HIF-1 Interacts with TRIM28 and DNA-PK to release paused RNA polymerase II and activate target gene transcription in response to hypoxia

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that acts as a regulator of oxygen (O2) homeostasis in metazoan species by binding to hypoxia response elements (HREs) and activating the transcription of hundreds of genes in response to reduced O2 availability. RNA polymerase II (Pol II) initiates transcription of many HIF target genes under non-hypoxic conditions but pauses after approximately 30–60 nucleotides and requires HIF-1 binding for release. Here we report that in hypoxic breast cancer cells, HIF-1 recruits TRIM28 and DNA-dependent protein kinase (DNA-PK) to HREs to release paused Pol II. We show that HIF-1α and TRIM28 assemble the catalytically-active DNA-PK heterotrimer, which phosphorylates TRIM28 at serine-824, enabling recruitment of CDK9, which phosphorylates serine-2 of the Pol II large subunit C-terminal domain as well as the negative elongation factor to release paused Pol II, thereby stimulating productive transcriptional elongation. Our studies reveal a molecular mechanism by which HIF-1 stimulates gene transcription and reveal that the anticancer effects of drugs targeting DNA-PK in breast cancer may be due in part to their inhibition of HIF-dependent transcription.

A Real-Time PCR Assay for Detection of Low Pneumocystis jirovecii Levels

Here we report a new real-time PCR assay using SYBR Green which provides higher sensitivity for the specific detection of low levels of Pneumocystis jirovecii. To do so, two primer sets were designed, targeting the family of genes that code for the most abundant surface protein of Pneumocystis spp., namely the major surface glycoproteins (Msg), and the mitochondrial large subunit rRNA (mtLSUrRNA) multicopy gene, simultaneously detecting two regions. PCR methods are instrumental in detecting these low levels; however, current nested-PCR methods are time-consuming and complex. To validate our new real-time Msg-A/mtLSUrRNA PCR protocol, we compared it with nested-PCR based on the detection of Pneumocystis mitochondrial large subunit rRNA (mtLSUrRNA), one of the main targets used to detect this pathogen. All samples identified as positive by the nested-PCR method were found positive using our new real-time PCR protocol, which also detected P. jirovecii in three nasal aspirate samples that were negative for both rounds of nested-PCR. Furthermore, we read both rounds of the nested-PCR results for comparison and found that some samples with no PCR amplification, or with a feeble band in the first round, correlated with higher Ct values in our real-time Msg-A/mtLSUrRNA PCR. This finding demonstrates the ability of this new single-round protocol to detect low Pneumocystis levels. This new assay provides a valuable alternative for P. jirovecii detection, as it is both rapid and sensitive.

Description of Cryptaphelenchus recticaudatus n. sp. (Aphelenchoidea: Ektaphelenchinae) in Pinus elliottii from the USA

Summary Cryptaphelenchus recticaudatus n. sp. is described and illustrated in Pinus elliottii from the USA. The new species is characterised by the female body 310-431 μm long with distinctly annulated cuticle, lateral fields with four lines, lip region separated from the body by a shallow depression, delicate stylet with small knobs, post-vulval uterine sac short, and rectum and anus invisible. Males are 228-314 μm long, spicules 9.8-12.4 μm long with a well-developed and broad condylus, and seven caudal papillae arranged as a single (P1) and pair (P2) of precloacal papillae plus two pairs of postcloacal papillae. Based upon the general female morphology, the new species most closely resembles C. baujardi and C. iranicus. The morphological differences with the aforementioned species and other species of the genus are discussed. The phylogenetic analyses based on small (SSU) and large subunit (LSU) D2-D3 expansion segments of ribosomal DNA of different individuals of the new species revealed that the new species fell into the Cryptaphelenchus clade in both SSU and LSU trees. The monophyly of the genus was retained after adding newly generated sequences of the new species.

Pollen Number and Ribosome Gene Expression Altered in a Genome-Editing Mutant of REDUCED POLLEN NUMBER1 Gene

The number of pollen grains varies within and between species. However, little is known about the molecular basis of this quantitative trait, in contrast with the many studies available on cell differentiation in the stamen. Recently, the first gene responsible for pollen number variation, REDUCED POLLEN NUMBER1 (RDP1), was isolated by genome-wide association studies of Arabidopsis thaliana and exhibited the signature of natural selection. This gene encodes a homolog of yeast Mrt4 (mRNA turnover4), which is an assembly factor of the large ribosomal subunit. However, no further data were available to link ribosome function to pollen development. Here, we characterized the RDP1 gene using the standard A. thaliana accession Col-0. The frameshift mutant, rdp1-3 generated by CRISPR/Cas9 revealed the pleiotropic effect of RDP1 in flowering, thus demonstrating that this gene is required for a broad range of processes other than pollen development. We found that the natural Col-0 allele conferred a reduced pollen number against the Bor-4 allele, as assessed using the quantitative complementation test, which is more sensitive than transgenic experiments. Together with a historical recombination event in Col-0, which was identified by sequence alignment, these results suggest that the coding sequence of RDP1 is the candidate region responsible for the natural phenotypic variation. To elucidate the biological processes in which RDP1 is involved, we conducted a transcriptome analysis. We found that genes responsible for ribosomal large subunit assembly/biogenesis were enriched among the differentially regulated genes, which supported the hypothesis that ribosome biogenesis is disturbed in the rdp1-3 mutant. Among the pollen-development genes, three key genes encoding basic helix-loop-helix (bHLH) transcription factors (ABORTED MICROSPORES (AMS), bHLH010, and bHLH089), as well as direct downstream genes of AMS, were downregulated in the rdp1-3 mutant. In summary, our results suggest a specialized function of ribosomes in pollen development through RDP1, which harbors natural variants under selection.

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.

Neodeightonia arengae sp. nov., Botryosphaeriaceous taxa on Arenga tremula (Arecaceae) from Guangdong, China

Microfungi associated with palm are a significant fungal group with a unique ecological niche and a broad distribution in tropical regions. Even though many fungal species have been reported from palm hosts, studies on fungi from Arenga tremula are considerably few. In this study, we isolated a saprobic Botryosphaeriaceae species on A. tremula, collected from Guangzhou, Guangdong Province, China. Morphological characteristics and phylogenetic analysis of the internal transcribed spacer (ITS), small subunit nuclear rRNA gene (SSU),part of the large subunit nuclear rRNA gene (LSU) and translation elongation factor 1−alpha gene (tef 1-α). Based on phylogenetic results and morphology we introduced Neodeightonia arengae sp. nov., with species description and illustrations. In addition, we provide a comparison of morphological characters of currently accepted Neodeightonia species. This is the first report of a Neodeightonia species associated with Arenga tremula and so represents an additional contribution to the knowledge of fungi associated with palm trees.

Calpain-mediated protein targets in cardiac mitochondria following ischemia–reperfusion

Calpain 1 and 2 (CPN1/2) are calcium-dependent cysteine proteases that exist in cytosol and mitochondria. Pharmacologic inhibition of CPN1/2 decreases cardiac injury during ischemia (ISC)–reperfusion (REP) by improving mitochondrial function. However, the protein targets of CPN1/2 activation during ISC–REP are unclear. CPN1/2 include a large subunit and a small regulatory subunit 1 (CPNS1). Genetic deletion of CPNS1 eliminates the activities of both CPN1 and CPN2. Conditional cardiomyocyte specific CPNS1 deletion mice were used in the present study to clarify the role of CPN1/2 activation in mitochondrial damage during ISC–REP with an emphasis on identifying the potential protein targets of CPN1/2. Isolated hearts from wild type (WT) or CPNS1 deletion mice underwent 25 min in vitro global ISC and 30 min REP. Deletion of CPNS1 led to decreased cytosolic and mitochondrial calpain 1 activation compared to WT. Cardiac injury was decreased in CPNS1 deletion mice following ISC–REP as shown by the decreased infarct size compared to WT. Compared to WT, mitochondrial function was improved in CPNS1 deletion mice following ischemia–reperfusion as shown by the improved oxidative phosphorylation and decreased susceptibility to mitochondrial permeability transition pore opening. H2O2 generation was also decreased in mitochondria from deletion mice following ISC–REP compared to WT. Deletion of CPNS1 also resulted in less cytochrome c and truncated apoptosis inducing factor (tAIF) release from mitochondria. Proteomic analysis of the isolated mitochondria showed that deletion of CPNS1 increased the content of proteins functioning in regulation of mitochondrial calcium homeostasis (paraplegin and sarcalumenin) and complex III activity. These results suggest that activation of CPN1 increases cardiac injury during ischemia–reperfusion by impairing mitochondrial function and triggering cytochrome c and tAIF release from mitochondria into cytosol.

RplI interacts with 5’ UTR of exsA to repress its translation and type III secretion system in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important opportunistic pathogen capable of causing variety of infections in humans. The type III secretion system (T3SS) is a critical virulence determinant of P. aeruginosa in the host infections. Expression of the T3SS is regulated by ExsA, a master regulator that activates the expression of all known T3SS genes. Expression of the exsA gene is controlled at both transcriptional and posttranscriptional levels. Here, we screened a P. aeruginosa transposon (Tn5) insertional mutant library and found rplI, a gene coding for the ribosomal large subunit protein L9, to be a repressor for the T3SS gene expression. Combining real-time quantitative PCR (qPCR), western blotting and lacZ fusion assays, we show that RplI controls the expression of exsA at the posttranscriptional level. Further genetic experiments demonstrated that RplI mediated control of the exsA translation involves 5’ untranslated region (5’ UTR). A ribosome immunoprecipitation assay and qPCR revealed higher amounts of a 24 nt fragment from exsA mRNA being associated with ribosomes in the ΔrplI mutant. An interaction between RplI and exsA mRNA harboring its 24 nt, but not 12 nt, 5’ UTR was confirmed by RNA Gel Mobility Shift and Microscale Thermophoresis assays. Overall, this study identifies the ribosomal large subunit protein L9 as a novel T3SS repressor that inhibits ExsA translation in P. aeruginosa.

Dothidea kunmingensis, a novel asexual species of Dothideaceae on Jasminum nudiflorum (winter jasmine) from Southwestern China

During a survey of saprobic microfungi in Southwest China, a coelomycetous fungus was found on dead twigs of Jasminum nudiflorum in Kunming, Yunnan Province. Based on a detailed morphological characterization coupled with multi-locus phylogenetic analyses, the fungus was identified as a new species in the genus Dothidea. Phylogenetic analyses using a combined matrix consisting of internal transcribed spacer (ITS), large subunit rRNA (LSU), small subunit rRNA (SSU), beta tubulin (tub2) and translation elongation factor-1 alpha (tef1-α) confirmed its placement in Dothideaceae and revealed a sister relationship to Dothidea eucalypti. The new species is characterized by pycnidial conidiomata, ampulliform or doliiform conidiogenous cells as well as aseptate, subglobose to ovoid, hyaline to pale-brown conidia. Comprehensive descriptions and illustrations are provided. Morphological characteristics of asexual morph taxa in Dothideaceae are also summarized and discussed.

Paraboeremia yungensis sp. nov., a new fungal species isolated from Las Yungas, South America, with promising tyrosinase production potential

In the context of a bioprospection programme for tyrosinase/L-DOPA- and melanin-producing fungal strains for biotechnological purposes, a hyperproducer isolate was obtained from Las Yungas rainforest, a relevant biodiverse ecoregion in North-Western Argentina. The selected strain was preliminarily identified as Paraboeremia sp. This is, to the best of our knowledge, the first native reported species of this genus in South America. Single-gene and multi-locus analyses of the internal transcribed spacer nuclear ribosomal RNA gene region (ITS), partial large subunit 28S nrDNA region (LSU), RNA polymerase II region (RPB2) and partial β-tubulin gene (TUB2) alignments were carried out to define the phylogenetic identity of this strain. As part of a polyphasic identification approach, these results were combined with morphological studies of active cultures growing on malt extract, oatmeal and potato dextrose agar plates. Incubation was performed under diverse conditions to stimulate sporulation for the subsequent micromorphological analysis. Microphotographs of pycnidia and conidia were taken with a scanning electron microscope. Maximum likelihood and Bayesian Inference analyses supported the location of the strain within the genus Paraboeremia, whilst morphological features allowed distinguishing it from previously described species within this genus. Based on the results herein reported, the new South-American species Paraboeremia yungensis is described and proposed.