Antibacterial activity of Ricinus communis plant extract against antibiotic resistant Helicobacter pylori and Gluconobacter oxydans isolated from fresh apple juices samples

Bacteria were isolated from samples of Fresh Apple juices from shops of three different localities of Lahore. Analysis of samples from Liberty, Anarkali and Yateem khana Markets show different levels of contamination. There were pathogenic and non-pathogenic bacteria in all samples and were identified by the morphological and biochemical tests. Most of the plasmids of pathogenic bacteria were 4kb in their molecular size. Ribotyping of 16S ribosomal RNA gene sequencing was done to confirm Helicobacter pylori strain and Gluconobacter oxydans. The highest sensitivity of 210mm was shown by Enterobacter sp. against Aztheromysine disk (15µg) while Micrococcus sp. was highly resistant against all of the Antibiotics applied. The antibiotic resistance of pathogenic bacteria was also checked against Ricinus communis plant's extracts, all isolated bacterial pathogens were resistant but only, E.coli was inhibited at 300µl of the extracts. Presence of pathogenic bacteria in Apple juice samples was due to contamination of sewage water in drinking water while some of these pathogenic bacteria came from Apple's tree and other from store houses of fruits.

Cryo-EM reconstruction of the human 40S ribosomal subunit at 2.15 Å resolution

The chemical modification of ribosomal RNA and proteins is critical for ribosome assembly, for protein synthesis and may drive ribosome specialization in development and disease. However, the inability to accurately visualize these modifications has limited mechanistic understanding of the role of these modifications in ribosome function. Here we report the 2.15 Å resolution cryo-EM reconstruction of the human 40S ribosomal subunit. We directly visualize post-transcriptional modifications within the 18S rRNA and post-translational modifications at the N-termini of two ribosomal proteins. Additionally, we interpret the solvation shells in the core regions of the 40S ribosomal subunit and reveal how potassium and magnesium ions establish both universally conserved and eukaryote-specific coordination to promote the stabilization and folding of key ribosomal elements. This work provides unprecedented structural details for the human 40S ribosomal subunit that will serve as an important reference for unraveling the functional role of ribosomal RNA modifications.

The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly

Ribosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

An RNA exosome subunit mediates cell-to-cell trafficking of a homeobox mRNA via plasmodesmata

mRNA migration through plasmodesmata In plants, certain transcription factors are produced in one cell but transported, sometimes as messenger RNA (mRNA), through plasmodesmata, channels between neighboring plant cells, where they act. This system helps to manage stem cell development. Kitagawa et al . now identify part of the machinery that manages this cell-to-cell transport. Transport of the mRNA encoding the KNOTTED1 homeobox transcription factor depends on Ribosomal RNA-Processing Protein 44 (AtRRP44A), which is a subunit of the RNA exosome. —PJH

High-grade ovarian cancer associated H/ACA snoRNAs promote cancer cell proliferation and survival

ABSTRACT Small nucleolar RNAs (snoRNAs) are an omnipresent class of non-coding RNAs involved in the modification and processing of ribosomal RNA (rRNA). As snoRNAs are required for ribosome production, the increase of which is a hallmark of cancer development, their expression would be expected to increase in proliferating cancer cells. However, assessing the nature and extent of snoRNAs’ contribution to cancer biology has been largely limited by difficulties in detecting highly structured RNA. In this study, we used a dedicated midsize non-coding RNA (mncRNA) sensitive sequencing technique to accurately survey the snoRNA abundance in independently verified high-grade serous ovarian carcinoma (HGSC) and serous borderline tumour (SBT) tissues. The results identified SNORA81, SNORA19 and SNORA56 as an H/ACA snoRNA signature capable of discriminating between independent sets of HGSC, SBT and normal tissues. The expression of the signature SNORA81 correlates with the level of ribosomal RNA (rRNA) modification and its knockdown inhibits 28S rRNA pseudouridylation and accumulation leading to reduced cell proliferation and migration. Together our data indicate that specific subsets of H/ACA snoRNAs may promote tumour aggressiveness by inducing rRNA modification and synthesis.

Multiple Cases of Bacterial Sequence Erroneously Incorporated Into Publicly Available Chloroplast Genomes

Public sequencing databases are invaluable resources to biological researchers, but assessing data veracity as well as the curation and maintenance of such large collections of data can be challenging. Genomes of eukaryotic organelles, such as chloroplasts and other plastids, are particularly susceptible to assembly errors and misrepresentations in these databases due to their close evolutionary relationships with bacteria, which may co-occur within the same environment, as can be the case when sequencing plants. Here, based on sequence similarities with bacterial genomes, we identified several suspicious chloroplast assemblies present in the National Institutes of Health (NIH) Reference Sequence (RefSeq) collection. Investigations into these chloroplast assemblies reveal examples of erroneous integration of bacterial sequences into chloroplast ribosomal RNA (rRNA) loci, often within the rRNA genes, presumably due to the high similarity between plastid and bacterial rRNAs. The bacterial lineages identified within the examined chloroplasts as the most likely source of contamination are either known associates of plants, or co-occur in the same environmental niches as the examined plants. Modifications to the methods used to process untargeted ‘raw’ shotgun sequencing data from whole genome sequencing efforts, such as the identification and removal of bacterial reads prior to plastome assembly, could eliminate similar errors in the future.

Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance

Alteration of antibiotic binding sites through modification of ribosomal RNA (rRNA) is a common form of resistance to ribosome-targeting antibiotics. The rRNA-modifying enzyme Cfr methylates an adenosine nucleotide within the peptidyl transferase center, resulting in the C-8 methylation of A2503 (m8A2503). Acquisition of cfr results in resistance to eight classes of ribosome-targeting antibiotics. Despite the prevalence of this resistance mechanism, it is poorly understood whether and how bacteria modulate Cfr methylation to adapt to antibiotic pressure. Moreover, direct evidence for how m8A2503 alters antibiotic binding sites within the ribosome is lacking. In this study, we performed directed evolution of Cfr under antibiotic selection to generate Cfr variants that confer increased resistance by enhancing methylation of A2503 in cells. Increased rRNA methylation is achieved by improved expression and stability of Cfr through transcriptional and post-transcriptional mechanisms, which may be exploited by pathogens under antibiotic stress as suggested by natural isolates. Using a variant that achieves near-stoichiometric methylation of rRNA, we determined a 2.2 Å cryo-electron microscopy structure of the Cfr-modified ribosome. Our structure reveals the molecular basis for broad resistance to antibiotics and will inform the design of new antibiotics that overcome resistance mediated by Cfr.

Species diversity, molecular phylogeny and ecological habits of Cyanosporus (Polyporales, Basidiomycota) with an emphasis on Chinese collections

Cyanosporus is a genus widely distributed in Asia, Europe, North America, South America and Oceania. It grows on different angiosperm and gymnosperm trees and can cause brown rot of wood. Blue-tinted basidiomata of Cyanosporus makes it easy to distinguish from other genera, but the similar morphological characters make it difficult to identify species within the genus. Phylogeny and taxonomy of Cyanosporus were carried out based on worldwide samples with an emphasis on Chinese collections, and the species diversity of the genus is updated. Four new species, C. flavus, C. rigidus, C. subungulatus and C. tenuicontextus, are described based on the evidence of morphological characters, distribution areas, host trees and molecular phylogenetic analyses inferred from the internal transcribed spacer (ITS) regions, the large subunit of nuclear ribosomal RNA gene (nLSU), the small subunit of nuclear ribosomal RNA gene (nSSU), the small subunit of mitochondrial rRNA gene (mtSSU), the largest subunit of RNA polymerase II (RPB1), the second largest subunit of RNA polymerase II (RPB2), and the translation elongation factor 1-α gene (TEF). Our study expanded the number of Cyanosporus species to 35 around the world including 23 species from China. Detailed descriptions of the four new species and the geographical locations of the Cyanosporus species in China are provided.

Evaluation of qPCR reference genes for taimen (Hucho taimen) under heat stress

As a powerful and attractive method for detecting gene expression, qRT-PCR has been broadly used in aquaculture research. Understanding the biology of taimen (Hucho taimen) has drawn increasing interest because of its ecological and economic value. Stable reference genes are required for the reliable quantification of gene expression, but such genes have not yet been optimized for taimen. In this study, the stability levels of 10 commonly used candidate reference genes were evaluated using geNorm, NormFinder, BestKeeper, and RefFinder. The expression levels of the 10 genes were detected using 240 samples from 48 experimental groups consisting of 40 individuals treated under four heat-stress conditions (18, 20, 22, and 24 °C) for 24 h and 26 °C for 4, 24, 48, and 72 h. Six tissues (blood, heart, brain, gill, skin, and liver) were collected from each individual. Ribosomal protein S29 (RPS29) and ribosomal protein L19 (RPL19) were the most stable genes among all of the samples, whereas 28S ribosomal RNA (28S rRNA), attachment region binding protein (ARBP), and 18S ribosomal RNA (18S rRNA) were the least stable. These results were verified by an expression analysis of taimen heat-stress genes (heat shock protein 60, hsp60, and heat shock protein 70, hsp70). In conclusion, RPS29 and RPL19 are the optimal reference genes for qRT-PCR analyses of taimen, irrespective of the tissue and experimental conditions. These results allow the reliable study of gene expression in taimen.

Type studies on two Paxillus species (Paxillaceae, Boletales) described from China

Types and recently collected samples of two Paxillus species namely P. rhytidophyllus and P. yunnanensis, originally described from southwestern China, were critically restudied based on morphology and molecular phylogenetic data of DNA sequences from the large subunit of the nuclear ribosomal RNA (nrLSU), the nuclear ribosomal internal transcribed spacer (ITS), and the translation elongation factor 1-α (tef1-α). The results showed that these two species belong to Boletinellus and Tricholomopsis, respectively. Thus, two new combinations, Boletinellus rhytidophyllus and Tricholomopsis yunnanensis are proposed. Boletinellus rhytidophyllus is characterized by a deeply decurrent and shallow hymenophore which is poroid-lamellate to alveolate, slightly thick-walled (0.6–1 μm) basidiospores, occasionally 2- to 4-spored basidia, rare or infrequent hymenial cystidia, and a trichodermal pileipellis. Tricholomopsis yunnanensis is characterized by a convex pileus densely covered by red-violet to red-brown fibrillose squamules, a yellowish stipe sparsely covered with red to red-brown fibrillose squamules, subglobose to broadly ellipsoid basidiospores, prominent large cheilocystidia measuring 60–195 × 11–39 μm, and a palisadic pileipellis. New descriptions and line drawings of these two species and their comparisons with allied taxa are presented.