Highly heterogeneous residual malaria risk in western Thailand

Over the past decades, the malaria burden in Thailand has substantially declined. Most infections now originate from the national border regions. In these areas, the prevalence of asymptomatic infections is still substantial and poses a challenge for the national malaria elimination program. To determine epidemiological parameters as well as risk factors for malaria infection in western Thailand, we carried out a cohort study in Kanchanaburi and Ratchaburi provinces on the Thailand-Myanmar border. Blood samples from 999 local participants were examined for malaria infection every 4 weeks between May 2013 and Jun 2014. Prevalence of Plasmodium falciparum and Plasmodium vivax was determined by quantitative PCR (qPCR) and showed a seasonal variation with values fluctuating from 1.7% to 4.2% for P. vivax and 0% to 1.3% for P. falciparum. Ninety percent of infections were asymptomatic. The annual molecular force of blood-stage infection (_molFOB) was estimated by microsatellite genotyping to be 0.24 new infections per person-year for P. vivax and 0.02 new infections per person-year for P. falciparum. The distribution of infections was heterogenous, that is, the vast majority of infections (>80%) were found in a small number of individuals (<8% of the study population) who tested positive at multiple timepoints. Significant risk factors were detected for P. vivax infections, including previous clinical malaria, occupation in agriculture and travel to Myanmar. In contrast, indoor residual spraying was associated with a protection from infection. These findings provide a recent landscape of malaria epidemiology and emphasize the importance of novel strategies to target asymptomatic and imported infections.     

Identifying ‘firebreaks’ to fragment dispersal networks of a marine parasite

Marine ecosystems are beset by disease outbreaks, and efficient strategies to control dispersal of pathogens are scarce. We tested whether introducing no-farming areas or ‘firebreaks’ could disconnect dispersal networks of a parasitic disease affecting the world’s largest marine fish farming industry (～1000 farms). Larval salmon lice (Lepeophtheirus salmonis) are released from and transported among salmon farms by ocean currents, creating inter-farm networks of louse dispersal. We used a state-of-the-art biophysical model to predict louse movement along the Norwegian coastline and network analysis to identify firebreaks to dispersal. At least one firebreak that fragmented the network into two large unconnected groups of farms was identified for all seasons. During spring, when wild salmon migrate out into the ocean, and louse levels per fish at farms must be minimised, two effective firebreaks were created by removing 13 and 21 farms (1.3% and 2.2% of all farms in the system) at ～61°N and 67°N, respectively. We have demonstrated that dispersal models coupled with network analysis can identify no-farming zones that fragment dispersal networks. Reduced dispersal pathways should lower infection pressure at farms, slow the evolution of resistance to parasite control measures, and alleviate infection pressure on wild salmon populations.     

Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage

CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids.

A major downside of double-strand break-mediated genome editing is the need to verify the genomic integrity of the targeted locus and confirm the absence of off-target indels. This study shows that in-trans paired nicking is a mutation-free CRISPR strategy to introduce precise knock-ins into human organoids; its genomic fidelity allows all knock-in cells to be pooled, accelerating the establishment of new organoid models.     

Two Peptidergic Drugs Increase the Synaptophysin Immunoreactivity in Brains of 24-month-old Rats

The brain-derived peptidergic drug Cerebrolysin has been found to support the survival of neurones in vitro and in vivo. Positive effects on learning and memory have been demonstrated in various animal models and also in clinical trails. In the present study, the effects of Cerebrolysin and its peptide preparation E021 on the synapse density in the hippocampus, the dentate gyrus and in the entorhinal cortex of 24-month-old rats were investigated. Rats received the drugs or saline for control for 19 consecutive days (2.5ml/kg per day). Slices of the brains were immunohistochemically stained with anti-synaptophysin, which is a specific marker of presynaptic terminals. Quantification of the synapse density was done by using light microscopy and a computerised image analysing system. Our results clearly showed that the rats benefit from the administration of both drugs, showing an enhancement in the number of synaptophysin-immunostained presynaptic terminals in the entorhinal cortex, the dentate gyrus, and also in the hippocampal subfields CA1, CA2, CA3 stratum lucidum and CA3 stratum radiatum. It can be assumed that these effects are the reason for improved cognitive performances of rats treated with Cerebrolysin and E021.     

2 new species of Stilestrongylus (Nematoda: Heligmonellidae) parasites of Peromyscus (Rodentia: Cricetidae) from Mexico

Stilestrongylus peromysci n. sp. collected from Peromyscus difficilis (Hidalgo state, México), differs from other species in the genus in number of the spines (30) in the synlophe (both sexes) and because the eighth ray arises from the root of the ninth ray; S. hidalguensis n. sp. parasitised Peromyscus sp. and differs from all other congeneric species in the presence of 24 spines in the male synlophe and in the arrangement of the bursal rays (2-2-1 in the right lobe and 2-3 in the left lobe). A key to the species of Stilestrongylus is provided.     

Electrospray ionization mass spectrometric analysis of blood for differentiation of species

The National Fish and Wildlife Forensic Laboratory is responsible for the determination of species of birds, reptiles, and mammals from the United States, as well as international species falling under the protection of CITES treaties. We have recently found electrospray ionization mass spectrometry to be an effective means of rapidly analyzing blood samples for species identification. Nearly 1000 individuals were analyzed which comprised 62 species represented by birds, mammals, and reptiles. Whole blood and dried blood samples were analyzed without purification to provide simultaneous molecular weights from the alpha- and beta-proteins present in each sample's hemoglobin. The combination of the two molecular weights for the hemoglobin proteins (i.e., alpha/beta-pairs) was used as species determining markers. In all, 133 distinctive alpha/beta-pairs were observed from the individuals analyzed. Despite the variability in the hemoglobins evaluated, 86% of these alpha/beta-pairs were found to be diagnostic for a particular species to the exclusion of all other species studied. No other single protein system studied by a single analytical technique can so effectively resolve species from a wide range of taxa as can the hemoglobin system when analyzed by electrospray ionization mass spectrometry.     

Regulation of apoptosis by the Ft1 protein, a new modulator of protein kinase B/Akt

The serine/threonine kinase protein kinase B (PKB)/Akt plays a central role in many cellular processes, including cell growth, glucose metabolism, and apoptosis. However, the identification and validation of novel regulators or effectors is key to future advances in understanding the multiple functions of PKB. Here we report the identification of a novel PKB binding protein, called Ft1, from a cDNA library screen using a green fluorescent protein-based protein-fragment complementation assay. We show that the Ft1 protein interacts directly with PKB, enhancing the phosphorylation of both of its regulatory sites by promoting its interaction with the upstream kinase PDK1. Further, the modulation of PKB activity by Ft1 has a strong effect on the apoptosis susceptibility of T lymphocytes treated with glucocorticoids. We demonstrate that this phenomenon occurs via a PDK1/PKB/GSK3/NF-ATc signaling cascade that controls the production of the proapoptotic hormone Fas ligand. The wide distribution of Ft1 in adult tissues suggests that it could be a general regulator of PKB activity in the control of differentiation, proliferation, and apoptosis in many cell types.