The development of <Emphasis Type="Italic">Gymnophallus australis</Emphasis> Szidat, 1962 (Digenea: Gymnophallidae) from the Patagonian coast (Argentina) from metacercaria to adult,with an amended diagnosis of <Emphasis Type="Italic">Gymnophallus</Emphasis> Odhner, 1905

Parvatrema australis (Szidat, 1962) Szidat, 1965 was described based on larval stages found in specimens of the mussel Mytilus edulis from the Buenos Aires Province, Argentina. Although Szidat later examined hundreds of mussels, this parasite has never been found again until now. In the present study, larval stages, including germinal sacs, found in mytilids from the Patagonian coast were identified as P. australis. Metacercariae were incubated in vitro at 39 degrees C in physiological solution for 18-20 hours, by which time 80% of the specimens had eggs. P. australis is redescribed, on the basis of infective metacercariae and adults obtained in the laboratory, and is reassigned to Gymnophallus Odhner, 1900, the genus in which it was originally described. The generic diagnosis of Gymnophallus is here amended to include as diagnostic characters the presence or absence of the lateral lips, the form and position of the vitellarium (compact or follicular) and the presence of a pars prostatica (i.e. prostatic cells open into proximal part of the ejaculatory duct). The validity of some characters (i.e. the presence of lateral lips of the oral sucker, the form of the vitellarium and excretory vesicle, the extent of the uterus) as diagnostic at the generic level within the family Gymnophallidae is discussed. It is proposed that the least unambiguous characters that can be used to distinguish gymnophallid genera include the position of the ovary, the presence of a ventral pit and a pars prostatica, and caecal diverticula.     

Transformation of Mexican lime with an intron-hairpin construct expressing untranslatable versions of the genes coding for the three silencing suppressors of Citrus tristeza virus confers complete resistance to the virus

Citrus tristeza virus (CTV), the causal agent of the most devastating viral disease of citrus, has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and/or intercellular level (p20 and p25) to overcome host antiviral defence. Previously, we showed that Mexican lime transformed with an intron-hairpin construct including part of the gene p23 and the adjacent 3' untranslated region displays partial resistance to CTV, with a fraction of the propagations from some transgenic lines remaining uninfected. Here, we transformed Mexican lime with an intron-hairpin vector carrying full-length, untranslatable versions of the genes p25, p20 and p23 from CTV strain T36 to silence the expression of these critical genes in CTV-infected cells. Three transgenic lines presented complete resistance to viral infection, with all their propagations remaining symptomless and virus-free after graft inoculation with CTV-T36, either in the nontransgenic rootstock or in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Inoculation with a divergent CTV strain led to partially breaking the resistance, thus showing the role of sequence identity in the underlying mechanism. Our results are a step forward to developing transgenic resistance to CTV and also show that targeting simultaneously by RNA interference (RNAi) the three viral silencing suppressors appears critical for this purpose, although the involvement of concurrent RNAi mechanisms cannot be excluded.     

Highly efficient protein misfolding cyclic amplification

Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrP(C) into PrP(Sc) in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrP(C) may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrP(C) into PrP(Sc) from ～10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrP(Sc) by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 1012-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrP(C) susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrP(Sc)in vitro.     

The Klebsiella pneumoniae wabG gene: role in biosynthesis of the core lipopolysaccharide and virulence

To determine the function of the wabG gene in the biosynthesis of the core lipopolysaccharide (LPS) of Klebsiella pneumoniae, we constructed wabG nonpolar mutants. Data obtained from the comparative chemical and structural analysis of LPS samples obtained from the wild type, the mutant strain, and the complemented mutant demonstrated that the wabG gene is involved in attachment to alpha-L-glycero-D-manno-heptopyranose II (L,D-HeppII) at the O-3 position of an alpha-D-galactopyranosyluronic acid (alpha-D-GalAp) residue. K. pneumoniae nonpolar wabG mutants were devoid of the cell-attached capsular polysaccharide but were still able to produce capsular polysaccharide. Similar results were obtained with K. pneumoniae nonpolar waaC and waaF mutants, which produce shorter LPS core molecules than do wabG mutants. Other outer core K. pneumoniae nonpolar mutants in the waa gene cluster were encapsulated. K. pneumoniae waaC, waaF, and wabG mutants were avirulent when tested in different animal models. Furthermore, these mutants were more sensitive to some hydrophobic compounds than the wild-type strains. All these characteristics were rescued by reintroduction of the waaC, waaF, and wabG genes from K. pneumoniae.     

Multiplex reactions for the molecular detection of predation on pest and nonpest invertebrates in agroecosystems

Species- and group-specific PCR primers were developed to study predation on pest and nonpest invertebrate species by generalist carabid predators in agroecosystems. To ensure the amplification of degraded DNA in predator gut samples, amplicons were designed to be less than 300 bp. Specificity of primers was assessed by cross-amplification against a panel of target and nontarget invertebrate species. The new primers were combined with previously published primers for slugs and collembolla in multiplex reactions to simultaneously screen each predator for the presence of multiple prey. All prey species were detected in a screen of the gut contents of field-caught predators.     

Plastid transformation of high‐biomass tobacco variety Maryland Mammoth for production of human immunodeficiency virus type 1 (HIV‐1) p24 antigen

Chloroplast transformation of the high‐biomass tobacco variety Maryland Mammoth has been assessed as a production platform for the human immunodeficiency virus type 1 (HIV‐1) p24 antigen. Maryland Mammoth offers the prospect of higher yields of intact functional protein per unit floor area of contained glasshouse per unit time prior to flowering. Two different transformation constructs, pZSJH1p24 (for the insertion of a native p24 cDNA between the rbcL and accD genes) and pZF5 (for the insertion of a chloroplast‐codon‐optimized p24 gene between trnfM and trnG) were examined for the production of p24. Plants generated with construct pZSJH1p24 exhibited a normal green phenotype, but p24 protein accumulated only in the youngest leaves (up to approximately 350 µg/g fresh weight or ~2.5% total soluble protein) and was undetectable in mature leaves. In contrast, some of the plants generated with pZF5 exhibited a yellow phenotype (pZF5‐yellow) with detectable p24 accumulation (up to approximately 450 µg/g fresh weight or ~4.5% total soluble protein) in all leaves, regardless of age. Total protein in pZF5‐yellow leaves was reduced by ~40%. The pZF5‐yellow phenotype was associated with recombination between native and introduced direct repeat sequences of the rbcL 3′ untransformed region in the plastid genome. Chloroplast‐expressed p24 was recognized by a conformation‐dependent monoclonal antibody to p24, and p24 protein could be purified from pZF5‐yellow leaves using a simple procedure, involving ammonium sulphate precipitation and ion‐exchange chromatography, without the use of an affinity tag. The purified p24 was shown to be full length with no modifications, such as glycosylation or phosphorylation, using N‐terminal sequencing and mass spectrometry.     

Correction to: d-Pinitol: a cyclitol with versatile biological and pharmacological activities

Phytochemistry Reviews - In the original publication.     

Modeling-Enabled Characterization of Novel NLRX1 Ligands

Nucleotide-binding domain and leucine-rich repeat containing (NLR) family are intracellular sentinels of cytosolic homeostasis that orchestrate immune and inflammatory responses in infectious and immune-mediated diseases. NLRX1 is a mitochondrial-associated NOD-like receptor involved in the modulation of immune and metabolic responses. This study utilizes molecular docking approaches to investigate the structure of NLRX1 and experimentally assesses binding to naturally occurring compounds from several natural product and lipid databases. Screening of compound libraries predicts targeting of NLRX1 by conjugated trienes, polyketides, prenol lipids, sterol lipids, and coenzyme A-containing fatty acids for activating the NLRX1 pathway. The ligands of NLRX1 were identified by docking punicic acid (PUA), eleostearic acid (ESA), and docosahexaenoic acid (DHA) to the C-terminal fragment of the human NLRX1 (cNLRX1). Their binding and that of positive control RNA to cNLRX1 were experimentally determined by surface plasmon resonance (SPR) spectroscopy. In addition, the ligand binding sites of cNLRX1 were predicted in silico and validated experimentally. Target mutagenesis studies demonstrate that mutation of 4 critical residues ASP677, PHE680, PHE681, and GLU684 to alanine resulted in diminished affinity of PUA, ESA, and DHA to NLRX1. Consistent with the regulatory actions of NLRX1 on the NF-κB pathway, treatment of bone marrow derived macrophages (BMDM)s with PUA and DHA suppressed NF-κB activity in a NLRX1 dependent mechanism. In addition, a series of pre-clinical efficacy studies were performed using a mouse model of dextran sodium sulfate (DSS)-induced colitis. Our findings showed that the regulatory function of PUA on colitis is NLRX1 dependent. Thus, we identified novel small molecules that bind to NLRX1 and exert anti-inflammatory actions.