Targeted amplification and MinION nanopore sequencing of key azole and echinocandin resistance determinants of clinically relevant Candida spp. from blood culture bottles

The objective of the study was to evaluate the use of targeted multiplex Nanopore MinION amplicon re-sequencing of key Candida spp. from blood culture bottles to identify azole and echinocandin resistance associated SNPs. Targeted PCR amplification of azole (ERG11 and ERG3) and echinocandin (FKS) resistance-associated loci was performed on positive blood culture media. Sequencing was performed using MinION nanopore device with R9.4.1 Flow Cells. Twenty-eight spiked blood cultures (ATCC strains and clinical isolates) and 12 prospectively collected positive blood cultures with candidaemia were included. Isolate species included Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Candida auris. SNPs that were identified on ERG and FKS genes using Snippy tool and CLC Genomic Workbench were correlated with phenotypic testing by broth microdilution (YeastOne™ Sensititre). Illumina whole-genome-sequencing and Sanger-sequencing were also performed as confirmatory testing of the mutations identified from nanopore sequencing data. There was a perfect agreement of the resistance-associated mutations detected by MinION-nanopore-sequencing compared to phenotypic testing for acquired resistance (16 with azole resistance; 3 with echinocandin resistance), and perfect concordance of the nanopore sequence mutations to Illumina and Sanger data. Mutations with no known association with phenotypic drug resistance and novel mutations were also detected.     

Circadian and Ultradian Rhythmicities in Very Premature Neonates Maintained in Incubators

Six very premature babies (born at 26–28 weeks gestational age) have been studied in hospital for 11–17 weeks, while in intensive care and in an incubator. Apart from suffering occasionally from the neonatal disorders of haemolytic jaundice and ‘respiratory distress of the newborn’, the babies were healthy and developed normally. Initially, the babies were continuously fed intravenously, and the lighting in the ward was on continuously. Routine care was given round the clock. When their medical condition permitted it, the babies were moved in their incubator to an adjacent ward, where they took frequent (2–4 hourly) small meals by mouth, the lighting was dimmed at night, and routine care tended to be given more in the daytime. Hourly recordings of insulated skin temperature were taken throughout the study, and it is the detection of rhythmicity in these measurements that has been the subject of the present study. The methods used were Phase-weighted Stacks, Phasor Walkout and Power Spectral Analysis. These methods have previously been used mainly in geophysical studies, and their value is that they can detect weak signals in noisy data and do not assume a particular waveform of any signal. Circadian rhythmicity was found in all babies for much of the time that were in the constant environment provided by the incubator. Ultradian rhythms were sometimes present also, but they were shorter-lived, and showed a wide range of changing periods, generally in excess of 8 h. When the babies were being treated for jaundice or respiratory distress, there was a tendency for the circadian rhythms to become weaker and for a broader spectrum of ultradian periods to appear. Placing babies in the 12 h : 12 h light : dark environment provided by the ward, and instituting feeding by mouth, had, in most cases, only modest effects upon either circadian or ultradian rhythms. Thus, circadian rhythms continued (but generally with a period not exactly equal to 24 h), and ultradian rhythms, when present, often did not show periods that could be related easily to feeding or care-giving. These results are discussed in terms of evidence for endogenous and exogenous origins of the observed rhythms, and of theories that have postulated the relationship between circadian and ultradian rhythms. It is concluded that the results from the present analyses are difficult to reconcile with the view that circadian rhythms develop from interactions between ultradian oscillators. We suggest that they indicate a matu-ration of the circadian system as a consequence of increasing associations between the circadian elements that are present in the suprachiasmatic nuclei and in other oscillators of the circadian system. The new analytical methods used here also indicate that the results obtained from time-frequency analysis depend to some extent upon the method used.     

Chewing lice from high‐altitude and migrating birds in Yunnan,China, with descriptions of two new species of Guimaraesiella

In total, 366 birds representing 55 species in 24 families and eight orders, were examined for chewing lice (Phthiraptera: Amblycera, Ischnocera) in two high‐altitude localities in Yunnan Province, China. In Ailaoshan, almost all of the birds examined were resident passeriforms, of which 36% were parasitized by chewing lice. In Jinshanyakou, most birds were on migration, and included both passerine and non‐passerine birds. Of the passerine birds caught in Jinshanyakou, only one bird (0.7%) was parasitized by chewing lice. The prevalence of Myrsidea and Brueelia‐complex lice on birds caught in Ailaoshan was higher than in previous reports. Of the chewing lice identifiable to species level, three represent new records for China: Actornithophilus hoplopteri (Mjöberg, 1910), Maculinirmus ljosalfar Gustafsson & Bush, 2017 and Quadraceps sinensis Timmermann, 1954. In total, 17 new host records are included, of which we describe two as new species in the Brueelia‐complex: Guimaraesiella (Cicchinella) ailaoshanensis sp. nov. ex Schoeniparus dubius dubius (Hume, 1874) and G. (C.) montisodalis sp. nov. ex Fulvetta manipurensis tonkinensis Delacour & Jabouille, 1930. This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:9FC3D8EE‐2CED‐4DBE‐A1DB‐471B71260D27 .     

Immunoregulatory T-cell defects in B-cell chronic lymphocytic leukemia: cause or consequence of the disease? The contributory role of decreased availability of interleukin 2 (IL-2)

Several phenotypic and functional defects have been described within the residual T-lymphocyte population of patients with B-cell chronic lymphocytic leukemia (B-CLL), particularly in those in the more advanced stages of the disease. In this study, we review these abnormalities and discuss their possible effects on the course of the illness. Particular emphasis is devoted to the role of interleukin 2 (IL-2) in B-CLL. Evidence is provided that the IL-2 released by B-CLL T-lymphocytes may be utilized by the neoplastic B-cell clone that expresses the IL-2 receptor and that decreased availability of IL-2 may play a contributory role in some of the T-cell defects encountered in B-CLL.     

The cucumber mosaic virus 1a protein regulates interactions between the 2b protein and ARGONAUTE 1 while maintaining the silencing suppressor activity of the 2b protein

The cucumber mosaic virus (CMV) 2b viral suppressor of RNA silencing (VSR) is a potent counter-defense and pathogenicity factor that inhibits antiviral silencing by titration of short double-stranded RNAs. It also disrupts microRNA-mediated regulation of host gene expression by binding ARGONAUTE 1 (AGO1). But in Arabidopsis thaliana complete inhibition of AGO1 is counterproductive to CMV since this triggers another layer of antiviral silencing mediated by AGO2, de-represses strong resistance against aphids (the insect vectors of CMV), and exacerbates symptoms. Using confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation assays we found that the CMV 1a protein, a component of the viral replicase complex, regulates the 2b-AGO1 interaction. By binding 2b protein molecules and sequestering them in P-bodies, the 1a protein limits the proportion of 2b protein molecules available to bind AGO1, which ameliorates 2b-induced disease symptoms, and moderates induction of resistance to CMV and to its aphid vector. However, the 1a protein-2b protein interaction does not inhibit the ability of the 2b protein to inhibit silencing of reporter gene expression in agroinfiltration assays. The interaction between the CMV 1a and 2b proteins represents a novel regulatory system in which specific functions of a VSR are selectively modulated by another viral protein. The finding also provides a mechanism that explains how CMV, and possibly other viruses, modulates symptom induction and manipulates host-vector interactions.