TNF-α mediates the stimulation of sclerostin expression in an estrogen-deficient condition

Obesity consists in fat accumulation leading to increase in adipose cells number and size. Adipocyte membrane biophysical properties are critical to maintain cellular viability in metabolically healthy obesity. This study investigated the effect of the genetic background and dietary protein restriction on fat tissue lipid composition, adipocyte membrane fluidity and water permeability using the pig as experimental model. Twenty-four male pigs from distinct genotypes, lean and obese, were fed on normal and reduced protein diets within a 2 × 2 factorial arrangement (two genotypes and two diets). Backfat thickness was twofold higher in obese than in lean pigs but unrelated to dietary protein level. In contrast, total fatty acids in the subcutaneous adipose tissue were dependent on both breed and diet, with increased lipid content promoted by the fatty genotype and by the restriction of dietary protein. Adipose membranes isolated from obese pig's subcutaneous fat tissue showed higher permeability to water, in line with an increased fluidity. Moreover, the reduced content of dietary protein influenced positively the fluidity of adipose membranes. Neither genotype nor diet affected total cholesterol concentration in the adipose membranes. Membrane-saturated fatty acids' content was influenced by genotype, while membrane-polyunsaturated fatty acids, particularly from the n-6 family, was influenced by diet. The ratio of oleic (18:1c9)/linoleic (18:2n-6) acids was positively correlated with membrane fluidity. All together, these findings reinforce the genetic background as a determinant player on adipose membrane biophysical properties and point to the dietary protein level as an important factor for subcutaneous lipid deposition as well as for regulation of membrane function, factors that may have impact on human obesity and metabolic syndrome.     

Bronchioalveolar stem cells increase after mesenchymal stromal cell treatment in a mouse model of bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) remains a major complication of prematurity resulting in significant morbidity and mortality. The pathology of BPD is multifactorial and leads to alveolar simplification and distal lung injury. Previous studies have shown a beneficial effect of systemic treatment with bone marrow-derived mesenchymal stromal cells (MSCs) and MSC-conditioned media (MSC-CM) leading to amelioration of the lung parenchymal and vascular injury in vivo in the hyperoxia murine model of BPD. It is possible that the beneficial response from the MSCs is at least in part due to activation of endogenous lung epithelial stem cells. Bronchioalveolar stem cells (BASCs) are an adult lung stem cell population capable of self-renewal and differentiation in culture, and BASCs proliferate in response to bronchiolar and alveolar lung injury in vivo. Systemic treatment of neonatal hyperoxia-exposed mice with MSCs or MSC-CM led to a significant increase in BASCs compared with untreated controls. Treatment of BASCs with MSC-CM in culture showed an increase in growth efficiency, indicating a direct effect of MSCs on BASCs. Lineage tracing data in bleomycin-treated adult mice showed that Clara cell secretory protein-expressing cells including BASCs are capable of contributing to alveolar repair after lung injury. MSCs and MSC-derived factors may stimulate BASCs to play a role in the repair of alveolar lung injury found in BPD and in the restoration of distal lung cell epithelia. This work highlights the potential important role of endogenous lung stem cells in the repair of chronic lung diseases.     

The Effects of Incidentally Learned Temporal and Spatial Predictability on Response Times and Visual Fixations during Target Detection and Discrimination

Responses are quicker to predictable stimuli than if the time and place of appearance is uncertain. Studies that manipulate target predictability often involve overt cues to speed up response times. However, less is known about whether individuals will exhibit faster response times when target predictability is embedded within the inter-trial relationships. The current research examined the combined effects of spatial and temporal target predictability on reaction time (RT) and allocation of overt attention in a sustained attention task. Participants responded as quickly as possible to stimuli while their RT and eye movements were measured. Target temporal and spatial predictability were manipulated by altering the number of: 1) different time intervals between a response and the next target; and 2) possible spatial locations of the target. The effects of target predictability on target detection (Experiment 1) and target discrimination (Experiment 2) were tested. For both experiments, shorter RTs as target predictability increased across both space and time were found. In addition, the influences of spatial and temporal target predictability on RT and the overt allocation of attention were task dependent; suggesting that effective orienting of attention relies on both spatial and temporal predictability. These results indicate that stimulus predictability can be increased without overt cues and detected purely through inter-trial relationships over the course of repeated stimulus presentations.     

Immunogenicity of a Trivalent Human Papillomavirus L1 DNA-Encapsidated,Non-Replicable Baculovirus Nanovaccine

Previously, we developed a non-replicating recombinant baculovirus coated with human endogenous retrovirus envelope protein (AcHERV) for enhanced cellular delivery of human papillomavirus (HPV) 16L1 DNA. Here, we report the immunogenicity of an AcHERV-based multivalent HPV nanovaccine in which the L1 segments of HPV 16, 18, and 58 genes were inserted into a single baculovirus genome of AcHERV. To test whether gene expression levels were affected by the order of HPV L1 gene insertion, we compared the efficacy of bivalent AcHERV vaccines with the HPV 16L1 gene inserted ahead of the 18L1 gene (AcHERV-HP16/18L1) with that of AcHERV with the HPV 18L1 gene inserted ahead of the 16L1 gene (AcHERV-HP18/16L1). Regardless of the order, the bivalent AcHERV DNA vaccines retained the immunogenicity of monovalent AcHERV-HP16L1 and AcHERV-HP18L1 DNA vaccines. Moreover, the immunogenicity of bivalent AcHERV-HP16/18L1 was not significantly different from that of AcHERV-HP18/16L1. In challenge tests, both bivalent vaccines provided complete protection against HPV 16 and 18 pseudotype viruses. Extending these results, we found that a trivalent AcHERV nanovaccine encoding HPV 16L1, 18L1, and 58L1 genes (AcHERV-HP16/18/58L1) provided high levels of humoral and cellular immunogenicity against all three subtypes. Moreover, mice immunized with the trivalent AcHERV-based nanovaccine were protected from challenge with HPV 16, 18, and 58 pseudotype viruses. These results suggest that trivalent AcHERV-HPV16/18/58L1 could serve as a potential prophylactic baculoviral nanovaccine against concurrent infection with HPV 16, 18, and 58.     

NsdD Is a Key Repressor of Asexual Development in Aspergillus nidulans

Asexual development (conidiation) of the filamentous fungus Aspergillus nidulans occurs via balanced activities of multiple positive and negative regulators. For instance, FluG (+) and SfgA (−) govern upstream regulation of the developmental switch, and BrlA (+) and VosA (−) control the progression and completion of conidiation. To identify negative regulators of conidiation downstream of FluG-SfgA, we carried out multicopy genetic screens using sfgA deletion strains. After visually screening >100,000 colonies, we isolated 61 transformants exhibiting reduced conidiation. Responsible genes were identified as AN3152 (nsdD), AN7507, AN2009, AN1652, AN5833, and AN9141. Importantly, nsdD, a key activator of sexual reproduction, was present in 10 independent transformants. Furthermore, deletion, overexpression, and double-mutant analyses of individual genes have led to the conclusion that, of the six genes, only nsdD functions in the FluG-activated conidiation pathway. The deletion of nsdD bypassed the need for fluG and flbA∼flbE, but not brlA or abaA, in conidiation, and partially restored production of the mycotoxin sterigmatocystin (ST) in the ΔfluG, ΔflbA, and ΔflbB mutants, suggesting that NsdD is positioned between FLBs and BrlA in A. nidulans. Nullifying nsdD caused formation of conidiophores in liquid submerged cultures, where wild-type strains do not develop. Moreover, the removal of both nsdD and vosA resulted in even more abundant development of conidiophores in liquid submerged cultures and high-level accumulation of brlA messenger (m)RNA even at 16 hr of vegetative growth. Collectively, NsdD is a key negative regulator of conidiation and likely exerts its repressive role via downregulating brlA.     

Comparison of the Accuracy of Two Conventional Phenotypic Methods and Two MALDI-TOF MS Systems with That of DNA Sequencing Analysis for Correctly Identifying Clinically Encountered Yeasts

We assessed the accuracy of species-level identification of two commercially available matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems (Bruker Biotyper and Vitek MS) and two conventional phenotypic methods (Phoenix 100 YBC and Vitek 2 Yeast ID) with that of rDNA gene sequencing analysis among 200 clinical isolates of commonly encountered yeasts. The correct identification rates of the 200 yeast isolates to species or complex (Candida parapsilosis complex, C. guilliermondii complex and C. rugosa complex) levels by the Bruker Biotyper, Vitek MS (using in vitro devices [IVD] database), Phoenix 100 YBC and Vitek 2 Yeast ID (Sabouraud''s dextrose agar) systems were 92.5%, 79.5%, 89%, and 74%, respectively. An additional 72 isolates of C. parapsilosis complex and 18 from the above 200 isolates (30 in each of C. parapsilosis, C. metapsilosis, and C. orthopsilosis) were also evaluated separately. Bruker Biotyper system could accurately identify all C. parapsilosis complex to species level. Using Vitek 2 MS (IVD) system, all C. parapsilosis but none of C. metapsilosis, or C. orthopsilosis could be accurately identified. Among the 89 yeasts misidentified by the Vitek 2 MS (IVD) system, 39 (43.8%), including 27 C. orthopsilosis isolates, could be correctly identified Using the Vitek MS Plus SARAMIS database for research use only. This resulted in an increase in the rate of correct identification of all yeast isolates (87.5%) by Vitek 2 MS. The two species in C. guilliermondii complex (C. guilliermondii and C. fermentati) isolates were correctly identified by cluster analysis of spectra generated by the Bruker Biotyper system. Based on the results obtained in the current study, MALDI-TOF MS systems present a promising alternative for the routine identification of yeast species, including clinically commonly and rarely encountered yeast species and several species belonging to C. parapsilosis complex, C. guilliermondii complex, and C. rugosa complex.     

Functional and Molecular Surveillance of Helicobacter pylori Antibiotic Resistance in Kuala Lumpur

Background

Helicobacter pylori is the etiological agent for diseases ranging from chronic gastritis and peptic ulcer disease to gastric adenocarcinoma and primary gastric B-cell lymphoma. Emergence of resistance to antibiotics possesses a challenge to the effort to eradicate H. pylori using conventional antibiotic-based therapies. The molecular mechanisms that contribute to the resistance of these strains have yet to be identified and are important for understanding the evolutional pattern and selective pressure imposed by the environment.

Methods and Findings

H. pylori was isolated from 102 patients diagnosed with gastrointestinal diseases, who underwent endoscopy at University Malaya Medical Centre (UMMC). The isolates were tested for their susceptibility on eleven antibiotics using Etest. Based on susceptibility test, 32.3% of the isolates were found to have primary metronidazole resistance; followed by clarithromycin (6.8%) and fluoroquinolones (6.8%). To further investigate the resistant strains, mutational patterns of gene rdxA, frxA, gyrA, gyrB, and 23S rRNA were studied. Consistent with the previous reports, metronidazole resistance was prevalent in the local population. However, clarithromycin, fluoroquinolone and multi-drug resistance were shown to be emerging. Molecular patterns correlated well with phenotypic data. Interestingly, multi-drug resistant (MDR) strains were found to be associated with higher minimum inhibitory concentration (MIC) than their single-drug resistant (SDR) counterparts. Most importantly, clarithromycin-resistant strains were suggested to have a higher incidence for developing multi-drug resistance.

Conclusion

Data from this study highlighted the urgency to monitor closely the prevalence of antibiotic resistance in the Malaysian population; especially that of clarithromycin and multi-drug resistance. Further study is needed to understand the molecular association between clarithromycin resistance and multi-drug resistance in H. pylori. The report serves a reminder that a strict antibiotic usage policy is needed in Malaysia and other developing countries (especially those where H. pylori prevalence remained high).     

Synergistic Signaling of Tumor Cell Invasiveness by Hepatocyte Growth Factor and Hypoxia

Hepatocyte growth factor (HGF) signaling promotes tumor invasiveness in renal cell carcinoma (RCC) and other cancers. In clear cell RCC, VHL loss generates pseudohypoxia that exacerbates HGF-driven invasion through β-catenin deregulation. Hypoxia also enhances HGF-driven invasiveness by papillary RCC cells, but in the absence of VHL, loss signaling integration involves three parallel routes: 1) hypoxia-induced reactive oxygen species production and decreased DUSP2 expression, leading to enhanced mitogen-activated protein kinase (MAPK) cascade activation; 2) reactive oxygen species-induced diacylglycerol production by phospholipase Cγ, leading to protein kinase C activation and increased protein phosphatase-2A activity, thereby suppressing HGF-induced Akt activation; and 3) a profound shift from HGF-enhanced, proliferation-oriented metabolism to autophagy-dependent invasion and suppression of proliferation. This tripartite signaling integration was not unique to RCC or HGF; in RCC cells, invasive synergy induced by the combination of hypoxia and epidermal growth factor occurred through the same mechanism, and in estrogen receptor-positive breast cancer cells, this mechanism was suppressed in the absence of estrogen. These results define the molecular basis of growth factor and hypoxia invasive synergy in VHL-competent papillary RCC cells, illustrate the plasticity of invasive and proliferative tumor cell states, and provide signaling profiles by which they may be predicted.     

A New Simple Method for Improving QTL Mapping Under Selective Genotyping

The selective genotyping approach, where only individuals from the high and low extremes of the trait distribution are selected for genotyping and the remaining individuals are not genotyped, has been known as a cost-saving strategy to reduce genotyping work and can still maintain nearly equivalent efficiency to complete genotyping in QTL mapping. We propose a novel and simple statistical method based on the normal mixture model for selective genotyping when both genotyped and ungenotyped individuals are fitted in the model for QTL analysis. Compared to the existing methods, the main feature of our model is that we first provide a simple way for obtaining the distribution of QTL genotypes for the ungenotyped individuals and then use it, rather than the population distribution of QTL genotypes as in the existing methods, to fit the ungenotyped individuals in model construction. Another feature is that the proposed method is developed on the basis of a multiple-QTL model and has a simple estimation procedure similar to that for complete genotyping. As a result, the proposed method has the ability to provide better QTL resolution, analyze QTL epistasis, and tackle multiple QTL problem under selective genotyping. In addition, a truncated normal mixture model based on a multiple-QTL model is developed when only the genotyped individuals are considered in the analysis, so that the two different types of models can be compared and investigated in selective genotyping. The issue in determining threshold values for selective genotyping in QTL mapping is also discussed. Simulation studies are performed to evaluate the proposed methods, compare the different models, and study the QTL mapping properties in selective genotyping. The results show that the proposed method can provide greater QTL detection power and facilitate QTL mapping for selective genotyping. Also, selective genotyping using larger genotyping proportions may provide roughly equivalent power to complete genotyping and that using smaller genotyping proportions has difficulties doing so. The R code of our proposed method is available on http://www.stat.sinica.edu.tw/chkao/.     

Secretome analysis of Magnaporthe oryzae using in vitro systems

Magnaporthe oryzae is a devastating blast fungal pathogen of rice (Oryza sativa L.) that causes dramatic decreases in seed yield and quality. During the early stages of infection by this pathogen, the fungal spore senses the rice leaf surface, germinates, and penetrates the cell via an infectious structure known as an appressorium. During this process, M. oryzae secretes several proteins; however, these proteins are largely unknown mainly due to the lack of a suitable method for isolating secreted proteins during germination and appressoria formation. We examined the secretome of M. oryzae by mimicking the early stages of infection in vitro using a glass plate (GP), PVDF membrane, and liquid culture medium (LCM). Microscopic observation of M. oryzae growth revealed appressorium formation on the GP and PVDF membrane resembling natural M. oryzae-rice interactions; however, appresorium formation was not observed in the LCM. Secreted proteins were collected from the GP (3, 8, and 24 h), PVDF membrane (24 h), and LCM (48 h) and identified by two-dimensional gel electrophoresis (2DE) followed by tandem mass spectrometry. The GP, PVDF membrane, and LCM-derived 2D gels showed distinct protein patterns, indicating that they are complementary approaches. Collectively, 53 nonredundant proteins including previously known and novel secreted proteins were identified. Six biological functions were assigned to the proteins, with the predominant functional classes being cell wall modification, reactive oxygen species detoxification, lipid modification, metabolism, and protein modification. The in vitro system using GPs and PVDF membranes applied in this study to survey the M. oryzae secretome, can be used to further our understanding of the early interactions between M. oryzae and rice leaves.