Glycosylinositol phosphorylceramides from Rosa cell cultures are boron‐bridged in the plasma membrane and form complexes with rhamnogalacturonan II

Boron (B) is essential for plant cell‐wall structure and membrane functions. Compared with its role in cross‐linking the pectic domain rhamnogalacturonan II (RG‐II), little information is known about the biological role of B in membranes. Here, we investigated the involvement of glycosylinositol phosphorylceramides (GIPCs), major components of lipid rafts, in the membrane requirement for B. Using thin‐layer chromatography and mass spectrometry, we first characterized GIPCs from Rosa cell culture. The major GIPC has one hexose residue, one hexuronic acid residue, inositol phosphate, and a ceramide moiety with a C₁₈ trihydroxylated mono‐unsaturated long‐chain base and a C₂₄ monohydroxylated saturated fatty acid. Disrupting B bridging (by B starvation in vivo or by treatment with cold dilute HCl or with excess borate in vitro) enhanced the GIPCs' extractability. As RG‐II is the main B‐binding site in plants, we investigated whether it could form a B‐centred complex with GIPCs. Using high‐voltage paper electrophoresis, we showed that addition of GIPCs decreased the electrophoretic mobility of radiolabelled RG‐II, suggesting formation of a GIPC–B–RG‐II complex. Last, using polyacrylamide gel electrophoresis, we showed that added GIPCs facilitate RG‐II dimerization in vitro. We conclude that B plays a structural role in the plasma membrane. The disruption of membrane components by high borate may account for the phytotoxicity of excess B. Moreover, the in‐vitro formation of a GIPC–B–RG‐II complex gives the first molecular explanation of the wall–membrane attachment sites observed in vivo. Finally, our results suggest a role for GIPCs in the RG‐II dimerization process.     

Resequencing studies of nonmodel organisms using closely related reference genomes: optimal experimental designs and bioinformatics approaches for population genomics

Decreasing costs of next‐generation sequencing (NGS) experiments have made a wide range of genomic questions open for study with nonmodel organisms. However, experimental designs and analysis of NGS data from less well‐known species are challenging because of the lack of genomic resources. In this work, we investigate the performance of alternative experimental designs and bioinformatics approaches in estimating variability and neutrality tests based on the site‐frequency‐spectrum (SFS) from individual resequencing data. We pay particular attention to challenges faced in the study of nonmodel organisms, in particular the absence of a species‐specific reference genome, although phylogenetically close genomes are assumed to be available. We compare the performance of three alternative bioinformatics approaches – genotype calling, genotype–haplotype calling and direct estimation without calling genotypes. We find that relying on genotype calls provides biased estimates of population genetic statistics at low to moderate read depth (2–8×). Genotype–haplotype calling returns more accurate estimates irrespective of the divergence to the reference genome, but requires moderate depth (8–20×). Direct estimation without calling genotypes returns the most accurate estimates of variability and of most SFS tests investigated, including at low read depth (2–4×). Studies without species‐specific reference genome should thus aim for low read depth and avoid genotype calling whenever individual genotypes are not essential. Otherwise, aiming for moderate to high depth at the expense of number of individuals, and using genotype–haplotype calling, is recommended.     

Chlamydia trachomatis infection and human papillomavirus in women with cervical neoplasia in Pernambuco-Brazil

Chlamydia trachomatis (CT) is the most common bacterial cause of sexually transmitted disease. High-risk human papillomavirus (HR-HPV) is considered the main etiological agent for cervical neoplasia. Evidences showed that the presence of co-infection of CT and HR-HPV plays a central role in the etiology of cervical intraepithelial neoplasia (CIN) and cervical cancer. The goals of this study were: evaluate the human papillomavirus (HPV) and CT prevalence among Brazilian women with abnormal cytology and provide the effect of this association on the severity of cervical neoplasia. The population of this study was composed by 142 women with incident histological incidence of CIN grades I, II, III or cervical cancer from Recife, Northeast of Brazil. The polymerase chain reaction method on a cervical brush specimen was used to detect both agents and the automatic sequencing method was used for HPV genotyping assay. The prevalence of HPV and CT was 100 and 24.65 %, respectively. Thirteen types of HPV were detected; HPV 16, 18, 31 and 33 were the most common. The most prevalent HPV types were HPV 16 and 18. A significant association between CT positive and HPV 16 infection was found (p < 0.0106; OR = 5.31; 95 % IC 1.59–17.67). In the study population, there was diversity of HPV infections, with high-risk types being the most common. Also, the data collected suggest that CT infection may play an important role in the natural history of HPV infection.     

The effect of celastrol,a triterpene with antitumorigenic activity,on conformational and functional aspects of the human 90 kDa heat shock protein Hsp90α, a chaperone implicated in the stabilization of the tumor phenotype

Background

Hsp90 is a molecular chaperone essential for cell viability in eukaryotes that is associated with the maturation of proteins involved in important cell functions and implicated in the stabilization of the tumor phenotype of various cancers, making this chaperone a notably interesting therapeutic target. Celastrol is a plant-derived pentacyclic triterpenoid compound with potent antioxidant, anti-inflammatory and anticancer activities; however, celastrol's action mode is still elusive.

Results

In this work, we investigated the effect of celastrol on the conformational and functional aspects of Hsp90α. Interestingly, celastrol appeared to target Hsp90α directly as the compound induced the oligomerization of the chaperone via the C-terminal domain as demonstrated by experiments using a deletion mutant. The nature of the oligomers was investigated by biophysical tools demonstrating that a two-fold excess of celastrol induced the formation of a decameric Hsp90α bound throughout the C-terminal domain. When bound, celastrol destabilized the C-terminal domain. Surprisingly, standard chaperone functional investigations demonstrated that neither the in vitro chaperone activity of protecting against aggregation nor the ability to bind a TPR co-chaperone, which binds to the C-terminus of Hsp90α, were affected by celastrol.

Conclusion

Celastrol interferes with specific biological functions of Hsp90α. Our results suggest a model in which celastrol binds directly to the C-terminal domain of Hsp90α causing oligomerization. However, the ability to protect against protein aggregation (supported by our results) and to bind to TPR co-chaperones are not affected by celastrol. Therefore celastrol may act primarily by inducing specific oligomerization that affects some, but not all, of the functions of Hsp90α.

General significance

To the best of our knowledge, this study is the first work to use multiple probes to investigate the effect that celastrol has on the stability and oligomerization of Hsp90α and on the binding of this chaperone to Tom70. This work provides a novel mechanism by which celastrol binds Hsp90α.     

Presence of fimH,mrkD, and irp2 Virulence Genes in KPC-2-Producing Klebsiella pneumoniae Isolates in Recife-PE,Brazil

Klebsiella pneumoniae strains can produce different virulence factors, such as fimbrial adhesins and siderophores, which are important in the colonization and development of the infection. The aims of this study were to determine the occurrence of fimH, mrkD, and irp2 virulence genes in 22 KPC-2-producing K. pneumoniae isolates as well as 22 not producing-KPC isolates, from patients from different hospitals in Recife-PE, Brazil, and also to analyze the clonal relationship of the isolates by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The genes were detected by PCR and DNA sequencing. The bla _KPC-2 gene was identified in 22 KPC-positive isolates. On analyzing the antimicrobial susceptibility profile of the isolates, it was detected that polymyxin and amikacin were the antimicrobials of best activity against K. pneumoniae. On the other hand, five isolates exhibited resistance to polymyxin. In the KPC-positive group, was observed a high rate of resistance to cephalosporins, followed by carbapenems. Molecular typing by ERIC-PCR detected 38 genetic profiles, demonstrating a multiclonal spread of the isolates analyzed. It was observed that the virulence genes irp2, mrkD, and fimH were seen to have together a higher frequency in the KPC-positive group. The accumulation of virulence genes of KPC-positive K. pneumoniae isolates, observed in this study, along with the multi-resistance impose significant therapeutic limitations on the treatment of infections caused by K. pneumoniae.     

Viral Cyclin promotes KSHV-induced cellular transformation and tumorigenesis by overriding contact inhibition

Kaposi sarcoma-associated herpesvirus (KSHV) is a tumor virus encoding several proto-oncogenes. However, the roles of these viral genes in KSHV-induced tumorigenesis have not been defined. In this study, we used a recently developed model of KSHV-induced cellular transformation and tumorigenesis combining with a reverse genetic system to examine the role of a KSHV latent gene vCyclin (ORF72), a cellular Cyclin D2 homolog, in KSHV-induced oncogenesis. Deletion of vCyclin did not affect cell proliferation and cell cycle progression at a low-density condition, when cells were at an active proliferation state. However, vCyclin mutant cells were contact-inhibited and arrested at G₁ phase at a high-density condition. As a result, vCyclin mutant cells formed less and smaller colonies in soft agar assay. Nude mice inoculated with vCyclin mutant cells had reduced tumor incidence and extended tumor latency and survival compared with mice inoculated with wild-type (WT) virus-infected cells. WT but not mutant virus effectively induced Cyclin-dependent kinase inhibitor p27/Kip1 Ser10 phosphorylation and cytoplasmic relocalization. shRNA knockdown of p27 released the blockage of the mutant cells from cell cycle arrest at G₁ phase at a high-density condition. Together, these results indicate that vCyclin primarily functions to enhance cellular transformation and tumorigenesis by promoting cell cycle progression and cell proliferation at a contact-inhibited condition.     

Deficient IFN Signaling by Myeloid Cells Leads to MAVS-Dependent Virus-Induced Sepsis

The type I interferon (IFN) signaling response limits infection of many RNA and DNA viruses. To define key cell types that require type I IFN signaling to orchestrate immunity against West Nile virus (WNV), we infected mice with conditional deletions of the type I IFN receptor (IFNAR) gene. Deletion of the Ifnar gene in subsets of myeloid cells resulted in uncontrolled WNV replication, vasoactive cytokine production, sepsis, organ damage, and death that were remarkably similar to infection of Ifnar ^−/− mice completely lacking type I IFN signaling. In Mavs^−/−×Ifnar^−/− myeloid cells and mice lacking both Ifnar and the RIG-I-like receptor adaptor gene Mavs, cytokine production was muted despite high levels of WNV infection. Thus, in myeloid cells, viral infection triggers signaling through MAVS to induce proinflammatory cytokines that can result in sepsis and organ damage. Viral pathogenesis was caused in part by massive complement activation, as liver damage was minimized in animals lacking complement components C3 or factor B or treated with neutralizing anti-C5 antibodies. Disease in Ifnar ^−/− and CD11c Cre⁺ Ifnar ^f/f mice also was facilitated by the proinflammatory cytokine TNF-α, as blocking antibodies diminished complement activation and prolonged survival without altering viral burden. Collectively, our findings establish the dominant role of type I IFN signaling in myeloid cells in restricting virus infection and controlling pathological inflammation and tissue injury.     

A new species of Lichtheimia (Mucoromycotina,Mucorales) isolated from Brazilian soil

During studies on Mucorales in semiarid and littoral dune areas in the northeast of Brazil, two cultures of an Absidia-like species were isolated from soil. They were characterized based on morphological, physiological and molecular data (5.8S and LSU rDNA sequences). The phylogenetic analyses of the isolates revealed that they belong to the Lichtheimiaceae and are closely related to species of Lichtheimia. The two isolates produced simple or branched, erect and circinate sporophores, occasionally with a septum under the sporangia, characteristics also common in Lichtheimia species. However, different from the described Lichtheimia species, the columellae of our isolates were mainly short hemispherical, never spatulate or elliptical and without projections. Sometimes, a long conical or bell shaped apophysis was found. Both isolates grew better at 30–35 °C, with no development at 42 °C, and giant cells were not observed. Based on the evidence of the analyzed datasets a new species of Lichtheimia is proposed.     

Chromosomal diversity in three species of electric fish (Apteronotidae,Gymnotiformes) from the Amazon Basin

Cytogenetic studies were carried out on samples of Parapteronotus hasemani, Sternarchogiton preto and Sternarchorhamphus muelleri (Apteronotidae, Gymnotiformes) from the Amazon basin. The first two species exhibited both a 2n = 52 karyotype, but differed in their karyotypic formulae, distribution of constitutive heterochromatin, and chromosomal location of the NOR. The third species, Sternarchorhamphus muelleri, was found to have a 2n = 32 karyotype. In all three species the DAPI and chromomycin A3 staining results were consistent with the C-banding results and nucleolar organizer region (NOR) localization. The 18S rDNA probe confirmed that there was only one pair of ribosomal DNA cistron bearers per species. The telomeric probe did not reveal interstitial telomeric sequences (ITS). The karyotypic differences among these species can be used for taxonomic identification. These data will be useful in future studies of these fishes and help understanding the phylogenetic relationships and chromosomal evolution of the Apteronotidae.