Phosphotungstic Acid-Eosin Combined with Hematoxylin as A Stain for Negri Bodies in Paraffin Sections

Experimentation with the Papanicolaou stain in this laboratory led to the discovery that the eosin, combined with phosphotungstic acid, was responsible for differential staining of Negri bodies. Eosin prepared as in EA 36 was used, but without the light green and Bismarck brown. Paraffin sections of hippocampus from brains of animal affected with rabies were fixed in 10% formol or in a mixture of 2 volumes of saturated aqueous HgCl₂ and 1 volume of absolute alcohol. They were stained first with hematoxylin and then with eosin. This procedure gave better results than staining with other types of eosin or by the original EA 36 mixture. The Negri bodies were well stained and their structure easily visible. The best results were obtained from material fixed with the HgCl₂ solution.     

Native LDL‐induced oxidative stress in human proximal tubular cells: multiple players involved

Dyslipidemia is a well‐established condition proved to accelerate the progression of chronic kidney disease leading to tubulo‐interstitial injury. However, the molecular aspects of the dyslipidemia‐induced renal damage have not been fully clarified and in particular the role played by low‐density lipoproteins (LDLs). This study aimed to examine the effects of native non‐oxidized LDL on cellular oxidative metabolism in cultured human proximal tubular cells. By means of confocal microscopy imaging combined to respirometric and enzymatic assays it is shown that purified native LDL caused a marked increase of cellular reactive oxygen species (ROS) production, which was mediated by activation of NADPH oxidase(s) and by mitochondrial dysfunction by means of a ROS‐induced ROS release mechanism. The LDL‐dependent mitochondrial alterations comprised inhibition of the respiratory chain activity, enhanced ROS production, uncoupling of the oxidative phosphorylation efficiency, collapse of the mtΔΨ, increased Ca²⁺ uptake and loss of cytochrome c. All the above LDL‐induced effects were completely abrogated by chelating extracellular Ca²⁺ as well as by inhibition of the Ca²⁺‐activated cytoplas‐mic phospholipase A2, NADPH oxidase and mitochondrial permeability transition. We propose a mechanicistic model whereby the LDL‐induced intracellular redox unbalance is triggered by a Ca²⁺ inward flux‐dependent commencement of cPLA2 followed by activation of a lipid‐ and ROS‐based cross‐talking signalling pathway. This involves first oxidants production via the plasmamembrane NADPH oxidase and then propagates downstream to mitochondria eliciting redox‐ and Ca²⁺‐dependent dysfunctions leading to cell‐harming conditions. These findings may help to clarify the mechanism of dyslipidemia‐induced renal damage and suggest new potential targets for specific therapeutic strategies to prevent oxidative stress implicated in kidney diseases.     

Humoral and cell-mediated immunity to pandemic H1N1 influenza in a Canadian cohort one year post-pandemic: implications for vaccination

We evaluated a cohort of Canadian donors for T cell and antibody responses against influenza A/California/7/2009 (pH1N1) at 8-10 months after the 2nd pandemic wave by flow cytometry and microneutralization assays. Memory CD8 T cell responses to pH1N1 were detectable in 58% (61/105) of donors. These responses were largely due to cross-reactive CD8 T cell epitopes as, for those donors tested, similar recall responses were obtained to A/California 2009 and A/PR8 1934 H1N1 Hviruses. Longitudinal analysis of a single infected individual showed only a small and transient increase in neutralizing antibody levels, but a robust CD8 T cell response that rose rapidly post symptom onset, peaking at 3 weeks, followed by a gradual decline to the baseline levels seen in a seroprevalence cohort post-pandemic. The magnitude of the influenza-specific CD8 T cell memory response at one year post-pandemic was similar in cases and controls as well as in vaccinated and unvaccinated donors, suggesting that any T cell boosting from infection was transient. Pandemic H1-specific antibodies were only detectable in approximately half of vaccinated donors. However, those who were vaccinated within a few months following infection had the highest persisting antibody titers, suggesting that vaccination shortly after influenza infection can boost or sustain antibody levels. For the most part the circulating influenza-specific T cell and serum antibody levels in the population at one year post-pandemic were not different between cases and controls, suggesting that natural infection does not lead to higher long term T cell and antibody responses in donors with pre-existing immunity to influenza. However, based on the responses of one longitudinal donor, it is possible for a small population of pre-existing cross-reactive memory CD8 T cells to expand rapidly following infection and this response may aid in viral clearance and contribute to a lessening of disease severity.     

Multiple roles of glucose-6-phosphatases in pathophysiology: State of the art and future trends

Background

The endoplasmic reticulum enzyme glucose-6-phosphatase catalyzes the hydrolysis of glucose-6-phosphate to glucose and inorganic phosphate. The enzyme is a part of a multicomponent system that includes several integral membrane proteins; the catalytic subunit (G6PC) and transporters for glucose-6-phosphate, inorganic phosphate and glucose. The G6PC gene family presently includes three members, termed as G6PC, G6PC2, and G6PC3. Although the three isoforms show a moderate amino acid sequence homology, their membrane topology and catalytic site are very similar. The isoforms are expressed differently in various tissues. Mutations in all three genes have been reported to be associated with human diseases.

Scope of review

The present review outlines the biochemical features of the G6PC gene family products, the regulation of their expression, their role in the human pathology and the possibilities for pharmacological interventions.

Major conclusions

G6PCs emerge as integrators of extra- and intracellular glucose homeostasis. Beside the well known key role in blood glucose homeostasis, the members of the G6PC family seem to play a role as sensors of intracellular glucose and of intraluminal glucose/glucose-6-phosphate in the endoplasmic reticulum.

General significance

Since mutations in the three G6PC genes can be linked to human pathophysiological conditions, the better understanding of their functioning in connection with genetic alterations, altered expression and tissue distribution has an eminent importance.     

Effectiveness of Community-Wide and Individual High-Risk Strategies to Prevent Diabetes: A Modelling Study

Background

Diabetes has been described as one of the most important threats to the health of developed countries. Effective population strategies to prevent diabetes have not been determined but two broad strategies have been proposed: “high-risk” and “community-wide” strategies.

Methods

We modelled the potential effectiveness of two strategies to prevent 10% of new cases of diabetes in Ontario, Canada over a 5-year period. The 5-year risk of developing physician-diagnosed diabetes was estimated for respondents to the Canadian Community Health Survey 2003 (CCHS 2.1, N = 26 232) using a validated and calibrated diabetes risk tool (Diabetes Population Risk Tool [DPoRT]). We estimated how many cases of diabetes could be prevented using two different strategies: a) a community-wide strategy that would uniformly reduce body mass index (BMI) in the entire population; and b) a high baseline risk strategy using either pharmacotherapy or lifestyle counselling to treat people who have an increased risk of developing diabetes.

Results

In 2003, the 5-year risk of developing diabetes was 4.7% (383 600 new diagnosed cases of diabetes in 8 189 000 Ontarians aged 20+) and risk was moderately diffused (0.5%, 3.1% and 17.9% risk in the 1^st, 5^th (median) and 10^th deciles of risk). A 10% reduction in new cases of diabetes would have been achieved under any of the following scenarios: if BMI was 3.5% lower in the entire population; if lifestyle counselling covered 32.2% of high-risk people (371 900 of 1 155 000 people with 5 year diabetes risk greater than 10%); or, if pharmacotherapy covered 65.2% of high-risk people.

Conclusions

Prevention using pharmacotherapy alone requires unrealistically high coverage levels to achieve modest population reduction in new diabetes cases. On the other hand, in recent years few jurisdictions have been able to achieve a reduction in BMI at the population level, let alone a reduction of BMI of 3.5%.     

Inhibition of Angiogenesis Mediated by Extremely Low-Frequency Magnetic Fields (ELF-MFs)

The formation of new blood vessels is an essential therapeutic target in many diseases such as cancer, ischemic diseases, and chronic inflammation. In this regard, extremely low-frequency (ELF) electromagnetic fields (EMFs) seem able to inhibit vessel growth when used in a specific window of amplitude. To investigate the mechanism of anti-angiogenic action of ELF-EMFs we tested the effect of a sinusoidal magnetic field (MF) of 2 mT intensity and frequency of 50 Hz on endothelial cell models HUVEC and MS-1 measuring cell status and proliferation, motility and tubule formation ability. MS-1 cells when injected in mice determined a rapid tumor-like growth that was significantly reduced in mice inoculated with MF-exposed cells. In particular, histological analysis of tumors derived from mice inoculated with MF-exposed MS-1 cells indicated a reduction of hemangioma size, of blood-filled spaces, and in hemorrhage. In parallel, in vitro proliferation of MS-1 treated with MF was significantly inhibited. We also found that the MF-exposure down-regulated the process of proliferation, migration and formation of tubule-like structures in HUVECs. Using western blotting and immunofluorescence analysis, we collected data about the possible influence of MF on the signalling pathway activated by the vascular endothelial growth factor (VEGF). In particular, MF exposure significantly reduced the expression and activation levels of VEGFR2, suggesting a direct or indirect influence of MF on VEGF receptors placed on cellular membrane. In conclusion MF reduced, in vitro and in vivo, the ability of endothelial cells to form new vessels, most probably affecting VEGF signal transduction pathway that was less responsive to activation. These findings could not only explain the mechanism of anti-angiogenic action exerted by MFs, but also promote the possible development of new therapeutic applications for treatment of those diseases where excessive angiogenesis is involved.     

Financing and current capacity for REDD+ readiness and monitoring,measurement, reporting and verification in the Congo Basin

This paper provides the first critical analysis of the financing and current capacity for REDD+ readiness in the Congo Basin, with a particular focus on the REDD+ component of national forest monitoring and measurement, reporting and verification (M&MRV). We focus on three areas of analysis: (i) general financing for REDD+ readiness especially M&MRV; (ii) capacity and information for REDD+ implementation and M&MRV; (iii) prospects and challenges for REDD+ and M&MRV readiness in terms of financing and capacity. For the first area of analysis, a REDD+ and M&MRV readiness financing database was created based on the information from the REDD+ voluntary database and Internet searches. For the second area of analysis, a qualitative approach to data collection was adopted (semi-structured interviews with key stakeholders, surveys and observations). All 10 countries were visited between 2010 and 2012. We find that: (i) a significant amount of REDD+ financing flows into the Congo Basin (±US$550 million or almost half of the REDD+ financing for the African continent); (ii) across countries, there is an important disequilibrium in terms of REDD+ and M&MRV readiness financing, political engagement, comprehension and capacity, which also appears to be a key barrier to countries receiving equal resources; (iii) most financing appears to go to smaller scale (subnational) REDD+ projects; (iv) four distinct country groups in terms of REDD+ readiness and M&MRV status are identified; and (v) the Congo Basin has a distinct opportunity to have a specific REDD+ financing window for large-scale and more targeted national REDD+ programmes through a specific fund for the region.     

Bone-marrow derived hematopoietic stem/progenitor cells express multiple isoforms of NADPH oxidase and produce constitutively reactive oxygen species

Consolidated evidence highlights the importance of redox signalling in poising the balance between self-renewal and differentiation in adult stem cells. The present study shows that human hematopoietic stem/progenitor cells (HSCs) constitutively generate low levels of hydrogen peroxide whose production is inhibited by DPI, apocynin, catalase, and LY294002 and scarcely stimulated by PMA. Moreover, it is shown that HSCs express at the mRNA and protein levels the catalytic subunits of NOX1, NOX2, and NOX4 isoforms of the NADPH oxidase family along with the complete battery of the regulatory subunits p22, p40, p47, p67, rac1, rac2, NOXO1, and NOXA1 as well as the splicing variant NOX2s and that the three NOX isoforms are largely co-expressed in the same HSC. These findings are interpreted in terms of a positive feed-back mechanism of NOXs activation enabling a fine tuning of the ROS level to be possibly used in redox-mediated signalling for growth and differentiation of HSCs.     

Diversity of natural products of the genera Curvularia and Bipolaris

Covering from 1963 to 2017.This review provides a summary of some secondary metabolites isolated from the genera Curvularia and Bipolaris from 1963 to 2017. The study has a broad objective. First to afford an overview of the structural diversity of these genera, classifying them depending on their chemical classes, highlighting individual examples of chemical structures. Also some information regarding their biological activities are presented. Several of the compounds reported here were isolated exclusively from endophytic and pathogenic strains in culture, while few from other sources such as sea Anemone and fish. Some secondary metabolites of the genus Curvularia and Bipolaris revealed a fascinating biological activities included: anti-malarial, anti-biofouling, anti-larval, anti-inflammatory, anti-oxidant, anti-bacterial, anti-fungal, anti-cancer, leishmanicidal and phytotoxicity. Herein, we presented a bibliography of the researches accomplished on the natural products of Curvularia and Bipolaris, which could help in the future prospecting of novel or new analogues of active metabolites from these two genera.