Direct electrochemical genosensing for multiple point mutation detection of Mycobacterium tuberculosis during the development of rifampin resistance

We present a robust and simple method for the direct detection of multiple point mutations in the Mycobacterium tuberculosis rpoB gene during the development of rifampin (RIF) resistance using an electrochemical genosensor. The device contained five different capture probes which are designed to hybridize with several sequence segments within the bacterial rpoB gene hotspot region. Point mutations were detected by monitoring the guanine oxidation with differential pulse voltammetry after hybridization between PCR amplicons and inosine modified capture probes at graphite surface. Changes in the peak voltage corresponding to guanine oxidation provide an electrochemical signal for hybridization that can be used to determine the presence of point mutations conferring rifampin resistance. The analytical parameters (sensitivity, selectivity and reproducibility) were evaluated. High selective discrimination against point mutation of bacteria at hot-spot region was observed. Several mutations were detected at several parts of the amplicon from 21 positive samples.     

Laboratory evaluation of Turkish entomopathogenic nematodes for suppression of the chestnut pests,Curculio elephas (Coleoptera: Curculionidae) and Cydia splendana (Lepidoptera: Tortricidae)

The lepidopteran, Cydia splendana, and the coleopteran, Curculio elephas, are the most serious pests of chestnut fruit in Turkey. We evaluated the biological control potential of three Turkish entomopathogenic nematode species, Steinernema feltiae, S. weiseri and Heterorhabditis bacteriophora, against the last instar larvae of C. splendana and C. elephas, both of which occur in the soil from fall (October–November) until mid-summer (August). The optimal temperature for infection, time to death of the hosts, and reproductive potential of the nematodes were determined at 10, 15, 20 and 25°C for both pest species. Cydia splendana was more susceptible to nematode infection than C. elephas. Temperature had a significant effect on the infectivity and development of entomopathogenic nematodes. The cold-adapted S. weiseri and S. feltiae were the most virulent species at 10 and 15°C, whereas the warm-adapted H. bacteriophora was the most effective at 20 and 25°C. In soil pot experiments conducted at 15°C, S. weiseri was the most virulent species against C. elephas and C. splendana. However, our data show that C. elephas larvae had a lower and C. splendana larvae had a higher susceptibility to the nematode species tested. Accordingly, we recommend that future efforts of using entomopathogenic nematodes, especially S. weiseri, be directed against C. splendana and that there be a continued effort to find more virulent nematode isolates against larvae of C. elephas.     

Modeling of polygalacturonase enzyme activity and biomass production by <Emphasis Type="Italic">Aspergillus sojae</Emphasis> ATCC 20235

Aspergillus sojae, which is used in the making of koji, a characteristic Japanese food, is a potential candidate for the production of polygalacturonase (PG) enzyme, which of a major industrial significance. In this study, fermentation data of an A. sojae system were modeled by multiple linear regression (MLR) and artificial neural network (ANN) approaches to estimate PG activity and biomass. Nutrient concentrations, agitation speed, inoculum ratio and final pH of the fermentation medium were used as the inputs of the system. In addition to nutrient conditions, the final pH of the fermentation medium was also shown to be an effective parameter in the estimation of biomass concentration. The ANN parameters, such as number of hidden neurons, epochs and learning rate, were determined using a statistical approach. In the determination of network architecture, a cross-validation technique was used to test the ANN models. Goodness-of-fit of the regression and ANN models was measured by the R ² of cross-validated data and squared error of prediction. The PG activity and biomass were modeled with a 5-2-1 and 5-9-1 network topology, respectively. The models predicted enzyme activity with an R ² of 0.84 and biomass with an R ² value of 0.83, whereas the regression models predicted enzyme activity with an R ² of 0.84 and biomass with an R ² of 0.69.     

Mutation and association analysis of the PVR and PVRL2 genes in patients with non-syndromic cleft lip and palate

Orofacial clefts (OFC; MIM 119530) are among the most common major birth defects. Here, we carried out mutation screening of the PVR and PVRL2 genes, which are both located at an OFC linkage region at 19q13 (OFC3) and are closely related to PVRL1, which has been associated with both syndromic and non-syndromic cleft lip and palate (nsCLP). We screened a total of 73 nsCLP patients and 105 non-cleft controls from the USA for variants in PVR and PVRL2, including all exons and encompassing all isoforms. We identified four variants in PVR and five in PVRL2. One non-synonymous PVR variant, A67T, was more frequent among nsCLP patients than among normal controls, but this difference did not achieve statistical significance.     

Gene networks in Drosophila melanogaster: integrating experimental data to predict gene function

Background

Discovering the functions of all genes is a central goal of contemporary biomedical research. Despite considerable effort, we are still far from achieving this goal in any metazoan organism. Collectively, the growing body of high-throughput functional genomics data provides evidence of gene function, but remains difficult to interpret.

Results

We constructed the first network of functional relationships for Drosophila melanogaster by integrating most of the available, comprehensive sets of genetic interaction, protein-protein interaction, and microarray expression data. The complete integrated network covers 85% of the currently known genes, which we refined to a high confidence network that includes 20,000 functional relationships among 5,021 genes. An analysis of the network revealed a remarkable concordance with prior knowledge. Using the network, we were able to infer a set of high-confidence Gene Ontology biological process annotations on 483 of the roughly 5,000 previously unannotated genes. We also show that this approach is a means of inferring annotations on a class of genes that cannot be annotated based solely on sequence similarity. Lastly, we demonstrate the utility of the network through reanalyzing gene expression data to both discover clusters of coregulated genes and compile a list of candidate genes related to specific biological processes.

Conclusions

Here we present the the first genome-wide functional gene network in D. melanogaster. The network enables the exploration, mining, and reanalysis of experimental data, as well as the interpretation of new data. The inferred annotations provide testable hypotheses of previously uncharacterized genes.     

High-throughput single cell arrays as a novel tool in biopreservation

Microwell array cytometry is a novel high-throughput experimental technique that makes it possible to correlate pre-stress cell phenotypes and post-stress outcomes with single cell resolution. Because the cells are seeded in a high density grid of cell-sized microwells, thousands of individual cells can be tracked and imaged through manipulations as extreme as freezing or drying. Unlike flow cytometry, measurements can be made at multiple time points for the same set of cells. Unlike conventional image cytometry, image analysis is greatly simplified by arranging the cells in a spatially defined pattern and physically separating them from one another. To demonstrate the utility of microwell array cytometry in the field of biopreservation, we have used it to investigate the role of mitochondrial membrane potential in the cryopreservation of primary hepatocytes.Even with optimized cryopreservation protocols, the stress of freezing almost always leads to dysfunction or death in part of the cell population. To a large extent, cell fate is dominated by the stochastic nature of ice crystal nucleation, membrane rupture, and other biophysical processes, but natural variation in the initial cell population almost certainly plays an important and under-studied role. Understanding why some cells in a population are more likely to survive preservation will be invaluable for the development of new approaches to improve preservation yields.For this paper, primary hepatocytes were seeded in microwell array devices, imaged using the mitochondrial dyes Rh123 or JC-1, cryopreserved for up to a week, rapidly thawed, and checked for viability after a short recovery period. Cells with a high mitochondrial membrane potential before freezing were significantly less likely to survive the freezing process, though the difference in short term viability was fairly small. The results demonstrate that intrinsic cell factors do play an important role in cryopreservation survival, even in the short term where extrinsic biophysical factors would be expected to dominate. We believe that microwell array cytometry will be an important tool for a wide range of studies in biopreservation and stress biology.     

Effect of black cumin (Nigella sativa) on heart rate, some hematological values, and pancreatic β-cell damage in cadmium-treated rats

This study was designed to investigate the effect of Nigella sativa (NS) on the heart rate, some hematological values, and pancreatic β-cell damage in cadmium (Cd)-treated rats. The rats were randomly grouped into one of three experimental groups: Control, Cd treated, and Cd+NS treated. Each group contained 10 animals. The Cd-treated and Cd+NS-treated groups were injected subcutaneously daily with CdCl₂ dissolved in isotonic NaCl in the amount of 2 mL/kg for 30 d, resulting in a dosage of 0.49 mg Cd/kg/d. The control group was injected with only isotonic NaCl (2 mL/kg/d) throughout the experiment (for 30 d). Three days prior to administration of CdCl₂, the Cd+NS-treated group received the daily intraperitoneal (ip) injection of 2 mL/kg NS until the end of the study; animals in all three groups were fasted for 12 h and blood samples were taken for the determination of the glucose and insulin levels, red blood cell (RBC) and white blood cell (WBC) counts, packet cell volume (PCV), and hemoglobin (Hb) concentration. The heart rates of rats were also measured by a direct writing electrocardiograph before the blood withdrawals. It was found that NS treatment increased the lowered insulin levels, RBC and WBC counts, PCV, and neutrophil percentage in Cd-treated rats. However, the WBC count of Cd-treated rats with NS treatment was still lower than those of control values. NS treatment also decreased the elevated heart rate and glucose concentration of Cd-treated rats. Pancreatic tissues were also harvested from the sacrificed animals for morphological and immunohistochemical examinations. Cd exposure alone caused a degeneration, necrosis, and weak degranulation in the β-cells of the pancreatic islets. In Cd+NS-treated rats, increased staining of insulin and preservation of islet cells were apparent. It is concluded that NS treatment might decrease the Cd-treated disturbances on heart rate, some hematological values, and pancreatic β-cell.     

Correlation of serum trace elements and melatonin levels to radiological, biochemical, and histological assessment of degeneration in patients with intervertebral disc herniation

The aim of our study was to assess the blood concentrations of some trace elements and melatonin (MLT) in patients with intervertebral disc herniation (IDH) and to investigate the interaction of histological and biochemical degeneration findings with aging. The present study was carried out on 13 subjects (8 women and 5 men) diagnosed with IDH. They were divided into three groups according to their ages. Nighttime serum MLT, zinc (Zn), and magnesium (Mg) levels were determined in all patients. In addition, computed tomography (CT) scan of the brain and magnetic resonance imaging examination of the lumbar spine were obtained in this study. The Zn level and Zn/Mg ratio showed a decline in patients with IDH with aging, whereas the serum Mg level and tissue hydroxyproline content increased. A positive correlation between serum Zn and MLT concentrations was found (r=0.104, p=0.734). In addition, there was a positive correlation between serum Zn level and Zn/Mg ratio (r=0.835 and p<0.01), and a negative correlation between serum Mg level and Zn/Mg ratio (r=−0.571, p<0.05). On CT study, both volume percentage of calcified pineal gland and density of calcification were found to increase progressively with advancing age. The results of semiquantitative evaluation of disc tissues of patients with IDH for histological degeneration findings showed that 66.7% of discs treated had slight degeneration in younger age group, but 75.0% and 100% of discs had moderate or marked degeneration in older age groups. Our data indicated that there is a close relationship between MLT and Zn or Mg levels in the serum samples of patients with IDH, and the levels of these elements might be affected by the presence of degeneration process and serum MLT level, or vice versa.     

Comparison of angiotensin-converting enzyme, malonaldehyde, zinc, and copper levels in preeclampsia

Preeclampsia is a syndrome of unknown etiopathogenesis. Recent studies carried out on preeclampsia have focused on the increase in free radicals in the feto-placental unit with poor perfusion. It is believed that the renin-angiotensin system (RAS) has a role in the poor perfusion of the placenta. It is uncertain whether there is a pre-existing impairment in RAS in pre-eclamptic pregnant women or not. In the present study, we measured angiotensin-converting enzyme (ACE), malonaldehyde (MDA), zinc, and copper levels in the placental tissue of 16 pre-eclamptic pregnant women and compared them with those in 20 healthy pregnant women. Whereas ACE activity and MDA were found to be high in the placentas of pre-eclamptic patients, zinc and copper levels were low and there was a negative correlation between ACE activity and zinc concentration. These findings suggest that high ACE activity might play a role in the increase in tissue hypoxia and consequent lipid peroxidation through vasoconstriction; zinc deficiency in the placental tissue might cause insufficiency of superoxide dismutase, an antioxidant enzyme. Furthermore, deficiency in placental zinc also plays a role in the biosynthesis of connective tissue, maintaining its integrity, which might have an impact on the structure of the spiral arteries