Cryptosporidium p30, a galactose/N-acetylgalactosamine-specific lectin, mediates infection in vitro

Cryptosporidium sp. cause human and animal diarrheal disease worldwide. The molecular mechanisms underlying Cryptosporidium attachment to, and invasion of, host cells are poorly understood. Previously, we described a surface-associated Gal/GalNAc-specific lectin activity in sporozoites of Cryptosporidium parvum. Here we describe p30, a 30-kDa Gal/GalNAc-specific lectin isolated from C. parvum and Cryptosporidium hominis sporozoites by Gal-affinity chromatography. p30 is encoded by a single copy gene containing a 906-bp open reading frame, the deduced amino acid sequence of which predicts a 302-amino acid, 31.8-kDa protein with a 22-amino acid N-terminal signal sequence. The p30 gene is expressed at 24-72 h after infection of intestinal epithelial cells. Antisera to recombinant p30 expressed in Escherichia coli react with an approximately 30-kDa protein in C. parvum and C. hominis. p30 is localized to the apical region of sporozoites and is predominantly intracellular in both sporozoites and intracellular stages of the parasite. p30 associates with gp900 and gp40, Gal/GalNAc-containing mucin-like glycoproteins that are also implicated in mediating infection. Native and recombinant p30 bind to Caco-2A cells in a dose-dependent, saturable, and Gal-inhibitable manner. Recombinant p30 inhibits C. parvum attachment to and infection of Caco-2A cells, whereas antisera to the recombinant protein also inhibit infection. Taken together, these findings suggest that p30 mediates C. parvum infection in vitro and raise the possibility that this protein may serve as a target for intervention.     

The polyhydroxyalkanoate genes of a stress resistant Antarctic Pseudomonas are situated within a genomic island

Pseudomonas sp. 14-3 is an Antarctic bacterium that shows high stress resistance in association with high polyhydroxybutyrate (PHB) production. In this paper genes involved in PHB biosynthesis (phaRBAC) were found within a genomic island named pha-GI. Numerous mobile elements or proteins associated with them, such as an integrase, insertion sequences, a bacterial group II intron, a complete Type I protein secretion system and IncP plasmid-related proteins were detected among the 28 ORFs identified in this large genetic element (32.3kb). The G+C distribution was not homogeneous, likely reflecting a mosaic structure that contains regions from diverse origins. pha-GI has strong similarities with genomic islands found in diverse Proteobacteria, including Burkholderiales species and Azotobacter vinelandii. The G+C content, phylogeny inference and codon usage analysis showed that the phaBAC cluster itself has a complex mosaic structure and indicated that the phaB and phaC genes were acquired by horizontal transfer, probably derived from Burkholderiales. These results describe for the first time a pha cluster located within a genomic island, and suggest that horizontal transfer of pha genes is a mechanism of adaptability to stress conditions such as those found in the extreme Antarctic environment.     

Bioconversion of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine into 2-phenylethanol by <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> in grape must cultures

A 2³ factorial design was employed to find the best conditions of pH, l-phenylalanine concentration and temperature for the production of 2-phenylethanol by Kluyveromyces marxianus CBS 6556. The cultivation was carried out on grape must, which contains a great amount of nitrogen compounds. Central composite design (CCD) was used for the analysis of treatment combinations. Results showed a second-degree polynomial regression model with good agreement of experimental data, with R ² = 0.92015 (p < 0.05). The maximum production of 2-phenylethanol was found at pH 7.0, temperature of 37 °C, and a concentration of 3.0 g of l-phenylalanine L⁻¹. Further experiments in bioreactors showed that oxygen concentration is also important to 2-phenylethanol production, with best results obtained at oxygen mass transfer rates of 2.0 h⁻¹.     

Radiosynthesis,radiochemical, and biological characterization of 177Lu-labeled diethylenetriamine penta-acetic acid

Diethylenetriamine penta-acetic acid (DTPA), when complexed with a gamma (γ)-emitter radioisotope like ^99mTc, is used for renal function diagnosis and many other diagnostic applications. The main aim of this study was to develop a novel and versatile single-step methodology for the synthesis of a new ¹⁷⁷Lu-labeled radiopharmaceutical with high radiochemical yield, which can be used for diagnostic purposes and therapeutic purposes also. The single and well-defined ¹⁷⁷Lu-DTPA complex was radiochemically characterized by paper chromatography, thin-layer chromatography, high-performance liquid chromatography, and electrophoresis techniques. Dependence of the labeling yield of ¹⁷⁷Lu-DTPA complex on different factors was studied in detail. Biological evaluation was also performed in a normal rabbit by developing images under a γ camera at various time intervals. More than 99% labeling yield was obtained by reacting DTPA with ¹⁷⁷Lu at specific conditions (pH 7.0, 15 minutes reaction time at 100 °C). ¹⁷⁷Lu-DTPA complex showed high stability both at room temperature and in vitro. Biodistribution studies in normal mice indicated the fractional renal uptake of intravenously administered ¹⁷⁷Lu-DTPA complex, which reached in the kidneys within 2-3 minutes. Scintigraphy showed rapid clearance from the body. Based on these results, we propose that ¹⁷⁷Lu-DTPA complex might be used as an ideal candidate for functional evaluation of kidneys and the urinary tract, especially when needed to be transported to long-range consumer sites, because of its suitable half-life.     

A native parasitic plant and soil microorganisms facilitate a native plant co‐occurrence with an invasive plant

Invasive plants often interact with antagonists that include native parasitic plants and pathogenic soil microbes, which may reduce fitness of the invaders. However, to date, most of the studies on the ecological consequences of antagonistic interactions between invasive plants and the resident biota focused only on pairwise interactions. A full understanding of invasion dynamics requires studies that test the effects of multiple antagonists on fitness of invasive plants and co‐occurring native plants. Here, we used an invasive plant Mikania micrantha, a co‐occurring native plant Coix lacryma‐jobi, and a native holoparasitic plant Cuscuta campestris to test whether parasitism on M. micrantha interacts with soil fungi and bacteria to reduce fitness of the invader and promote growth of the co‐occurring native plant. In a factorial setup, M. micrantha and C. lacryma‐jobi were grown together in pots in the presence versus absence of parasitism on M. micrantha by C. campestris and in the presence versus absence of full complements of soil bacteria and fungi. Fungicide and bactericide were used to suppress soil fungi and bacteria, respectively. Findings show that heavy parasitism by C. campestris caused the greatest reduction in M. micrantha biomass when soil fungi and bacteria were suppressed. In contrast, the co‐occurring native plant C. lacryma‐jobi experienced the greatest increase in biomass when grown with heavily parasitized M. micrantha and in the presence of a full complement of soil fungi and bacteria. Taken together, our results suggest that selective parasitism on susceptible invasive plants by native parasitic plants and soil microorganisms may diminish competitive ability of invasive plants and facilitate native plant coexistence with invasive plants.     

Identification of a homozygous splice site mutation in the dynein axonemal light chain 4 gene on 22q13.1 in a large consanguineous family from Pakistan with congenital mirror movement disorder

Mirror movements (MRMV) are involuntary movements on one side of the body that mirror voluntary movements on the opposite side. Congenital mirror movement disorder is a rare, typically autosomal-dominant disorder, although it has been suspected that some sporadic cases may be due to recessive inheritance. Using a linkage analysis and a candidate gene approach, two genes have been implicated in congenital MRMV disorder to date: DCC on 18q21.2 (MRMV1), which encodes a netrin receptor, and RAD51 on 15q15.1 (MRMV2), which is involved in the maintenance of genomic integrity. Here, we describe a large consanguineous Pakistani family with 11 cases of congenital MRMV disorder reported across five generations, with autosomal recessive inheritance likely. Sanger sequencing of DCC and RAD51 did not identify a mutation. We then employed microarray genotyping and autozygosity mapping to identify a shared region of homozygosity-by-descent among the affected individuals. We identified a large autozygous region of ~3.3 Mb on chromosome 22q13.1 (Chr22:36605976?39904648). We used Sanger sequencing to exclude several candidate genes within this region, including DMC1 and NPTXR. Whole exome sequencing was employed, and identified a splice site mutation in the dynein axonemal light chain 4 gene, DNAL4. This splice site change leads to skipping of exon 3, and omission of 28 amino acids from DNAL4 protein. Linkage analysis using Simwalk2 gives a maximum Lod score of 6.197 at this locus. Whether or how DNAL4 function may relate to the function of DCC or RAD51 is not known. Also, there is no suggestion of primary ciliary dyskinesis, situs inversus, or defective sperm in affected family members, which might be anticipated given a putative role for DNAL4 in axonemal-based dynein complexes. We suggest that DNAL4 plays a role in the cytoplasmic dynein complex for netrin-1-directed retrograde transport, and in commissural neurons of the corpus callosum in particular. This, in turn, could lead to faulty cross-brain wiring, resulting in MRMV.     

Probing druggability and biological function of essential proteins in Leishmania combining facilitated null mutant and plasmid shuffle analyses

Leishmania parasites cause important human morbidity and mortality. Essential Leishmania genes escape genetic assessment by loss‐of‐function analyses due to lethal null mutant phenotypes, even though these genes and their products are biologically most significant and represent validated drug targets. Here we overcome this limitation using a facilitated null mutant approach applied for the functional genetic analysis of the MAP kinase LmaMPK4. This system relies on the episomal expression of the target gene from vector pXNG that expresses the Herpes simplex virus thymidine kinase gene thus rendering transgenic parasites susceptible for negative selection using the antiviral drug ganciclovir. Using this system we establish the genetic proof of LmaMPK4 as essential kinase in promastigotes. LmaMPK4 structure/function analysis by plasmid shuffle allowed us to identify regulatory kinase sequence elements relevant for chemotherapeutic intervention. A partial null mutant, expressing an MPK4 derivative with altered ATP‐binding properties, showed defects in metacyclogenesis, establishing a first link of MPK4 function to parasite differentiation. The approaches presented here are broadly applicable to any essential gene in Leishmania thus overcoming major bottlenecks for their functional genetic analysis and their exploitation for structure‐informed drug development.     

Replication of the association of a MET variant with autism in a Chinese Han population

Background

Autism is a common, severe and highly heritable neurodevelopmental disorder in children, affecting up to 100 children per 10,000. The MET gene has been regarded as a promising candidate gene for this disorder because it is located within a replicated linkage interval, is involved in pathways affecting the development of the cerebral cortex and cerebellum in ways relevant to autism patients, and has shown significant association signals in previous studies.

Principal Findings

Here, we present new ASD patient and control samples from Heilongjiang, China and use them in a case-control and family-based replication study of two MET variants. One SNP, rs38845, was successfully replicated in a case-control association study, but failed to replicate in a family-based study, possibly due to small sample size. The other SNP, rs1858830, failed to replicate in both case-control and family-based studies.

Conclusions

This is the first attempt to replicate associations in Chinese autism samples, and our result provides evidence that MET variants may be relevant to autism susceptibility in the Chinese Han population.     

Protective immunity to Listeria monocytogenes infection mediated by recombinant Listeria innocua harboring the VGC locus

In this study we propose a novel bacterial vaccine strategy where non-pathogenic bacteria are complemented with traits desirable for the induction of protective immunity. To illustrate the proof of principle of this novel vaccination strategy, we use the model organism of intracellular immunity Listeria. We introduced a, low copy number BAC-plasmid harbouring the virulence gene cluster (vgc) of L. monocytogenes (Lm) into the non-pathogenic L. innocua (L.inn) strain and examined for its ability to induce protective cellular immunity. The resulting strain (L.inn::vgc) was attenuated for virulence in vivo and showed a strongly reduced host detrimental inflammatory response compared to Lm. Like Lm, L.inn::vgc induced the production of Type I Interferon's and protection was mediated by Listeria-specific CD8(+) T cells. Rational vaccine design whereby avirulent strains are equipped with the capabilities to induce protection but lack detrimental inflammatory effects offer great promise towards future studies using non-pathogenic bacteria as vectors for vaccination.