Rac1 activation can generate untemplated, lamellar membrane ruffles.

BACKGROUND: Membrane protrusions that occur on the dorsal surface of a cell are an excellent experimental system to study actin machinery at work in a living cell. Small GTPase Rac1 controls the membrane protrusions that form and encapsulate extracellular volumes to perform pinocytic or phagocytic functions. RESULTS: Here, capitalizing on rapid volumetric imaging capabilities of lattice light-sheet microscopy (LLSM), we describe optogenetic approaches using photoactivable Rac1 (PA-Rac1) for controlled ruffle generation. We demonstrate that PA-Rac1 activation needs to be continuous, suggesting a threshold local concentration for sustained actin polymerization leading to ruffling. We show that Rac1 activation leads to actin assembly at the dorsal surface of the cell membrane that result in sheet-like protrusion formation without any requirement of a template. Further, this approach can be used to study the complex morpho-dynamics of the protrusions or to investigate specific proteins that may be enriched in the ruffles. Deactivating PA-Rac1 leads to complex contractile processes resulting in formation of macropinosomes. Using multicolour imaging in combination with these approaches, we find that Myo1e specifically is enriched in the ruffles. CONCLUSIONS: Combining LLSM and optogenetics enables superior spatial and temporal control for studying such dynamic mechanisms. Demonstrated here, the techniques implemented provide insight into the complex nature of the molecular interplay involved in dynamic actin machinery, revealing that Rac1 activation can generate untemplated, lamellar protrusions.

Rapid whole cell imaging reveals a calcium-APPL1-dynein nexus that regulates cohort trafficking of stimulated EGF receptors.

The endosomal system provides rich signal processing capabilities for responses elicited by growth factor receptors and their ligands. At the single cell level, endosomal trafficking becomes a critical component of signal processing, as exemplified by the epidermal growth factor (EGF) receptors. Activated EGFRs are trafficked to the phosphatase-enriched peri-nuclear region (PNR), where they are dephosphorylated and degraded. The details of the mechanisms that govern the movements of stimulated EGFRs towards the PNR, are not completely known. Here, exploiting the advantages of lattice light-sheet microscopy, we show that EGFR activation by EGF triggers a transient calcium increase causing a whole-cell level redistribution of Adaptor Protein, Phosphotyrosine Interacting with PH Domain And Leucine Zipper 1 (APPL1) from pre-existing endosomes within one minute, the rebinding of liberated APPL1 directly to EGFR, and the dynein-dependent translocation of APPL1-EGF-bearing endosomes to the PNR within ten minutes. The cell spanning, fast acting network that we reveal integrates a cascade of events dedicated to the cohort movement of activated EGF receptors. Our findings support the intriguing proposal that certain endosomal pathways have shed some of the stochastic strategies of traditional trafficking and have evolved processes that provide the temporal predictability that typify canonical signaling.

Atypical optic neuritis: An overview.

Optic neuritis (ON) refers to conditions that involve inflammation of the optic nerve. Various autoantibodies have been found, which are associated with central nervous system inflammatory disorders and have provided much information about the immune targets and mechanisms that impact the prognosis, treatment, and recurrence of atypical ON. Therefore, neurologists and ophthalmologists together should work to find out clinical, laboratory, and imaging findings that may provide important clues to the etiology of atypical ON and its management. Various biomarkers have been identified to confirm and distinguish atypical optic neuritis from others. The purpose of this review is to present the current scenario of atypical ON and its clinical management.

Chemodenitrification in the cryoecosystem of Lake Vida, Victoria Valley, Antarctica.

Lake Vida, in the Victoria Valley of East Antarctica, is frozen, yet harbors liquid brine (~20% salt, >6 times seawater) intercalated in the ice below 16 m. The brine has been isolated from the surface for several thousand years. The brine conditions (permanently dark, -13.4 °C, lack of O2 , and pH of 6.2) and geochemistry are highly unusual. For example, nitrous oxide (N2 O) is present at a concentration among the highest reported for an aquatic environment. Only a minor 17 O anomaly was observed in N2 O, indicating that this gas was predominantly formed in the lake. In contrast, the 17 O anomaly in nitrate (NO3-) in Lake Vida brine indicates that approximately half or more of the NO3- present is derived from atmospheric deposition. Lake Vida brine was incubated in the presence of 15 N-enriched substrates for 40 days. We did not detect microbial nitrification, dissimilatory reduction of NO3- to ammonium (NH4+), anaerobic ammonium oxidation, or denitrification of N2 O under the conditions tested. In the presence of 15 N-enriched nitrite (NO2-), both N2 and N2 O exhibited substantial 15 N enrichments; however, isotopic enrichment declined with time, which is unexpected. Additions of 15 N-NO2- alone and in the presence of HgCl2 and ZnCl2 to aged brine at -13 °C resulted in linear increases in the δ15 N of N2 O with time. As HgCl2 and ZnCl2 are effective biocides, we interpret N2 O production in the aged brine to be the result of chemodenitrification. With this understanding, we interpret our results from the field incubations as the result of chemodenitrification stimulated by the addition of 15 N-enriched NO2- and ZnCl2 and determined rates of N2 O and N2 production of 4.11-41.18 and 0.55-1.75 nmol L-1  day-1 , respectively. If these rates are representative of natural production, the current concentration of N2 O in Lake Vida could have been reached between 6 and 465 years. Thus, chemodenitrification alone is sufficient to explain the high levels of N2 O present in Lake Vida.

Medical students and interns' knowledge about and attitude towards homosexuality.

BACKGROUND AND RATIONALE: Medical professionals' attitude towards homosexuals affects health care offered to such patients with a different sexual orientation. There is absence of literature that explores the attitudes of Indian medical students or physicians towards homosexuality. AIM: This study aimed to evaluate Indian medical students and interns' knowledge about homosexuality and attitude towards homosexuals. MATERIALS AND METHODS: After IEC approval and written informed consent, a cross-sectional study was conducted on a purposive sample of undergraduate medical students and interns studying in one Indian medical college. The response rate was 80.5%. Only completely and validly filled responses (N = 244) were analyzed. The participants filled the Sex Education and Knowledge about Homosexuality Questionnaire (SEKHQ) and the Attitudes towards Homosexuals Questionnaire (AHQ). SEKHQ consisted of 32 statements with response chosen from 'true', 'false', or 'don't know'. AHQ consisted of 20 statements scorable on a 5-point Likert scale. Multiple linear regression was used to find the predictors of knowledge and attitude. RESULTS: Medical students and interns had inadequate knowledge about homosexuality, although they endorsed a neutral stance insofar as their attitude towards homosexuals is concerned. Females had more positive attitudes towards homosexuals. Knowledge emerged as the most significant predictor of attitude; those having higher knowledge had more positive attitudes. CONCLUSION: Enhancing knowledge of medical students by incorporation of homosexuality related health issues in the curriculum could help reduce prejudice towards the sexual minority and thus impact their future clinical practice.

Electro Convulsive Therapy in Psychiatric Manifestations in Wilson's Disease.

Wilson's disease occurs due to an inborn error of metabolism. Psychiatric symptoms are often the first manifestation of the disease and can obscure the diagnosis. There are five neuropsychiatric symptoms clusters established for Wilson's disease patients: Behavior and/or personality disorders, mood disorders, cognitive deficits, psychotic manifestations, and others. The frequency with which psychiatric manifestations appears in Wilson's disease remains vague. However, whenever they occur, they need to be correctly identified and treated. Though encouraging results have been obtained in controlling psychiatric manifestations of Wilson's with psychotropic medications, some sub-group of patients fail to respond to any therapy. We aim at finding options for controlling psychosis in these patients with electro convulsive therapy (ECT). A patient exhibiting rare neuropsychiatric manifestations of Wilson's and who is not responding to psychotropic medication was considered for ECT. Considerable control over psychiatric manifestations with ECT was observed and later treated with maintenance ECTs for relapse control.

Design and synthesis of 6,7-dimethoxyquinazoline analogs as multi-targeted ligands for α1- and AII-receptors antagonism.

Multiple-targeted ligands can have certain advantages for the management of hypertension which has multiple controls. Molecules with dual bioactivities are available in literature for treating metabolic disorders like diabetes, hypertension and hypercholesterolemia. After scrutinizing the SAR of prazosin-type α1-blockers and AII-antagonists it was planned to develop dual α1- and AII-antagonists. Five series of quinazoline derivatives were synthesized and evaluated as dual α1- and AII-antagonists on rat aortic strips for the blockade of known α1- and AII-agonist mediated contractions. Many compounds showed balanced activity on both the receptors but compound (22) was found to be the most active derivative having higher antagonistic activity on both the receptors. In the in vivo experiments the chosen compound (22) was slightly less active than prazosin but was found to be equipotent to losartan. These findings shed a new light on the structural requirements for both α1- as well as AII-receptor antagonists.

Revelation on the potency of α(1) -blockers - parallel blockade of angiotensin II receptor: a new finding.

CONTEXT: The problem of hypertension has gained enormous proportions in the past decade. Multifactorial etiology and complex pathophysiology of the disease has rendered the treatment of the disease a hard task. Sympathetic nervous system and the renin-angiotensin-aldosterone system are primary contributors of blood pressure homeostasis. OBJECTIVE: Structural similarities were identified among AT(1) and α(1)-antagonists, initiating a speculation that α(1)-antagonists could possibly block the AT(1) receptor and vice-versa. METHODS: To corroborate this speculation, we screened prototypical α(1)-antagonists such as prazosin, doxazosin, and terazosin for antagonism of angiotensin II on rat aortic strips. We also examined the AT(1) antagonists losartan, valsartan, and olmesartan for their possible antagonistic effect, on contractions of rat aortic strips induced by phenylephrine. RESULTS: To our astonishment, we found that prazosin and its analogs which have been reported to have α(1)-antagonistic activity only, were able to shift concentration response curves of angiotensin II. CONCLUSION: Our findings suggest that the potent antihypertensive effect of prazosin-type α(1)-antagonists is not purely due to α(1)-receptor blocking activity of these compounds but also due to blockade of AT(1) receptors. This finding may lead to the development of more potent dual inhibitors which would prove to be of immense value in the control of the scourge of hypertension.

Successful treatment of active haemorrhage from a duodenal diverticulum using surgicel (absorbable haemostat): a case report.

Haemorrhage is one of the rare but serious complications of duodenal diverticula. Current methods of treatment include: endoscopy with injection therapy or hemoclip application and diverticulectomy. In this paper we present the case of a 61 year old man with life threatening haemorrhage who was managed successfully with gentle packing of a bleeding duodenal diverticulum using SURGICEL (Absorbable Haemostat). This appears to be a simple and effective way of dealing with the problem especially in situations where other methods are ineffective or inapplicable. Early surgical intervention before the development of any coagulopathy increases the chances of a successful outcome. It has not been possible to find a similar report from a thorough literature search.

Distinguishing nitrous oxide production from nitrification and denitrification on the basis of isotopomer abundances.

The intramolecular distribution of nitrogen isotopes in N2O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N2O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N2O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 +/- 1.2 per thousand, 32.5 +/- 0.6 per thousand, and 35.6 +/- 1.4 per thousand for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N2O from ammonia oxidation by N. europaea (31.4 +/- 4.2 per thousand) was similar to that produced during hydroxylamine oxidation (33.5 +/- 1.2 per thousand) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 +/- 1.7 per thousand), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N2O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (-0.6 +/- 1.9 per thousand and -0.5 +/- 1.9 per thousand, respectively) were similar to those during nitrate reduction (-0.5 +/- 1.9 per thousand and -0.5 +/- 0.6 per thousand, respectively), indicating no influence of either substrate on site preference. Site preferences of approximately 33 per thousand and approximately 0 per thousand are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N2O.

Isotopic ((13)C) analysis of dissolved organic carbon in stream water using an elemental analyzer coupled to a stable isotope ratio mass spectrometer.

A technique for measurement of the stable isotope composition of dissolved organic carbon (DOC) in stream water, using an elemental analyzer (EA) coupled to an isotope ratio mass spectrometer (IRMS), is described. Stream water samples were concentrated by rotary evaporation, acidified to remove dissolved inorganic carbon (DIC), and dried in silver cups prior to analysis. Precision was evaluated with standards (alanine and humic acid), and with stream water samples with varying (13)C enrichment. Standards and samples were also prepared in sealed quartz tubes for high-temperature combustion (HTC) and analyzed by dual inlet for comparison. The delta(13)C values of natural abundance standards and samples measured by the two techniques differed by

Nitrogen isotopomer site preference of N2O produced by Nitrosomonas europaea and Methylococcus capsulatus Bath.

The relative importance of individual microbial pathways in nitrous oxide (N(2)O) production is not well known. The intramolecular distribution of (15)N in N(2)O provides a basis for distinguishing biological pathways. Concentrated cell suspensions of Methylococcus capsulatus Bath and Nitrosomonas europaea were used to investigate the site preference of N(2)O by microbial processes during nitrification. The average site preference of N(2)O formed during hydroxylamine oxidation by M. capsulatus Bath (5.5 +/- 3.5 per thousand) and N. europaea (-2.3 +/- 1.9 per thousand) and nitrite reduction by N. europaea (-8.3 +/- 3.6 per thousand) differed significantly (ANOVA, f((2,35)) = 247.9, p = 0). These results demonstrate that the mechanisms for hydroxylamine oxidation are distinct in M. capsulatus Bath and N. europaea. The average delta(18)O-N(2)O values of N(2)O formed during hydroxylamine oxidation for M. capsulatus Bath (53.1 +/- 2.9 per thousand) and N. europaea (-23.4 +/- 7.2 per thousand) and nitrite reduction by N. europaea (4.6 +/- 1.4 per thousand) were significantly different (ANOVA, f((2,35)) = 279.98, p = 0). Although the nitrogen isotope value of the substrate, hydroxylamine, was similar in both cultures, the observed fractionation (delta(15)N) associated with N(2)O production via hydroxylamine oxidation by M. capsulatus Bath and N. europaea (-2.3 and 26.0 per thousand, respectively) provided evidence that differences in isotopic fractionation were associated with these two organisms. The site preferences in this study are the first measured values for isolated microbial processes. The differences in site preference are significant and indicate that isotopomers provide a basis for apportioning biological processes producing N(2)O.

Effect of honey on the growth of and acid production by human intestinal Bifidobacterium spp.: an in vitro comparison with commercial oligosaccharides and inulin.

Five human intestinal Bifidobacterium spp., B. longum, B. adolescentis, B. breve, B. bifidum, and B. infantis, were cultured in reinforced clostridial medium (control) and in reinforced clostridial medium supplemented with 5% (wt/vol) honey, fructooligosaccharide (FOS), galactooligosaccharide (GOS), and inulin. Inoculated samples were incubated anaerobically at 37degrees C for 48 h. Samples were collected at 12-h intervals and examined for specific growth rate. Levels of fermentation end products (lactic and acetic acids) were measured by high-pressure liquid chromatography. Honey enhanced the growth of the five cultures much like FOS, GOS, and inulin did. Honey, FOS, GOS, and inulin were especially effective (P < 0.05) in sustaining the growth of these cultures after 24 h of incubation as compared with the control treatment. Overall, the effects of honey on lactic and acetic acid production by intestinal Bifidobacterium spp. were similar to those of FOS, GOS, and inulin.

Growth and viability of commercial Bifidobacterium spp. in honey-sweetened skim milk.

Two commercial Bifidobacterium bifidum (Bf-1 and Bf-6) were cultured in 12% (wt/vol) reconstituted nonfat dry milk (NDM) containing 5% (wt/wt) honey, sucrose, fructose, or glucose. Inoculated samples were incubated anaerobically at 37 degrees C for 48 h. Samples were collected at 12-h intervals and examined for (i) specific growth rate, (ii) pH, and (iii) levels of fermentation end products (lactic and acetic acids) as measured by high-performance liquid chromatography (HPLC). Viability of the organisms during 28 days of refrigerated storage at 4 degrees C was also assessed at 7-day intervals. Growth promotion and acid production were greatest when Bf-1 and Bf-6 were grown in the presence of honey. For both Bf-1 and Bf-6, retention of viability was greatest up to 14 days of refrigerated storageat 4 degrees C when they were grown and stored in the presence of honey compared to other sweeteners.

Polyclonal-antibody-based ELISA to detect milk alkaline phosphatase.

Polyclonal antibodies (PAb) prepared against bovine milk alkaline phosphatase (ALP) were used to develop a competitive indirect (CI) ELISA. Anti-ALP PAb were specific for milk ALP and did not react with ALP from E. coli or bovine and calf intestinal mucosa. Anti-ALP PAb were 20% cross-reactive with bovine placenta ALP. The anti-ALP antibodies also did not recognize bovine serum albumin, acid glycoprotein, ovalbumin, ferritin, and casein, although some cross-reactivity was observed with whey protein isolate. Anti-ALP PAbs reacted with deglycosylated native ALP, but did not recognize ALP denatured at 100 degrees C in 2% SDS or deglycosylated denatured ALP. When buffered solutions of milk ALP were heated at 70 degrees C, ALP activity decreased at a faster rate than ALP content determined by CI-ELISA. The ELISA differentiated between native and heat denatured ALP. Further studies are warranted to determine if an ELISA can be used to verify pasteurization of fluid milk.