Sperm membrane fatty acid composition in the Eastern grey kangaroo (Macropus giganteus), koala (Phascolarctos cinereus), and common wombat (Vombatus ursinus) and its relationship to cold shock injury and cryopreservation success

Marsupial spermatozoa tolerate cold shock well, but differ in cryopreservation tolerance. In an attempt to explain these phenomena, the fatty acid composition of the sperm membrane from caput and cauda epididymides of the Eastern grey kangaroo, koala, and common wombat was measured and membrane sterol levels were measured in cauda epididymidal spermatozoa. While species-related differences in the levels of linolenic acid (18:3, n-6) and arachidonic acid (20:4, n-6) were observed in caput epididymal spermatozoa, these differences failed to significantly alter the ratio of unsaturated/saturated membrane fatty acids. However in cauda epididymidal spermatozoa, the ratio of unsaturated/saturated membrane fatty acids in koala and kangaroo spermatozoa was approximately 7.6 and 5.2, respectively; substantially higher than any other mammalian species so far described. Koala spermatozoal membranes had a higher ratio of unsaturated/saturated membrane fatty acids than that of wombat spermatozoa (t = 3.81; df = 4; p < or = 0.02); however, there was no significant difference between wombat and kangaroo spermatozoa. The highest proportions of DHA (22:6, n-3), the predominant membrane fatty acid in cauda epididymidal spermatozoa, were found in wombat and koala spermatozoa. While species-related differences in membrane sterol levels (cholesterol and desmosterol) were observed in cauda epididymidal spermatozoa, marsupial membrane sterol levels are very low. Marsupial spermatozoal membrane analyses do not support the hypothesis that a high ratio of saturated/unsaturated membrane fatty acids and low membrane sterol levels predisposes spermatozoa to cold shock damage. Instead, cryogenic tolerance appears related to DHA levels.     

Hydraulic properties of layered soils influence survival of Rhodes grass (<Emphasis Type="Italic">Chloris gayana</Emphasis> Kunth.) during water stress

Survival of vegetation on soil-capped mining wastes is often impaired during dry seasons due to the limited amount of water stored in the shallow soil capping. Growth and survival of Rhodes grass (Chloris gayana) during soil drying on various layered capping sequences constructed of combinations of topsoil, subsoil, seawater-neutralised residue sand and low grade bauxite was determined in a glasshouse. The aim was to describe the survival of Rhodes grass in terms of plant and soil water relationships. The soil water characteristic curve and soil texture analysis was a good predictor of plant survival. The combination of soil with a high water holding capacity and low soil water diffusivity (e.g. subsoil with high clay contents) with soil having a high water holding capacity and high diffusivity (e.g. residue sand) gave best survival during drying down (up to 88 days without water), whereas topsoil and low grade bauxite were unsuitable (plants died within 18–39 days). Clayey soil improved plant survival by triggering a water stress response during peak evaporative water demand once residue sand dried down and its diffusivity fell below a critical range. Thus, for revegetation in seasonally dry climates, soil capping should combine one soil with low diffusivity and one or more soils with high total water holding capacity and high diffusivity.     

Two modes of gating during late Na+ channel currents in frog sartorius muscle

Na+ currents were measured during 0.4-s depolarizing pulses using the cell-attached variation of the patch-clamp technique. Patches on Cs-dialyzed segments of sartorius muscle of Rana pipiens contained an estimated 25-500 Na+ channels. Three distinct types of current were observed after the pulse onset: a large initial surge of inward current that decayed within 10 ms (early currents), a steady "drizzle" of isolated, brief, inward unitary currents (background currents), and occasional "cloudbursts" of tens to hundreds of sequential unitary inward currents (bursts). Average late currents (background plus bursts) were 0.12% of peak early current amplitude at -20 mV. 85% of the late currents were carried by bursting channels. The unit current amplitude was the same for all three types of current, with a conductance of 10.5 pS and a reversal potential of +74 mV. The magnitudes of the three current components were correlated from patch to patch, and all were eliminated by slow inactivation. We conclude that all three components were due to Na+ channel activity. The mean open time of the background currents was approximately 0.25 ms, and the channels averaged 1.2 openings for each event. Neither the open time nor the number of openings of background currents was strongly sensitive to membrane potential. We estimated that background openings occurred at a rate of 0.25 Hz for each channel. Bursts occurred once each 2,000 pulses for each channel (assuming identical channels). The open time during bursts increased with depolarization to 1-2 ms at -20 mV, whereas the closed time decreased to less than 20 ms. The fractional open time during bursts was fitted with m infinity 3 using standard Na+ channel models. We conclude that background currents are caused by a return of normal Na+ channels from inactivation, while bursts are instances where the channel's inactivation gate spontaneously loses its function for prolonged periods.     

Kinetics of veratridine action on Na channels of skeletal muscle

Veratridine bath-applied to frog muscle makes inactivation of INa incomplete during a depolarizing voltage-clamp pulse and leads to a persistent veratridine-induced Na tail current. During repetitive depolarizations, the size of successive tail currents grows to a plateau and then gradually decreases. When pulsing is stopped, the tail current declines to zero with a time constant of approximately 3 s. Higher rates of stimulation result in a faster build-up of the tail current and a larger maximum value. I propose that veratridine binds only to open channels and, when bound, prevents normal fast inactivation and rapid shutting of the channel on return to rest. Veratridine-modified channels are also subject to a "slow" inactivation during long depolarizations or extended pulse trains. At rest, veratridine unbinds with a time constant of approximately 3 s. Three tests confirm these hypotheses: (a) the time course of the development of veratridine-induced tail currents parallels a running time integral of gNa during the pulse; (b) inactivating prepulses reduce the ability to evoke tails, and the voltage dependence of this reduction parallels the voltage dependence of h infinity; (c) chloramine-T, N-bromoacetamide, and scorpion toxin, agents that decrease inactivation in Na channels, each greatly enhance the tail currents and alter the time course of the appearance of the tails as predicted by the hypothesis. Veratridine-modified channels shut during hyperpolarizations from -90 mV and reopen on repolarization to -90 mV, a process that resembles normal activation gating. Veratridine appears to bind more rapidly during larger depolarizations.     

Using wintergreen oil for mounting mosquito larvae: a safer alternative to xylene

Permanent mounting of fourth instar mosquito larvae is essential for identifying Aedes spp. This procedure requires extensive exposure to xylene, a clearing agent in the mounting process. We investigated wintergreen oil as a substitute for xylene. Five hundred larvae were mounted on slides to evaluate shrinkage or expansion of specimens after clearing using xylene or wintergreen oil. We examined the ventral brush and siphonal hair tufts for species identification and for preservation of morphological characteristics after clearing specimens in xylene or wintergreen oil. Shrinkage of the length of whole larvae and width of the head, thorax and abdomen after mounting was significantly greater after clearing with xylene than with wintergreen oil. The length of the comb scale nearest the ventral brush was similar for both clearing agents. The clarity of the specimens after mounting was improved by clearing with wintergreen oil, but the integrity of the ventral brush and siphonal hair tufts were similar for both clearing agents.     

Community-acquired MRSA and pig-farming

Background

Female genital tuberculosis is an uncommon disease that is rarely diagnosed in developed countries.

Case presentation

A 61-year-old postmenopausal woman who had undergone surgery and treated with adjuvant chemotherapy for infiltrating ductal carcinoma of the breast five years ago, presented with bloody vaginal discharge, fatigue, weight loss, and low grade fevers at night for two months. Histological examination of the endometrium, done based on the suspicion of a second primary cancer due to the tamoxifen therapy, revealed a granulomatous reaction. Liquid and solid mycobacterial cultures of the tissues were performed. Although the acid fast staining was negative, the liquid culture was positive for Mycobacterium tuberculosis. Involvement of other systems was not detected. The patient was treated with a three-drug antituberculosis regimen for 9 months and recovered fully.

Conclusion

Female genital tuberculosis is a rare but curable disease that should be included in the differential diagnosis of women with menstrual problems. Early diagnosis is important and may prevent unnecessary invasive procedures for the patient.     

Characterization of T-Cell Responses in Macaques Immunized with a Single Dose of HIV DNA Vaccine

The optimization of immune responses (IR) induced by HIV DNA vaccines in humans is one of the great challenges in the development of an effective vaccine against AIDS. Ideally, this vaccine should be delivered in a single dose to immunize humans. We recently demonstrated that the immunization of mice with a single dose of a DNA vaccine derived from pathogenic SHIV_KU2 (Δ4SHIV_KU2) induced long-lasting, potent, and polyfunctional HIV-specific CD8⁺ T-cell responses (G. Arrode, R. Hegde, A. Mani, Y. Jin, Y. Chebloune, and O. Narayan, J. Immunol. 178:2318-2327, 2007). In the present work, we expanded the characterization of the IR induced by this DNA immunization protocol to rhesus macaques. Animals immunized with a single high dose of Δ4SHIV_KU2 DNA vaccine were monitored longitudinally for vaccine-induced IR using multiparametric flow cytometry-based assays. Interestingly, all five immunized macaques developed broad and polyfunctional HIV-specific T-cell IR that persisted for months, with an unusual reemergence in the blood following an initial decline but in the absence of antibody responses. The majority of vaccine-specific CD4⁺ and CD8⁺ T cells lacked gamma interferon production but showed high antigen-specific proliferation capacities. Proliferative CD8⁺ T cells expressed the lytic molecule granzyme B. No integrated viral vector could be detected in mononuclear cells from immunized animals, and this high dose of DNA did not induce any detectable autoimmune responses against DNA. Taken together, our comprehensive analysis demonstrated for the first time the capacity of a single high dose of HIV DNA vaccine alone to induce long-lasting and polyfunctional T-cell responses in the nonhuman primate model, bringing new insights for the design of future HIV vaccines.The development of a vaccine that substantially decreases the viral load set points and reduces the transmission of HIV-1 appears to be the long-term solution to control the persistently growing epidemic of this virus in the world (10). In the past, vaccines against challenging infectious diseases, including smallpox, polio, measles, and yellow fever, have been the most effective strategies for fighting these human pandemics. However, and unlike these traditional vaccines that mostly rely on the production of neutralizing antibodies (Abs) for protection from pathogenic infections, the control of HIV infection strongly depends on the development of high-frequency, broadly targeted, polyfunctional T-cell responses specific to the virus (11, 28, 45). Live-attenuated simian immunodeficiency virus (SIV)/HIV vaccines so far have been the best inducers of potent T-cell responses that correlate with protection against AIDS following challenge with pathogenic strains in nonhuman primate (NHP) models (24, 39, 47, 61), although the exact correlates of such protection remain to be fully delineated. However, the persistence, integration, and possible reversion to pathogenic forms of these replication-competent vaccines comprise a risk that will not be acceptable for their use in humans.Instead, the use of DNA-based vaccines as a strategy to induce protective responses to control infectious diseases, including HIV-1/AIDS, is very attractive, based on its safety, the absence of infection even in immunocompromised recipients, and its capacity to induce both humoral and T-cell immune responses. For many years, numerous plasmid DNAs encoding HIV proteins have been developed and tested in animal models, and some of them have been tested in humans (14, 18, 42, 49). However, unlike that in rodents, the immune responses induced in humans and NHPs by these DNA vaccines were dramatically weak despite successive immunizations with multiple doses of DNA (30). To circumvent this limitation, new strategies currently are used to improve the immunogenicity of DNA vaccines, including the incorporation of signal-to-target dendritic cells (43), the codon optimization of HIV antigens (Ag) (14), the coexpression of adjuvant (15), and new tools that optimize the delivery of DNA in target cells in the muscle (34).We have developed a noninfectious DNA vaccine derived from the highly pathogenic SHIV_KU2 expressing seven proteins of HIV under the control of the SIV 5′ long terminal repeat (LTR) promoter (35). This design mimics the natural expression of the viral proteins and leads to the formation of numerous viral-like particles that are extruded out of expressing cells (4). Repeated low-dose immunizations with this vaccine without heterologous boost protected macaques from progression to AIDS following challenge with pathogenic SHIV. However, enzyme-linked immunospot (ELISPOT) assay responses to HIV antigens before challenge were sporadic and weak (35, 54). In contrast, T-cell responses specific to HIV antigens induced by our construct in immunized mice were substantially higher (21). Using the mouse model, we developed a more sensitive immunity-monitoring assay that measures proliferative capacity, cytotoxic potential, and other immune functions (gamma interferon [IFN-γ] and interleukin-2 [IL-2] secretion) and provides more robust indications regarding the immunogenicity induced by the vaccine. We reported that the intramuscular immunization of mice with a single dose of this HIV DNA vaccine induced long-lasting and polyfunctional CD8⁺ T-cell responses directed against all HIV antigens expressed by the construct. Interestingly, in the absence of any additional immunization, we observed a primary peak of immune responses (IR) within 2 to 4 weeks postinfection (p.i.), followed by a contraction phase and then the late reemergence of responses after 14 to 20 weeks p.i. and lasting until the end of the experiment (more than 63 weeks p.i.). This is a typical pattern of vaccine-specific T-cell responses induced by nonpersistent vectors that progressively elicit secondary lymphoid tissue-based memory T cells as the expressed antigen becomes rare (9, 38, 58). Importantly, the major proportion of these HIV-specific CD8⁺ T cells was not producing IFN-γ but proliferated vigorously following antigen stimulation and produced the lytic molecule granzyme B (5). The contribution of this type of antigen-specific T cell to viral control remains to be fully elucidated.The surprising lack of efficacy of the human STEP trial conducted by Merck using the Ad-5 vectors expressing HIV antigens that elicit sustained effector T-cell responses has been disappointing for strategies designed to induce T-cell responses to prevent HIV-1 infections (29, 48, 53, 59). However, we learned from this failure that the characteristics of the T-cell responses induced by candidate vaccines could be critical for immediate as well as long-term protection (20). To address this issue, we developed a multiparametric flow-cytometric assay in the NHP model that was similar to that used in the mouse model. Using this assay, we performed a longitudinal characterization of HIV-specific T-cell IR induced in rhesus macaques immunized with a single high dose of Δ4SHIV_KU2 DNA vaccine given intramuscularly (i.m.). We also assessed the antibody responses against HIV antigens. We also addressed potential safety concerns, since this is the first report using one high dose of DNA in NHP, and we tested the animals for the integration of the vaccine genome into that of the circulating mononuclear cells and assessed whether anti-DNA antibodies were produced in all immunized monkeys.