Novel and recurrent LDLR gene mutations in Pakistani hypercholesterolemia patients

The majority of patients with the autosomal dominant disorder familial hypercholesterolemia (FH) carry novel mutations in the low density lipoprotein receptor (LDLR) that is involved in cholesterol regulation. In different populations the spectrum of mutations identified is quite different and to date there have been only a few reports of the spectrum of mutations in FH patients from Pakistan. In order to identify the causative LDLR variants the gene was sequenced in a Pakistani FH family, while high resolution melting analysis followed by sequencing was performed in a panel of 27 unrelated sporadic hypercholesterolemia patients. In the family a novel missense variant (c.1916T > G, p.(V639G)) in exon 13 of LDLR was identified in the proband. The segregation of the identified nucleotide change in the family and carrier status screening in a group of 100 healthy subjects was done using restriction fragment length polymorphism analysis. All affected members of the FH family carried the variant and none of the non-affected members nor any of the healthy subjects. In one of the sporadic cases, two sequence changes were detected in exon 9, one of these was a recurrent missense variant (c.1211C > T; p.T404I), while the other was a novel substitution mutation (c.1214 A > C; N405T). In order to define the allelic status of this double heterozygous individual, PCR amplified fragments were cloned and sequenced, which identified that both changes occurred on the same allele. In silico tools (PolyPhen and SIFT) were used to predict the effect of the variants on the protein structure, which predicted both of these variants to have deleterious effect. These findings support the view that there will be a novel spectrum of mutations causing FH in patients with hypercholesterolaemia from Pakistan.     

Characterization of Erg K+ Channels in ��- and ��-Cells of Mouse and Human Islets

Voltage-gated eag-related gene (Erg) K⁺ channels regulate the electrical activity of many cell types. Data regarding Erg channel expression and function in electrically excitable glucagon and insulin producing cells of the pancreas is limited. In the present study Erg1 mRNA and protein were shown to be highly expressed in human and mouse islets and in α-TC6 and Min6 cells α- and β-cell lines, respectively. Whole cell patch clamp recordings demonstrated the functional expression of Erg1 in α- and β-cells, with rBeKm1, an Erg1 antagonist, blocking inward tail currents elicited by a double pulse protocol. Additionally, a small interference RNA approach targeting the kcnh2 gene (Erg1) induced a significant decrease of Erg1 inward tail current in Min6 cells. To investigate further the role of Erg channels in mouse and human islets, ratiometric Fura-2 AM Ca²⁺-imaging experiments were performed on isolated α- and β-cells. Blocking Erg channels with rBeKm1 induced a transient cytoplasmic Ca²⁺ increase in both α- and β-cells. This resulted in an increased glucose-dependent insulin secretion, but conversely impaired glucagon secretion under low glucose conditions. Together, these data present Erg1 channels as new mediators of α- and β-cell repolarization. However, antagonism of Erg1 has divergent effects in these cells; to augment glucose-dependent insulin secretion and inhibit low glucose stimulated glucagon secretion.Voltage-gated eag-related gene (Erg)² potassium (K⁺) channels are part of the larger family of voltage dependent K⁺ (Kv) channels (1). Three channel isoforms Erg1, Erg2, and Erg3 have been discovered (2, 3), and they differ by their activation and inactivation voltage dependence, gating properties, and pharmacological profile (4–7). Erg channels control cellular activity by controlling the repolarization of the action potential (AP). In atrial cells and ventricular myocytes, Erg regulates plateau formation and AP repolarization, as blocking Erg channels increases AP length (8, 9). These channels are also strongly involved in the pacemaking activity of cardiac cells (10, 11). Interestingly, a rare congenital heart condition, the inherited form of long QT syndrome is caused by mutations of Erg channel genes (9, 12). Erg channels also control the resting membrane potential in various cell types. For example, in neurons of the medial vestibular nucleus, blocking Erg channels produce an increase in AP discharge or in smooth muscle cells, blocking Erg channels mediates depolarization up to 20 mV (13–15). Hormone secretion studies also demonstrated the involvement of Erg channels in the secretion of prolactin from neurons of the anterior pituitary. Thyrotropin-releasing factor decreases Erg current, which depolarizes neurons and thereby stimulates prolactin secretion (16, 17).In the pancreas, Kv channels and more specifically Kv2.1, regulate insulin secretion by controlling the repolarization of β-cell membrane potential (18–20), although the contribution of this isoform in humans has recently been questioned (21). In α-cells, Kv2.1 and Kv1.4 channels repolarize the membrane potential (22, 23); however, the involvement of Kv channels in the secretion of glucagon is yet to be investigated. One study showed that Erg1, -2, and -3 are expressed in rat α- and β-cells and the rat insulinoma cell line, INS-1, and that they are involved in decreasing membrane potential. Blocking Erg channels with the channel antagonist E4031 increases insulin secretion from INS1 cells (24); however, definitive data regarding the role of Erg channels in insulin and glucagon secretion is limited.Therefore this study aimed to define the functions of Erg channels in α- and β-cells. We found that Erg1 channels are strongly expressed in pancreatic α- and β-cells. Pharmacological and genetic manipulation combined with whole cell recordings in pancreatic cell lines and primary islet cells determined that Erg1 produces a functional current in α- and β-cells. Blocking Erg1 increased intracellular calcium ([Ca²⁺]_i) in mouse β-cells, but only in a minority of mouse and human α-cells. Secretion studies using isolated mouse islets demonstrated that Erg1 are negative regulators of insulin secretion, but positive regulators of glucagon secretion, suggesting distinct roles for Erg1 in β- and α-cells.     

Dopaminergic inhibition of secretin-stimulated choleresis by increased PKC-gamma expression and decrease of PKA activity

To determine the role and mechanisms of action by which dopaminergic innervation modulates ductal secretion in bile duct-ligated rats, we determined the expression of D1, D2, and D3 dopaminergic receptors in cholangiocytes. We evaluated whether D1, D2 (quinelorane), or D3 dopaminergic receptor agonists influence basal and secretin-stimulated choleresis and lumen expansion in intrahepatic bile duct units (IBDU) and cAMP levels in cholangiocytes in the absence or presence of BAPTA-AM, chelerythrine, 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine (H7), or rottlerin. We evaluated whether 1) quinelorane effects on ductal secretion were associated with increased expression of Ca(2+)-dependent PKC isoforms and 2) increased expression of PKC causes inhibition of PKA activity. Quinelorane inhibited secretin-stimulated choleresis in vivo and IBDU lumen space, cAMP levels, and PKA activity in cholangiocytes. The inhibitory effects of quinelorane on secretin-stimulated ductal secretion and PKA activity were blocked by BAPTA-AM, chelerythrine, and H7. Quinelorane effects on ductal secretion were associated with activation of the Ca(2+)-dependent PKC-gamma but not other PKC isoforms. The dopaminergic nervous system counterregulates secretin-stimulated ductal secretion in experimental cholestasis.     

Antibacterial cobalt(II), nickel(II) and zinc(II) complexes of nicotinic acid-derived Schiff-bases

Nicotinic acid derived Schiff bases and their transition metal [cobalt(II), nickel(II) and zinc(II)] complexes have been prepared and characterized by physical, spectral and analytical data. The Schiff bases act as deprotonated tridentate ligands for the complexation of the above mentioned metal ions. These complexes, possessing the general formula [M(L)2] [where M = Co(II), Ni(II) and Zn(II) and L = HL1-HL4] showed an octahedral geometry of the metal ions. For determining the effect of metal ions upon chelation, the Schiff bases and their complexes have been screened for antibacterial activity against several pathogenic strains of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The new metal derivatives reported here were more bactericidal against one or more bacterial species as compared to the uncomplexed Schiff bases.     

Barriers,Facilitators and Priorities for Implementation of WHO Maternal and Perinatal Health Guidelines in Four Lower-Income Countries: A GREAT Network Research Activity

BackgroundHealth systems often fail to use evidence in clinical practice. In maternal and perinatal health, the majority of maternal, fetal and newborn mortality is preventable through implementing effective interventions. To meet this challenge, WHO’s Department of Reproductive Health and Research partnered with the Knowledge Translation Program at St. Michael’s Hospital (SMH), University of Toronto, Canada to establish a collaboration on knowledge translation (KT) in maternal and perinatal health, called the GREAT Network (Guideline-driven, Research priorities, Evidence synthesis, Application of evidence, and Transfer of knowledge). We applied a systematic approach incorporating evidence and theory to identifying barriers and facilitators to implementation of WHO maternal heath recommendations in four lower-income countries and to identifying implementation strategies to address these.MethodsWe conducted a mixed-methods study in Myanmar, Uganda, Tanzania and Ethiopia. In each country, stakeholder surveys, focus group discussions and prioritization exercises were used, involving multiple groups of health system stakeholders (including administrators, policymakers, NGOs, professional associations, frontline healthcare providers and researchers).ResultsDespite differences in guideline priorities and contexts, barriers identified across countries were often similar. Health system level factors, including health workforce shortages, and need for strengthened drug and equipment procurement, distribution and management systems, were consistently highlighted as limiting the capacity of providers to deliver high-quality care. Evidence-based health policies to support implementation, and improve the knowledge and skills of healthcare providers were also identified. Stakeholders identified a range of tailored strategies to address local barriers and leverage facilitators.ConclusionThis approach to identifying barriers, facilitators and potential strategies for improving implementation proved feasible in these four lower-income country settings. Further evaluation of the impact of implementing these strategies is needed.     

Phytoavailability and Leachability of Heavy Metals from Contaminated Soil Treated with Composted Livestock Manure

A study was carried out with maize as the test crop to investigate the bioavailability and leachability of heavy metals (HM) from HM-contaminated soils treated with composted manure. The application of composted manure increased the maize shoot growth by 32.3% and root growth by 30.5% compared with fresh manure. The concentration of HM in maize shoot varied in the order lead (Pb) > nickel (Ni) > zinc (Zn) > chromium (Cr) > copper (Cu) > cadmium (Cd). Whether for the shoot or root, the heavy metal concentrations decreased as the length of manure composting increased, but concentrations were higher in the root than the shoot. Composting decreased the bioavailability and leachability of HM and, hence, their export to the environment more effectively than direct use of fresh manure. Thus, the use of composted manure in place of fresh manure in polluted soils would be more beneficial for mitigating HM pollution.     

Ultrastructure and histochemistry of the digestive tract of juvenile Paramphistomum epiclitum (Paramphistomidae: Digenea) during migration in Indian ruminants.

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and 1992. Ultrastructure and histochemistry of the digestive tract of juvenile Paramphistomum epiclitum (Paramphistomidae: Digenea) during migration in Indian ruminants. International Journal for Parasitology 22: 1089–1101. The digestive tract of juvenile Paramphistomum epiclitum consists of a foregut with a highly muscular terminal pharynx and an oesophagus, which leads to a pair of unbranched and blind-ending intestinal caeca. A syncytium lining the foregut is continuous with the external tegument and displays similar sensory papillae and secretory bodies (T1 and T2). A third type of secretory body (T3) is confined to the oesophageal cytons of newly excysted juveniles and is first evident in the syncytium by day 14 of migration. An epithelium lining the caeca is composed of a single layer of morphologically uniform cells whose apical surface is amplified by microvilli. Dense secretions synthesized in the caecal epithelium of mature cercariae are released during migration by a mechanism resembling modified apocrine discharge. The caecal epithelium of migrating juveniles undergoes a 10-fold increase in surface amplification (irrespective of growth) during its transition from a primarily secretory tissue to one apparently specialized for absorption.     

Influence of Frequently Used Chemical Insecticides on Mycoflora Carried by Common Housefly, <Emphasis Type="Italic">Musca domestica</Emphasis> L.

The housefly, Musca domestica L. (Diptera: Muscidae), is a major medical and veterinary insect pest. It serves as a vector of many pathogenic microorganisms causing spoilage of food and diseases in human and animals. Use of chemical insecticides is adapted as a principal tool to manage housefly. Insecticides have many unforeseen ecological consequences including effects on non-target organisms. In the present study, we have assessed the effects of 10 different synthetic insecticides on the growth of mycoflora associated with the external body of the housefly by using poison food technique. Our results reveled that all synthetic insecticides enhanced the growth. Surprisingly, in most of the cases, mycelial growth of fungi was significantly increased at high concentration as compared with lower concentration. This study provides useful information about the dangerous effects of synthetic insecticides on environment by increasing the spread of various non-target pathogenic, mycotoxigenic, and food spoiling fungi, carried by houseflies.     

Endophytic bacteria enhance remediation of tannery effluent in constructed wetlands vegetated with Leptochloa fusca

The use of constructed wetlands (CWs) is a promising approach for the remediation of wastewater. The present study aims to develop a plant–bacteria system within CWs for the efficient remediation of tannery effluent. In a vertical-flow CW vegetated with Leptochloa fusca (Kallar grass), a consortium of three different endophytic bacteria, Pantoea stewartii ASI11, Microbacterium arborescens HU33, and Enterobacter sp. HU38, was used for bioaugmentation. CWs vegetated with only L. fusca had the potential to remediate tannery effluent, but augmentation with endophytic bacteria enhanced the growth of L. fusca while aiding in the removal of both organic and inorganic pollutants from the tannery effluent. Moreover, the bacterial augmentation decreased toxicity in the effluent as well. A higher number of chromium (Cr)-resistant bacteria were isolated from the rhizosphere and endosphere of L. fusca inoculated with the endophytes than from uninoculated plants. Due to promising bioremediation and detoxification potential of L. fusca, it is reported for the first time as a potential candidate to develop effective CWs for the remediation of polluted effluents in combination with pollutant-degrading endophytic bacteria.     

Comparative trial of different anti-bacterial combinations with propolis and ciprofloxacin on Pseudomonas keratitis in rabbits

The aim of the current study was to evaluate the effects of five different treatment combinations to find out whether propolis could be an alternative or an adjunctive treatment, in experimental Pseudomonas aeruginosa keratitis. Intrastromal P. aeruginosa strains were given to both eyes of 20 young New Zealand white rabbits. The rabbits were randomly divided equally into five treatment groups; ciprofloxacin and dexamethasone drops (C+D), ciprofloxacin drop (C), ciprofloxacin and propolis drops (C+P), propolis drop (P), 3% ethanol drop (control), respectively. Directly before the first treatment and 108 h after inoculation, the eyes were examined by slit lamp to assess the corneal opacity and rabbits were sacrificed for bacterial count. The mean corneal opacity scores and the mean bacterial counts log cfu/ml were significantly different in the treatment groups (P=0.001; ANOVA). According to post hoc tests for both the mean bacterial counts and corneal opacity scores, C+D, C, C+P groups were found to be statistically the same (P>0.05), and although the P group had significantly better scores than the control group it did not reach the scores of the rest of the treatment groups (P<0.01). We conclude that propolis may be a useful adjunctive agent but should not be regarded as a replacement for traditional antibiotic therapy for P. aeruginosa keratitis in rabbits.