Modifications to United States Environmental Protection Agency Methods 1622 and 1623 for Detection of Cryptosporidium Oocysts and Giardia Cysts in Water

Collaborative and in-house laboratory trials were conducted to evaluate Cryptosporidium oocyst and Giardia cyst recoveries from source and finished-water samples by utilizing the Filta-Max system and U.S. Environmental Protection Agency (EPA) methods 1622 and 1623. Collaborative trials with the Filta-Max system were conducted in accordance with manufacturer protocols for sample collection and processing. The mean oocyst recovery from seeded, filtered tap water was 48.4% ± 11.8%, while the mean cyst recovery was 57.1% ± 10.9%. Recovery percentages from raw source water samples ranged from 19.5 to 54.5% for oocysts and from 46.7 to 70.0% for cysts. When modifications were made in the elution and concentration steps to streamline the Filta-Max procedure, the mean percentages of recovery from filtered tap water were 40.2% ± 16.3% for oocysts and 49.4% ± 12.3% for cysts by the modified procedures, while matrix spike oocyst recovery percentages ranged from 2.1 to 36.5% and cyst recovery percentages ranged from 22.7 to 68.3%. Blinded matrix spike samples were analyzed quarterly as part of voluntary participation in the U.S. EPA protozoan performance evaluation program. A total of 15 blind samples were analyzed by using the Filta-Max system. The mean oocyst recovery percentages was 50.2% ± 13.8%, while the mean cyst recovery percentages was 41.2% ± 9.9%. As part of the quality assurance objectives of methods 1622 and 1623, reagent water samples were seeded with a predetermined number of Cryptosporidium oocysts and Giardia cysts. Mean recovery percentages of 45.4% ± 11.1% and 61.3% ± 3.8% were obtained for Cryptosporidium oocysts and Giardia cysts, respectively. These studies demonstrated that the Filta-Max system meets the acceptance criteria described in U.S. EPA methods 1622 and 1623.     

In situ detection of expression of thegus reporter gene in transgenic plants: ten years of blue genes

Among the methods now available to localize the sites of gene expression in plant materials, reporter genes based on thegus (uidA) gene ofEscherichia coli, which encodes a -glucuronidase (E.C. 3.2.1.31; GUS), have been the most widely used during the last ten years. The apparent simplicity of the histochemical GUS assay has been a major factor in the increase in articles usinggus genes. However, over the last four years, there have been occasional reports expressing doubts concerning the specificity of the observed localizations based on discrepancies between results obtained with GUS histochemistry and immunocytochemistry and/orin situ hybridization. This brief review compares the results obtained with immunocytochemistry with those obtained with various GUS substrates for histochemical studies. Certain sources of artefact are discussed, as are the limits that should be imposed on interpretation of GUS histochemistry results at the organ, tissue and cell levels.     

Regulation of principal cell pH by Na/H exchange in rabbit cortical collecting tubule

Summary Changes in intracellular pH (pH _i ) were measured using the pH indicator, BCECF, in principal cells from split opened cortical collecting tubules (CCTs) derived from rabbits maintained on a normal diet. This monolayer preparation has the advantage of allowing us to visualize the morphological differences in the two major cell types in this nephron segment under transmitted light. The visual identification of the cell types was verified using emission measurements taken from single principal and intercalated cells in the opened tubule which had been exposed to fluorescein isothiocyanate (FITC)-labeled peanut lectin. We confirmed the existence of an amiloride-sensitive Na/H exchange process activated during intracellular acidosis in principal cells. In addition, the exchanger was active under basal conditions and over a wide range of pH _i . Because the exchanger was active under basal conditions we tested the hypothesis that changes in intracellular Na (Na _i ) would alter pH _i in a predictable way. Maneuvers designed to alter Na _i were without significant effects within a 10-min time frame. Specifically, addition of 100 m ouabain to increase Na _i or exposure of the tubules to 10^–5 m amiloride to decrease luminal Na entry and reduce Na _i did not have an effect on pH _i . In some experiments we did observe however, after a 30-min exposure to ouabain, a small decrease in pH _i . These results suggest that Na/H exchange is a major regulator of pH _i in principal cells. However, regulation of Na transport by changes in pH _i in principal cells of rabbit CCT via the activity of a Na/H exchanger do not seem to contribute to the feedback control of Na transport.This work was supported by U.S. Public Health Service grants DK27847 to L.G. Palmer and DK11489 to E.E. Windhager.     

Characterization of Polar and Non‐Polar Compounds of House Edible Bird's Nest (EBN) from Johor,Malaysia

This work investigated the polar (PC: protein, amino acid and metabolite) and non‐polar (NPC: fatty acid) compounds and bioactivity characteristics of the EBN harvested from the state of Johor in Malaysia. The electrophoretic gels exhibited 15 protein bands (16–173 kD) with unique protein profile. Amino acids analysis by AccQ?Tag method revealed 18 types of amino acids in EBN. Metabolite profiling was performed using High‐Performance Liquid Chromatography coupled with Quadrupole Time‐of‐Flight Mass Spectrometer (HPLC‐QTOF/MS) technique and a total of 54 compounds belonging to different groups were detected and identified. These findings help to uncover the relation of therapeutic activity of EBN. The EBN was further extracted with AcOEt and BuOH. The AcOEt extract was fractionated into three fractions (F₁?F₃), and the high triglyceride content in F₂ was verified by gC‐FID. The three groups of fatty acids discovered in EBN are 48.43 % of poly‐unsaturated (PUFA), 25.35 % of saturated fatty acids (SFA) and 24.74 % of mono‐unsaturated fat (MUFA). This is the first time to report results ofEBN, BuOH, and AcOEt extracts and of fraction F₂ (TEBN) on their analysis for their antioxidant activities by DPPH, ABTS and catalase assay and for their paraoxonase and anti‐tyrosinase activities. The results showed that TEBN exhibited the significant bioactivity in all assays. These findings suggest that TEBN is a good source for natural bioactive compounds in promoting body vigor. Current work widened the content of EBN especially on the triglyceride and also marked the content of specific location (Johor, Malaysia) of EBN origin.     

Signaling of Ankle Joint Position by Receptors in Different Muscles

Plots were made of multiunit activity versus ankle joint position for receptors in each of the 12 muscles crossing the cat ankle joint, except peroneus tertius, by recording from populations of afferent fibers in muscle nerves. The discharge was measured 15 or 30 sec after terminating the movements that altered the position of the joint. These recordings were dominated by large-spike activity that would be expected to originate mainly from primary spindle endings. Seven of the 12 muscles also cross other joints. Their responses at a given ankle joint position were so altered by changes in the position of the knee or toe joints that they could not reliably signal the position of the ankle joint. As judged from multiunit recording, receptors in each of the five muscles specific to the ankle joint were influenced by more than one axis of ankle joint displacement.

Single-unit recording from dorsal root filaments was used to determine whether primary or secondary spindle receptors in soleus and tibialis anterior could selectively signal one axis of ankle joint rotation. Individual soleus receptors were tested both on the flexion extension axis and with a combined adduction–eversion movement.

For 38 of the 70 soleus receptors examined (54%), firm adduction–eversion produced a level of activity greater than that caused by 10° of flexion, and for 77% the level of activity was greater than that caused by 5° of flexion. For 168 of the 184 tibialis anterior receptors studied (91%), firm abduction inversion produced a level of activity greater than that caused by 10° of extension. Thus few receptors were found that responded exclusively to one axis of rotation.

One way in which the position of the ankle joint could be specified in the face of multiaxial receptor activity is by examining the receptor discharge from more than one muscle. A suggestion for how the nervous system might do this is given in the discussion.     

Efficiency of Cell-Free and Cell-Associated Virus in Mucosal Transmission of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus

Effective strategies are needed to block mucosal transmission of human immunodeficiency virus type 1 (HIV-1). Here, we address a crucial question in HIV-1 pathogenesis: whether infected donor mononuclear cells or cell-free virus plays the more important role in initiating mucosal infection by HIV-1. This distinction is critical, as effective strategies for blocking cell-free and cell-associated virus transmission may be different. We describe a novel ex vivo model system that utilizes sealed human colonic mucosa explants and demonstrate in both the ex vivo model and in vivo using the rectal challenge model in rhesus monkeys that HIV-1-infected lymphocytes can transmit infection across the mucosa more efficiently than cell-free virus. These findings may have significant implications for our understanding of the pathogenesis of mucosal transmission of HIV-1 and for the development of strategies to prevent HIV-1 transmission.     

Relief of Autoinhibition Enhances Vta1 Activation of Vps4 via the Vps4 Stimulatory Element

The endosomal sorting complexes required for transport (ESCRTs) impact multiple cellular processes including multivesicular body sorting, abscission, and viral budding. The AAA-ATPase Vps4 is required for ESCRT function, and its full activity is dependent upon the co-factor Vta1. The Vta1 carboxyl-terminal Vta1 SBP1 Lip5 (VSL) domain stimulates Vps4 function by facilitating oligomerization of Vps4 into its active state. Here we report the identification of the Vps4 stimulatory element (VSE) within Vta1 that is required for additional stimulation of Vps4 activity in vitro and in vivo. VSE activity is autoinhibited in a manner dependent upon the unstructured linker region joining the amino-terminal microtubule interacting and trafficking domains and the carboxyl-terminal VSL domain. The VSE is also required for Vta1-mediated Vps4 stimulation by ESCRT-III subunits Vps60 and Did2. These results suggest that ESCRT-III binding to the Vta1 microtubule interacting and trafficking domains relieves linker region autoinhibition of the VSE to produce maximal activation of Vps4 during ESCRT function.     

Environmental Synthesis of Few Layers Graphene Sheets Using Ultrasonic Exfoliation with Enhanced Electrical and Thermal Properties

In this paper, we report how few layers graphene that can be produced in large quantity with low defect ratio from exfoliation of graphite by using a high intensity probe sonication in water containing liquid hand soap and PVP. It was founded that the graphene powder obtained by this simple exfoliation method after the heat treatment had an excellent exfoliation into a single or layered graphene sheets. The UV-visible spectroscopy, FESEM, TEM, X-ray powder diffraction and Raman spectroscopy was used to analyse the graphene product. The thermal diffusivity of the samples was analysed using a highly accurate thermal-wave cavity photothermal technique. The data obtained showed excellent enhancement in the thermal diffusivity of the graphene dispersion. This well-dispersed graphene was then used to fabricate an electrically conductive polymer-graphene film composite. The results demonstrated that this low cost and environmental friendly technique allowed to the production of high quality layered graphene sheets, improved the thermal and electrical properties. This may find use in the wide range of applications based on graphene.