Isolation and molecular identification of microorganisms isolated from soils contaminated with heavy metals in Mosul city

This research is concerned with organisms isolated from soils contaminated with heavy metals in industrial and residential areas in the city of Mosul, the center of Nineveh Governorate, and the diagnosis of these organisms using molecular biology technique. Samples were collected from four locations in the city between the industrial area and residential neighborhoods. Soil samples were analyzed and dilutions were prepared, then the dilutions were grown on potato extract and dextrose (PDA) medium for the development of fungi and Nutrient agar for bacterial development. The dilutions were planted by casting method by three replications, then the process of purifying the fungal and bacterial colonies was carried out using the traditional methods. For the purpose of diagnosing these pure colonies using PCR technique, colonies of fungi were grown on the medium of PDA, and bacteria were grown on the medium of nutritious broth. As a result, nine fungal species were diagnosed, two of them are new undiagnosed genera that have been registered in the gene bank, four of them contain genetic mutations, and three of them are known and previously diagnosed fungi. As for bacteria, two new strains were isolated and registered in the gene bank among the four types that were diagnosed. And some of these genera exhibited severe resistance to antibiotics, while others showed moderate resistance, in contrast to the control, which was very sensitive to antibiotics.

Validation of a Novel Molecular Assay to the Diagnostic of COVID-19 Based on Real Time PCR with High Resolution Melting

With the emergence of the Covid-19 pandemic, the world faced an unprecedented need for RT-qPCR-based molecular diagnostic tests, leading to a lack of kits and inputs, especially in developing countries. Hence, the costs for commercial kits and inputs were overrated, stimulating the development of alternative methods to detect SARS-CoV-2 in clinical specimens. The availability of the complete SARS-CoV-2 genome at the beginning of the pandemic facilitated the development of specific primers and standardized laboratory protocols for Covid-19 molecular diagnostic. High-sensitive and cost-effective molecular biology technique based on the Melting Temperature differences between purine and pyrimidine bases can be used to the detection and genotyping of pathogens in clinical specimens. Here, a RT-qPCR assays with High Resolution Melting (HRM-RTqPCR) was developed for different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states in Brazil, in comparison to a commercial TaqMan RT-qPCR assay, as gold standard. The sensitivity of the HRM-RTqPCR assays targeting N, RdRp and E were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 targets, and diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, the HRM-RTqPCR emerges as an alternative and low-cost methodology to increase the molecular diagnostic of patients suspicious for Covid-19, especially in restricted-budget laboratories.

Isolation, identification and investigation some biochemical properties of lactic acid bacteria from “Mam sac chua” and “Com me” in Tien Giang province

The objective of this study was isolate, identify and investigate some biochemical properties of strains of lactic acid bacteria from “com me” and “mam sac chua” in Tien Giang province. Nineteen strains of LAB were isolated from four “com me” and three “mam sac chua” samples. They have characterized of lactic acid bacteria such as: halo rings in MRS agar environment added 0,85% CaCO3, rod-shaped cells, Gram positive, catalate and oxidase negative. All 19 strains of LAB were able to produce lactic acid in MRS broth (1,01 – 2,23 mg/ mL after 24 hours). Three strains of LAB were isolated from “com me” were able to produce lactic acid in MRS broth at salt concentration of 0, 2, 4 and 6% (0,57 – 1,29 mg/ mL after 24 hours). In particular, strains of LAB were coded ML3 and ML4 produced the highest lactic acid and VB strain was the most salinity tolerance. Therefore, these three srains were choosed to identify species by molecular biology technique. The results of identification were Staphylococcus piscifermentans VB, Lactobacillus plantarum ML3 and Lactobacillus plantarum ML4 because they are 99% homologous to S. piscifermentans and L. plantarum.

MeltingPlot, a user-friendly online tool for epidemiological investigation using High Resolution Melting data

Background The rapid identification of pathogen clones is pivotal for effective epidemiological control strategies in hospital settings. High Resolution Melting (HRM) is a molecular biology technique suitable for fast and inexpensive pathogen typing protocols. Unfortunately, the mathematical/informatics skills required to analyse HRM data for pathogen typing likely limit the application of this promising technique in hospital settings. Results MeltingPlot is the first tool specifically designed for epidemiological investigations using HRM data, easing the application of HRM typing to large real-time surveillance and rapid outbreak reconstructions. MeltingPlot implements a graph-based algorithm designed to discriminate pathogen clones on the basis of HRM data, producing portable typing results. The tool also merges typing information with isolates and patients metadata to create graphical and tabular outputs useful in epidemiological investigations and it runs in a few seconds even with hundreds of isolates. Availability: https://skynet.unimi.it/index.php/tools/meltingplot/. Conclusions The analysis and result interpretation of HRM typing protocols can be not trivial and this likely limited its application in hospital settings. MeltingPlot is a web tool designed to help the user to reconstruct epidemiological events by combining HRM-based clustering methods and the isolate/patient metadata. The tool can be used for the implementation of HRM based real time large scale surveillance programs in hospital settings.

Proposal of a Model for Periodontal Prevention During Orthodontic Treatment

Background: Patients in orthodontic therapy often have an increase in gingival plaque and also in the periodontal bacterial load. The goal of the present work is to propose a model for the management of the periodontal tissues of these patients. Methods: 1) Patient 1 month after the insertion of the brackets is subjected to a test with molecular biology technique; 2) The patient is instructed to use a water jet daily, using it 2 minutes on both arches 2 times a day for a month; 3) The previously described microbiological test is performed again; 4) The reports obtained are compared and delivered to the patient after illustrating the problems; 5) if the percentage of periodontal pathogens exceeds 5% of the total, continue with phase 2 and 3, until the lowering of the same; 6) if lower, the test will be repeated after 6 months. Results: We reported 2 examples of the prevention model proposed in which patients improved significantly. Conclusions. The protocol we propose aims to limit periodontal damage possibly caused by orthodontic treatment. The proposed model could be the basis of clinical trials that also include a comparison with a control group.

Biodiversity and Dynamics (Rate of Change) of Bacterial Communities Involved in the Biodegradation of Petroleum Refinery Sludge in Contaminated Soils

Aim: The study assessed the bio treatability of the petroleum refinery sludge in contaminated soils by indigenous bacterial communities and the effects of the sludge contamination and bio stimulants on the biodiversity and dynamics (rate of change) of the bacterial communities involved in the biodegradation of the sludge, using the molecular biology technique, Denaturing Gradient Gel Electrophoresis (DGGE). Study Design: The randomnized block design was used for the study. Place and duration of the Study: The research was conducted in the biology laboratory of Flinders University, Adelaide, South Australia. Methodology: The percentage of total petroleum hydrocarbons (TPH) degraded and the bacterial load in the test microcosms was assessed tri-weekly for 12 weeks. The percentage TPH was assessed using Gas chromatography, while the bacterial count was determined as gene copies using the culture independent molecular tool, quantitative real-time PCR (qRt-PCR) analysis. The effects of the experimental treatments on the biodiversity and dynamics (rate of change) of the bacterial communities involved in the biodegradation of the sludge in the soils was determined by the culture-independent molecular biology technique, DGGE. Moving Windows Analysis (MWA) and Shannon Weaver diversity index were used to determine the dynamics (rate of change) and biodiversity of the bacterial communities respectively. Results: Results obtained for the Moving Window Analysis (MWA) which is used to determine the dynamics (Dy), or rate of change of the bacterial communities, showed that, the 1% and 5% sludge contaminated soils biostimulated with compost, recorded the highest Dy of 86.0 ± 1.90% and 87.0 ± 2.20% respectively.NPK biostimilated soil microcosms however recorded a lower Dy of 33.75± 3.20 and 32.50 ± 4.68% for 1% and 5% sludge contamination respectively. The biodiversity of the bacterial communities expressed as Shannon -Weaver index (H1), recorded the highest value of 2.76 ±0.02 for the compost biostimulated microcosm in the 1% sludge treatment, while for the 5% sludge contamination, the treatment with NPK and surfactant enhanced the bacterial biodiversity most with a value of 2.76 ±0.07%. In the test soils with 1% sludge contamination, bio stimulation with NPK gave the highest % TPH degradation (78.25%) while the treatment with NPK and Triton-X 100 had the highest TPH degradation (46.55%) for the 5% sludge contaminated soils. There was insignificant difference in the % sludge degradation between the control and other treatments at P > 0.05 and F = 4.07 for the 1% sludge treated soils, while for the soils treated with 5% sludge there was significant difference between the control and other treatments at P < 0.05 and F= 4.07. Conclusion: Bacteria species identified in the sludge by molecular biology techniques included; Pseudomonas sp. ITRI77, Uncultured Thauera sp., Uncultured Pseudomonas sp., Flavobacterium sp., Bacillaceae bacterium, Uncultured soil bacterium, Clostridium sp., most of which are Gram negative. Biostimulation with compost enhanced a higher biodiversity (H i) and dynamics (Dy) of the bacterial communities involved in the biodegradation of the sludge. Though the NPK treated soils enhanced the biodegradation of the sludge most, degradation started declining by the 9th week while that of compost continued to rise steadily till the 12th week. Results obtained indicate that compost is as good as NPK in the biodegradation of petroleum sludge especially at 1% sludge contamination, since there was no statistical difference between the % TPH degraded and the use of compost is environmentally friendly and economically sustainable.

Genetic Engineering and Editing of Plants: An Analysis of New and Persisting Questions

Genetic engineering is a molecular biology technique that enables a gene or genes to be inserted into a plant's genome. The first genetically engineered plants were grown commercially in 1996, and the most common genetically engineered traits are herbicide and insect resistance. Questions and concerns have been raised about the effects of these traits on the environment and human health, many of which are addressed in a pair of 2008 and 2009 Annual Review of Plant Biology articles. As new science is published and new techniques like genome editing emerge, reanalysis of some of these issues, and a look at emerging issues, is warranted. Herein, an analysis of relevant scientific literature is used to present a scientific perspective on selected topics related to genetic engineering and genome editing.

Overview of the Total Bacteria and Number of Streptococcus mutans in the Saliva of Children with High Caries Activity

Introduction: Microorganisms play a crucial role in the early stage of caries. Streptococcus mutans produces lactic acid, which enhances the risk of caries. Polymerase chain reaction (PCR) is a molecular biology technique used in the amplification and replication of DNA. PCR exhibits high specificity and a rapid detection time, as well as differentiation between microorganisms. Based on a previous study, there is a significant correlation between S. mutans in saliva and the prevalence of caries. Objectives: To provide an overview of the total bacteria and number of S. mutans in the saliva of children aged between 5 and 10 years at the Department of Pediatric, Faculty of Dentistry, Trisakti University, Jakarta, Indonesia. Methods: Saliva sample from children (n = 28, aged 5-10 years old) was collected from healthy children. 2-3 mL of a saliva samples were collected and cultured by using TYS media for total bacterial count. The number of S. mutans in the saliva of these children was counted by real-time PCR methods by using a fluorescence chemical compound green SYBR using 16S rRNA gene specific primers for S. mutans. Results: The average number of total bacterial colony count in saliva was 9.965 ± 2.078 log10 CFU/mL, and the average number of S. mutans in the saliva of children was 8.303 ± 2.351 log10 CFU/mL. Conclusion: The number of S. mutans in the saliva of children in Department of Pediatric, Faculty of Dentistry, Trisakti University was categorized is in the high-risk.

Biodiversity and medicinal uses of globe artichoke (Cynara scolymus L.) plant

Globe artichoke (Cynara scolymus L.) is a vegetable crop native of the Mediterranean basin and it is grown mainly for its immature flower buds. The edible parts of artichoke have important nutrition values to their content of inulin, fibers and minerals. Furthermore, artichoke is recognized as a medicinal plant where leaves and heads are rich source of polyphenolic compounds. It is recommended for the treatment of gallstones, liver disease or damage and poor liver function. The pharmaceutical properties of artichoke are linked to their special chemical composition, which includes high levels of polyphenols such as cynarin, along with its biosynthetic precursor chlorogenic acid. Globe artichoke is characterized by a wide range of biodiversity which represents a prerequisite for genetic improvement. However, there are many factors including biotic and abiotic stresses threatening the genetic resources of globe artichoke. Such factors could result in genetic erosion due to crop failure and loss of cultivars. Therefore, there is a great need to preserve the globe artichoke diversity for future genetic improvement. Because most cultivars are highly heterozygous, maintenance of globe artichoke germplasm in seed form is restricted. Otherwise, conservation in the field presents major drawbacks, which limit its efficacy and threaten the safety of germplasm conserved in this way. The application of biotechnology tools, i.e. plant tissue culture and molecular biology technique can provide reliable methods for supporting the exploitation and conservation of globe artichoke biodiversity. Furthermore, in vitro culture techniques could be used for the controlled production of artichoke secondary metabolites. This article discusses the biodiversity, conservation and nutritional and medicinal aspects of globe artichoke plant. J. Biodivers. Conserv. Bioresour. Manag. 2019, 5(1): 39-54

NLRP3, IL-1β, and Caspase-1 Gene Transcript Identification and Expression by QCM-D in Obese Children

Background. Obesity in children is highly prevalent in Mexican population. Adipose tissue has been related to specific pro- and anti-inflammatory cytokine and inflammasome gene and protein expression patterns. Actually, there is no existing biosensor for detecting gene expression patterns in children with obesity. The quartz crystal microbalance with dissipation monitoring (QCM-D) has been used as a transducer for DNA biosensor design. Results. In this study, the gene expression pattern of IL-1β, NLRP3, and CASPASE-1 in children with obesity was successfully determined by means of QCM-D. Gene expression patterns were validated with those obtained by quantitative polymerase chain reaction (qPCR), a validated molecular biology technique for gene expression quantification. QCM-D analysis of the detected mass corresponding results for each of the genes showed a major detected mass for IL-1β, followed by similar NLRP3 and constitutive gene 18S deposited mass and a smaller deposited mass for CASPASE-1. Surprisingly, when comparing mRNA gene expression results for NLRP3, IL-1β, and CASPASE-1 obtained with qPCR and QCM-D, similar patterns were found, revealing greatest expression of IL-1β, followed by NLRP3, with CASPASE-1 being the molecule of least expression in the group of children with obesity. AFM images illustrate the step-by-step changes that took place on the quartz surface. Conclusions. QCM-D proved successfully for determining the gene transcripts and expression of NLRP3, IL-1β, and CASPASE-1 in children with obesity, with similar results validated by qPCR. “QCM-D decreases detection costs compared with a validated molecular biology technique.” The QCM-D biosensor developed by our group was successful for gene expression determination; in the future, it can be used for molecular diagnosis.