Thermal requirement for development of Sancassania rodionovi (Acari: Acaridae) on mushrooms

The free-living mite species Sancassania rodionovi (Zachvatkin) (Acari: Acaridae), is a serious pest of mushrooms in Iran. Studies were conducted to examine the development of this mite in relation to temperature on two mushroom species: Agaricus bisporus Lange (button mushroom) and Pleurotus ostreatus Kummer (oyster mushroom). The developmental time of this acarid mite was studied at eight constant temperatures, ranging from 5 to 40 degrees C, and developmental rates were modeled as a function of temperature. Sancassania rodionovi completed immature development in 17.35 +/- 0.58 and 20.17 +/- 0.88 d at 25 degrees C on button and oyster mushrooms, respectively. When the mite fed on button mushroom, the rate of development increased gradually from 10 to 35 degrees C. Using a linear model, the developmental zero was estimated to be 3.50 degrees C with a thermal constant of 357.14 degree-days. The Logan 10, Briere 1, and Thermodynamic models adequately described the data for this mite and yielded R2 values >0.95; these models provided estimates of optimum temperature for development of 33.244, 32.145, and 32.148 degrees C, respectively. Understanding the influence of temperature on development of S. rodionovi is discussed with respect to pest management in mushroom production.     

Increased resistance of neutrophils to deformation upon cooling and rate of recovery on rewarming

Increase in the resistance to deformation of neutrophils upon exposure to the cold may impair their passage through microvessels. However, the potential for such rheological changes to cause prolonged microvascular obstruction in cooled tissue will depend on whether and at what rate the neutrophils recover on rewarming. We tested the ability of neutrophils to pass through micropore filters, and found that neutrophils cooled to 10 degrees C for 10-20 minutes could block either 5 microm or 8 microm pore filters. On return to 37 degrees C, flow resistance remained impaired briefly but recovered over about 5 minutes. The kinetics of changes in flow resistance in the cold and on rewarming were linked to kinetics of actin polymerisation during these periods. However, they were not closely linked to distortion of cell shape in the cold, which recovered only slowly with rewarming. The results suggest that while rigid neutrophils might occlude capillaries in cold tissue, mechanical obstruction should not be long-lived on rewarming. Moreover, rigid neutrophils washed out of cold tissue should experience only temporary mechanical trapping in remote tissues.     

A dominant-negative approach that prevents diphthamide formation confers resistance to Pseudomonas exotoxin A and diphtheria toxin

Diphtheria toxin (DT), Pseudomonas aeruginosa Exotoxin A (ETA) and cholix toxin from Vibrio cholerae share the same mechanism of toxicity; these enzymes ADP-rybosylate elongation factor-2 (EF-2) on a modified histidine residue called diphthamide, leading to a block in protein synthesis. Mutant Chinese hamster ovary cells that are defective in the formation of diphthamide have no distinct phenotype except their resistance to DT and ETA. These observations led us to predict that a strategy that prevents the formation of diphthamide to confer DT and ETA resistance is likely to be safe. It is well documented that Dph1 and Dph2 are involved in the first biochemical step of diphthamide formation and that these two proteins interact with each other. We hypothesized that we could block diphthamide formation with a dominant negative mutant of either Dph1 or Dph2. We report in this study the first cellular-targeted strategy that protects against DT and ETA toxicity. We have generated Dph2(C-), a dominant-negative mutant of Dph2, that could block very efficiently the formation of diphthamide. Cells expressing Dph2(C-) were 1000-fold more resistant to DT than parental cells, and a similar protection against Pseudomonas exotoxin A was also obtained. The targeting of a cellular component with this approach should have a reduced risk of generating resistance as it is commonly seen with antibiotic treatments.     

Multilocus typing of Cryptosporidium spp. and Giardia duodenalis from non-human primates in China

Non-human primates (NHPs) are commonly infected with Cryptosporidium spp. and Giardia duodenalis. However, molecular characterisation of these pathogens from NHPs remains scarce. In this study, 2,660 specimens from 26 NHP species in China were examined and characterised by PCR amplification of 18S rRNA, 70 kDa heat shock protein (hsp70) and 60 kDa glycoprotein (gp60) gene loci for Cryptosporidium; and 1,386 of the specimens by ssrRNA, triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh) gene loci for Giardia. Cryptosporidium was detected in 0.7% (19/2660) specimens of four NHP species including rhesus macaques (0.7%), cynomolgus monkeys (1.0%), slow lorises (10.0%) and Francois’ leaf monkeys (6.7%), belonging to Cryptosporidium hominis (14/19) and Cryptosporidium muris (5/19). Two C. hominis gp60 subtypes, IbA12G3 and IiA17 were observed. Based on the tpi locus, G. duodenalis was identified in 2.2% (30/1,386) of specimens including 2.1% in rhesus macaques, 33.3% in Japanese macaques, 16.7% in Assam macaques, 0.7% in white-headed langurs, 1.6% in cynomolgus monkeys and 16.7% in olive baboons. Sequence analysis of the three targets indicated that all of the Giardia-positive specimens belonged to the zoonotic assemblage B. Highest sequence polymorphism was observed at the tpi locus, including 11 subtypes: three known and eight new ones. Phylogenetic analysis of the subtypes showed that most of them were close to the so-called subtype BIV. Intragenotypic variations at the gdh locus revealed six types of sequences (three known and three new), all of which belonged to so-called subtype BIV. Three specimens had co-infection with C. hominis (IbA12G3) and G. duodenalis (BIV). The presence of zoonotic genotypes and subtypes of Cryptosporidium spp. and G. duodenalis in NHPs suggests that these animals can potentially contribute to the transmission of human cryptosporidiosis and giardiasis.     

MicroRNAs in hereditary and sporadic premature aging syndromes and other laminopathies

Hereditary and sporadic laminopathies are caused by mutations in genes encoding lamins, their partners, or the metalloprotease ZMPSTE24/FACE1. Depending on the clinical phenotype, they are classified as tissue‐specific or systemic diseases. The latter mostly manifest with several accelerated aging features, as in Hutchinson–Gilford progeria syndrome (HGPS) and other progeroid syndromes. MicroRNAs are small noncoding RNAs described as powerful regulators of gene expression, mainly by degrading target mRNAs or by inhibiting their translation. In recent years, the role of these small RNAs has become an object of study in laminopathies using in vitro or in vivo murine models as well as cells/tissues of patients. To date, few miRNAs have been reported to exert protective effects in laminopathies, including miR‐9, which prevents progerin accumulation in HGPS neurons. The recent literature has described the potential implication of several other miRNAs in the pathophysiology of laminopathies, mostly by exerting deleterious effects. This review provides an overview of the current knowledge of the functional relevance and molecular insights of miRNAs in laminopathies. Furthermore, we discuss how these discoveries could help to better understand these diseases at the molecular level and could pave the way toward identifying new potential therapeutic targets and strategies based on miRNA modulation.     

Design and Synthesis of Fluoroacylshikonin as an Anticancer Agent

A series of shikonin derivatives, selectively acylated by various fluorinated carboxylic acids at the side chain of shikonin, were synthesized and their anticancer activity evaluated, in which eight compounds are reported for the first time. Among all the compounds tested, compound S7 showed the most potent anticancer activity against B16‐F10 (malignant melanoma cells), MG63 (human osteosarcoma cells), and A549 (lung cancer cells) with IC₅₀ 0.39 ± 0.01, 0.72 ± 0.04 and 0.58 ± 0.02 µmol/L. Docking simulation of compound S7 was carried out to position S7 into a tubulin active site to determine the probable binding conformation. All the results suggested that compound S7 may be a potential anticancer agent. Chirality 25:757–762, 2013. © 2013 Wiley Periodicals, Inc.     

Synthesis of TOAC spin-labeled proteins and reconstitution in lipid membranes

A procedure is described for the synthetic incorporation into membrane proteins of the non-natural amino acid TOAC (2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxylic acid), which is coupled rigidly to the alpha-carbon, providing direct detection of peptide backbone dynamics by electron paramagnetic resonance (EPR). Also included is a protocol for the functional reconstitution of the spin-labeled protein in lipid vesicles. This protocol can be completed in 17 d.     

Human immunodeficiency virus-specific gamma interferon enzyme-linked immunospot assay responses targeting specific regions of the proteome during primary subtype C infection are poor predictors of the course of viremia and set point

It is unknown whether patterns of human immunodeficiency virus (HIV)-specific T-cell responses during acute infection may influence the viral set point and the course of disease. We wished to establish whether the magnitude and breadth of HIV type 1 (HIV-1)-specific T-cell responses at 3 months postinfection were correlated with the viral-load set point at 12 months and hypothesized that the magnitude and breadth of HIV-specific T-cell responses during primary infection would predict the set point. Gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay responses across the complete proteome were measured in 47 subtype C HIV-1-infected participants at a median of 12 weeks postinfection. When corrected for amino acid length and individuals responding to each region, the order of recognition was as follows: Nef > Gag > Pol > Rev > Vpr > Env > Vpu > Vif > Tat. Nef responses were significantly (P < 0.05) dominant, targeted six epitopic regions, and were unrelated to the course of viremia. There was no significant difference in the magnitude and breadth of responses for each protein region with disease progression, although there was a trend of increased breadth (mean, four to seven pools) in rapid progressors. Correlation of the magnitude and breadth of IFN-γ responses with the viral set point at 12 months revealed almost zero association for each protein region. Taken together, these data demonstrate that the magnitude and breadth of IFN-γ ELISPOT assay responses at 3 months postinfection are unrelated to the course of disease in the first year of infection and are not associated with, and have low predictive power for, the viral set point at 12 months.     

In Vitro Evaluation of a Soluble Leishmania Promastigote Surface Antigen as a Potential Vaccine Candidate against Human Leishmaniasis

PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection.