Quantitative HIV-1 proviral DNA detection: a multicentre analysis

Despite the widespread use of molecular biology techniques, standardized methods for the measurement of HIV-1 proviral DNA are currently lacking and several discordant results are still present in different studies. To assess the clinical meaning of the proviral DNA load, a study group comprising seven different laboratories was set up to standardize a HIV-1 proviral DNA quantification method able to assess the DNA proviral load of the most relevant circulating HIV-1 subtypes. Reference samples (24 cellular samples infected with HIV-1 clade B, and 40 samples of peripheral blood mononuclear cells containing different concentrations of plasmids expressing different HIV-1 clades) were distributed and tested blindly. All laboratories employed hTERT gene as housekeeping gene and primers within the gag gene to quantify different HIV-1 clades. Inter-laboratory results did not differ statistically but showed only minor variations concerning HIV-1 DNA amounts and different HIV clades, with a good agreement among the laboratories participating in the study. Since test standardization represents a key step for future application in clinical practice, further studies of the patients' samples are in progress to establish the real meaning and utility of the proviral DNA load for clinical management of HIV-1 infected patients.     

Microbiota of the <Emphasis Type="Italic">Planalto de Bolona</Emphasis>: an artisanal cheese produced in uncommon environmental conditions in the Cape Verde Islands

The present study aimed to evaluate the dominant microbial community naturally present in the Planalto de Bolona cheese, produced in the Cape Verde Islands. Samples of milk, curd and cheese from two different producers were examined through culture-dependent and independent-methods. Traditional plating and genetic identification of lactic acid bacteria (LAB) and yeast isolates were carried out. Moreover, DNA and RNA extracted directly from samples were subjected to Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). Concerning the LAB population, a total of 278 isolates were identified: Lactococcus lactis subsp. lactis and Enterococcus faecium represented the most isolated species. Regarding yeasts, the analysis of isolates throughout the manufacturing period showed a consistent presence of the genus Candida. Divergences in species detection between culture-dependent and culture-independent methods were observed, as well as between DNA and RNA analysis. PCR-DGGE underlined high heterogeneity among bacterial species while yeast microbiota was dominated by Aureobasidium pullulans at DNA level. The obtained results represent a first approach in the understanding of the Planalto de Bolona cheese microbial ecology and consequently may constitute a first step towards the full comprehension of the microbiota of this artisanal cheese produced in unusual environmental conditions in the Cape Verde Islands.     

Erratum to: Extracellular protein production and morphogenesis of <Emphasis Type="Italic">Lentinula edodes</Emphasis> in submerged culture

Along with a brief review of Lentinula edodes (shiitake mushroom) submerged cultivation history within the framework of important extracellular proteins biosynthesis, this study contains the authors’ own results. The possibility of regulating the lectin activity of shiitake using the synthetic components is shown. The time course of lectin production in culture liquid of L. edodes in different media under submerged culture conditions was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and the pH of the culture medium. A relationship between the chemical composition of nutrient medium, the activity of extracellular lectins of L. edodes, and the formation of pigmented mycelial film in liquid culture has been found. The formulation of medium, on which the brown mycelial film appears in several days of submerged cultivation, is proposed. The results obtained make a contribution to the present notion of biochemical processes that give rise to the occurrence of the aforesaid morphological structure of shiitake. Finally, two extracellular lectins from the submerged culture of L. edodes have been isolated and purified to homogeneity. Their physicochemical properties and composition have been studied.     

Extracellular protein production and morphogenesis of <Emphasis Type="Italic">Lentinula edodes</Emphasis> in submerged culture

Along with a brief review of Lentinula edodes (shiitake mushroom) submerged cultivation history within the framework of important extracellular proteins biosynthesis, this study contains the authors’ own results. The possibility of regulating the lectin activity of shiitake using the synthetic components is shown. The time course of lectin production in culture liquid of L. edodes in different media under submerged culture conditions was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and the pH of the culture medium. A relationship between the chemical composition of nutrient medium, the activity of extracellular lectins of L. edodes, and the formation of pigmented mycelial film in liquid culture has been found. The formulation of medium, on which the brown mycelial film appears in several days of submerged cultivation, is proposed. The results obtained make a contribution to the present notion of biochemical processes that give rise to the occurrence of the aforesaid morphological structure of shiitake. Finally, two extracellular lectins from the submerged culture of L. edodes have been isolated and purified to homogeneity. Their physicochemical properties and composition have been studied.     

Bacterial and Protozoal Communities and Fatty Acid Profile in the Rumen of Sheep Fed a Diet Containing Added Tannins

This study evaluated the effects of tannins on ruminal biohydrogenation (BH) due to shifts in the ruminal microbial environment in sheep. Thirteen lambs (45 days of age) were assigned to two dietary treatments: seven lambs were fed a barley-based concentrate (control group) while the other six lambs received the same concentrate with supplemental quebracho tannins (9.57% of dry matter). At 122 days of age, the lambs were slaughtered, and the ruminal contents were subjected to fatty acid analysis and sampled to quantify populations of Butyrivibrio fibrisolvens, which converts C_18:2 c9-c12 (linoleic acid [LA]) to C_18:2 c9-t11 (rumenic acid [RA]) and then RA to C_18:1 t11 (vaccenic acid [VA]); we also sampled for Butyrivibrio proteoclasticus, which converts VA to C_18:0 (stearic acid [SA]). Tannins increased (P < 0.005) VA in the rumen compared to the tannin-free diet. The concentration of SA was not affected by tannins. The SA/VA ratio was lower (P < 0.005) for the tannin-fed lambs than for the controls, suggesting that the last step of the BH process was inhibited by tannins. The B. proteoclasticus population was lower (−30.6%; P < 0.1), and B. fibrisolvens and protozoan populations were higher (+107% and +56.1%, respectively; P < 0.05) in the rumen of lambs fed the tannin-supplemented diet than in controls. These results suggest that quebracho tannins altered BH by changing ruminal microbial populations.The fatty acid profile of the meat and milk of ruminants is strongly affected by diet (2, 15). When ingested, the dietary polyunsaturated fatty acids (PUFA) undergo a process known as biohydrogenation (BH) carried out by ruminal microorganisms (20). During the BH of C_18:2(n-6) (linoleic acid [LA]) and C_18:3(n-3) (linolenic acid [LNA]) a number of C_18:1 and C_18:2 isomers are formed (6). The last step in the BH process leads to the formation of C_18:0 (stearic acid [SA]). Among the intermediate products formed during this process, the isomer C_18:2 c9t11 (rumenic acid [RA]) is active in preventing cancer in mammals (17). Only a small amount of the RA found in meat and milk originates during BH. It is produced to a larger extent in muscle and mammary glands from the desaturation of C_18:1 t11 (vaccenic acid [VA], another intermediate of ruminal BH) by the action of Δ⁹-desaturase enzyme (41, 43).Ruminal BH is carried out mostly by bacteria belonging to the Butyrivibrio genus (38). Butyrivibrio fibrisolvens has the capacity to convert LA to RA and RA to VA, while Butyrivibrio proteoclasticus (previously classified as Clostridium proteoclasticum [35]) hydrogenates VA to SA (38, 39). According to Or-Rashid et al. (37), ruminal protozoa also play a role in BH by converting LA to RA. However, this issue is still controversial, as Devillard et al. (11) have reported that protozoa do not have the capability of hydrogenating LA. The proportion of BH intermediates in the rumen can vary depending on changes in ruminal microbial populations (7, 51). Changes in ruminal fatty acid profiles are also reflected in intramuscular fatty acid composition (48, 52).Tannins are phenolic compounds that are widespread in plants. When ingested by ruminants in large amounts, tannins can reduce the activity and the proliferation of ruminal microorganisms (34). Tannins from Lotus corniculatus (33) or from Acacia spp. (12) reduce the proliferation of B. proteoclasticus B316^T and B. proteoclasticus P18, respectively. Durmic et al. (12) reported that VA increased and SA decreased when extracts from Acacia iteaphylla, which contains condensed tannins (1), were incubated in vitro with sheep ruminal fluid inoculated with B. fibrisolvens JW11 and B. proteoclasticus P18 strains. In two recent in vitro studies, the inclusion of tannins in fermentor systems containing bovine ruminal fluid inhibited the conversion of VA to SA, while no effect was detected on RA production (21, 47). These results have been also confirmed in vivo in the rumen of sheep fed a diet with 4.0% dry matter (DM) quebracho tannin (48). However, to date there is no in vivo study focusing on the effects of dietary tannins on the proliferation of the microorganisms involved in ruminal BH.We assessed whether dietary tannins may affect the BH pathway via changes in bacterial and protozoal ruminal populations. We gave particular emphasis to B. fibrisolvens and B. proteoclasticus. We also assayed the production of conjugated linoleic acids (CLAs) by linoleic acid isomerase (LA-I) enzyme.     

Cyclophilin D in mitochondrial pathophysiology

Cyclophilins are a family of peptidyl-prolyl cis–trans isomerases whose enzymatic activity can be inhibited by cyclosporin A. Sixteen cyclophilins have been identified in humans, and cyclophilin D is a unique isoform that is imported into the mitochondrial matrix. Here we shall (i) review the best characterized functions of cyclophilin D in mitochondria, i.e. regulation of the permeability transition pore, an inner membrane channel that plays an important role in the execution of cell death; (ii) highlight new regulatory interactions that are emerging in the literature, including the modulation of the mitochondrial F1FO ATP synthase through an interaction with the lateral stalk of the enzyme complex; and (iii) discuss diseases where cyclophilin D plays a pathogenetic role that makes it a suitable target for pharmacologic intervention.     

The antinociceptive effect of acetylsalicylic acid is differently affected by a CB1 agonist or antagonist and involves the serotonergic system in rats

AimsCombinations of non-steroidal anti-inflammatory drugs (NSAIDs) and cannabinoids are promising because of their potential synergistic effects in analgesia, resulting in a reduction in dosage and minimizing adverse reactions. The analgesic effect of acetylsalicylic acid (ASA), probably due to a central mechanism, also implicates changes in the central monoaminergic system. Therefore, we decided to evaluate the antinociceptive interaction between the CB₁ receptor agonist, HU210, and ASA in tests involving central pain in rats as well as the implication of the central serotonergic system thereon.Main methodsThe selective CB₁ antagonist SR141716A and the potent cannabinoid agonist HU210 were evaluated alone and in combination with ASA in both algesimetric tests (hot-plate and formalin tests) and for 5-HT activity and 5-HT₂ receptor density and affinity.Key findingsASA or HU210 alone showed a dose-dependent effect in both tests. HU210, at an inactive dose, significantly increased the antinociceptive effect of the sub-active dose of ASA. SR141716A (1.5 mg/kg i.p.) was ineffective per se and failed to modify antinociception induced by the HU210 plus ASA combination in either test. HU210 plus ASA significantly decreased the 5-HIAA/5-HT ratio and the 5-HT₂ receptor density in the frontal cortex, changes not antagonized by SR141716A.SignificanceThe present study provides evidence that mutual potentiation of the antinociceptive effects of HU210 and ASA may, at least partly, depend on serotonergic mechanisms, with an indirect participation of cannabinodiergic mechanism. In conclusion, combinations of low doses of cannabinoids and NSAIDs may be of interest from the therapeutic point of view.     

Heavy metal distribution in superficial sedimenta ta Saco, Gulf of Cariaco, Sucre, Venezuela

The Gulf of Cariaco is a marine ecosystem with high primary productivity, which gives it an ecological and socioeconomic importance. Nevertheless, anthropogenic activities around the Gulf produce wastes that are deposited directly or by runoff into the sediments, and consequently, increases concentrations of metals in this ecosystem. The objective of this study was to determine the distribution of cadmium, copper, lead, manganese, nickel and zinc in geochemical fractions of surface sediments, using modified BCR sequential extraction procedure. The concentrations were measured using flame atomic absorption spectroscopy. In addition, the contents of soluble and exchangeable metals associated to carbonate fractions, determined by BCR, were compared with those determined by the method of Campanella. Samples were collected in 12 stations during June 2007. The applied methodologies were evaluated with a certified reference material of marine sediments (HISS-1) and the results indicated that these methods provide adequate accuracy and precision for the extraction of metals. The total metal concentrations (microg g(-1)) were, Cd: < limit of detection (LD)-5.0; Pb: 1.79-60.41; Cu: no detected (ND)-42.18; Zn: 25.13-104.57; Mn: 66.31-80.29 and Ni: 3.29-24.58. Cd, Cu, Ni and Pb at several stations, exceeded the Canadian Sediment Quality Guidelines of the Lowest Effect Levels (LEL). Cadmium was identified as being the most mobile of the elements, having the highest concentrations in soluble and exchangeable cations and carbonates. However, Pb, Cu, Mn and Zn levels were found highly associated to organic matter and sulfide fractions. The methods did not show significant statistical differences for the extraction of soluble and exchangeable cations and the metals associated to carbonate fraction. There are several significant correlations between heavy metals, which suggest their common origin.     

Protection against Pseudomonas aeruginosa lung infection in mice by recombinant OprF-pulsed dendritic cell immunization

Background

Porcine reproductive and respiratory syndrome (PRRS) has now been widely recognized as an economically important disease. The objective of this study was to compare the molecular and biological characteristics of porcine reproductive and respiratory syndrome virus (PRRSV) field isolates in China to those of the modified live virus (MLV) PRRS vaccine and its parent strain (ATCC VR2332).

Results

Five genes (GP2, GP3, GP4, GP5 and NSP2) of seven isolates of PRRSV from China, designated LS-4, HM-1, HQ-5, HQ-6, GC-2, GCH-3 and ST-7/2008, were sequenced and analyzed. Phylogenetic analyses based on the nucleotide sequence of the ORF2-5 and NSP2 showed that the seven Chinese isolates belonged to the same genetic subgroup and were related to the North American PRRSV genotype. Comparative analysis with the relevant sequences of another Chinese isolate (BJ-4) and North American (VR2332 and MLV) viruses revealed that these isolates have 80.8-92.9% homology with VR-2332, and 81.3-98.8% identity with MLV and 80.7-92.9% with BJ-4. All Nsp2 nonstructural protein of these seven isolates exhibited variations (a 29 amino acids deletion) in comparison with other North American PRRSV isolates. Therefore, these isolates were novel strain with unique amino acid composition. However, they all share more than 97% identity with other highly pathogenic Chinese PRRSV strains. Additionally, there are extensive amino acid (aa) mutations in the GP5 protein and the Nsp2 protein when compared with the previous isolates.

Conclusions

These results might be useful to study the genetic diversity of PRRSV in China and to track the infection sources as well as for vaccines development.