In vivo phosphorylation of isocitrate lyase inEscherichia coli andAcinetobacter calcoaceticus

Isocitrate lyase inEscherichia coli and inAcinetobacter calcoaceticus is phosphorylated when the cells are grown with acetate as the sole carbon source in low-phosphate mineral salts medium containing³²P inorganic phosphate. The level of³²P incorporation into the enzyme in both microorganisms appears to be constant throughout the entire growth cycle. Further, theresults of immunoblots and rocket immunoelectrophoresis suggest that the amount of isocitrate lyase protein, although at different levels in each microorganism, also remains constant throughout the growth cycle.     

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.     

A cross-talk between epithelium and endothelium mediates human alveolar–capillary injury during SARS-CoV-2 infection

COVID-19, caused by SARS-CoV-2, is an acute and rapidly developing pandemic, which leads to a global health crisis. SARS-CoV-2 primarily attacks human alveoli and causes severe lung infection and damage. To better understand the molecular basis of this disease, we sought to characterize the responses of alveolar epithelium and its adjacent microvascular endothelium to viral infection under a co-culture system. SARS-CoV-2 infection caused massive virus replication and dramatic organelles remodeling in alveolar epithelial cells, alone. While, viral infection affected endothelial cells in an indirect manner, which was mediated by infected alveolar epithelium. Proteomics analysis and TEM examinations showed viral infection caused global proteomic modulations and marked ultrastructural changes in both epithelial cells and endothelial cells under the co-culture system. In particular, viral infection elicited global protein changes and structural reorganizations across many sub-cellular compartments in epithelial cells. Among the affected organelles, mitochondrion seems to be a primary target organelle. Besides, according to EM and proteomic results, we identified Daurisoline, a potent autophagy inhibitor, could inhibit virus replication effectively in host cells. Collectively, our study revealed an unrecognized cross-talk between epithelium and endothelium, which contributed to alveolar–capillary injury during SARS-CoV-2 infection. These new findings will expand our understanding of COVID-19 and may also be helpful for targeted drug development.Subject terms: Mechanisms of disease, Viral infection     

A new species of Galearis (Orchidinae: Orchidaceae) from Sichuan,China

A new species, Galearis huanglongensis Q.W.Meng & Y.B.Luo, is described and illustrated. It is similar to Galearis cyclochila (Franch. & Sav.) Soó and Galearis diantha (Schltr.) P.F.Hunt, but differs in having a short spur, two elliptical lateral stigma lobes and distinctly separated bursicles. This new species is known only from the type locality, the Huanglong Valley, Songpan County, western Sichuan, China, growing amongst mosses under alpine shrubs at an elevation of about 3000 m. Based on two years of observations of its population size, the species was categorized as critically endangered CR (B1a, B2a) according to the World Conservation Union (IUCN) Red List Categories and Criteria, Version 3.1. The micromorphology of pollinia and seeds was observed by scanning electron microscopy and compared with that of G. cyclochila and G. diantha. The results supported G. huanglongensis Q.W.Meng & Y.B.Luo as a new species. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158 , 689–695.     

Spatial and temporal factors associated with nest survival of Gray Flycatchers in managed ponderosa pine forests

Gray Flycatchers (Empidonax wrightii) breed in a variety of habitats in the arid and semi‐arid regions of the western United States, but little is known about their breeding biology, especially in the northern portion of their range where they nest in ponderosa pine (Pinus ponderosa) forests. From May to July 2014 and 2015, we conducted surveys for singing male Gray Flycatchers along the eastern slope of the Cascade Range in Washington, U.S.A, monitored flycatcher nests, and quantified nest‐site vegetation. We used a logistic‐exposure model fit within a Bayesian framework to model the daily survival probability of flycatcher nests. During the 2 yr of our study, we monitored 141 nests, with 93% in ponderosa pines. Mean clutch size was 3.6 eggs and the mean number of young fledged per nest was 3.2. Predation accounted for 90% of failed nests. We found a positive association between daily nest survival and both nest height and distance of nest substrates from the nearest tree. Flycatchers that locate their nests higher above the ground and further from adjacent trees may be choosing the safest alternative because higher nests may be less exposed to terrestrial predators and nests in trees that are farther from other trees may be less exposed to arboreal predators such as jays (Corvidae) that may forage in patches with connected canopies. Nests in trees farther from other trees may also allow earlier detection of approaching predators and thus aid in nest defense.     

Compatible Models of Carbon Content of Individual Trees on a Cunninghamia lanceolata Plantation in Fujian Province,China

We tried to establish compatible carbon content models of individual trees for a Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) plantation from Fujian province in southeast China. In general, compatibility requires that the sum of components equal the whole tree, meaning that the sum of percentages calculated from component equations should equal 100%. Thus, we used multiple approaches to simulate carbon content in boles, branches, foliage leaves, roots and the whole individual trees. The approaches included (i) single optimal fitting (SOF), (ii) nonlinear adjustment in proportion (NAP) and (iii) nonlinear seemingly unrelated regression (NSUR). These approaches were used in combination with variables relating diameter at breast height (D) and tree height (H), such as D, D²H, DH and D&H (where D&H means two separate variables in bivariate model). Power, exponential and polynomial functions were tested as well as a new general function model was proposed by this study. Weighted least squares regression models were employed to eliminate heteroscedasticity. Model performances were evaluated by using mean residuals, residual variance, mean square error and the determination coefficient. The results indicated that models with two dimensional variables (DH, D²H and D&H) were always superior to those with a single variable (D). The D&H variable combination was found to be the most useful predictor. Of all the approaches, SOF could establish a single optimal model separately, but there were deviations in estimating results due to existing incompatibilities, while NAP and NSUR could ensure predictions compatibility. Simultaneously, we found that the new general model had better accuracy than others. In conclusion, we recommend that the new general model be used to estimate carbon content for Chinese fir and considered for other vegetation types as well.     

Purification of (Ca2+-Mg2+)-ATPase from rat liver plasma membranes

The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.