A conditional export system provides new insights into protein export in Plasmodium falciparum-infected erythrocytes

The human malarial parasite Plasmodium falciparum exports determinants of virulence and pathology to destinations within its host erythrocyte, including the cytoplasm, the plasma membrane and membrane profiles of parasite origin termed Maurer's clefts. While there is some information regarding the signals that allot proteins for export, the trafficking route itself has remained largely obscure, partly due to technical limitations in following protein trafficking with time. To overcome these shortcomings, we have established a conditional protein export system in P. falciparum, based on the previously described conditional aggregation domain (CAD domain) that self-aggregates in the endoplasmic reticulum in a manner that is reversible by the addition of a small molecule. By fusing the CAD domain to the first 80 amino acids of STEVOR and full-length PfSBP1, we were able to control export of a soluble and a transmembrane protein to the erythrocyte cytosol and the Maurer's clefts respectively. The conditional export system allowed us to study the temporal sequence of events of protein export and identify intermediate steps. We further explored the potential of the conditional export system in identifying factors that interact with exported proteins en route. Our data provide evidence for a physical interaction of exported proteins with the molecular chaperone PfBiP during early export steps.     

TomoJ: tomography software for three-dimensional reconstruction in transmission electron microscopy

Background  

Transmission electron tomography is an increasingly common three-dimensional electron microscopy approach that can provide new insights into the structure of subcellular components. Transmission electron tomography fills the gap between high resolution structural methods (X-ray diffraction or nuclear magnetic resonance) and optical microscopy. We developed new software for transmission electron tomography, TomoJ. TomoJ is a plug-in for the now standard image analysis and processing software for optical microscopy, ImageJ.     

Ebola virus glycoprotein 1: identification of residues important for binding and postbinding events

The filoviruses Ebola virus (EBOV) and Marburg virus (MARV) are responsible for devastating hemorrhagic fever outbreaks. No therapies are available against these viruses. An understanding of filoviral glycoprotein 1 (GP1) residues involved in entry events would facilitate the development of antivirals. Towards this end, we performed alanine scanning mutagenesis on selected residues in the amino terminus of GP1. Mutant GPs were evaluated for their incorporation onto feline immunodeficiency virus (FIV) particles, transduction efficiency, receptor binding, and ability to be cleaved by cathepsins L and B. FIV virions bearing 39 out of 63 mutant glycoproteins transduced cells efficiently, whereas virions bearing the other 24 had reduced levels of transduction. Virions pseudotyped with 23 of the poorly transducing GPs were characterized for their block in entry. Ten mutant GPs were very poorly incorporated onto viral particles. Nine additional mutant GPs (G87A/F88A, K114A/K115A, K140A, G143A, P146A/C147A, F153A/H154A, F159A, F160A, and Y162A) competed poorly with wild-type GP for binding to permissive cells. Four of these nine mutants (P146A/C147A, F153A/H154A, F159A, and F160A) were also inefficiently cleaved by cathepsins. An additional four mutant GPs (K84A, R134A, D150A, and E305/E306A) that were partially defective in transduction were found to compete effectively for receptor binding and were readily cleaved by cathepsins. This finding suggested that this latter group of mutants might be defective at a postbinding, cathepsin cleavage-independent step. In total, our study confirms the role of some GP1 residues in EBOV entry that had previously been recognized and identifies for the first time other residues that are important for productive entry.     

A proteomics analysis of cell signaling alterations in colorectal cancer

To gain further insight into alterations in cellular pathways, tumor profiling, and marker discovery in colorectal cancer (CRC) we used a new antibody microarray specific for cell signaling. Soluble protein extracts were prepared from paired tumor/normal biopsies of 11 patients diagnosed with colorectal carcinoma at different stages; four liver carcinomas were used as a reference. Antibody microarray analysis identified 46 proteins that were differentially expressed between normal colorectal epithelium and adenocarcinoma. These proteins gave a specific signature for CRC, different from other tumors, as well as a panel of novel markers and potential targets for CRC. Twenty-four proteins were validated by using a specific colorectal cancer tissue microarray and immunoblotting analysis. Together with some previously well known deregulated proteins in CRC (beta-catenin, c-MYC, or p63), we found new potential markers preferentially expressed in CRC tumors: cytokeratin 13, calcineurin, CHK1, clathrin light chain, MAPK3, phospho-PTK2/focal adhesion kinase (Ser-910), and MDM2. CHK1 antibodies were particularly effective in discriminating between tumoral and normal mucosa in CRC. Moreover a global picture of alterations in signaling pathways in CRC was observed, including a significant up-regulation of different components of the epidermal growth factor receptor and Wnt/beta-catenin pathways and the down-regulation of p14(ARF). The experimental approach described here should be applicable to other pathologies and neoplastic processes.     

Ground wheat straw as a substitute for portions of oak wood chips used in shiitake (Lentinula edodes) substrate formulae

Oak woodchips, used for production of shiitake Lentinula edodes (Berk) Pegler, are increasingly difficult to obtain due to dwindling supplies. We investigated the effect of adding ground wheat straw as a substitute for portions of oak woodchips in substrate formulae on mushroom yield and size. We also determined the effect of mushroom cropping on relative feed value (RFV) by chemical analysis of the substrate at spawning (AS) and after cropping (AC). Three formulae containing 0%, 8% and 16% ground wheat straw and 52%, 44% and 36% oak sawdust, respectively, were bulk pasteurized (111 degrees C for 20 min) in an autoclaving mixer, subjected to spawn run (21 d), browning (28 d) and a production cycle of three breaks (38 d). Mean (4 crops) mushroom yields were 11% higher when 8% wheat straw was used in the medium and 19% higher when 16% wheat straw was substituted for portions of oak sawdust. There were no significant differences in mushroom sizes between any of the treatments. Relative feed values of shiitake substrates AC increased more dramatically as more wheat straw was added to the formulae. Using mature alfalfa (full bloom) as a base value of 100%, RFVs for substrate AS were 98%, 92%, and 92% for 0%, 8% and 16% straw, respectively; RFVs AC were 118%, 120% and 133%, respectively. Substrate AC containing 16% straw had a RFV comparable to corn silage (well-eared). Fat contents of the substrates decreased by 50-62% AC, whereas potassium contents decreased by 40%. Use of ground wheat straw in synthetic medium would not only increase mushroom yield by up to 19%, but may help alleviate periodic shortages of oak sawdust. In addition, growers would avoid the added expense of aging the wheat straw (for 8-12 week) as is typically done for oak sawdust in the industry. This is the first report of RFVs for spent shiitake substrate (SSS) predicting its excellent potential for use as animal feed.     

Lack of species-specificity in mammalian sperm chemotaxis

Attraction of spermatozoa by way of chemotaxis to substances secreted from the egg or its surrounding cells has been demonstrated in marine species, amphibians, and mammals. This process is species- or family-specific in marine invertebrates: a chemoattractant for one marine species is usually not recognized by another species or by a member of another family. It is not known whether this selectivity is also the rule in other phyla. Furthermore, it is not at all obvious that such selectivity would be advantageous to species with internal fertilization. Here, using a directionality-based assay for chemotaxis, we studied in vitro the chemotactic response of human and rabbit spermatozoa to human, rabbit, and bovine egg-related factors. We found that spermatozoa from each of the two sources responded similarly well to egg-related factors obtained from any of the three species examined. These results indicate lack of chemotaxis-related, species specificity between these species, suggesting that their sperm chemoattractants are common or very similar. The findings further suggest that mammals do not rely on species specificity of sperm chemotaxis for avoidance of interspecies fertilization.     

Influence of maternal stress on uranium-induced developmental toxicity in rats

It has been demonstrated that uranium is an embryo/fetal toxicant when given orally or subcutaneously to pregnant mice. On the other hand, maternal stress has been shown to enhance the developmental toxicity of a number of metals. In this study, maternal toxicity and developmental effects of a concurrent exposure to uranyl acetate dihydrate (UAD) and restraint stress were evaluated in rats. Four groups of pregnant animals were given subcutaneous injections of UAD at 0.415 and 0.830 mg/kg/day on Days 6 to 15 of gestation. Animals in two of these groups were also subjected to restraint for 2 hr/day during the same gestational days. Control groups included restrained and unrestrained pregnant rats not exposed to UAD. Cesarean sections were performed on gestation Day 20, and the fetuses were weighed and examined for malformations and variations. Maternal toxicity and embryotoxicity were noted at 0.830 mg/kg/day of UAD, while fetotoxicity was evidenced at 0.415 and 0.830 mg/kg/day of UAD by significant reductions in fetal body weight and increases in the total number of skeletally affected fetuses. No teratogenic effects were noted in any group. Maternal restraint enhanced uranium-induced embryo/fetal toxicity only at 0.830 mg/kg/day, a dose that was also significantly toxic to the dams. As in previous studies with other metals, maternal stress enhances uranium-induced developmental toxicity at uranium doses that are highly toxic to the dams; however, at doses that are less acutely toxic the role of maternal stress would not be significant.     

Differential Role of Autophagy in CD4 T Cells and Macrophages during X4 and R5 HIV-1 Infection

Background

HIV-1 can infect and replicate in both CD4 T cells and macrophages. In these cell types, HIV-1 entry is mediated by the binding of envelope glycoproteins (gp120 and gp41, Env) to the receptor CD4 and a coreceptor, principally CCR5 or CXCR4, depending on the viral strain (R5 or X4, respectively). Uninfected CD4 T cells undergo X4 Env-mediated autophagy, leading to their apoptosis, a mechanism now recognized as central to immunodeficiency.

Methodology/Principal Findings

We demonstrate here that autophagy and cell death are also induced in the uninfected CD4 T cells by HIV-1 R5 Env, while autophagy is inhibited in productively X4 or R5-infected CD4 T cells. In contrast, uninfected macrophages, a preserved cell population during HIV-1 infection, do not undergo X4 or R5 Env-mediated autophagy. Autophagosomes, however, are present in macrophages exposed to infectious HIV-1 particles, independently of coreceptor use. Interestingly, we observed two populations of autophagic cells: one highly autophagic and the other weakly autophagic. Surprisingly, viruses could be detected in the weakly autophagic cells but not in the highly autophagic cells. In addition, we show that the triggering of autophagy in macrophages is necessary for viral replication but addition of Bafilomycin A1, which blocks the final stages of autophagy, strongly increases productive infection.

Conclusions/Significance

Taken together, our data suggest that autophagy plays a complex, but essential, role in HIV pathology by regulating both viral replication and the fate of the target cells.     

Modeling Dynamic Introduction of Chikungunya Virus in the United States

Chikungunya is a mosquito-borne viral infection of humans that previously was confined to regions in central Africa. However, during this century, the virus has shown surprising potential for geographic expansion as it invaded other countries including more temperate regions. With no vaccine and no specific treatment, the main control strategy for Chikungunya remains preventive control of mosquito populations. In consideration for the risk of Chikungunya introduction to the US, we developed a model for disease introduction based on virus introduction by one individual. Our study combines a climate-based mosquito population dynamics stochastic model with an epidemiological model to identify temporal windows that have epidemic risk. We ran this model with temperature data from different locations to study the geographic sensitivity of epidemic potential. We found that in locations with marked seasonal variation in temperature there also was a season of epidemic risk matching the period of the year in which mosquito populations survive and grow. In these locations controlling mosquito population sizes might be an efficient strategy. But, in other locations where the temperature supports mosquito development all year the epidemic risk is high and (practically) constant. In these locations, mosquito population control alone might not be an efficient disease control strategy and other approaches should be implemented to complement it. Our results strongly suggest that, in the event of an introduction and establishment of Chikungunya in the US, endemic and epidemic regions would emerge initially, primarily defined by environmental factors controlling annual mosquito population cycles. These regions should be identified to plan different intervention measures. In addition, reducing vector: human ratios can lower the probability and magnitude of outbreaks for regions with strong seasonal temperature patterns. This is the first model to consider Chikungunya risk in the US and can be applied to other vector borne diseases.     

Motor unit recruitment strategy of knee antagonist muscles in a step-wise, increasing isometric contraction

The purpose of this study was to determine if differences exist between the control strategies of two antagonist thigh muscles during knee flexion and extension muscular coactivation. Surface myoelectric signal (MES) of the quadriceps (rectus femoris) and the hamstrings (semitendinosus) were obtained from both muscles while performing step-wise increasing contractions during flexion and extension with the knee at 1.57 rad of flexion (90 degrees). The median frequency of the power density spectrum, which is related to the average muscle fiber action potential conduction velocity and therefore to motor unit recruitment, was calculated from each MES. The results suggest that, in all the subjects tested, when the muscle acts as antagonist most motor units are recruited up to 50% of the maximal voluntary force, whereas when the muscle acts as antagonist motor units are recruited up to 40% of the maximal voluntary force. The force range past 40–50% of the maximal force is also characterized by differences between the agonist/antagonist.