Losses of both products of the Cdkn2a/Arf locus contribute to asbestos-induced mesothelioma development and cooperate to accelerate tumorigenesis

The CDKN2A/ARF locus encompasses overlapping tumor suppressor genes p16(INK4A) and p14(ARF), which are frequently co-deleted in human malignant mesothelioma (MM). The importance of p16(INK4A) loss in human cancer is well established, but the relative significance of p14(ARF) loss has been debated. The tumor predisposition of mice singly deficient for either Ink4a or Arf, due to targeting of exons 1α or 1β, respectively, supports the idea that both play significant and nonredundant roles in suppressing spontaneous tumors. To further test this notion, we exposed Ink4a(+/-) and Arf(+/-) mice to asbestos, the major cause of MM. Asbestos-treated Ink4a(+/-) and Arf(+/-) mice showed increased incidence and shorter latency of MM relative to wild-type littermates. MMs from Ink4a(+/-) mice exhibited biallelic inactivation of Ink4a, loss of Arf or p53 expression and frequent loss of p15(Ink4b). In contrast, MMs from Arf(+/-) mice exhibited loss of Arf expression, but did not require loss of Ink4a or Ink4b. Mice doubly deficient for Ink4a and Arf, due to deletion of Cdkn2a/Arf exon 2, showed accelerated asbestos-induced MM formation relative to mice deficient for Ink4a or Arf alone, and MMs exhibited biallelic loss of both tumor suppressor genes. The tumor suppressor function of Arf in MM was p53-independent, since MMs with loss of Arf retained functional p53. Collectively, these in vivo data indicate that both CDKN2A/ARF gene products suppress asbestos carcinogenicity. Furthermore, while inactivation of Arf appears to be crucial for MM pathogenesis, the inactivation of both p16(Ink4a) and p19(Arf) cooperate to accelerate asbestos-induced tumorigenesis.     

Intranasal immunization with recombinant HA and mast cell activator C48/80 elicits protective immunity against 2009 pandemic H1N1 influenza in mice

Background

Pandemic influenza represents a major threat to global health. Vaccination is the most economic and effective strategy to control influenza pandemic. Conventional vaccine approach, despite being effective, has a number of major deficiencies including limited range of protection, total dependence on embryonated eggs for production, and time consuming for vaccine production. There is an urgent need to develop novel vaccine strategies to overcome these deficiencies.

Methodology/Principal Findings

The major objective of this work was to develop a novel vaccine strategy combining recombinant haemagglutinin (HA) protein and a master cell (MC) activator C48/80 for intranasal immunization. We demonstrated in BALB/c mice that MC activator C48/80 had strong adjuvant activity when co-administered with recombinant HA protein intranasally. Vaccination with C48/80 significantly increased the serum IgG and mucosal surface IgA antibody responses against HA protein. Such increases correlated with stronger and durable neutralizing antibody activities, offering protection to vaccinated animals from disease progression after challenge with lethal dose of A/California/04/2009 live virus. Furthermore, protected animals demonstrated significant reduction in lung virus titers, minimal structural alteration in lung tissues as well as higher and balanced production of Th1 and Th2 cytokines in the stimulated splenocytes when compared to those without C48/80.

Conclusions/Significance

The present study demonstrates that the novel vaccine approach of combining recombinant HA and mucosal adjuvant C48/80 is safe and effective in eliciting protective immunity in mice. Future studies on the mechanism of action of C48/80 and potential combination with other vaccine strategies such as prime and boost approach may help to induce even more potent and broad immune responses against viruses from various clades.     

Effects of simulated microgravity on embryonic stem cells

There have been many studies on the biological effects of simulated microgravity (SMG) on differentiated cells or adult stem cells. However, there has been no systematic study on the effects of SMG on embryonic stem (ES) cells. In this study, we investigated various effects (including cell proliferation, cell cycle distribution, cell differentiation, cell adhesion, apoptosis, genomic integrity and DNA damage repair) of SMG on mouse embryonic stem (mES) cells. Mouse ES cells cultured under SMG condition had a significantly reduced total cell number compared with cells cultured under 1 g gravity (1G) condition. However, there was no significant difference in cell cycle distribution between SMG and 1G culture conditions, indicating that cell proliferation was not impaired significantly by SMG and was not a major factor contributing to the total cell number reduction. In contrast, a lower adhesion rate cultured under SMG condition contributed to the lower cell number in SMG. Our results also revealed that SMG alone could not induce DNA damage in mES cells while it could affect the repair of radiation-induced DNA lesions of mES cells. Taken together, mES cells were sensitive to SMG and the major alterations in cellular events were cell number expansion, adhesion rate decrease, increased apoptosis and delayed DNA repair progression, which are distinct from the responses of other types of cells to SMG.     

Molecular characterization of a Fus3/Kss1 type MAPK from Puccinia striiformis f. sp. tritici, PsMAPK1

Puccinia striiformis f. sp. tritici (Pst) is an obligate biotrophic fungus that causes the destructive wheat stripe rust disease worldwide. Due to the lack of reliable transformation and gene disruption method, knowledge about the function of Pst genes involved in pathogenesis is limited. Mitogen-activated protein kinase (MAPK) genes have been shown in a number of plant pathogenic fungi to play critical roles in regulating various infection processes. In the present study, we identified and characterized the first MAPK gene PsMAPK1 in Pst. Phylogenetic analysis indicated that PsMAPK1 is a YERK1 MAP kinase belonging to the Fus3/Kss1 class. Single nucleotide polymerphisms (SNPs) and insertion/deletion were detected in the coding region of PsMAPK1 among six Pst isolates. Real-time RT-PCR analyses revealed that PsMAPK1 expression was induced at early infection stages and peaked during haustorium formation. When expressed in Fusarium graminearum, PsMAPK1 partially rescued the map1 mutant in vegetative growth and pathogenicity. It also partially complemented the defects of the Magnaporthe oryzae pmk1 mutant in appressorium formation and plant infection. These results suggest that F. graminearum and M. oryzae can be used as surrogate systems for functional analysis of well-conserved Pst genes and PsMAPK1 may play a role in the regulation of plant penetration and infectious growth in Pst.     

Rosiglitazone inhibits transforming growth factor-β1 mediated fibrogenesis in ADPKD cyst-lining epithelial cells

Background

Interstitial fibrosis plays an important role in progressive renal dysfunction in autosomal dominant polycystic kidney disease (ADPKD). In our previous studies, we confirmed that PPAR-γ agonist, rosiglitazone could protect renal function and prolong the survival of a slowly progressive ADPKD animal model by reducing renal fibrosis. However, the mechanism remains unknown.

Methods

Primary culture epithelial cells pretreated with TGF-β1 were incubated with rosiglitazone. Extracellular matrix proteins were detected using real-time PCR and Western blotting. MAPK and Smad2 phosphorylation were measured with western blot. ERK1/2 pathway and P38 pathway were inhibited with the specific inhibitors PD98059 and SB203580. The Smad2 pathway was blocked with the siRNA. To address whether PPAR-γ agonist-mediated inhibition of TGF-β1–induced collagen type I expression was mediated through a PPAR-γ dependent mechanism, genetic and pharmaceutical approaches were used to block the activity of endogenous PPARγ.

Results

TGF-β1-stimulated collagen type I and fibronectin expression of ADPKD cyst-lining epithelia were inhibited by rosiglitazone in a dosage-dependent manner. Smad2, ERK1/2 and P38 pathways were activated in response to TGF-β1; however, TGF-β1 had little effect on JNK pathway. Rosiglitazone suppressed TGF-β1 induced Smad2 activation, while ERK1/2 and P38MAPK signals remained unaffected. Rosiglitazone could also attenuate TGF-β1-stimulated collagen type I and fibronectin expression in primary renal tubular epithelial cells, but had no effect on TGF-β1–induced activation of Smad2, ERK1/2 and P38 pathways. There was no crosstalk between the Smad2 and MAPK pathways in ADPKD cyst-lining epithelial cells. These inhibitory effects of rosiglitazone were reversed by the PPARγ specific antagonist GW9662 and PPARγ siRNA.

Conclusion

ADPKD cyst-lining epithelial cells participate in TGF-β1 mediated fibrogenesis. Rosiglitazone could suppress TGF-β1–induced collagen type I and fibronectin expression in ADPKD cyst-lining epithelia through modulation of the Smad2 pathway. Our study may provide therapeutic basis for clinical applications of rosiglitazone in retarding the progression of ADPKD.     

Genomic polymorphism of the pandemic A (H1N1) influenza viruses correlates with viral replication, virulence, and pathogenicity in vitro and in vivo

The novel pandemic A (H1N1) virus was first identified in Mexico in April 2009 and quickly spread worldwide. Like all influenzas, the H1N1 strain-specific properties of replication, virulence, and pathogenicity are a result of the particular genomic sequence and concerted expression of multiple genes. Thus, specific mutations may support increased virulence and may be useful as biomarkers of potential threat to human health. We performed comparative genomic analysis of ten strains of the 2009 pandemic A (H1N1) influenza viruses to determine whether genotypes associated with clinical phenotypes, which ranged from mild to severe illness and up to lethal. Virus replication capacity was tested for each strain in vitro using cultured epithelial cells, while virulence and pathogenicity were investigated in vivo using the BALB/c mouse model. The results indicated that A/Sichuan/1/2009 strain had significantly higher replication ability and virulence than the other strains, and five unique non-synonymous mutations were identified in important gene-encoding sequences. These mutations led to amino acid substitutions in HA (L32I), PA (A343T), PB1 (K353R and T566A), and PB2 (T471M), and may be critical molecular determinants for replication, virulence, and pathogenicity. Our results suggested that the replication capacity in vitro and virulence in vivo of the 2009 pandemic A (H1N1) viruses were not associated with the clinical phenotypes. This study offers new insights into the transmission and evolution of the 2009 pandemic A (H1N1) virus.     

Serum early prostate cancer antigen (EPCA) level and its association with disease progression in prostate cancer in a Chinese population

Background

Early prostate cancer antigen (EPCA) has been shown a prostate cancer (PCa)-associated nuclear matrix protein, however, its serum status and prognostic power in PCa are unknown. The goals of this study are to measure serum EPCA levels in a cohort of patients with PCa prior to the treatment, and to evaluate the clinical value of serum EPCA.

Methods

Pretreatment serum EPCA levels were determined with an ELISA in 77 patients with clinically localized PCa who underwent radical prostatectomy and 51 patients with locally advanced or metastatic disease who received primary androgen deprivation therapy, and were correlated with clinicopathological variables and disease progression. Serum EPCA levels were also examined in 40 healthy controls.

Results

Pretreatment mean serum EPCA levels were significantly higher in PCa patients than in controls (16.84±7.60 ng/ml vs. 4.12±2.05 ng/ml, P<0.001). Patients with locally advanced and metastatic PCa had significantly higher serum EPCA level than those with clinically localized PCa (22.93±5.28 ng/ml and 29.41±8.47 ng/ml vs. 15.17±6.03 ng/ml, P = 0.014 and P<0.001, respectively). Significantly elevated EPCA level was also found in metastatic PCa compared with locally advanced disease (P<0.001). Increased serum EPCA levels were significantly and positively correlated with Gleason score and clinical stage, but not with PSA levels and age. On multivariate analysis, pretreatment serum EPCA level held the most significantly predictive value for the biochemical recurrence and androgen-independent progression among pretreatment variables (HR = 4.860, P<0.001 and HR = 5.418, P<0.001, respectively).

Conclusions

Serum EPCA level is markedly elevated in PCa. Pretreatment serum EPCA level correlates significantly with the poor prognosis, showing prediction potential for PCa progression.     

The mitochondrial genome of Baylisascaris procyonis

Background

Baylisascaris procyonis (Nematoda: Ascaridida), an intestinal nematode of raccoons, is emerging as an important helminthic zoonosis due to serious or fatal larval migrans in animals and humans. Despite its significant veterinary and public health impact, the epidemiology, molecular ecology and population genetics of this parasite remain largely unexplored. Mitochondrial (mt) genomes can provide a foundation for investigations in these areas and assist in the diagnosis and control of B. procyonis. In this study, the first complete mt genome sequence of B. procyonis was determined using a polymerase chain reaction (PCR)-based primer-walking strategy.

Methodology/Principal Findings

The circular mt genome (14781 bp) of B. procyonis contained 12 protein-coding, 22 transfer RNA and 2 ribosomal RNA genes congruent with other chromadorean nematodes. Interestingly, the B. procyonis mtDNA featured an extremely long AT-rich region (1375 bp) and a high number of intergenic spacers (17), making it unique compared with other secernentean nematodes characterized to date. Additionally, the entire genome displayed notable levels of AT skew and GC skew. Based on pairwise comparisons and sliding window analysis of mt genes among the available 11 Ascaridida mtDNAs, new primer pairs were designed to amplify specific short fragments of the genes cytb (548 bp fragment) and rrnL (200 bp fragment) in the B. procyonis mtDNA, and tested as possible alternatives to existing mt molecular beacons for Ascaridida. Finally, phylogenetic analysis of mtDNAs provided novel estimates of the interrelationships of Baylisasaris and Ascaridida.

Conclusions/Significance

The complete mt genome sequence of B. procyonis sequenced here should contribute to molecular diagnostic methods, epidemiological investigations and ecological studies of B. procyonis and other related ascaridoids. The information will be important in refining the phylogenetic relationships within the order Ascaridida and enriching the resource of markers for systematic, population genetic and evolutionary biological studies of parasitic nematodes of socio-economic importance.