Validation of the isothermal Schistosoma haematobium Recombinase Polymerase Amplification (RPA) assay,coupled with simplified sample preparation,for diagnosing female genital schistosomiasis using cervicovaginal lavage and vaginal self-swab samples

BackgroundFemale genital schistosomiasis (FGS) is a neglected and disabling gynecological disease that can result from infection with the parasitic trematode Schistosoma haematobium. Accurate diagnosis of FGS is crucial for effective case management, surveillance and control. However, current methods for diagnosis and morbidity assessment can be inaccessible to those at need, labour intensive, costly and unreliable. Molecular techniques such as PCR can be used to reliably diagnose FGS via the detection of Schistosoma DNA using cervicovaginal lavage (CVL) samples as well as lesser-invasive vaginal self-swab (VSS) and cervical self-swab samples. PCR is, however, currently unsuited for use in most endemic settings. As such, in this study, we assessed the use of a rapid and portable S. haematobium recombinase polymerase amplification (Sh-RPA) isothermal molecular diagnostic assay, coupled with simplified sample preparation methodologies, to detect S. haematobium DNA using CVL and VSS samples provided by patients in Zambia.Methodology/Principal findingsVSS and CVL samples were screened for FGS using a previously developed Sh-RPA assay. DNA was isolated from VSS and CVL samples using the QIAamp Mini kit (n = 603 and 527, respectively). DNA was also isolated from CVL samples using two rapid and portable DNA extraction methods: 1) the SpeedXtract Nucleic Acid Kit (n = 223) and 2) the Extracta DNA Tissue Prep Kit (n = 136). Diagnostic performance of the Sh-RPA using VSS DNA extacts (QIAamp Mini kit) as well as CVL DNA extracts (QIAamp Mini kit, SpeedXtract Nucleic Acid Kit and Extracta DNA Tissue Prep Kit) was then compared to a real-time PCR reference test.Results suggest that optimal performance may be achieved when the Sh-RPA is used with PuVSS samples (sensitivity 93.3%; specificity 96.6%), however no comparisons between different DNA extraction methods using VSS samples could be carried out within this study. When using CVL samples, sensitivity of the Sh-RPA ranged between 71.4 and 85.7 across all three DNA extraction methods when compared to real-time PCR using CVL samples prepared using the QIAamp Mini kit. Interestingly, of these three DNA extraction methods, the rapid and portable SpeedXtract method had the greatest sensitivity and specificity (85.7% and 98.1%, respectively). Specificity of the Sh-RPA was >91% across all comparisons.Conclusions/SignificanceThese results supplement previous findings, highlighting that the use of genital self-swab sampling for diagnosing FGS should be explored further whilst also demonstrating that rapid and portable DNA isolation methods can be used to detect S. haematobium DNA within clinical samples using RPA. Although further development and assessment is needed, it was concluded that the Sh-RPA, coupled with simplified sample preparation, shows excellent promise as a rapid and sensitive diagnostic tool capable of diagnosing FGS at the point-of-care in resource-poor schistosomiasis-endemic settings.     

Recognition and specificity determinants of the human cbx chromodomains

The eight mammalian Cbx proteins are chromodomain-containing proteins involved in regulation of heterochromatin, gene expression, and developmental programs. They are evolutionarily related to the Drosophila HP1 (dHP1) and Pc (dPc) proteins that are key components of chromatin-associated complexes capable of recognizing repressive marks such as trimethylated Lys-9 and Lys-27, respectively, on histone H3. However, the binding specificity and function of the human homologs, Cbx1-8, remain unclear. To this end we employed structural, biophysical, and mutagenic approaches to characterize the molecular determinants of sequence contextual methyllysine binding to human Cbx1-8 proteins. Although all three human HP1 homologs (Cbx1, -3, -5) replicate the structural and binding features of their dHP counterparts, the five Pc homologs (Cbx2, -4, -6, -7, -8) bind with lower affinity to H3K9me3 or H3K27me3 peptides and are unable to distinguish between these two marks. Additionally, peptide permutation arrays revealed a greater sequence tolerance within the Pc family and suggest alternative nonhistone sequences as potential binding targets for this class of chromodomains. Our structures explain the divergence of peptide binding selectivity in the Pc subfamily and highlight previously unrecognized features of the chromodomain that influence binding and specificity.     

Scheduled food hastens re-entrainment more than melatonin does after a 6-h phase advance of the light-dark cycle in rats

Circadian desynchrony occurs when individuals are exposed to abrupt phase shifts of the light-dark cycle, as in jet lag. For reducing symptoms and for speeding up resynchronization, several strategies have been suggested, including scheduled exercise, exposure to bright light, drugs, and especially exogenous melatonin administration. Restricted feeding schedules have shown to be powerful entraining signals for metabolic and hormonal daily cycles, as well as for clock genes in tissues and organs of the periphery. This study explored in a rat model of jet lag the contribution of exogenous melatonin or scheduled feeding on the re-entrainment speed of spontaneous general activity and core temperature after a 6-h phase advance of the light-dark cycle. In a first phase, the treatment was scheduled for 5 days prior to the phase shift, while in a second stage, the treatment was simultaneous with the phase advance of the light-dark cycle. Melatonin administration and especially scheduled feeding simultaneous with the phase shift improved significantly the re-entrainment speed. The evaluation of the free-running activity and temperature following the 5-day treatment proved that both exogenous melatonin and specially scheduled feeding accelerated re-entrainment of the SCN-driven general activity and core temperature, respectively, with 7, 5 days (p < 0.01) and 3, 3 days (p < 0.001). The present results show the relevance of feeding schedules as entraining signals for the circadian system and highlight the importance of using them as a strategy for preventing internal desynchrony.     

Novel aggregate formation of a frame-shift mutant protein of tissue-nonspecific alkaline phosphatase is ascribed to three cysteine residues in the C-terminal extension. Retarded secretion and proteasomal degradation

In the majority of hypophosphatasia patients, reductions in the serum levels of alkaline phosphatase activity are caused by various missense mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. A unique frame-shift mutation due to a deletion of T at cDNA number 1559 [TNSALP (1559delT)] has been reported only in Japanese patients with high allele frequency. In this study, we examined the molecular phenotype of TNSALP (1559delT) using in vitro translation/translocation system and COS-1 cells transiently expressing this mutant protein. We showed that the mutant protein not only has a larger molecular size than the wild type enzyme by approximately 12 kDa, reflecting an 80 amino acid-long extension at its C-terminus, but that it also lacks a glycosylphosphatidylinositol anchor. In support of this, alkaline phosphatase activity of the cells expressing TNSALP (1559delT) was localized at the juxtanucleus position, but not on the cell surface. However, only a limited amount of the newly synthesized protein was released into the medium and the rest was polyubiquitinated, followed by degradation in the proteasome. SDS/PAGE and analysis by sucrose-density-gradient analysis indicated that TNSALP (1559delT) forms a disulfide-bonded high-molecular-mass aggregate. Interestingly, the aggregate form of TNSALP (1559delT) exhibited a significant enzyme activity. When all three cysteines at positions of 506, 521 and 577 of TNSALP (1559delT) were replaced with serines, the aggregation disappeared and instead this modified mutant protein formed a noncovalently associated dimer, strongly indicating that these cysteine residues in the C-terminal region are solely responsible for aggregate formation by cross-linking the catalytically active dimers. Thus, complete absence of TNSALP on cell surfaces provides a plausible explanation for a severe lethal phenotype of a homozygote hypophosphatasia patient carrying TNSALP (1559delT).     

Acorn cake during the Holocene: experimental reconstruction of its preparation in the western Pyrenees,Iberia

In many agricultural and hunter-gatherer communities, edible wild plants are still a relevant food source, although their use has been, in many cases, undervalued. In this sense, acorns have been known as a foodstuff in written sources since antiquity, as well as from scientific analyses in archaeobotany and ethnobotany. We have tried to reconstruct the chaîne opératoire in the preparation of acorn cake, with experiments using materials similar to those potentially used in the western Pyrenees in the past. Furthermore, we have considered some ethnographic data which are available for this area. The results show that the treatment of pre-selected acorns by raw-roasting and leaching is feasible and we emphasise their nutritional value. The results of the experiments improved considerably with the use of pottery and optimal storage conditions. In addition, several by-products useful for other daily tasks were generated which may have been used as well in the past.     

Ultrastructural analysis and identification of envelope proteins of "Candidatus Chloracidobacterium thermophilum" chlorosomes

Chlorosomes are sac-like, light-harvesting organelles that characteristically contain very large numbers of bacteriochlorophyll (BChl) c, d, or e molecules. These antenna structures occur in chlorophototrophs belonging to some members of the Chlorobi and Chloroflexi phyla and are also found in a recently discovered member of the phylum Acidobacteria, "Candidatus Chloracidobacterium thermophilum." "Ca. Chloracidobacterium thermophilum" is the first aerobic organism discovered to possess chlorosomes as light-harvesting antennae for phototrophic growth. Chlorosomes were isolated from "Ca. Chloracidobacterium thermophilum" and subjected to electron microscopic, spectroscopic, and biochemical analyses. The chlorosomes of "Ca. Chloracidobacterium thermophilum" had an average size of ～100 by 30 nm. Cryo-electron microscopy showed that the BChl c molecules formed folded or twisted, sheet-like structures with a lamellar spacing of ～2.3 nm. Unlike the BChls in the chlorosomes of the green sulfur bacterium Chlorobaculum tepidum, concentric cylindrical nanotubes were not observed. Chlorosomes of "Ca. Chloracidobacterium thermophilum" contained a homolog of CsmA, the BChl a-binding, baseplate protein; CsmV, a protein distantly related to CsmI, CsmJ, and CsmX of C. tepidum, which probably binds a single [2Fe-2S] cluster; and five unique polypeptides (CsmR, CsmS, CsmT, CsmU, and a type II NADH dehydrogenase homolog). Although "Ca. Chloracidobacterium thermophilum" is an aerobe, energy transfer among the BChls in these chlorosomes was very strongly quenched in the presence of oxygen (as measured by quenching of fluorescence emission). The combined analyses showed that the chlorosomes of "Ca. Chloracidobacterium thermophilum" possess a number of unique features but also share some properties with the chlorosomes found in anaerobic members of other phyla.     

Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos.

Peptide growth factors may play a role in patterning of the early embryo, particularly in the induction of mesoderm. We have explored the role of fibroblast growth factor (FGF) in early Xenopus development by expressing a dominant negative mutant form of the FGF receptor. Using a functional assay in frog oocytes, we found that a truncated form of the receptor effectively abolished wild-type receptor function. Explants from embryos expressing this dominant negative mutant failed to induce mesoderm in response to FGF. In whole embryos the mutant receptor caused specific defects in gastrulation and in posterior development, and overexpression of a wild-type receptor could rescue these developmental defects. These results demonstrate that the FGF signaling pathway plays an important role in early embryogenesis, particularly in the formation of the posterior and lateral mesoderm.     

Kinetic and mechanistic analysis of Trypanosoma cruzi trans-sialidase reveals a classical ping-pong mechanism with acid/base catalysis

The trans-sialidase from Trypanosoma cruzi catalyzes the transfer of a sialic acid moiety from sialylated donor substrates to the terminal galactose moiety of lactose and lactoside acceptors to yield alpha-(2,3)-sialyllactose or its derivatives with net retention of anomeric configuration. Through kinetic analyses in which the concentrations of two different donor aryl alpha-sialoside substrates and the acceptor substrate lactose were independently varied, we have demonstrated that this enzyme follows a ping-pong bi-bi kinetic mechanism. This is supported for both the native enzyme and a mutant (D59A) in which the putative acid/base catalyst has been replaced by the demonstration of the half-reaction in which a sialyl-enzyme intermediate is formed. Mass spectrometric analysis of the protein directly demonstrates the formation of a covalent intermediate, while the observation of release of a full equivalent of p-nitrophenol by the mutant in a pre-steady state burst provides further support. The active site nucleophile is confirmed to be Tyr342 by trapping of the sialyl-enzyme intermediate using the D59A mutant and sequencing of the purified peptic peptide. The role of D59 as the acid/base catalyst is confirmed by chemical rescue studies in which activity is restored to the D59A mutant by azide and a sialyl azide product is formed.