Evidence of RIP (repeat-induced point mutation) in transposase sequences of Aspergillus oryzae

A DNA methyl-binding column was used to isolate genomic fragments enriched for DNA-methylation from Aspergillus parasiticus. One of the isolated sequences presented 67% identity at the protein level with the transposase from the transposable element Tan1 of Aspergillus niger var. awamori, and was found to be present in at least 20 copies in the Aspergillus oryzae database. Analysis of four copies showed evidence of C:G to T:A transitions in at least 98.2% of the mutations found over a 1,032-1,180 bp region spanning a large part of the transposase sequence. Using copy specific primers three sequences were amplified from a different strain of A. oryzae and a similar pattern of C:G to T:A transitions was found. These transitions are similar to those observed in RIP, in Neurospora crassa, where cytosine-methylation is believed to be involved. Using methylation-sensitive Southern blotting, no evidence of methylation was found in the transposase sequences in these two A. oryzae strains as well as one A. parasiticus and one Aspergillus flavus strain.     

Gap junctional intercellular communication and cytoskeletal organization in chondrocytes in suspension in an ultrasound trap

Particles or cells suspended in an appropriately designed ultrasound standing wave field can be aggregated at a node to form a single monolayer in a plane that can be interrogated microscopically. The approach is applied here to investigate the temporal development of F-actin and Cx43 distribution and of gap junctional intercellular communication in 2-D chondrocyte aggregates (monolayers) rapidly and synchronously formed and held in suspension in an ultrasound trap. Development of the F-actin cytoskeleton in the confluent single layer of 'cuboidal' cells forming the aggregate was completed within 1 h. Chondrocytes levitated in the trap synchronously formed functional gap junctions (as assessed by CMFDA dye transfer assays) in less than 1 h of initiation of cell-cell contact in the trap. It was shown that Cx43 gene expression was retained in isolated chondrocytes in suspension. Preincubation of cells with the protein synthesis inhibitor cycloheximide caused a six-fold decrease in Cx43 accumulation (as assessed by immunofluorescence) at the interfaces of chondrocytes in the aggregate. It is shown that the ultrasound trap provides an approach to studying the early stages of cytoskeletal and gap junction development as cells progress from physical aggregation, through molecular adhesion, to display the intracellular consequences of receptor interactions.     

Linking teratogen information service and birth defects registry databases to improve knowledge of birth defect status

BACKGROUND: Although teratogen information services (TISs) obtain maternal exposure information from their callers, such services often do not know if the pregnancies were affected by a birth defect. This study attempted to improve the completeness of this information for Texas Teratogen Information Service (TTIS) callers by linking their records with the Texas Birth Defects Registry (TBDR) and Texas birth certificates (TBCs). METHODS: A total of 344 expectant mothers called TTIS with expected dates of delivery between 1 January 2000 and 31 December 2001. These pregnancies were linked with TBDR and TBC data. The percentages of pregnancies with known birth defect information both before and after the linkage were compared. RESULTS: The TTIS originally collected birth defect status information for 101 of the 344 callers (29.4%) and 0.6% of all 344 callers or 2.0% of callers with birth defect status information had a pregnancy affected by a birth defect. Linking TTIS records with TBDR and TBC data helped to raise the percentage of callers with birth defect status information from 29.4% to 71.5%. Among those callers, the percentage known to have birth defects increased from 2.0% to 4.1%. The sensitivity of TTIS follow-up calls in identifying birth defects was 50%, and the specificity was 100%. CONCLUSIONS: Linking TTIS caller records with TBDR and TBC data significantly increased both the percentage of pregnancies with birth defect status information and the percentage of pregnancies identified as affected by birth defects. Such linkage may be a good approach by which TISs can increase the completeness of their birth defect status information.     

Little evidence for intralocus sexual conflict over the optimal intake of nutrients for life span and reproduction in the black field cricket Teleogryllus commodus

There is often large divergence in the effects of key nutrients on life span (LS) and reproduction in the sexes, yet nutrient intake is regulated in the same way in males and females given dietary choice. This suggests that the sexes are constrained from feeding to their sex‐specific nutritional optima for these traits. Here, we examine the potential for intralocus sexual conflict (IASC) over optimal protein and carbohydrate intake for LS and reproduction to constrain the evolution of sex‐specific nutrient regulation in the field cricket, Teleogryllus commodus. We show clear sex differences in the effects of protein and carbohydrate intake on LS and reproduction and strong positive genetic correlations between the sexes for the regulated intake of these nutrients. However, the between‐sex additive genetic covariance matrix had very little effect on the predicted evolutionary response of nutrient regulation in the sexes. Thus, IASC appears unlikely to act as an evolutionary constraint on sex‐specific nutrient regulation in T. commodus. This finding is supported by clear sexual dimorphism in the regulated intake of these nutrients under dietary choice. However, nutrient regulation did not coincide with the nutritional optima for LS or reproduction in either sex, suggesting that IASC is not completely resolved in T. commodus.     

Transmission and diversity of Schistosoma haematobium and S. bovis and their freshwater intermediate snail hosts Bulinus globosus and B. nasutus in the Zanzibar Archipelago,United Republic of Tanzania

BackgroundThe Zanzibar Archipelago (Pemba and Unguja islands) is targeted for the elimination of human urogenital schistosomiasis caused by infection with Schistosoma haematobium where the intermediate snail host is Bulinus globosus. Following multiple studies, it has remained unclear if B. nasutus (a snail species that occupies geographically distinct regions on the Archipelago) is involved in S. haematobium transmission on Zanzibar. Additionally, S. haematobium was thought to be the only Schistosoma species present on the Zanzibar Archipelago until the sympatric transmission of S. bovis, a parasite of ruminants, was recently identified. Here we re-assess the epidemiology of schistosomiasis on Pemba and Unguja together with the role and genetic diversity of the Bulinus spp. involved in transmission.Methodology/Principal findingsMalacological and parasitological surveys were conducted between 2016 and 2019. In total, 11,116 Bulinus spp. snails were collected from 65 of 112 freshwater bodies surveyed. Bulinus species identification were determined using mitochondrial cox1 sequences for a representative subset of collected Bulinus (n = 504) and together with archived museum specimens (n = 6), 433 B. globosus and 77 B. nasutus were identified. Phylogenetic analysis of cox1 haplotypes revealed three distinct populations of B. globosus, two with an overlapping distribution on Pemba and one on Unguja. For B. nasutus, only a single clade with matching haplotypes was observed across the islands and included reference sequences from Kenya. Schistosoma haematobium cercariae (n = 158) were identified from 12 infected B. globosus and one B. nasutus collected between 2016 and 2019 in Pemba, and cercariae originating from 69 Bulinus spp. archived in museum collections. Schistosoma bovis cercariae (n = 21) were identified from seven additional B. globosus collected between 2016 and 2019 in Pemba. By analysing a partial mitochondrial cox1 region and the nuclear ITS (1–5.8S-2) rDNA region of Schistosoma cercariae, we identified 18 S. haematobium and three S. bovis haplotypes representing populations associated with mainland Africa and the Indian Ocean Islands (Zanzibar, Madagascar, Mauritius and Mafia).Conclusions/SignificanceThe individual B. nasutus on Pemba infected with S. haematobium demonstrates that B. nasutus could also play a role in the local transmission of S. haematobium. We provide preliminary evidence that intraspecific variability of S. haematobium on Pemba may increase the transmission potential of S. haematobium locally due to the expanded intermediate host range, and that the presence of S. bovis complicates the environmental surveillance of schistosome infections.     

A mouse infection model and long-term lymphatic endothelium co-culture system to evaluate drugs against adult Brugia malayi

The development of new drugs targeting adult-stage lymphatic filarial nematodes is hindered by the lack of a robust long-term in vitro culture model. Testing potential direct-acting and anti-Wolbachia therapeutic candidates against adult lymphatic filariae in vitro requires their propagation via chronic infection of gerbils. We evaluated Brugia malayi parasite burden data from male Mongolian gerbils compared with two immune-deficient mouse strains highly susceptible to B. malayi: CB.17 Severe-Combined Immmuno-Deficient (SCID) and interleukin-4 receptor alpha, interleukin-5 double knockout (IL-4Rα^-/-IL-5^-/-) mice. Adult worms generated in IL-4Rα^-/-IL-5^-/- mice were tested with different feeder cells (human embryonic kidney cells, human adult dermal lymphatic endothelial cells and human THP-1 monocyte differentiated macrophages) and comparative cell-free conditions to optimise and validate a long-term in vitro culture system. Cultured parasites were compared against those isolated from mice using motility scoring, metabolic viability assay (MTT), ex vivo microfilariae release assay and Wolbachia content by qPCR. A selected culture system was validated as a drug screen using reference anti-Wolbachia (doxycycline, ABBV-4083 / flubentylosin) or direct-acting compounds (flubendazole, suramin). BALB/c IL-4Rα^-/-IL-5^-/- or CB.17 SCID mice were superior to Mongolian gerbils in generating adult worms and supporting in vivo persistence for periods of up to 52 weeks. Adult females retrieved from BALB/c IL-4Rα^-/-IL-5^-/- mice could be cultured for up to 21 days in the presence of a lymphatic endothelial cell co-culture system with comparable motility, metabolic activity and Wolbachia titres to those maintained in vivo. Drug studies confirmed significant Wolbachia depletions or direct macrofilaricidal activities could be discerned when female B. malayi were cultured for 14 days. We therefore demonstrate a novel methodology to generate adult B. malayi in vivo and accurately evaluate drug efficacy ex vivo which may be adopted for drug screening with the dual benefit of reducing overall animal use and improving anti-filarial drug development.     

Nucleotide sequence and fine structural analysis of the Corynebacterium glutamicum hom-thrB operon

The complete nucleotide sequence of the Corynebacterium glutamicum hom-thrB operon has been determined and the structural genes and promoter region mapped. A polypeptide of Mr 46,136 is encoded by hom and a polypeptide of Mr 32,618 is encoded by thrB. Both predicted protein sequences show amino acid sequence homology to their counterparts in Escherichia coli and Bacillus subtilis. The promoter region has been mapped by S1-nuclease and deletion analysis. Located between -88, RNA start site and -219 (smallest deletion clone with complete activity) are sequence elements similar to those found in E. coli and B. subtilis promoters. Although there are no obvious attenuator-like structures in the 5'-untranslated region, there is a dyad-symmetry element, which may act as an operator.