Performed Polymerase Chain Reaction Research Articles

Prevalence study of SNP c.421G>T in the ADAMTS2 gene responsible for dermatosparaxis in White Dorper sheep in Brazil

Abstract: Dermatosparaxis is an autosomal recessive disorder of connective tissue; the disorder is clinically characterized by skin fragility and hyperextensibility. Dermatosparaxis in White Dorper sheep is caused by a single nucleotide polymorphism (SNP) (c.421G>T) in the ADAM metalloproteinase with thrombospondin type 1 motif, 2 (ADAMTS2) gene. The aim of this study was to investigate the prevalence of this SNP in a White Dorper herd in São Paulo state, Brazil. In this study, we collected blood DNA samples from 303 White Dorper sheep and performed polymerase chain reaction to amplify the SNP region. The samples were sequenced to determine the presence of the SNP in the ADAMTS2 gene. The SNP prevalence in the studied population was 15.5%; this finding indicates that more effective control measures should be used to prevent the inheritance of SNP c.421G>T in the ADAMTS2 gene in Brazilian White Dorper herds.

Molecular detection of Babesia spp. in ticks sampled from asymptomatic dogs in the area of some Belgrade municipalities

Babesiosis of domestic animals is a vector transmissible and clinically significant disease, caused by protozoa of genus Babesia and Theileria. Possible causative agents for this disease in dogs in Europe are: Babesia canis, B. gibsoni, B. vogeli and B. microti-like. Diagnostics of babesiosis of dogs was for a long time based on the visual inspection of stained blood smear under a microscope, while today there have been increasingly used molecular methods of detection in precise, species diagnostics. The objective of this work was molecular detection of the cause of babesiosis of dogs in the ticks sampled from asymptomatic dogs in the region of some Belgrade municipalities, all for better understanding of epizootiological situation. From three sites in Belgrade, there were collected 49 ticks, sampled from the dogs with no symptoms. There was carried out the determination of the ticks, and after that, DNA was isolated for molecular examination. First, there was performed Polymerase Chain Reaction (PCR), for determining the species of the genus Babesia, and after that there was also carried out the determining of polymorphism in the length of restriction fragments (RFLP) for the purpose of the causative agent species determination. Out of the total number of the examined ticks, 18,34% were positive on Babesia spp. By RFLP method, in two cases (4,08%) B. Gibsoni was identified, while in 7 cas?es (14,92%) there were no restriction sites for the used enzymes, what suggests that most likely it was B. canis. The ticks positive on the cause of babesiosis were: Dermacentor reticulatus (4 cases), Rhipicephalus sanguineus (4 cases) i Ixodes ricinus (1 case). This work confirms the presence of Babesia spp. in the ticks sampled from asmptomatic dogs on the teritory of Belgrade as well as the significance of PCR-RFLP method in diagnostics and identification of the causative agent of babesiosis in dogs. For the first time in Serbia, there was determined the presence of B. gibsoni in ticks (Species Rhipicephalus sanguineus)

Isolation and Characterization of Bacteriophage against Methicillin Resistant Staphylococcus aureus

Methicillin resistant Staphylococcus aureus (MRSA) is a major human pathogen responsible for several life threatening conditions. MRSA have the ability to acquire resistance to several antimicrobial agents and phage therapy is one potential option to treat this pathogen. The aim of the study was to isolate and characterize bacteriophages effective against a wide range of methicillin-resistant Staphylococcus aureus (MRSA). A mixture of ten MRSA isolates was used for the isolation of phage from wastewater treatment plants. Three phages were selected for further characterization. All three phages belong to the Siphoviridae family and have long non-contractile flexible tails. The three phages showed a wide host range against S. aureus. Phages E¸SA1 and E¸SA2 were resistant to a pH range from 4-10 while E¸SA3 has a pH range from 3-11. DNA from all three phages was resistant to digestion by endonuclease enzymes such as EcoRI and AccI. There was a high degree of mosaicism among the three virulent phages and with their ancestor phages of Siphoviridae due to their non-uniform access to the common genetic pool by horizontal gene transfer and recombination. Since some of the staphylococcal toxins are phage encoded, the presence of genes for such toxins was tested by performing polymerase chain reaction and all three phages lacked genes for any of the staphylococcal toxins, including staphylococcal enterotoxins (sea, seb, sec and see), exfoliating toxins (eta and etb) and the toxic shock syndrome toxin (tst), therefore these bacteriophage are suitable candidates for future use in phage therapy against MRSA.

Screening and evaluation of chronic and occult Hepatitis B in chemo - radiotherapy patients with cancer.

Background:Hepatitis B virus infection (HBV) and its complications is one of the most serious problems of the health system in many parts of the world. In the present study, we will assess chronic and occult HBV and isolated anti-Hepatitis B core antigen whose screening and evaluation is not routine in different populations.Materials and Methods:This descriptive analytical study was conducted on 213 patients undergoing chemotherapy - radiotherapy referred to the hematology - oncology clinics of Isfahan, Iran in 2012. In order to determine the serum levels of hepatitis B surface antigen (HbSAg), Hepatitis B Antigen and Antibody (HBCAb), aspartate aminotransferase (AST), alanine transaminase (ALT) and Alkaline phosphatase (ALK.P), venous blood samples were obtained. If the HBCAb sample was positive, another sample of the serum was sent to the laboratory to perform polymerase chain reaction and to determine viral load.Results:The mean age of the patients was 47.7 ± 9 years, with an age range of 27 -73 years; 98 (46%) and 115 (54%) cases were male and female, respectively, with mean age of 51.9 ± 8.3 and 44.1 ± 8.1 years, and there was no significant difference (P < 0.001). The mean level of liver enzymes including AST, ALT and ALK.P were 34.2 ± 36.02, 38.9 ± 47.1 and 252.1 ± 234.7, respectively. Two cases were HbSAg positive (0.9%) and six cases were HBCAb positive (2.8%) and HbSAg negative. Three cases had a high viral load at the rate of starting treatment among positive anti-HBC patients.Conclusion:Because occult hepatitis is investigated less commonly in routine studies, it seems that screening and evaluating its prevalence is useful in the management of patients.

Incidence and Differential Diagnosis of Human Herpes Virus-6 Associated Encephalitis/Myelitis in Allogeneic Stem Cell Transplantation That Mimics Calcineurin Inhibitor-Induced Pain Syndrome

Background: Human herpes virus-6 (HHV6)-associated limbic encephalitis/myelitis is rare but life-threatening nervous system complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). When the allo-HSCT recipients present encephalitis-associated manifestations such as short-term memory dysfunction, disorientation, consciousness disturbance and seizures, it is essential to immediately perform polymerase chain reaction (PCR) analysis for detection of HHV6 viral DNA in the cerebrospinal fluid (CSF) for the definite diagnosis. However, if reactivation of HHV6 is confined to the spinal cord but not cerebrum, these patients lack encephalitis-like symptoms but manifest only sensory nerves-related symptoms such as severe unexplained pain, dysesthesia and prutitus, leading to the delayed examination of HHV6 DNA detection in CSF. As a result, the patients with HHV6-associated myelitis can be sometimes misdiagnosed as having calcineurin inhibitor (CI)-induced pain syndrome (CIPS), since such manifestations are commonly observed in these two syndromes. In this study, we evaluate incidence and clinical features of HHV6 encephalitis/myelitis and CIPS after allo-HSCT.Method: We retrospectively reviewed the medical records of 435 patients who underwent allo-HSCT between 2002 and 2014 in our facility. HHV6-associated encephalitis/myelitis was directly proved when HHV6 DNA was detected by PCR in CSF. For those patients who were unable to undergo lumbar puncture because of severe thrombocytopenia or a deteriorated general condition, we diagnosed HHV6-associated encephalitis/myelitis if they satisfied more than 2 of the following 3 criteria: (1) typical clinical manifestations as described above; (2) detection of HHV6 DNA in peripheral blood; or (3) limbic encephalopathy based on the selective involvement of the medial temporal lobe on magnetic resonance imaging (MRI). CIPS was diagnosed using three factors: (a) typical clinical manifestations including unexplained severe pain and cutaneous pruritus without skin rash; and (b) not detection of HHV6 DNA in CSF; or (c) abnormal findings at X-ray, MRI, or bone scintigraphy at joint of knee and foot.Results: Twenty-five patients were diagnosed as having HHV-6 encephalitis/myelitis with a cumulative incidence of 5.7%. Median onset was on day 19 after transplantation. HHV-6 encephalitis/myelitis was documented in 15 of 99 cord blood transplant (CBT) recipients (15.2%) and 10 of 336 recipients (3.0%) transplanted with bone marrow or peripheral blood stem cells. This result suggests that a higher incidence of HHV-6 encephalitis/myelitis occurring in CBT recipients. Four patients manifested typical symptoms at the onset of HHV6-associated encephalitis. However, 11 patients presented with dysesthesia and pruritus, described as typical manifestations of patients with CIPS, and the remaining 10 showed both symptoms. Six of 11 patients with CIPS-like symptom also exhibited dysautonomia (bladder and rectal disturbance and sinus tachycardia) and/or abdominal pain. Positive results for brain MRI scans (limbic encephalitis) were observed in 9 of 14 patients (64.3%) who developed encephalitis-type symptoms, which were not found in the 11 patients who had CIPS-like symptoms; none presenting with CIPS-like symptoms had positive results for spinal MRI as well. On the other hand, 8 patients (1.8%) were diagnosed as having CIPS on the 5 to 91 days (madian 22 days) posttransplant. For graft-versus-host disease (GVHD) prophylaxis, 2 patients received cyclosporine, and 6 received tacrolimus. CI concentrations in these patients were maintained within the target ranges at onset of the pain. Abnormal findings at X-ray and MRI were not observed in all patients, but only one patient showed abnormal findings at joint of hand, finger and ankle in bone scintigraphy. For the treatment of CIPS, in 7 patients dosage of CI was reduced, whereas in one CI was switched into another one. Clinical symptoms in all of these patients were improved and exacerbation of GVHD was not seen.Conclusion: Detection of HHV6 DNA in CSF is crucial to make a differential diagnosis of HHV6 myelitis and CIPS. Transplantation physicians should be aware that CIPS-like dysesthesia and pruritus might be early manifestations to suggest the reactivation of HHV6, especially for patients who develop myelitis. DisclosuresAkashi:Asahi Kasei: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Chugai: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Consultancy, Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau.

New Epidemiological and Clinical Signatures of 18 Pathogens from Respiratory Tract Infections Based on a 5-Year Study.

BackgroundRespiratory tract infections (RTIs) are a heavy burden on society. However, due to the complex etiology of RTIs, the clinical diagnosis, treatment, and prevention of these infections remain challenging, especially in developing countries.MethodsTo determine the epidemiological and clinical characteristics of 18 respiratory pathogens, we analyzed 12,502 patients with acute respiratory infections (ARIs) by performing polymerase chain reaction (PCR) on patient pharyngeal swabs.ResultsSamples positive for at least 1 pathogen were obtained from 48.42% of the total patients. Of these pathogen-positive patients, 17.99% were infected with more than 1 pathogen. Of the 18 pathogens analyzed, four were detected with a positive detection rate (PDR) > 5%: influenza A virus (IAV) > respiratory syncytial virus (RSV) >Mycoplasma pneumoniae (MP) > human coronavirus (HCoV). The pathogens with the 4 highest co-infection rates (CIRs) were as follows: HCoV > human bocavirus (HBoV) > enterovirus (EV) > parainfluenza virus (PIV). The overall positive detection rate (PDR) varied significantly according to patient age, the season and year of detection, and the disease subgroup, but not according to patient sex. The individual PDRs of the pathogens followed 3 types of distributions for patient sex, 4 types of distributions for patient age, 4 types of seasonal distributions, 2 types of seasonal epidemic trends, 4 types of yearly epidemic trends, and different susceptibility distributions in the disease subgroups. Additionally, the overall CIR showed significantly different distributions according to patient sex, patient age, and the disease subgroup, whereas the CIRs of individual pathogens suggested significant preference characteristics.ConclusionIAV remains the most common pathogen among the pathogens analyzed. More effort should be directed toward the prevention and control of pathogens that show a trend of increasing incidence such as HCoV, human adenovirus (ADV), and RSV. Although clinically distinguishing specific pathogens responsible for RTIs is difficult, the epidemiological and clinical characteristics of the various RTI-causing agents could provide clues for clinicians, thereby informing decisions regarding prevention and medication and guiding appropriate public health strategies.

Identification of the bacteriocin produced by cheese isolate Lactobacillus paraplantarum FT259 and its potential influence on Listeria monocytogenes biofilm formation

Lactic acid bacteria (LAB) are known by their technological properties, such as the inhibition of spoilage and pathogenic bacteria. The aims of this study were: (i) to evaluate the influence of Lactobacillus paraplantarum FT259 on biofilm formation by Listeria monocytogenes by using culture method and fluorescence in situ hybridization (FISH) and, (ii) to partially characterize the bacteriocin produced by this strain isolated from Brazilian semi-hard cheese by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). We also performed polymerase chain reactions with primers for plantaricin NC8, plantaricin S and plantaricin W structural genes followed by DNA sequencing to possibly identify the bacteriocin produced. Results of microscopy assays demonstrated the complex and heterogenous structure of the biofilm formed by co-culture of both strains. SDS-PAGE analysis indicated that the partially purified bacteriocin presented a molecular mass around 3900 Da and, PCR-DNA sequencing detected the plantaricin NC8 gene. To our knowledge, this is the first report on the inhibitory effect of an L. paraplantarum strain on L. monocytogenes biofilms, as well as the formation of biofilm by this LAB species. In addition, we have demonstrated L. paraplantarum FT259 harbored the gene for plantaricin NC8 production, which may be responsible by the antimicrobial activity observed.

Underestimation of foraging behaviour by standard field methods in malaria vector mosquitoes in southern Africa.

BackgroundDefining the anopheline mosquito vectors and their foraging behaviour in malaria endemic areas is crucial for disease control and surveillance. The standard protocol for molecular identification of host blood meals in mosquitoes is to morphologically identify fed mosquitoes and then perform polymerase chain reaction (PCR), precipitin tests, or ELISA assays. The purpose of this study was to determine the extent to which the feeding rate and human blood indices (HBIs) of malaria vectors were underestimated when molecular confirmation by PCR was performed on both visually fed and unfed mosquitoes.MethodsIn association with the Southern Africa International Centers of Excellence in Malaria Research (ICEMR), mosquito collections were performed at three sites: Choma district in southern Zambia, Nchelenge district in northern Zambia, and Mutasa district in eastern Zimbabwe. All anophelines were classified visually as fed or unfed, and tested for blood meal species using PCR methods. The HBIs of visually fed mosquitoes were compared to the HBIs of overall PCR confirmed fed mosquitoes by Pearson’s Chi-Square Test of Independence.ResultsThe mosquito collections consisted of Anopheles arabiensis from Choma, Anopheles funestus s.s., Anopheles gambiae s.s. and Anopheles leesoni from Nchelenge, and An. funestus s.s. and An. leesoni from Mutasa. The malaria vectors at all three sites had large human blood indices (HBI) suggesting high anthropophily. When only visually fed mosquitoes tested by PCR for blood meal species were compared to testing those classified as both visually fed and unfed mosquitoes, it was found that the proportion blooded was underestimated by up to 18.7%. For most Anopheles species at each site, there was a statistically significant relationship (P < 0.05) between the HBIs of visually fed mosquitoes and that of the overall PCR confirmed fed mosquitoes.ConclusionThe impact on HBI of analysing both visually fed and unfed mosquitoes varied from site to site. This discrepancy may be due to partial blood feeding behaviour by mosquitoes, digestion of blood meals, sample condition, and/or expertise of entomology field staff. It is important to perform molecular testing on all mosquitoes to accurately characterize vector feeding behaviour and develop interventions in malaria endemic areas.

Molecular beacon-loaded polymeric nanoparticles for non-invasive imaging of mRNA expression.

Assessment of intracellular mRNA expression is invaluable for understanding cellular signaling activities, identifying disease stages, and monitoring the gene expression pattern of therapeutic cells during their culture, expansion and/or differentiation process. Previous methods suffer from the need to disrupt the biological samples to perform polymerase chain reaction analysis which can be laborious, fragmented and destructive. Herein, we develop a mRNA nanosensor based on the sustained release of mRNA-specific molecular beacons (probes that fluoresce upon hybridization) from the biodegradable poly(d,l-lactide-co-glycolide) nanoparticles. Post cellular internalization, the particles gradually degrade and release the encapsulated probes which are initially weakly fluorescent. When the released probes meet and hybridize with target mRNA, they restore pre-quenched fluorescence. By virtue of quantifying the fluorescence intensity, we can estimate the cellular mRNA expression. As a case study, β-actin mRNA expression in mesenchymal stem cells cultured on a 3D matrix was monitored and compared with those cultured on a 2D plate for one week. Critically, the observed expression profile shows a great correlation with the established quantitative polymerase chain reaction analysis.

Use of Polymerase Chain Reaction for Identification of the Pathogen and Management of Potato Soft Rot with Zinc Application

Effect of different Zinc doses was investigated against Erwinia carotovora ssp. atroseptica, the potato blackleg/soft rot causing organism, during 2009 and 2010 in Department of Plant Pathology and Institute of Biotechnology and Genetic Engineering, The University of Agriculture, Peshawar-Pakistan. Out of 200 tested samples, 21 of them were proved to be Eca. However, these tentative Eca isolates showed some characteristics which were unexpected for Eca. We, therefore, decided to perform Polymerase Chain Reaction using Eca-specific primers, Eca1F and Eca2R for confirm identification. For disease management, at the time of sowing, pots containing 5 kg sterilized soil were applied with Zinc in four different treatments i.e. 8 mg, 10 mg, 12 mg and 14 mg along with one control. Results indicated that 12 mg (4.8 kg Zn ha-1) were better doses in controlling the disease up to 73% and increasing the yield up to 117% as compared to control plants.

Study on Leishmania infection in cats from Ahar, East Azerbaijan Province and North West Iran by parasitological, serological and molecular methods

ABSTRACT Objective To study Leishmania infection in cats and its potential role in transmission of the disease to human by parasitological, serological and molecular methods in Ahar District, East Azerbaijan Province. Methods In this study, 65 cats from different parts of Ahar Province were trapped. The cats were anesthetized with chloroform and blood samples were taken from jugular vein and tested by direct agglutination test. Spleen and liver smear samples were prepared in order to microscopically examine these organs, and also cultured in Novy-MacNeal-Nicolle and Roswell Park Memorial Institute 1 640 media. Finally, spleen tissue DNA was extracted to perform polymerase chain reaction analysis. Results In direct agglutination test, 4 (6%) cats had a positive titer, while 14 (22%) cats had a titer of 1:80 which was suspected for an infection and 47 (72%) cats were negative. Culture results were negative and in polymerase chain reaction no amplification was observed. Conclusions We found no case of feline visceral leishmaniasis. It needs more extensive studies by using a larger number of cats to firmly establish leishmaniasis in this area.

Association study of H2AFZ with schizophrenia in a Japanese case-control sample.

It is widely accepted that malfunction of the N-methyl-D-aspartate (NMDA)-type glutamate receptor may be involved in the pathophysiology of schizophrenia. Several recent microRNA (miRNA) studies have demonstrated that the expression of the glutamate system-related miR-132 and miR-212 is changed in postmortem schizophrenic brains. Here we attempted to obtain further insight into the relationships among schizophrenia, the NMDA receptor, the molecular cascades controlled by these miRNAs and commonly predicted target genes of the two miRNAs. We focused on the H2AFZ (encoding H2A histone family, member Z) gene, whose expression was shown in our screening study to be modified by a schizophrenomimetic NMDA antagonist, phencyclidine. By performing polymerase chain reaction with fluorescent signal detention using the TaqMan system, we examined four tag single nucleotide polymorphisms (SNPs; SNP01-04) located around and within the H2AFZ gene for their genetic association with schizophrenia. The subjects were a Japanese cohort (2,012 patients with schizophrenia and 2,170 control subjects). We did not detect any significant genetic association of these SNPs with schizophrenia in this cohort. However, we observed a significant association of SNP02 (rs2276939) in the male patients with schizophrenia (allelic P = 0.003, genotypic P = 0.008). A haplotype analysis revealed that haplotypes consisting of SNP02-SNP03 (rs10014424)-SNP04 (rs6854536) also showed a significant association in the male patients with schizophrenia (P = 0.018). These associations remained significant even after correction for multiple testing. The present findings suggest that the H2AFZ gene may be a susceptibility factor in male subjects with schizophrenia, and that modification of the H2AFZ signaling pathway warrants further study in terms of the pathophysiology of schizophrenia.

Vibrio cholerae Detection in Water and Wastewater by Polymerase Chain Reaction Assay

Background: Vibrio cholerae is a significant human pathogen worldwide and annually causes some cases of deaths. Contaminated water plays an important role in transmission of this pathogen, which indicates the importance of early diagnosis. Objectives: The current study aimed to perform Polymerase Chain Reaction (PCR) on water and wastewater samples to determine the detection limit for Vibrio cholerae. Materials and Methods: PCR was performed on the DNA extracted from Vibrio cholerae of the contaminated water and wastewater using ctxA gene specific primers. The accuracy of PCR method to detect these bacteria was also assessed. Results: The result of PCR performed on the extracted DNA showed a specific 241 base pair band. The limit of bacterial detection for water and wastewater were 40 cfu/mL and 81 cfu/mL, respectively. Conclusions: In the current study, PCR performance using the ctxA gene specific primers to detect Vibrio cholerae was found highly accurate and specific.

Mutations in the B30.2 domain of pyrin and the risk of ankylosing spondylitis in the Chinese Han population: A case–control study

Ankylosing spondylitis (AS) and familial Mediterranean fever (FMF) are a common autoimmune disease and a classic autoinflammatory disease, respectively. Mediterranean fever (MEFV) encodes the pyrin protein and is the causal disease gene in FMF. This protein is an important regulator of innate immunity and may play a key role in the development of AS. To identify the mutations in the B30.2 domain of pyrin and to uncover the relationships between these mutations and AS risk in the Chinese Han population, we extracted genomic DNA from the peripheral blood of 200 AS patients and 200 matched controls and performed polymerase chain reactions (PCRs) and direct sequencing on those samples. Statistical analysis indicated that only Met694Val (rs61752717) in the B30.2 domain of pyrin could affect the risk of AS (P=0.042; odds ratio [OR]=5.103; 95% confidence interval [CI]=1.111–23.437 for the model of Met (M) vs. Val (V), P=0.040; OR=5.211; 95% CI=1.127–24.091 for the model of MM vs. MV+VV). Moreover, M694V is significantly associated with a higher level of erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) in AS patients. Our results are the first to suggest that the M694V allele of the pyrin was associated with AS risk in the Chinese Han population and that this mutation may be associated with the inflammatory response in the development of AS.

Association of MOV10L1 gene polymorphisms and male infertility in azoospermic men with complete maturation arrest.

The present research was undertaken to study probable genetic variations of MOV10L1 in 30 infertile men that had complete maturation arrest in their spermatocyte levels and 70 fertile men as the control group. We performed polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) on extracted DNAs and sequencing was used to confirm the results. Identified polymorphisms in the MOV10L1 were further subjected to a haplotype analysis. We identified eight single nucleotide polymorphisms (SNPs): one missense (rs2272837) and four nonsense polymorphisms (rs2272836, rs11704548, rs2272838, rs138271) in the exonic sequences and three polymorphisms (rs12170772, rs2272840, rs17248147) in the intronic regions. With the exception of rs2272838, there was a statistically significant association in all polymorphisms between study population (P < 0.05). The result of haplotyping analysis showed ten possible haplotypes, from which five were significantly increased in infertile patients compared with the control group. Our results suggest that MOV10L1 gene polymorphisms in the studied infertile males with complete maturation arrest are linked to infertility.

Accumulation of Lactic Acid during Biodigestion of Municipal Solid Waste Leachate and Identification of Indigenous Lactic Acid Bacteria in Leachate

AbstractLactic acid accumulation is a common phenomenon in animal bodies during anaerobic respiration, but it is rarely found in natural systems like cattle manure digestion plants or anaerobic municipal sewage sludge bioreactors. The objective of this study was to demonstrate lactic acid production and accumulation during anaerobic digestion of municipal solid waste (MSW) leachate. Three reactors were set up containing cow dung, cow dung plus leachate, and only leachate to carry out anaerobic digestions. The amount of lactic acid accumulated in the reactor containing cow dung only was much less in comparison to the reactor containing cow dung plus leachate. This may be because of the suppression of sulphate reducing bacteria (SRB) and other groups of bacteria which utilize lactic acid, by some component in MSW leachate. The accumulated lactic acid might have come from various bacterial sources. For the identification of responsible organisms, the authors performed polymerase chain reaction (PCR), cloning...

Pattern of Tuber melanosporum extramatrical mycelium expansion over a 20-year chronosequence in Quercus ilex-truffle orchards

Successful cultivation of black truffle (Tuber melanosporum) requires a long-term investment and the maintenance of the symbiosis throughout its preproductive and productive years. Monitoring the symbiosis over time is challenging, as it requires methods that can detect the belowground proliferation of the fungus associated with its host tree. In this study, we used a chronosequence design to study the expansion pattern of this fungus as the host tree grows. We hypothesize that this expansion can be estimated by monitoring T. melanosporum DNA from soil beneath host trees of different ages (3, 5, 7, 10, 14, and 20years old) and at different distances from the trunk of the trees (40, 100, and 200cm). We also wished to evaluate the presences of Tuber brumale and Tuber indicum, potentially problematic truffle species, in these plantations. To detect the mycelium of T. melanosporum in these soils, we extracted DNA and performed polymerase chain reaction (PCR) with Tuber species-specific primers, and to estimate DNA amount, we measured relative band intensities from the amplicons in agarose gels. Both age and distance were related to T. melanosporum DNA quantity, which was more abundant in the oldest age classes, reaching a plateau in 5-7years. At 40cm from the tree, there were no differences in T. melanosporum DNA amounts in orchards of different ages, but at 100 and 200cm, younger orchards had less T. melanosporum DNA. We did not detect DNA from T. brumale or T. indicum in any of our samples.

Prevalence of methicillin resistance and virulence determinants of &amp;lt;i&amp;gt;Staphylococcus aureus&amp;lt;/i&amp;gt; in diabetic foot ulcers

Background: Diabetic foot ulceration (DFU) is a multifactorial process and is responsible for considerable morbidity and contributes to the increasing cost of health care worldwide. The diagnosis and identification of these ulcers remains a complex problem. Bacterial infection is promoted in the diabetic foot wound by decreased vascular supply and impaired host immune response. As conventional clinical microbiological methods are time-consuming and only identifies about 1% of the wound microbiota, detection of bacteria present in DFUs using molecular methods is highly advantageous and efficient. The aim of this study was to assess the virulence and methicillin resistance profiles of Staphylococcus aureus detected in DFUs using DNA-based methods. Methods: A total of 223 swab samples were collected from 30 patients from March to October 2012. Bacterial DNA was extracted from the swab samples using standard procedures and was used to perform polymerase chain reaction (PCR) using specific oligonucleotide primers. The products were visualized using agarose gel electrophoresis. Results: S. aureus was detected in 44.8% of samples. 25% of the S. aureus was methicillin-resistant S. aureus harboring the mecA gene. The alpha-toxin gene was present in 85% of the S. aureus positive samples. 61% of the S. aureus present in DFU samples harbored the exfoliatin factor A gene. Both the fibronectin factor A and fibronectin factor B gene were detected in 71% and 74% of the S. aureus positive samples. Conclusions: DNA-based detection and characterization of bacteria in DFUs are rapid and efficient and can assist in accurate, targeted antibiotic therapy of DFU infections. The majority of S. aureus detected in this study were highly virulent and also resistant to methicillin. Further studies are required to understand the role of S. aureus in DFU trajectory.

Angiopoietin-like 4 is involved in the poor angiogenic potential of high glucose-insulted bone marrow stem cells.

Background and ObjectivesDiabetes is reported to reduce the function or number of progenitor cells. We compared the gene expression patterns of bone marrow-derived mesenchymal stem cells from diabetic (DM-BMCs) and healthy (non-DM-BMCs) rats and suggested Angiopoietin-like 4 (Angptl4) could be a responsible factor for impaired angiogenesis of DM-BMCs.Subjects and MethodsBMCs were isolated from DM or non-DM rat, and in vitro angiogenesis activity was compared by tube formation assay on Matrigel and complementary deoxyribonucleic acid expression was analyzed by microarray with or without oxytocin treatment. Human BMCs (hBMCs) were treated with high glucose, and were performed polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Angptl4 plasmid DNA and micro ribonucleic acid-132 (miR-132) were transfected to immortalized hBMCs.ResultsIn vitro angiogenesis assay showed the impaired tube formation in DM-BMCs, and slightly recovery by oxytocin treatment. Angptl4, an adipokine, was upregulated in DM-BMCs compared to non-DM-BMCs. Oxytocin treatment reduced Angptl4 in DM-BMCs. In hBMCs, overexpression of Angptl4 attenuated the tube formation. In addition to Angptl4, miR-132 was increased by high glucose treatment. Collectively, high glucose resulted in impaired tube formation through miR-132 induction and Angptl4 upregulation in BMCs.ConclusionOur results show that the angiogenic activity of BMCs is impaired by high glucose stress, which would be mediated by Angptl4 and miR-132.

Analysis of DNA Polymorphism in SRY Gene of Madura Cattle Populations

The aim of this research was to assess the SRY-gene polymorphism in Madura cattle populations and the hybrids of Madura cow with Limousin bull. We investigated the specific markers of the paternally transmitted Y-chromosome for the direct detection of male-mediated introgression. We performed polymerase chain reaction and restriction fragment length polymorphisme (PCR– RFLP) to identify the polymorphism of Y-chromosomes in 17 Madura cattle from Pamekasan district, 4 conserved Madura cattle from Sapudi Island and 22 Limousin x Madura crossbred cattle from Pamekasan. A 211bp PCR product were cut with PstI and BfaI restriction enzyme. The result showed all the Madura cattle types were identified by PstI resrtiction enzyme performed into two fragments DNA. The polymorphisme of Y-chromosome was recognized by BfaI restriction enzymes at the position 432bp and 544bp. Using BfaI restriction enzyme, this study identified 68.18% of Limousin x Madura crossbred cattle was in the sequence of Y-chromosome. The high numbers of BfaI site restriction in the sequence of Y-chromosome due to the introduction Limousin bull by artificial insemination. For those, the preservation of pure Madura cattle as one of animal genetics resources in Indonesia should be a concern.