Novel properties of recombinant Sso7d-Taq DNA polymerase purified using aqueous two-phase extraction: Utilities of the enzyme in viral diagnosis.

Using Sso7d from Sulfolobus solfataricus as the DNA binding protein fused to Taq DNA polymerase at its amino terminus, we report the hyper-expression and a novel purification methodology of Sso7d-Taq polymerase (S-Taq) using aqueous two-phase extraction system followed by Ni-affinity chromatography. The utility of such a fusion enzyme in carrying out PCR of human genes from whole blood directly and in detecting hepatitis B virus from clinical samples is demonstrated in this article. We present data on the enhanced thermo-stability of S-Taq DNA polymerase over Taq DNA polymerase and also provide evidence of its higher stability with detergents in comparison to Taq polymerase. The purified S-Taq protein showed acceptable limits of host genomic DNA levels without the use of DNases and other DNA precipitating agents and shows promising potential for use in PCR based diagnostics, in-situ PCR's and forensic science.

Medical Management of Oral Lichen Planus: A Systematic Review.

INTRODUCTION: Oral Lichen Planus (OLP) is a chronic inflammatory, T-cell-mediated autoimmune oral mucosal disease with unclear aetiology. The clinical management of OLP poses considerable difficulties to the oral physician. AIM: The aim was to assess the efficacy of any form of intervention used to medically manage OLP. MATERIALS AND METHODS: We searched and analysed the following databases (from January 1990 to December 2014):- Cochrane Oral Health Group Trials Register, Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE and EMBASE. All Randomised Controlled Trials (RCTs) for the medical management of OLP which compared active treatment with placebo or between active treatments were considered in this systematic review. Participants of any age, gender or race having symptomatic OLP (including mixed forms), unconnected to any identifiable cause (e.g. lichenoid drug reactions) and confirmed by histopathology have been included. Interventions of all types, including topical treatments or systemic drugs of variable dosage, duration & frequency of delivery have been considered. All the trials identified were appraised by five review authors and the data for all the trials were synthesised using specifically designed data extraction form. Binary data has been presented as risk ratios (RR) with 95% confidence intervals (CI) and continuous data as mean differences (MD) with 95% CIs. RESULTS: A total of 35 RCTs were included in this systematic review on medical management of OLP. No strong evidence suggesting superiority of any specific intervention in reducing pain and clinical signs of OLP were shown by the RCTs included here. CONCLUSION: Future RCTs on a larger scale, adopting standardized outcome assessing parameters should be considered.

Decreasing mechanical ventilator support in medically fragile children with bronchopulmonary dysplasia: A step-by-step weaning protocol at a pediatric long term extended care facility.

PURPOSE: Prolonged mechanical ventilation (PMV) in medically fragile children is commonly used in pediatric long term extended care facilities (P-LTEC). Currently, PMV weaning is performed in an unstandardized fashion. Without an official protocol, patients are subjected to delayed weaning, infection, increased mortality, and difficulty obtaining placement at adult group homes. A step-wise approach may help these children wean from PMV effectively. METHODS: A retrospective chart review of five tracheostomized children with bronchopulmonary dysplasia was conducted. RESULTS: A 5-step weaning protocol was created using data collected retrospectively. First, pressure control ventilator settings were decreased until rate = 10, fraction of inspired oxygen = 30% and pressure support = 6-10. Second, continuous positive airway pressure (CPAP) was trialed while awake with ventilator at night. Third, CPAP was continued for 24 hours. Fourth, tracheostomy collar (TC) was trialed while awake, with CPAP at night. Lastly, TC was continued for 24 hours. Advancing to Step 2 required the most time, likely secondary to episodic illnesses, with a mean of 31.2 months. The process required 3.2 months to advance to Step 3, 1.6 months to achieve Step 4, and 2.6 months to attain Step 5. CONCLUSION: Using the data obtained in this case series an official protocol could be created to wean P-LTEC residents from PMV, with reasonable expectations of the process.

Treating Every Needle in the Haystack: Hyperammonemic Encephalopathy and Severe Malnutrition After Bariatric Surgery-A Case Report and Review of the Literature.

Neurologic complications are not uncommon following bariatric surgery. Hyperammonemic encephalopathy (HAE) due to an acquired or unmasked urea cycle deficit is among the rarest of these. Pediatric nutrition support specialists are familiar with recognizing urea cycle deficits, but adult specialists may not be. Here we present a case of a patient initially misdiagnosed with cirrhosis who presented with recurrent HAE 4 years after Roux-en-Y gastric bypass. She was diagnosed with a proximal urea cycle deficit and severe protein calorie malnutrition. The patient recovered with specialized nutrition and medical support targeting this condition. A literature review indicates multiple fatalities from this condition, indicating the importance of early diagnosis and appropriate nutrition support.

Receptor binding enables botulinum neurotoxin B to sense low pH for translocation channel assembly.

Botulinum neurotoxins (BoNTs, serotypes A-G), elaborated by Clostridium botulinum, can induce lethal paralysis and are classified as Category A bioterrorism agents. However, how BoNTs translocate from endosomes into the cytosol of neurons to gain access to their intracellular targets remains enigmatic. We discovered that binding to the ganglioside GT1b, a toxin coreceptor, enables BoNT/B to sense low pH, undergo a significant change in secondary structure, and transform into a hydrophobic oligomeric membrane protein. Imaging of the toxin on lipid bilayers using atomic force microscopy revealed donut-shaped channel-like structures that resemble other protein translocation assemblies. Toosendanin, a drug with therapeutic effects against botulism, inhibited GT1b-dependent BoNT/B oligomerization and in parallel truncated BoNT/B single-channel conductance, suggesting that oligomerization plays a role in the translocation reaction. Thus, BoNT/B functions as a coincidence detector for receptor and low pH to ensure spatial and temporal accuracy for toxin conversion into a translocation channel.

Phase separation in lipid bilayers triggered by low pH.

Endocytosis involves the capture of membrane from the cell surface in the form of vesicles, which become rapidly acidified to about pH 5. Here we show using atomic force microscopy (AFM) imaging that this degree of acidification triggers phase separation in lipid bilayers containing mixed acyl chains (e.g. palmitoyl/oleoyl) or complex mixtures (e.g. total brain extract) but not in bilayers containing only lipids with unsaturated chains (e.g. dioleoyl). Since mixed-chain lipids are major constituents of the outer leaflet of the plasma membrane, the type of phase separation reported here might support protein clustering and signaling during endocytosis.

The endophilin N-BAR domain perturbs the structure of lipid bilayers.

Endophilin A is a key player in clathrin-mediated endocytosis at nerve terminals and is essential for the maintenance of synaptic transmission. Endophilin consists of two regions: an SH3 domain that interacts with other endocytotic proteins and an N-BAR domain that binds and bends membranes. Here, we used atomic force microscopy (AFM) under fluid to examine the interaction of the endophilin N-BAR domain with planar supported lipid bilayers, under conditions that closely mimic the environment in which this protein normally operates. We found that when bound to lipid bilayers, the N-BAR domain formed aggregates of various sizes. The N-BAR domain also perturbed the structure of the planar bilayer, at a low concentration (0.15 microM) causing bilayer thinning, and at a 10-fold higher concentration (1.5 microM) forming thin slivers from the bilayer sheet. This bilayer sculpting effect crucially involved the central appendage domain. Reduced hydrophobicity in this domain, caused by the A66D mutation, almost abolished the ability of the endophilin N-BAR domain to bind to supported bilayers. In contrast, increased hydrophobicity, caused by the A66W mutation, switched the bilayer sculpting effect of the N-BAR domain from sliver formation to vesiculation. By following the action of the endophilin N-BAR domain under near-physiological conditions, we have been able to provide additional insights into its membrane binding and bending mechanism.

Myelin basic protein and myelin protein 2 act synergistically to cause stacking of lipid bilayers.

Saltatory conduction of nerve impulses along axonal membranes depends on the presence of a multilayered membrane, myelin, that wraps around the axon. Myelin basic protein (MBP) and myelin protein 2 (P2) are intimately involved in the generation of the myelin sheath. They are also implicated in a number of neurological diseases, including autoimmune diseases of both the central and peripheral nervous systems. Here, we have used atomic force microsopy (AFM) to study the effects of MBP and P2 on lipid bilayers. MBP in association with a mica substrate appeared unstructured, and tended to coat the mica surface in the form of a monolayer. In contrast, P2 appeared as discrete particles, with molecular volumes consistent with the formation of both monomers and dimers. Either MBP or P2, at micromolar concentrations, caused stacking of brain lipid bilayers. This stacking effect was significantly potentiated when both proteins were added together. Bilayers composed of phosphatidylcholine (PC) and phosphatidylserine (PS) were stacked by MBP, provided that cholesterol was also present; in contrast, P2 did not stack PC/PS/cholesterol bilayers. Hence, the bilayer stacking effects of the two proteins have different lipid requirements.