High-speed shaking of frozen blood clots for extraction of human and malaria parasite DNA

<p>Abstract</p> <p>Background</p> <p>Frozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome.</p> <p>Methods</p> <p>High-speed shaking of clot samples in a cell disruptor manufactured for homogenization of tissue and faecal specimens was evaluated for processing frozen blood clots for DNA extraction. The method was compared to two commercial clot protocols based on a chemical kit and centrifugation through a plastic sieve, followed by the same DNA extraction protocol. Blood clots with different levels of parasitaemia (1-1,000 p/μl) were prepared from parasite cultures to assess sensitivity of PCR detection. In addition, clots retrieved from serum samples collected within two epidemiological studies in Kenya (n = 630) were processed by high speed shaking and analysed by PCR for detection of malaria parasites and the human α-thalassaemia gene.</p> <p>Results</p> <p>High speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of <it>P. falciparum </it>parasites and the human thalassaemia gene was the same as samples optimally collected with an anticoagulant. The commercial clot protocol and centrifugation through a sieve failed to fully dissolve the clots and resulted in lower sensitivity of PCR detection.</p> <p>Conclusions</p> <p>High speed shaking was a simple and efficacious method for homogenizing frozen blood clots before DNA purification and resulted in PCR templates of high quality both from humans and malaria parasites. This novel method enables genetic studies from stored blood clots.</p

Child with Deletion 9p Syndrome Presenting with Craniofacial Dysmorphism, Developmental Delay, and Multiple Congenital Malformations

A 4-month-old Sri Lankan male child case with a de novo terminal deletion in the p22 → pter region of chromosome 9 is described. The child presented with craniofacial dysmorphism, developmental delay, and congenital malformations in agreement with the consensus phenotype. A distinctive feature observed in this child was complete collapse of the left lung due to malformation of lung tissue. Cytogenetic studies confirmed terminal deletion of the short arm of chromosome 9 distal to band p22 [46,XY,del(9)(p22 → pter)]. This is the first reported case of a de novo deletion 9p syndrome associated with pulmonary hypoplasia. This finding contributes to the widening of the spectrum of phenotypic features associated with deletion 9p syndrome

Genetic diversity of Leishmania amazonensis strains isolated in northeastern Brazil as revealed by DNA sequencing, PCR-based analyses and molecular karyotyping

Abstract\ud \ud \ud \ud Background\ud \ud Leishmania (Leishmania) amazonensis infection in man results in a clinical spectrum of disease manifestations ranging from cutaneous to mucosal or visceral involvement. In the present study, we have investigated the genetic variability of 18 L. amazonensis strains isolated in northeastern Brazil from patients with different clinical manifestations of leishmaniasis. Parasite DNA was analyzed by sequencing of the ITS flanking the 5.8 S subunit of the ribosomal RNA genes, by RAPD and SSR-PCR and by PFGE followed by hybridization with gene-specific probes.\ud \ud \ud \ud Results\ud \ud ITS sequencing and PCR-based methods revealed genetic heterogeneity among the L. amazonensis isolates examined and molecular karyotyping also showed variation in the chromosome size of different isolates. Unrooted genetic trees separated strains into different groups.\ud \ud \ud \ud Conclusion\ud \ud These results indicate that L. amazonensis strains isolated from leishmaniasis patients from northeastern Brazil are genetically diverse, however, no correlation between genetic polymorphism and phenotype were found.We thank Lucile FloeterWinter for critical reading of the manuscript and Artur T.L. de Queiroz for initial help with phylogenetic analysis. This work is supported by grants from CNPq, FAPESB and PAPES/FIOCRUZ. J.P.C. de Oliveira was supported by a CNPq fellowship; C.I.O. and F.M.C.F were supported by a FAPESB fellowship. AAC, AB, and CIO are senior investigators from CNPq. AB is a senior investigator for Instituto de Investigação em Imunologia (iii).We thank Lucile Floeter-Winter for critical reading of the manuscript and Artur T.L. de Queiroz for initial help with phylogenetic analysis. This work is supported by grants from CNPq, FAPESB and PAPES/FIOCRUZ. J.P.C. de Oliveira was supported by a CNPq fellowship; C.I.O. and F.M.C.F were supported by a FAPESB fellowship. AAC, AB, and CIO are senior investigators from CNPq. AB is a senior investigator for Instituto de Investigação em Imunologia (iii)

Association of kidney disease measures with risk of renal function worsening in patients with type 1 diabetes

Background: Albuminuria has been classically considered a marker of kidney damage progression in diabetic patients and it is routinely assessed to monitor kidney function. However, the role of a mild GFR reduction on the development of stage 653 CKD has been less explored in type 1 diabetes mellitus (T1DM) patients. Aim of the present study was to evaluate the prognostic role of kidney disease measures, namely albuminuria and reduced GFR, on the development of stage 653 CKD in a large cohort of patients affected by T1DM. Methods: A total of 4284 patients affected by T1DM followed-up at 76 diabetes centers participating to the Italian Association of Clinical Diabetologists (Associazione Medici Diabetologi, AMD) initiative constitutes the study population. Urinary albumin excretion (ACR) and estimated GFR (eGFR) were retrieved and analyzed. The incidence of stage 653 CKD (eGFR &lt; 60 mL/min/1.73 m2) or eGFR reduction &gt; 30% from baseline was evaluated. Results: The mean estimated GFR was 98 \ub1 17 mL/min/1.73m2 and the proportion of patients with albuminuria was 15.3% (n = 654) at baseline. About 8% (n = 337) of patients developed one of the two renal endpoints during the 4-year follow-up period. Age, albuminuria (micro or macro) and baseline eGFR &lt; 90 ml/min/m2 were independent risk factors for stage 653 CKD and renal function worsening. When compared to patients with eGFR &gt; 90 ml/min/1.73m2 and normoalbuminuria, those with albuminuria at baseline had a 1.69 greater risk of reaching stage 3 CKD, while patients with mild eGFR reduction (i.e. eGFR between 90 and 60 mL/min/1.73 m2) show a 3.81 greater risk that rose to 8.24 for those patients with albuminuria and mild eGFR reduction at baseline. Conclusions: Albuminuria and eGFR reduction represent independent risk factors for incident stage 653 CKD in T1DM patients. The simultaneous occurrence of reduced eGFR and albuminuria have a synergistic effect on renal function worsening

The Hv1 proton channel responds to mechanical stimuli

The voltage-gated proton channel, Hv1, is expressed in tissues throughout the body and plays important roles in pH homeostasis and regulation of NADPH oxidase. Hv1 operates in membrane compartments that experience strong mechanical forces under physiological or pathological conditions. In microglia, for example, Hv1 activity is potentiated by cell swelling and causes an increase in brain damage after stroke. The channel complex consists of two proton-permeable voltage-sensing domains (VSDs) linked by a cytoplasmic coiled-coil domain. Here, we report that these VSDs directly respond to mechanical stimuli. We find that membrane stretch facilitates Hv1 channel opening by increasing the rate of activation and shifting the steady-state activation curve to less depolarized potentials. In the presence of a transmembrane pH gradient, membrane stretch alone opens the channel without the need for strong depolarizations. The effect of membrane stretch persists for several minutes after the mechanical stimulus is turned off, suggesting that the channel switches to a “facilitated” mode in which opening occurs more readily and then slowly reverts to the normal mode observed in the absence of membrane stretch. Conductance simulations with a six-state model recapitulate all the features of the channel’s response to mechanical stimulation. Hv1 mechanosensitivity thus provides a mechanistic link between channel activation in microglia and brain damage after stroke

Flagellar ion channels of sperm: similarities and differences between species

Motility and fertilization potential of mammalian sperm are regulated by ion homeostasis which in turn is under tight control of ion channels and transporters. Sperm intracellular pH, membrane voltage and calcium concentration ([Ca2+]i) are all important for sperm activity within the female reproductive tract. While all mammalian sperm are united in their goal to find and fertilize an egg, the molecular mechanisms they utilize for this purpose are diverse and differ between species especially on the level of ion channels. Recent direct recording from sperm cells of different species indicate the differences between rodent, non-human primate, ruminant, and human sperm on the basic levels of their ion channel regulation. In this review we summarize the current knowledge about ion channel diversity of the animal kingdom and concentrate our attention on flagellar ion channels of mammalian sperm