue based on the number of bands detected using TTGE. In addition, statistical differences in bacterial community structure were found between the foregut and hindgut tissue of L. gigas. The dominant phylogenetic affiliations of the gonad bacteria, as determined using 16S rRNA gene sequencing, belong to Ralstonia (50%). The possible involvement of this genus in the reproduction and development of the conch is discussed. On the other hand, the bacterial phylotypes from foregut and hindgut included members of Alphaproteobactera (12.5%), Betaproteobacteria (12.5%), Gammaproteobacteria (12.5%), Bacilli (31.25%), Clostridia (6.25%), Actinobacteria (6.25%), Mollicutes (6.25%) and Deinococci (6.25%) classes. Knowing the composition of the gonad and foregut and hindgut bacteria of L. gigas is the first step toward exploring the proper management of this species, as well as provides useful information to future researches that allow a better understanding of the role of these bacterial populations in the health and reproductive rate of L. gigas.

Characterization of lactic acid bacterial communities associated with a traditional Colombian cheese: Double cream cheese

Culture-dependent and Independent methods such as temporal temperature gradient electrophoresis (TTGE) and evaluation of the technological properties of five strains representing isolated groups, were used to evaluate the bacterial composition of three traditional double cream cheese companies in Colombia-South America. The results showed that fat, protein, moisture, and titratable acidity were significantly different between cheeses. Genetic analysis of these bacterial communities revealed a low level of diversity, based on the TTGE analysis. The dominant phylogenetic affiliations were grouped into four genera Enterococcus, Lactobacillus, Leuconostoc, and Lactococcus, related to acidification processes and formation of aromas and flavours in the cheese. The characterization of lactic acid bacteria (LAB) isolates showed predominance of the genera Leuconostoc and Weissella. These bacteria are related to the development of certain features such as texture and production of antimicrobial metabolites. In the evaluation of technological properties Weissella viridescens and Weissella paramesenteroides displayed greater pH reduction at 24 h, Pediococcus acidilactici, Leuconostoc mesenteroides and Leuconostoc citreum presented diacetyl production. The combination of different approaches allowed inferring the presence of a dominant population of LAB present in this cheese, this results complements the information needed for the establishment of a product protected under the status of Designation of Origin.

Diversity of the Bacterial Microbiota of Anopheles Mosquitoes from Binh Phuoc Province, Vietnam.

The naturally acquired microbiota of Anopheles can influence vector’s susceptibility to Plasmodium and its capacity to transmit them. Microbiota modification is a new challenge to limit disease transmission but it still needs advanced knowledges on bacterial community in Anopheles, especially in wild and infected specimens from diverse origin and species. Bacterial culture and 16S rRNA gene-PCR associated to Temporal Temperature Gradient Electrophoresis (TTGE) were applied to explore the bacterial diversity in the abdomen of 100 wild specimens (eight Anopheles species) collected in the Binh Phuoc Province, Vietnam. Culture and PCR-TTGE were complementary. The bacterial richness of the mosquito collection encompassed 105 genera belonging to seven phyla, mostly Proteobacteria, Firmicutes, and Actinobacteria. Staphylococcus, Clostridium, and Bacillus in Firmicutes were the most prevalent genera. However, Proteobacteria represented by 57 genera was the most diversified phylum in Anopheles microbiota. The high overall of Anopheles-associated bacteria is confirmed with, to our knowledge, 51 genera described for the first time in Anopheles microbiota. However, the diversity per specimen was low with average diversity index and the average Shannon–Wiener score (H) of 4.843 and 5.569, respectively. The most represented bacterial genera were present in <30% of the specimens. Consequently, the core microbiota share by Anopheles from Binh Phuoc was very narrow, suggesting that Anopheles microbiota was greatly influenced by local environments. The repertory of bacterial genera in two specimens of An. dirus and An. pampanai naturally infected by Plasmodium vivax was also described as preliminary results. Finally, this study completed the repertory of bacteria associated to wild Anopheles. Anopheles associated-bacteria appeared specimen-dependent rather than mosquitoe species- or group-dependent. Their origin and the existence of Anopheles-specific bacterial taxa are discussed.

Characterization of bacterial diversity associated with calcareous deposits and drip-waters, and isolation of calcifying bacteria from two Colombian mines

Bacterial carbonate precipitation has implications in geological processes and important biotechnological applications. Bacteria capable of precipitating carbonates have been isolated from different calcium carbonate deposits (speleothems) in caves, soil, freshwater and seawater around the world. However, the diversity of bacteria from calcareous deposits in Colombia, and their ability to precipitate carbonates, remains unknown. In this study, conventional microbiological methods and molecular tools, such as temporal temperature gradient electrophoresis (TTGE), were used to assess the composition of bacterial communities associated with carbonate deposits and drip-waters from two Colombian mines. A genetic analysis of these bacterial communities revealed a similar level of diversity, based on the number of bands detected using TTGE. The dominant phylogenetic affiliations of the bacteria, determined using 16S rRNA gene sequencing, were grouped into two phyla: Proteobacteria and Firmicutes. Within these phyla, seven genera were capable of precipitating calcium carbonates: Lysinibacillus, Bacillus, Strenotophomonas, Brevibacillus, Methylobacterium, Aeromicrobium and Acinetobacter. FTIR and SEM/EDX were used to analyze calcium carbonate crystals produced by isolated Acinetobacter gyllenbergii. The results showed that rhombohedral and angular calcite crystals with sizes of 90μm were precipitated. This research provides information regarding the presence of complex bacterial communities in secondary carbonate deposits from mines and their ability to precipitate calcium carbonate from calcareous deposits of Colombian mines.

On the bacterial communities associated with the corrosion product layer during the early stages of marine corrosion of carbon steel

Carbon steel coupons were immersed for 1–8 weeks at ∼1 m deep in a French harbor of the Atlantic coast. The resulting corrosion product layers were characterized by μ-Raman spectroscopy. They consisted of an inner stratum of sulfated green rust covered by an outer stratum of lepidocrocite (predominantly), goethite and magnetite. Mackinawite FeS was detected after one month of immersion, but only locally. The bacterial characterization of these layers was coupled to this analysis. The diversity of the culturable bacteria grown in liquid media directly from biofilms or isolated on agar plates was evaluated by 16S rRNA gene sequencing. The microbial diversity was also estimated, without cultivation and after Temporal Temperature Gradient Electrophoresis (TTGE) separation. A moderate biodiversity was found in all cases and different bacteria associated with the redox cycles of Fe and S were identified. Roseobacter, Erythrobacter and Bacillus were dominant among culturable bacteria whereas Sulfurimonas autotrophica, a sulfur-oxidizing bacterium, was detected among the total bacterial community at all immersion times. After one month of immersion, sulfide-producing bacteria were detected, e.g. Desulfovibrio profundus in liquid mixed cultures and Sulfurospirillum arcachonense in the total bacterial community, in agreement with the local identification of FeS by μ-Raman spectroscopy.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Allelic Variants Relate to Shifts in Faecal Microbiota of Cystic Fibrosis Patients

IntroductionIn this study we investigated the effects of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene variants on the composition of faecal microbiota, in patients affected by Cystic Fibrosis (CF). CFTR mutations (F508del is the most common) lead to a decreased secretion of chloride/water, and to mucus sticky secretions, in pancreas, respiratory and gastrointestinal tracts. Intestinal manifestations are underestimated in CF, leading to ileum meconium at birth, or small bowel bacterial overgrowth in adult age.MethodsThirty-six CF patients, fasting and under no-antibiotic treatment, were CFTR genotyped on both alleles. Faecal samples were subjected to molecular microbial profiling through Temporal Temperature Gradient Electrophoresis and species-specific PCR. Ecological parameters and multivariate algorithms were employed to find out if CFTR variants could be related to the microbiota structure.ResultsPatients were classified by two different criteria: 1) presence/absence of F508del mutation; 2) disease severity in heterozygous and homozygous F508del patients. We found that homozygous-F508del and severe CF patients exhibited an enhanced dysbiotic faecal microbiota composition, even within the CF cohort itself, with higher biodiversity and evenness. We also found, by species-specific PCR, that potentially harmful species (Escherichia coli and Eubacterium biforme) were abundant in homozygous-F508del and severe CF patients, while beneficial species (Faecalibacterium prausnitzii, Bifidobacterium spp., and Eubacterium limosum) were reduced.ConclusionsThis is the first report that establishes a link among CFTR variants and shifts in faecal microbiota, opening the way to studies that perceive CF as a ‘systemic disease’, linking the lung and the gut in a joined axis.

Inoculation of PAH-degrading strains of Fusarium solani and Arthrobacter oxydans in rhizospheric sand and soil microcosms: microbial interactions and PAH dissipation

Very little is known about the influence of bacterial-fungal ecological interactions on polycyclic aromatic hydrocarbon (PAH) dissipation in soils. Fusarium solani MM1 and Arthrobacter oxydans MsHM11 can dissipate PAHs in vitro. We investigated their interactions and their effect on the dissipation of three PAHs-phenanthrene (PHE), pyrene (PYR) and dibenz(a,h)anthracene (DBA)-in planted microcosms, in sterile sand or non-sterile soil. In sterile sand microcosms planted with alfalfa, the two microbes survived and grew, without any significant effect of co-inoculation. Co-inoculation led to the dissipation of 46% of PHE after 21days. In soil microcosms, whether planted with alfalfa or not, both strains persisted throughout the 46days of the experiment, without any effect of co-inoculation or of alfalfa, as assessed by real-time PCR targeting taxon-level indicators, i.e. Actinobacteria 16S rDNA and the intergenic transcribed spacer specific to the genus Fusarium. The microbial community was analyzed by temporal temperature gradient electrophoresis and real-time PCR targeting bacterial and fungal rDNA and PAH-ring hydroxylating dioxygenase genes. These communities were modified by PAH pollution, which selected PAH-degrading bacteria, by the presence of alfalfa and, concerning the bacterial community, by inoculation. PHE and PYR concentrations significantly decreased (91 and 46%, respectively) whatever the treatment, but DBA concentration significantly decreased (30%) in planted and co-inoculated microcosms only.

Clostridium leptum group bacteria abundance and diversity in the fecal microbiota of patients with inflammatory bowel disease: a case–control study in India

BackgroundAlterations in the fecal bacterial flora occur in inflammatory bowel disease (IBD). We examined the abundance and diversity of Clostridium leptum group, an important group of carbohydrate-fermenting bacteria, in the feces of patients with IBD and compared them with healthy controls.MethodsSeventeen healthy controls (HC), 20 patients with Crohn’s disease (CD) and 22 patients with ulcerative colitis (UC) participated in the study. DNA extracted from fecal samples was amplified by PCR targeting 16S rRNA gene sequences specific to C. leptum group. The PCR product was subjected to temporal temperature gradient electrophoresis (TTGE) and the number and position of individual bands were noted and diversity was estimated. The identity of bands at different positions was confirmed by cloning and sequencing. Real time quantitative PCR with Mesa Green, targeted at specific 16S rRNA gene sequences, was used to quantitate C. leptum group and its most prominent constituent, Faecalibacterium prausnitzii.ResultsTwenty five different operational taxonomic units (OTUs, equivalent to species) were identified constituting the C. leptum group in these participants. Their sequences were deposited in GenBank [accession numbers GQ465348 to GQ465370]. OTU number was significantly reduced in CD (7.7±3.7, mean±SD) and UC (9.0±3.0) compared to HC (11.9±2.2) (P=0.0005). The Simpson D index of alpha diversity was not significantly different between the three groups. Total numbers of C. leptum group bacteria and F. prausnitzii were reduced in both CD and UC compared to HC (P=0.0036 and P<0.0001 respectively). Disease activity did not influence numbers of C. leptum or F. prausnitzii in patients with CD or UC.ConclusionC. leptum numbers and diversity were significantly reduced in both CD and UC suggesting that alterations noted were not specific to one disease. This could contribute to reduced short chain fatty acid production in IBD.

Changes in Bowel Microbiota Induced by Feeding Weanlings Resistant Starch Stimulate Transcriptomic and Physiological Responses

The ability to predictably engineer the composition of bowel microbial communities (microbiota) using dietary components is important because of the reported associations of altered microbiota composition with medical conditions. In a synecological study, weanling conventional Sprague-Dawley rats (21 days old) were fed a basal diet (BD) or a diet supplemented with resistant starch (RS) at 5%, 2.5%, or 1.25% for 28 days. Pyrosequencing of 16S rRNA genes and temporal temperature gradient electrophoresis (TTGE) profiles in the colonic digesta showed that rats fed RS had altered microbiota compositions due to blooms of Bacteroidetes and Actinobacteria. The altered microbiota was associated with changes in colonic short-chain fatty acid (SCFA) concentrations, colonic-tissue gene expression (Gsta2 and Ela1), and host physiology (serum metabolite profiles and colonic goblet cell numbers). Comparisons between germ-free and conventional rats showed that transcriptional and serum metabolite differences were mediated by the microbiota and were not the direct result of diet composition. Altered transcriptomic and physiological responses may reflect the young host's attempts to maintain homeostasis as a consequence of exposure to a new collection of bacteria and their associated biochemistry.

Molecular Genetic Characterization of β-Thalassemia and Sickle Cell Syndrome in the Albanian Population

β-Thalassemia (β-thal) is a major public health problem in Albania as it is in many Mediterranean countries. We determined the different β-thal alleles that are present in the Albanian population by using the temporal temperature gradient electrophoresis (TTGE) method because of its high throughput, cost-effectiveness, sensitivity and simplicity. DNA from blood of 68 patients with β-thal, 26 with sickle cell anemia or sickle cell β-thal, 54 parents of these patients and 14 heterozygotes related to these families. We found the IVS-I-110 (G>A), codon 39 (C>T), IVS-I-6 (T>C), IVS-I-1 (G>A) and codon 44 (–C) mutations that accounted for nearly 90% of the β-thal alleles. Their frequencies were similar to those found in other studies in the Albanian population. This method has permitted the detection of heterozygotes for β-thal in this population and offers a prenatal diagnosis with a probability of 90% accuracy.

Effect of Thymus vulgaris essential oil on intestinal bacterial microbiota of rainbow trout, Oncorhynchus mykiss (Walbaum) and bacterial isolates

The application of natural and innocuous compounds has potential in aquaculture as an alternative to antibiotics. We evaluated the effect of diet supplementation with Thymus vulgaris essential oil (TVEO) on the allochthonous microbial composition of rainbow trout. DNA was extracted directly from the intestinal contents, and the V3-V4 regions of the 16S rRNA genes were amplified by PCR. The bacterial composition was analysed using temporal temperature gradient electrophoresis (TTGE). No significant changes (P>0.05) were detected in the TTGE profiles of TVEO-treated trout compared with the controls. The Dice similarity index revealed a high stability (Cs >70%) of the intestinal microbiota in both groups during the 5-week period. Sequence analyses of the TTGE bands revealed the same bacterial composition in both groups, with most bacteria belonging to the Proteobacteria and Firmicutes phyla. The in vitro antibacterial activity of TVEO was assessed using a range of normal intestinal isolates and fish pathogens. The inhibitory concentrations for all the tested bacteria were higher than the TVEO levels used in trout, which may explain the in vivo results.

Comparative assessment of mutations in mtDNA of x-irradiated mice using mismatch-specific endonuclease and temporal temperature gradient electrophoresis

Mutations in brain mtDNA of mice X-irradiated to 5 Gy were determined using a mismatch-specific endonuclease (CEL I) and by temporal temperature gradient gel electrophoresis (TTGE). The CEL I nuclease method allows simultaneous analysis of multiple DNA samples and proves to yield better results than TTGE; it is more sensitive, quite reproducible, and economical, requiring no complicated equipment. The CEL I nuclease assay for mtDNA mutations in the brain of x-irradiated mice has shown that the amount of mutant mtDNA copies is markedly reduced (2–3 times) from the 8th to the 28th day after irradiation, while the overall amount of mtDNA copies in the brain tissue remains fairly constant over this period, though lower than in the control. It can be suggested that mutant mtDNA copies are eliminated from the tissues of irradiated animals in the postirradiation period.

Molecular detection of the ruminal bacterium,Butyrivibrio fibrisolvens, in feces from rural residents of southern India

Objective: This study was undertaken to determine whether Butyrivibrio fibrisolvens, a butyrate-producing bacterium present in the rumen of cattle, could be detected in the feces of healthy southern Indians. Subjects and methods: Feces were collected from 25 children and 25 adults from a village and from 25 children living in a town in southern India. Four cow dung samples were also collected from the village. DNA was extracted and real-time PCR was carried out using primers targeted at 16S rDNA sequences peculiar to B. fibrisolvens. Amplicons were subjected to temporal temperature gradient electrophoresis (TTGE) and DNA sequencing. Phylogenetic analysis was done. Results: B. fibrisolvens rDNA was detected in feces of 13 adults (52%) and 15 children (60%) from the village, but not in urban children. In TTGE, amplicons from children and from cattle migrated as a single band, with only one sample showing a second band. Sequencing revealed 12 novel sequences (accessed in GenBank, 8 from children, 4 from dung) closely similar to the ATCC strain of B. fibrisolvens. Conclusions: B. fibrisolvens, a microorganism found in cattle, colonizes the human gastrointestinal tract in rural southern India.

Application of PCR-TTGE and PCR-RFLP for Intraspecific and Interspecific Characterization of the Genus Saccharomyces Using Actin Gene (ACT1) Primers

In this work the actin gene was used to establish phylogenetic relationships of wider and more diffuse species of the genus Saccharomyces in food ecology by temporal temperature gradient electrophoresis (TTGE) and amplified restriction fragment length polymorphism (RFLP) analysis. Results for DNA RFLP analysis varied considerably, and some enzymes showed a high intra- and interspecific power; however, comparison of experimental results with those provided by the National Center for Biotechnology Information database disclosed a number of interesting variations. Only some experimental results matched the theoretical ones. A theoretical study of melting temperatures using available information from partial sequences of the actin gene was done. Several Saccharomyces species and strains could be distinguished using different TTGE melting points. Some degree of discrimination was achieved under different conditions, in that the Saccharomyces strains tested were separated into groups like the results obtained by PCR-RFLP.

Associations between cigarette smoking and mitochondrial DNA abnormalities in buccal cells

DNA alterations in mitochondria are believed to play a role in carcinogenesis and are found in smoking-related cancers. We sought to replicate earlier findings for the association of smoking with increased mitochondrial DNA (mtDNA) content in buccal cells and further hypothesized that there would be an increased number of somatic mtDNA mutations in smokers. Buccal cells and blood lymphocytes were studied from 42 healthy smokers and 30 non-smokers. Temporal temperature gradient electrophoresis screening and sequencing was used to identify mtDNA mutations. The relative mtDNA content was determined by real-time polymerase chain reaction. Assuming that mtDNA in lymphocytes represents the inherited sequence, it was found that 31% of smokers harbored at least one somatic mtDNA mutation in buccal cells with a total of 39 point mutations and 8 short deletions/insertions. In contrast, only 23% of non-smokers possessed mutations with a total of 10 point mutations and no insertions/deletions detected. mtDNA somatic mutation density was higher in smokers (0.68/10 000 bp per person) than in non-smokers (0.2/10 000 bp per person). There was a statistically significant difference in the pattern of homoplasmy and heteroplasmy mutation changes between smokers and non-smokers. Whereas non-smokers had the most mutations in D-loop region (70%), smokers had mutations in both messenger RNA encoding gene (36%) and D-loop region (49%). The mean ratio of buccal cells to lymphocytes of mtDNA content in smokers was increased (2.81) when compared with non-smokers (0.46). These results indicate that cigarette smoke exposure affects mtDNA in buccal cells of smokers. Additional studies are needed to determine if mitochondrial mutation assays provide new or complementary information for estimating cigarette smoke exposure at the cellular level or as a cancer risk biomarker.

Evaluation of fungal aerosols using Temporal Temperature Gradient Electrophoresis (TTGE) and comparison with culture

Information obtained from fungal air samples can assist in the assessment of health hazards and can be useful in proactive indoor air quality monitoring. The objective of the present study was to evaluate the PCR–TTGE technique for the analysis of fungal diversity in the air. Eleven air samples were collected in five different sites using the bioimpactor CIP 10-M (Arelco). After a 2 hours sampling period, the collection liquid was recovered for subsequent cultivation and PCR–TTGE. A set of three fungi-specific primers (Fungcont 1, Fungcont 2 + GC and Fungcont 3) was designed for the partial amplification of the 18S rRNA gene. The amplification was obtained in a single reaction tube by a semi-nested PCR. For identification, the TTGE bands were extracted and sequenced. PCR–TTGE allowed the clear separation of amplicons corresponding to distinct fungal species (both Ascomycota and Basidiomycota) that may be encountered in air. The number of fungal taxa detected after culture was systematically higher than the number of taxa found using PCR–TTGE. However, few fungal species were detected by PCR–TTGE and not by cultivation, suggesting that the combination of these approaches may provide a better analysis of fungal diversity in air samples than either method alone.

Comprehensive genetic analysis of the cystic fibrosis transmembrane conductance regulator from dried blood specimens – Implications for newborn screening

PurposeIn the United States, approximately 1/3,700 babies is born with cystic fibrosis each year. The >1,300 documented sequence variants pose a challenge for detection of cystic fibrosis through genetic screening. To investigate whether comprehensive characterization of the cystic fibrosis gene is feasible using dried newborn blood specimens, we modified the whole blood Ambry Test™: CF and determined its sensitivity by testing DNA from individuals with cystic fibrosis who still had unknown mutations after commercial mutation panel testing. MethodsDNA from 42 archived newborn dried blood specimens of affected Hispanic, African-American and Caucasian individuals in California was analyzed by temporal temperature gradient electrophoresis screening and targeted sequencing, and by gross deletion analysis. ResultsExcluding two specimens that could not be analyzed due to poor DNA quality, we report a 100% sensitivity and clinical detection rate in the remaining 40 patients. Eighty-three mutations representing 40 different variants were detected, including 8 novel mutations. ConclusionsThis study demonstrates the feasibility of temporal temperature gradient electrophoresis-based full sequence analysis and targeted sequencing from DNA in newborn blood specimens. The Ambry Test™: CF, as an additional step in cystic fibrosis newborn screening models, can be used to dramatically reduce the number of cystic fibrosis carrier sweat test referrals.

TP53 and KRAS2 Mutations in Plasma DNA of Healthy Subjects and Subsequent Cancer Occurrence: A Prospective Study

In cancer patients, plasma often contains mutant DNA released by cancer cells. We have assessed the significance of plasma DNA mutations for subsequent cancer development in healthy subjects in a large longitudinal prospective study. The European Prospective Investigation into Cancer and Nutrition study was analyzed with a nested case-control design. Cases were nonsmokers or ex-smokers for >10 years and newly diagnosed with lung, bladder, or upper aerodigestive tract cancers or leukemia accrued after a median follow-up of 6.3 years. Controls were matched 2:1 for follow-up, age, sex, area of recruitment, and smoking status. KRAS2 mutations were detected by mutant-enriched PCR and sequencing (n = 1,098). TP53 mutations were detected by denaturing high-performance liquid chromatography, temporal temperature gradient electrophoresis, and sequencing (n = 550). KRAS2 or TP53 mutations were detected in 13 of 1,098 (1.2%) and 20 of 550 (3.6%) subjects, respectively, 16 of whom developed cancer on average after 18.3 months of follow-up. Among 137 subjects who developed bladder cancer, 5 had KRAS2 mutations [odds ratio (OR), 4.25; 95% confidence interval (95% CI), 1.27-14.15] and 7 had TP53 mutations (OR, 1.81; 95% CI, 0.66-4.97). There was a nonsignificant trend for association between TP53 mutations and bulky adducts in lymphocyte DNA (OR, 2.78; 95% CI, 0.64-12.17). This is the first report of TP53 or KRAS2 mutations in the plasma of healthy subjects in a prospective study, suggesting that KRAS2 mutation is detectable ahead of bladder cancer diagnosis. TP53 mutation may be associated with environmental exposures. These observations have implications for monitoring early steps of carcinogenesis.

Diabetic induction of mitochondrial DNA (mtDNA) mutations

Cardiovascular disease is the leading cause of mortality in noninsulin-dependent diabetes mellitus patients (NIDDM). Originally thought to be a metabolic problem, widespread systemic complications are now recognized. Diabetic-induced chronic hyperglycemia can lead to increased oxidative stress and may be the common underlying cause of cellular damage. Packaged into nucleosomes, genomic DNA is more protected from oxidative damage than the relatively naked mitochondrial DNA. Damage within mtDNA is further exacerbated by the greater production of reactive oxygen species (ROS) by the electron transport chain. We have utilized the temporal temperature gradient electrophoresis (TTGE) method to analyze myocardial mtDNA mutations in the Goto-Kakizaki (GK) diabetic rat. Heart mitochondria were isolated by differential centrifugation and mtDNA isolated. Specific regions of the myocardial mtDNA were amplified by PCR and loaded onto a TTGE gel for mutation analysis. Mobility shifts were observed in different regions indicating mtDNA mutations including one 544bp region of the cytochrome oxidase III (COX3). Direct sequencing confirmed the presence of mutations in GK diabetic (3 month old: 6 errors in 2 of 6 rats) but not Wistar controls (3 month old: 0 errors in 6 rats), and the number of errors in the GK rats increased with age (9 month old: 27 errors in 6 of 6 rats). Mutations included basepair substitutions and insertions, either of which altered the 3′ end of the COX3 coding sequence. The accumulation of mtDNA mutations and concomitant mitochondrial dysfunction may be the underlying pathology for many complications of diabetes, including vascular dysfunction, renal failure, and congestive heart failure. Supported in part by NIH PO1HL43023 and the New York Medical College Research Endowment Fund.

Microbiological study of lactic acid fermentation of Caper berries by molecular and culture-dependent methods.

Fermentation of capers (the fruits of Capparis sp.) was studied by molecular and culture-independent methods. A lactic acid fermentation occurred following immersion of caper berries in water, resulting in fast acidification and development of the organoleptic properties typical of this fermented food. A collection of 133 isolates obtained at different times of fermentation was reduced to 75 after randomly amplified polymorphic DNA (RAPD)-PCR analysis. Isolates were identified by PCR or 16S rRNA gene sequencing as Lactobacillus plantarum (37 isolates), Lactobacillus paraplantarum (1 isolate), Lactobacillus pentosus (5 isolates), Lactobacillus brevis (9 isolates), Lactobacillus fermentum (6 isolates), Pediococcus pentosaceus (14 isolates), Pediococcus acidilactici (1 isolate), and Enterococcus faecium (2 isolates). Cluster analysis of RAPD-PCR patterns revealed a high degree of diversity among lactobacilli (with four major groups and five subgroups), while pediococci clustered in two closely related groups. A culture-independent analysis of fermentation samples by temporal temperature gradient electrophoresis (TTGE) also indicated that L. plantarum is the predominant species in this fermentation, in agreement with culture-dependent results. The distribution of L. brevis and L. fermentum in samples was also determined by TTGE, but identification of Pediococcus at the species level was not possible. TTGE also allowed a more precise estimation of the distribution of E. faecium, and the detection of Enterococcus casseliflavus (which was not revealed by the culture-dependent analysis). Results from this study indicate that complementary data from molecular and culture-dependent analysis provide a more accurate determination of the microbial community dynamics during caper fermentation.