Aspergillus nidulans ArfB plays a role in endocytosis and polarized growth

Filamentous fungi undergo polarized growth throughout most of their life cycles. The Spitzenkörper is an apical organelle composed primarily of vesicles that is unique to filamentous fungi and is likely to act as a vesicle supply center for tip growth. Vesicle assembly and trafficking are therefore important for hyphal growth. ADP ribosylation factors (Arfs), a group of small GTPase proteins, play an important role in nucleating vesicle assembly. Little is known about the role of Arfs in filamentous hyphal growth. We found that Aspergillus nidulans is predicted to encode six Arf family proteins. Analysis of protein sequence alignments suggests that A. nidulans ArfB shares similarity with ARF6 of Homo sapiens and Arf3p of Saccharomyces cerevisiae. An arfB null allele (arfB disrupted by a transposon [arfB::Tn]) was characterized by extended isotropic growth of germinating conidia followed by cell lysis or multiple, random germ tube emergence, consistent with a failure to establish polarity. The mutant germ tubes and hyphae that do form initially meander abnormally off of the axis of polarity and frequently exhibit dichotomous branching at cell apices, consistent with a defect in polarity maintenance. FM4-64 staining of the arfB::Tn strain revealed that another phenotypic characteristic seen for arfB::Tn is a reduction and delay in endocytosis. ArfB is myristoylated at its N terminus. Green fluorescent protein-tagged ArfB (ArfB::GFP) localizes to the plasma membrane and endomembranes and mutation (ArfB^G2A::GFP) of the N-terminal myristoylation motif disperses the protein to the cytoplasm rather than to the membranes. These results demonstrate that ArfB functions in endocytosis to play important roles in polarity establishment during isotropic growth and polarity maintenance during hyphal extension.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

COX‐2‐mediated Inflammation in Fat Is Crucial for Obesity‐linked Insulin Resistance and Fatty Liver

The aim was to examine the role of cyclooxygenase (COX)‐2‐mediated inflammation in the development of obese linked insulin resistance and fatty liver. The rats were fed separately regular diet (CONT), high‐fat diet (HFD) ad libitum, or energy restrictedly for 12 weeks. Rats fed HFD ad libitum were further divided into three subgroups co‐treated with vehicle (HFa), or a selective COX‐2 inhibitor celecoxib (HFa‐Cel) or mesulid (HFa‐Mes). Euglycemic hyperinsulinemic clamp (EHC) experiment was performed at the end of study. Another set of rats with similar grouping was further divided into those with a 4, 8, or 12‐week intervention period for hepatic sampling. Body weight was increased significantly and similarly in HFa, HFa‐Cel, and HFa‐Mes. Time‐dependent increases in plasma insulin, glucose, 8‐isoprostanes, leptin levels, homeostasis model assessment of insulin resistance (HOMA‐IR) and hepatic triglyceride contents shown in HFa were significantly reversed in HFa‐Cel and HFa‐Mes. During EHC period, the reduction in stimulation of whole body glucose uptake, suppression of hepatic glucose production and metabolic clearance rate of insulin shown in HFa were significantly reversed in HFa‐Cel and HFa‐Mes. The enhanced COX‐2 and tumor necrosis factor‐α (TNF‐α) but attenuated PPAR‐γ and C/EBP‐α mRNA expressions in epididymal fat shown in HFa were significantly reversed in HFa‐Cel and HFa‐Mes. The increases in average cell size of adipocytes and CD68 positive cells shown in HFa were also significantly reversed in HFa‐Cel and HFa‐Mes. Our findings suggest that COX‐2 activation in fat inflammation is important in the development of insulin resistance and fatty liver in high fat induced obese rats.     

Phototrophic Phylotypes Dominate Mesothermal Microbial Mats Associated with Hot Springs in Yellowstone National Park

The mesothermal outflow zones (50-65°C) of geothermal springs often support an extensive zone of green and orange laminated microbial mats. In order to identify and compare the microbial inhabitants of morphologically similar green-orange mats from chemically and geographically distinct springs, we generated and analyzed small-subunit ribosomal RNA (rRNA) gene amplicons from six mesothermal mats (four previously unexamined) in Yellowstone National Park. Between three and six bacterial phyla dominated each mat. While many sequences bear the highest identity to previously isolated phototrophic genera belonging to the Cyanobacteria, Chloroflexi, and Chlorobi phyla, there is also frequent representation of uncultured, unclassified members of these groups. Some genus-level representatives of these dominant phyla were found in all mats, while others were unique to a single mat. Other groups detected at high frequencies include candidate divisions (such as the OP candidate clades) with no cultured representatives or complete genomes available. In addition, rRNA genes related to the recently isolated and characterized photosynthetic acidobacterium "Candidatus Chloracidobacterium thermophilum" were detected in most mats. In contrast to microbial mats from well-studied hypersaline environments, the mesothermal mats in this study accrue less biomass and are substantially less diverse, but have a higher proportion of known phototrophic organisms. This study provides sequences appropriate for accurate phylogenetic classification and expands the molecular phylogenetic survey of Yellowstone microbial mats.     

Tumour suppressive function and modulation of programmed cell death 4 (PDCD4) in ovarian cancer

Background

Programmed cell death 4 (PDCD4), originally identified as the neoplastic transformation inhibitor, was attenuated in various cancer types. Our previous study demonstrated a continuous down-regulation of PDCD4 expression in the sequence of normal-borderline-malignant ovarian tissue samples and a significant correlation of PDCD4 expression with disease-free survival. The objective of the current study was to further investigate the function and modulation of PDCD4 in ovarian cancer cells.

Principal Findings

We demonstrated that ectopic PDCD4 expression significantly inhibited cell proliferation by inducing cell cycle arrest at G₁ stage and up-regulation of cell cycle inhibitors of p27 and p21. Cell migration and invasion were also inhibited by PDCD4. PDCD4 over-expressing cells exhibited elevated phosphatase and tensin homolog (PTEN) and inhibited protein kinase B (p-Akt). In addition, the expression of PDCD4 was up-regulated and it was exported to the cytoplasm upon serum withdrawal treatment, but it was rapidly depleted via proteasomal degradation upon serum re-administration. Treatment of a phosphoinositide 3-kinase (PI3K) inhibitor prevented the degradation of PDCD4, indicating the involvement of PI3K-Akt pathway in the modulation of PDCD4.

Conclusion

PDCD4 may play a critical function in arresting cell cycle progression at key checkpoint, thus inhibiting cell proliferation, as well as suppressing tumour metastasis. The PI3K-Akt pathway was implied to be involved in the regulation of PDCD4 degradation in ovarian cancer cells. In response to the stress condition, endogenous PDCD4 was able to shuttle between cell compartments to perform its diverted functions.     

Electrostatic contribution of surface charge residues to the stability of a thermophilic protein: benchmarking experimental and predicted pKa values

Optimization of the surface charges is a promising strategy for increasing thermostability of proteins. Electrostatic contribution of ionizable groups to the protein stability can be estimated from the differences between the pKa values in the folded and unfolded states of a protein. Using this pKa-shift approach, we experimentally measured the electrostatic contribution of all aspartate and glutamate residues to the stability of a thermophilic ribosomal protein L30e from Thermococcus celer. The pKa values in the unfolded state were found to be similar to model compound pKas. The pKa values in both the folded and unfolded states obtained at 298 and 333 K were similar, suggesting that electrostatic contribution of ionizable groups to the protein stability were insensitive to temperature changes. The experimental pKa values for the L30e protein in the folded state were used as a benchmark to test the robustness of pKa prediction by various computational methods such as H++, MCCE, MEAD, pKD, PropKa, and UHBD. Although the predicted pKa values were affected by crystal contacts that may alter the side-chain conformation of surface charged residues, most computational methods performed well, with correlation coefficients between experimental and calculated pKa values ranging from 0.49 to 0.91 (p<0.01). The changes in protein stability derived from the experimental pKa-shift approach correlate well (r = 0.81) with those obtained from stability measurements of charge-to-alanine substituted variants of the L30e protein. Our results demonstrate that the knowledge of the pKa values in the folded state provides sufficient rationale for the redesign of protein surface charges leading to improved protein stability.     

Molecular dissection of the response of a rice leucine-rich repeat receptor-like kinase (LRR-RLK) gene to abiotic stresses

Leucine-rich repeat (LRR) receptor-like kinase (RLK) proteins play key roles in a variety of biological pathways. In a previous study, we analyzed the members of the rice LRR-RLK gene family using in silico analysis. A total of 23 LRR-RLK genes were selected based on the expression patterns of a genome-wide dataset of microarrays. The Oryza sativa gamma-ray induced LRR-RLK1 (OsGIRL1) gene was highly induced by gamma irradiation. Therefore, we studied its expression pattern in response to various different abiotic and phytohormone treatments. OsGIRL1 was induced on exposure to abiotic stresses such as salt, osmotic, and heat, salicylic acid (SA), and abscisic acid (ABA), but exhibited downregulation in response to jasmonic acid (JA) treatment. The OsGIRL1 protein was clearly localized at the plasma membrane. The truncated proteins harboring juxtamembrane and kinase domains (or only harboring a kinase domain) exhibited strong autophosphorylation. The biological function of OsGIRL1 was investigated via heterologous overexpression of this gene in Arabidopsis plants subjected to gamma-ray irradiation, salt stress, osmotic stress, and heat stress. A hypersensitive response was observed in response to salt stress and heat stress, whereas a hyposensitive response was observed in response to gamma-ray treatment and osmotic stress. These results provide critical insights into the molecular functions of the rice LRR-RLK genes as receptors of external signals.     

C‐terminal β‐strand swapping in a consensus‐derived fibronectin Type III scaffold

The crystal structures of six different fibronectin Type III consensus‐derived Tencon domains, whose solution properties exhibit no, to various degrees of, aggregation according to SEC, have been determined. The structures of the five variants showing aggregation reveal 3D domain swapped dimers. In all five cases, the swapping involves the C‐terminal β‐strand resulting in the formation of Tencon dimers in which the target‐binding surface is blocked. All of the variants differ in sequence in the FG loop, which is the hinge loop in the β‐strand‐swapped dimers. The six tencon variants have between 0 and 5 residues inserted between positions 77 and 78 in the FG loop. Analysis of the structures suggests that a non‐glycine residue at position 77 and insertions of <4 residues may destabilize the β‐turn in the FG loop promoting β‐strand swapping. Swapped dimers with an odd number of inserted residues may be less stable, particularly if they contain proline residues, because they cannot form perfect β‐bridges in the FG regions that link the swapped dimers. The Tencon β‐swapped variants with the longest FG sequences are observed to form higher order hexameric or helical oligomeric structures in the crystal correlating well with the aggregation properties of these domains observed in solution. Understanding the structural basis for domain‐swapped dimerization and oligomerization will support engineering efforts of the Tencon domain to produce variants with desired biophysical properties. Proteins 2014; 82:1359–1369. © 2013 Wiley Periodicals, Inc.     

DNA barcoding of Orchidaceae in Korea

Species of Orchidaceae are under severe threat of extinction mainly due to overcollection and habitat destruction; accurate identification of orchid species is critical in conservation biology and sustainable utilization of orchids as plant resources. We examined 647 sequences of the cpDNA regions rbcL, matK, atpF‐atpH IGS, psbK‐psbI IGS and trnH‐psbA IGS from 89 orchid species (95 taxa) and four outgroup taxa to develop an efficient DNA barcode for Orchidaceae in Korea. The five cpDNA barcode regions were successfully amplified and sequenced for all chlorophyllous taxa, but the amplification and sequencing of the same regions in achlorophyllous taxa produced variable results. psbK‐psbI IGS showed the highest mean interspecific K2P distance (0.1192), followed by matK (0.0803), atpF‐atpH IGS (0.0648), trnH‐psbA IGS (0.0460) and rbcL (0.0248). The degree of species resolution for individual barcode regions ranged from 60.5% (rbcL) to 83.5% (trnH‐psbA IGS). The degree of species resolution was significantly enhanced in multiregion combinations of the five barcode regions. Of the 26 possible combinations of the five regions, six provided the highest degree of species resolution (98.8%). Among these, a combination of atpF‐atpH IGS, psbK‐psbI IGS and trnH‐psbA IGS, which comprises the least number of DNA regions, is the best option for barcoding of the Korean orchid species.