Escalating Plasmodium falciparum antifolate drug resistance mutations in Macha, rural Zambia

Background

Artemisinin and its derivatives have been used for falciparum malaria treatment in China since late 1970s. Monotherapy and uncontrolled use of artemisinin drugs were common practices for a long period of time. In vitro tests showed that the susceptibility of Plasmodium falciparum to artemisinins was declining in China. A concern was raised about the resistance to artemisinins of falciparum malaria in the country. It has been reported that in vitro artemisinin resistance was associated with the S769N mutation in the PfATPase6 gene. The main purpose of this study was to investigate whether that mutation has occurred in field isolates from China.

Methods

Plasmodium falciparum field isolates were collected in 2006–2007 from Hainan and Yunnan provinces, China. A nested PCR-sequencing assay was developed to analyse the genotype of the PfATPase6 S769N polymorphism in the P. falciparum field isolates.

Results

The genotyping results of six samples could not be obtained due to failure of PCR amplification, but no S769N mutation was detected in any of the 95 samples successfully analysed.

Conclusion

The results indicate that the S769N mutation in the PfATPase6 gene is not present in China, suggesting that artemisinin resistance has not yet developed, but the situation needs to be watched very attentively.     

Exercise training delays cardiac dysfunction and prevents calcium handling abnormalities in sympathetic hyperactivity-induced heart failure mice.

Exercise training (ET) is a coadjuvant therapy in preventive cardiology. It delays cardiac dysfunction and exercise intolerance in heart failure (HF); however, the molecular mechanisms underlying its cardioprotection are poorly understood. We tested the hypothesis that ET would prevent Ca(2+) handling abnormalities and ventricular dysfunction in sympathetic hyperactivity-induced HF mice. A cohort of male wild-type (WT) and congenic alpha(2A)/alpha(2C)-adrenoceptor knockout (alpha(2A)/alpha(2C)ARKO) mice with C57BL6/J genetic background (3-5 mo of age) were randomly assigned into untrained and exercise-trained groups. ET consisted of 8-wk swimming session, 60 min, 5 days/wk. Fractional shortening (FS) was assessed by two-dimensional guided M-mode echocardiography. The protein expression of ryanodine receptor (RyR), phospho-Ser(2809)-RyR, sarcoplasmic reticulum Ca(2+) ATPase (SERCA2), Na(+)/Ca(2+) exchanger (NCX), phospholamban (PLN), phospho-Ser(16)-PLN, and phospho-Thr(17)-PLN were analyzed by Western blotting. At 3 mo of age, no significant difference in FS and exercise tolerance was observed between WT and alpha(2A)/alpha(2C)ARKO mice. At 5 mo, when cardiac dysfunction is associated with lung edema and increased plasma norepinephrine levels, alpha(2A)/alpha(2C)ARKO mice presented reduced FS paralleled by decreased SERCA2 (26%) and NCX (34%). Conversely, alpha(2A)/alpha(2C)ARKO mice displayed increased phospho-Ser(16)-PLN (76%) and phospho-Ser(2809)-RyR (49%). ET in alpha(2A)/alpha(2C)ARKO mice prevented exercise intolerance, ventricular dysfunction, and decreased plasma norepinephrine. ET significantly increased the expression of SERCA2 (58%) and phospho-Ser(16)-PLN (30%) while it restored the expression of phospho-Ser(2809)-RyR to WT levels. Collectively, we provide evidence that improved net balance of Ca(2+) handling proteins paralleled by a decreased sympathetic activity on ET are, at least in part, compensatory mechanisms against deteriorating ventricular function in HF.     

Assembly of a Marine Viral Metagenome after Physical Fractionation

Metagenomic analyses of marine viruses generate an overview of viral genes present in a sample, but the percentage of the resulting sequence fragments that can be reassembled is low and the phenotype of the virus from which a given sequence derives is usually unknown. In this study, we employed physical fractionation to characterize the morphological and genomic traits of a subset of uncultivated viruses from a natural marine assemblage. Viruses from Kāne‘ohe Bay, Hawai‘i were fractionated by equilibrium buoyant density centrifugation in a cesium chloride (CsCl) gradient, and one fraction from the CsCl gradient was then further fractionated by strong anion-exchange chromatography. One of the fractions resulting from this two-dimensional separation appeared to be dominated by only a few virus types based on genome sizes and morphology. Sequences generated from a shotgun clone library of the viruses in this fraction were assembled into significantly more numerous contigs than have been generated with previous metagenomic investigations of whole DNA viral assemblages with comparable sequencing effort. Analysis of the longer contigs (up to 6.5 kb) assembled from our metagenome allowed us to assess gene arrangement in this subset of marine viruses. Our results demonstrate the potential for physical fractionation to facilitate sequence assembly from viral metagenomes and permit linking of morphological and genomic data for uncultivated viruses.     

Attractiveness of dung beetles (Coleoptera: Scarabaeinae) to faeces from native mammals in different trophic guilds

Dung beetles are predominantly coprophagous and use mammalian faeces as their main food resource and for offspring rearing. However, these resources are generally ephemeral and scarce. The objective of this study was to verify the attractiveness to dung beetles of the faeces of nine native mammal species of the Cerrado biome in different trophic guilds. This study was conducted during the rainy season (October 2016 to March 2017) in two sensu stricto areas of the Cerrado in the Brasília National Park (PNB), Federal District, Brazil. Pitfalls containing faeces of nine mammals native to the region were used as bait (from three herbivores, three carnivores and three omnivores) for the collection of dung beetles. We collected 203 individuals and 19 species, and the most abundant species were Uroxys aff. thoracalis, Oxysternon palemo, Coprophanaeus spitzi and Diabroctis mirabilis. The most attractive faecal bait was from the omnivorous Chrysocyon brachyurus (32.67% of the individuals and 13 species). This study reveals variation in the attraction of dung beetles to native mammal dung of the Cerrado, with more abundance in the faeces of omnivores and carnivores and less in the faeces of herbivores. The species composition in the carnivore baits did not differ from that of the omnivore baits. These data indicate that changes in Scarabaeinae assemblages result from changes in mammal species such as those that have occurred in the Cerrado biome with the conversion to cattle production, which in recent years has caused intense fragmentation of habitats, expansion of agricultural and livestock activities and the introduction of exotic species. The mammal C. brachyurus, vulnerable to extinction in Brazil, should be a focus for future research, since its faeces support a great diversity of dung beetles in the Cerrado.     

Differences in virulence between isolates of feline Sporotrichosis

Sporotrichosis is a chronic subcutaneous mycosis caused by Sporothrix schenckii. This work aimed to evaluate the virulence of two different isolates of S. schenckii from cutaneous (CUT) and systemic (SYS) forms of feline sporotrichosis. A standard inoculum with 2 × 10³ yeast cells/ml was prepared from each of the isolates. The experimental infection was carried out with 0.1 ml of the inoculum from both isolates and then injected in the paw pads of Swiss albino mice of groups CUT and SYS. The clinical evolution of the disease and the diameter of the lesion at the inoculated sites were evaluated during nine weeks. Four necropsies were done to collect material from the lesions (p < 0.01). Group CUT demonstrated a more evident clinical evolution of the disease from week two to week five; large lesions in the paw pad on week four (p < 0.01); and a higher incidence of lesions in other parts of the body (p < 0.01) than group SYS (p < 0.01). S. schenckii was isolated from the inoculated site in groups SYS and CUT until days 30 and 45, respectively. Granulomas with yeast cells usually localized in the central area were observed in histopathology sections on days 15 and 30 post-inoculations. Those yeast cells decreased on day 45 being absent on day 62 when tissue repair initiated. The results showed that distinct clinical isolates of S. schenckii cause significant differences in the clinical evolution of sporotrichosis.     

Constitutive expression of alpha interferon by skin dendritic cells confers resistance to infection by foot-and-mouth disease virus

The role of dendritic cells (DC) in the initiation of immune responses against foot-and-mouth disease virus (FMDV) is poorly understood. We analyzed the innate response of freshly isolated swine skin DC to the virus and show a rapid induction of beta interferon (IFN-beta) mRNA but not IFN-alpha mRNA. However, these DC secreted both IFN-alpha and IFN-beta proteins in response to live virus but not killed virus. Furthermore, the surface expression of swine major histocompatibility complex class II (SLA II) or CD80/CD86 molecules and antigen processing functions were not affected by FMDV exposure. Given the demonstrated sensitivity of FMDV to IFN-alpha/beta, there was no productive or nonproductive infection of these cells. Finally, freshly isolated skin DC constitutively expressed intracellular IFN-alpha protein in the absence of stimulation, with no detectable secretion of the cytokine until virus exposure. In situ analysis of these DC showed that these cells express and store IFN-alpha in uninfected animals. This is the first demonstration of the constitutive expression of IFN-alpha in resident, tissue-derived DC and indicates that skin DC can play an important role in the innate immune response of swine to viral infections.     

Concerted vs. sequential. Two activation patterns of vast arrays of intracellular Ca2+ channels in muscle

To signal cell responses, Ca(2+) is released from storage through intracellular Ca(2+) channels. Unlike most plasmalemmal channels, these are clustered in quasi-crystalline arrays, which should endow them with unique properties. Two distinct patterns of local activation of Ca(2+) release were revealed in images of Ca(2+) sparks in permeabilized cells of amphibian muscle. In the presence of sulfate, an anion that enters the SR and precipitates Ca(2+), sparks became wider than in the conventional, glutamate-based solution. Some of these were "protoplatykurtic" (had a flat top from early on), suggesting an extensive array of channels that activate simultaneously. Under these conditions the rate of production of signal mass was roughly constant during the rise time of the spark and could be as high as 5 microm(3) ms(-1), consistent with a release current >50 pA since the beginning of the event. This pattern, called "concerted activation," was observed also in rat muscle fibers. When sulfate was combined with a reduced cytosolic [Ca(2+)] (50 nM) these sparks coexisted (and interfered) with a sequential progression of channel opening, probably mediated by Ca(2+)-induced Ca(2+) release (CICR). Sequential propagation, observed only in frogs, may require parajunctional channels, of RyR isoform beta, which are absent in the rat. Concerted opening instead appears to be a property of RyR alpha in the amphibian and the homologous isoform 1 in the mammal.     

CstF-64 and 3′-UTR cis-element determine Star-PAP specificity for target mRNA selection by excluding PAPα

Almost all eukaryotic mRNAs have a poly (A) tail at the 3′-end. Canonical PAPs (PAPα/γ) polyadenylate nuclear pre-mRNAs. The recent identification of the non-canonical Star-PAP revealed specificity of nuclear PAPs for pre-mRNAs, yet the mechanism how Star-PAP selects mRNA targets is still elusive. Moreover, how Star-PAP target mRNAs having canonical AAUAAA signal are not regulated by PAPα is unclear. We investigate specificity mechanisms of Star-PAP that selects pre-mRNA targets for polyadenylation. Star-PAP assembles distinct 3′-end processing complex and controls pre-mRNAs independent of PAPα. We identified a Star-PAP recognition nucleotide motif and showed that suboptimal DSE on Star-PAP target pre-mRNA 3′-UTRs inhibit CstF-64 binding, thus preventing PAPα recruitment onto it. Altering 3′-UTR cis-elements on a Star-PAP target pre-mRNA can switch the regulatory PAP from Star-PAP to PAPα. Our results suggest a mechanism of poly (A) site selection that has potential implication on the regulation of alternative polyadenylation.     

Effects of prenatal diclofenac sodium exposure on newborn testis: a histomorphometric study

Diclofenac sodium (DS) is used primarily to treat fever and to alleviate pain and inflammation. We investigated the effects of DS exposure during gestation on the testes of rat pups to investigate the safety of its use during the prenatal period. Pregnant rats were separated into control, saline, low dose, medium dose and high dose groups. DS was given between weeks 15 and 21 of gestation. Total numbers of spermatogonia and Sertoli cells were counted in the testes of 7-day-old male rats using the physical disector method. By the end of the study, the total number of Sertoli cells was decreased significantly in a dose dependent manner in the medium and high dose groups compared to controls. No significant differences were found in the total number of spermatogonia in the control, saline and low dose DS groups. Medium and high dose DS administration reduced the total number of spermatogonia compared to other groups. We suggest that prenatal administration of DS can cause deleterious effects on the testis development, especially in high doses.