The legumain McPAL1 from Momordica cochinchinensis is a highly stable Asx-specific splicing enzyme

Legumains, also known as asparaginyl endopeptidases (AEPs), cleave peptide bonds after Asn/Asp (Asx) residues. In plants, certain legumains also have ligase activity that catalyzes biosynthesis of Asx-containing cyclic peptides. An example is the biosynthesis of MCoTI-I/II, a squash family-derived cyclic trypsin inhibitor, which involves splicing to remove the N-terminal prodomain and then N-to-C-terminal cyclization of the mature domain. To identify plant legumains responsible for the maturation of these cyclic peptides, we have isolated and characterized a legumain involved in splicing, McPAL1, from Momordica cochinchinensis (Cucurbitaceae) seeds. Functional studies show that recombinantly expressed McPAL1 displays a pH-dependent, trimodal enzymatic profile. At pH 4 to 6, McPAL1 selectively catalyzed Asp-ligation and Asn-cleavage, but at pH 6.5 to 8, Asn-ligation predominated. With peptide substrates containing N-terminal Asn and C-terminal Asp, such as is found in precursors of MCoTI-I/II, McPAL1 mediates proteolysis at the Asn site and then ligation at the Asp site at pH 5 to 6. Also, McPAL1 is an unusually stable legumain that is tolerant of heat and high pH. Together, our results support that McPAL1 is a splicing legumain at acidic pH that can mediate biosynthesis of MCoTI-I/II. We purport that the high thermal and pH stability of McPAL1 could have applications for protein engineering.     

The oncogenic role of Epstein–Barr virus‐encoded microRNAs in Epstein–Barr virus‐associated gastric carcinoma

Epstein–Barr virus (EBV) infection is detected in various epithelial malignancies, such as nasopharyngeal carcinoma (NPC) and gastric cancer (GC). EBV comprises some unique molecular features and encodes viral genes and microRNAs (miRNAs) by its own DNA sequence. EBV genes are required to maintain latency and contribute to oncogenic property. miRNAs encoded by EBV have been shown to contribute to initiation and progression of EBV‐related malignancies. By a number of genomic profiling studies, some EBV miRNAs were confirmed to be highly expressed in EBV‐associated gastric cancer (EBVaGC) samples and cell lines. The majority host targets of the EBV miRNAs are important for promoting cell growth and inhibiting apoptosis, facilitating cell survival and immune evasion. However, the integrated molecular mechanisms related to EBV miRNAs remain to be investigated. In this review, we summarized the crucial role of EBV miRNAs in epithelial malignancies, especially in EBVaGC. Collectively, EBV miRNAs play a significant role in the viral and host gene regulation network. Understanding the comprehensive potential targets and relevant functions of EBV miRNAs in gastric carcinogenesis might provide better clinical translation.     

Habitat selection by threatened burrowing spiders (Araneae: Atypidae,Eresidae) of central Europe: evidence base for conservation management

Habitat selection in sedentary, long-lived burrowing spiders is a key life-history event that directly affects their reproductive success. In this study, we analyzed the role of the chemical and physical properties of soil in habitat selection by threatened temperate burrowing spiders. We examined 296 burrows of three Atypus spp. and three Eresus spp. at 68 sites in Czechia. We found that the study species were associated with soils that have high or very high cation exchange capacity, which allows the presence of a stable and humid microclimate in their burrows. We found that specific bedrock types can be used as predictors of the presence of particular study species. All Eresus spp. avoided compacted soils. However, when present in soils with very low penetration resistance, they were limited to sites with high soil cohesion. The burrows of all study species were located at well-drained sites. The study species seemed to be selective for a steppe-like character of their microhabitat but not necessarily for its southward orientation. We found the study species even at sites where steppes were present in the early 1950s and that underwent later afforestation but not extensive landscaping or plowing. What types of disturbances can be sustained by the study species and how long it takes the decreased populations to recover after, e.g., trampling associated with grazing, remain to be investigated.     

South Asia as a Reservoir for the Global Spread of Ciprofloxacin-Resistant Shigella sonnei: A Cross-Sectional Study

BackgroundAntimicrobial resistance is a major issue in the Shigellae, particularly as a specific multidrug-resistant (MDR) lineage of Shigella sonnei (lineage III) is becoming globally dominant. Ciprofloxacin is a recommended treatment for Shigella infections. However, ciprofloxacin-resistant S. sonnei are being increasingly isolated in Asia and sporadically reported on other continents. We hypothesized that Asia is a primary hub for the recent international spread of ciprofloxacin-resistant S. sonnei.ConclusionsThis study suggests that a single clone, which is widespread in South Asia, is likely driving the current intercontinental surge of ciprofloxacin-resistant S. sonnei and is capable of establishing endemic transmission in new locations. Despite being limited in geographical scope, our work has major implications for understanding the international transfer of antimicrobial-resistant pathogens, with S. sonnei acting as a tractable model for studying how antimicrobial-resistant Gram-negative bacteria spread globally.     

Multiplex PCR and Next Generation Sequencing for the Non-Invasive Detection of Bladder Cancer

Background

Highly sensitive and specific urine-based tests to detect either primary or recurrent bladder cancer have proved elusive to date. Our ever increasing knowledge of the genomic aberrations in bladder cancer should enable the development of such tests based on urinary DNA.

Methods

DNA was extracted from urine cell pellets and PCR used to amplify the regions of the TERT promoter and coding regions of FGFR3, PIK3CA, TP53, HRAS, KDM6A and RXRA which are frequently mutated in bladder cancer. The PCR products were barcoded, pooled and paired-end 2 x 250 bp sequencing performed on an Illumina MiSeq. Urinary DNA was analysed from 20 non-cancer controls, 120 primary bladder cancer patients (41 pTa, 40 pT1, 39 pT2+) and 91 bladder cancer patients post-TURBT (89 cancer-free).

Results

Despite the small quantities of DNA extracted from some urine cell pellets, 96% of the samples yielded mean read depths >500. Analysing only previously reported point mutations, TERT mutations were found in 55% of patients with bladder cancer (independent of stage), FGFR3 mutations in 30% of patients with bladder cancer, PIK3CA in 14% and TP53 mutations in 12% of patients with bladder cancer. Overall, these previously reported bladder cancer mutations were detected in 86 out of 122 bladder cancer patients (70% sensitivity) and in only 3 out of 109 patients with no detectable bladder cancer (97% specificity).

Conclusion

This simple, cost-effective approach could be used for the non-invasive surveillance of patients with non-muscle-invasive bladder cancers harbouring these mutations. The method has a low DNA input requirement and can detect low levels of mutant DNA in a large excess of normal DNA. These genes represent a minimal biomarker panel to which extra markers could be added to develop a highly sensitive diagnostic test for bladder cancer.     

Molecular Epidemiology of P. vivax in Iran: High Diversity and Complex Sub-Structure Using Neutral Markers,but No Evidence of Y976F Mutation at pvmdr1

BackgroundMalaria remains endemic at low levels in the south-eastern provinces of Iran bordering Afghanistan and Pakistan, with the majority of cases attributable to P. vivax. The national guidelines recommend chloroquine (CQ) as blood-stage treatment for uncomplicated P. vivax, but the large influx of imported cases enhances the risk of introducing CQ resistance (CQR).ConclusionsThe contrasting low versus high diversity in the two sub-populations (K1 and K2) infers that a combination of local transmission and cross-border malaria from higher transmission regions shape the genetic make-up of the P. vivax population in south-eastern Iran. There was no molecular evidence of CQR amongst the local or imported cases, but ongoing clinical surveillance is warranted.     

Temperature-activity relationships of cation activation and ouabain inhibition of (Na+ + K+)-ATPase.

The nonlinear temperature-activity relationship of membrane preparations of (Na⁺ + K⁺)-ATPase gives rise to discontinuities in Arrhenius plots of this enzyme. The different apparent energies of activation of (Na⁺ + K⁺) — ATPase which are observed above and below the critical temperature of the system have been considered to result from different conformational forms of the enzyme protein. Because both activation of (Na⁺ + K⁺)-ATPase by cations, and its specific inhibition by cardiac glycosides may be influenced by the conformational form of the enzyme protein, we have reexamined the effect of temperature upon the activation energy of the system under the different experimental conditions of cation activation and ouabain inhibition.Our results indicate that the activation of (Na⁺ + K⁺)-ATPase by cations, is less influenced by change in temperature than is inhibition of the enzyme by ouabain. In addition, mild lipolysis by phospholipase-A had a marked effect upon the ouabain-dependent response of the enzyme to temperature, but not upon the cation-dependent response. The effect of phospholipase-A can be overcome by reincubation of the treated preparation with phosphatidyl serine.We conclude that the ouabain-dependent temperature effects of (Na⁺ + K⁺)-ATPase are more dependent upon the integrity and nature of the membrane lipids than are the cation-dependent responses. It is possible that phosphatidyl serine plays a unique role in this regard.