Prognostic prediction of the immunohistochemical expression of p16 and p53 in cutaneous melanoma: a comparison of two populations from different geographical regions

p16INK4a and p53 are tumor-suppressor genes frequently altered in various malignancies, including cutaneous melanoma. The purpose of the study was to establish the prognostic value of immunohistochemical expression of p16INK4a a and p53 in sporadic cutaneous melanoma (CM) in two regions with a high-risk for melanoma in Italy and Ecuador. Immunohistochemical staining of p16 and p53 was performed in samples of primary CM from 82 patients with Stage I and II melanoma according to the American Joint Committee on Cancer (AJCC) staging system. Survival differences between categories of p16 or p53 expression were analyzed using the product-limit procedure (Kaplan-Meier method, log-rank test). Clinical variables (gender, age, tumor location, Clark's level, thickness) were correlated with survival and p16 or p53 expression. p16 nuclear immunoreactivity was observed in 85% of Italian patients compared to 48.7% of Ecuadorians; a small number of cases showed p53 immunoreactivity in both populations. Only nuclear p16 expression exhibited a significant correlation with survival (Italians p=0.001, Ecuadorians p=0.017) but did not appear to correlate with any clinicopathological parameter. No significant difference was observed in survival with regard to p53 expression or cytoplasmic p16. Our results demonstrate that nuclear expression of p16 can be considered a molecular prognostic factor in patients with sporadic CM and indicate its importance as a clinical marker.     

Allosteric activation mechanism of the alpha1beta2gamma2 gamma-aminobutyric acid type A receptor revealed by mutation of the conserved M2 leucine

A conserved leucine residue in the midpoint of the second transmembrane domain (M2) of the ligand-activated ion channel family has been proposed to play an important role in receptor activation. In this study, we assessed the importance of this leucine in the activation of rat alpha1beta2gamma2 GABA receptors expressed in Xenopus laevis oocytes by site-directed mutagenesis and two-electrode voltage clamp. The hydrophobic conserved M2 leucines in alpha1(L263), beta2(L259), and gamma2(L274) subunits were mutated to the hydrophilic amino acid residue serine and coexpressed in all possible combinations with their wild-type and/or mutant counterparts. The mutation in any one subunit decreased the EC(50) and created spontaneous openings that were blocked by picrotoxin and, surprisingly, by the competitive antagonist bicuculline. The magnitudes of the shifts in GABA EC(50) and picrotoxin IC(50) as well as the degree of spontaneous openings were all correlated with the number of subunits carrying the leucine mutation. Simultaneous mutation of the GABA binding site (beta2Y157S; increased the EC(50)) and the conserved M2 leucine (beta2L259S; decreased the EC(50)) produced receptors with the predicted intermediate agonist sensitivity, indicating the two mutations affect binding and gating independently. The results are discussed in light of a proposed allosteric activation mechanism.     

Production of haploids and doubled haploids in oil palm

Background

Studies on host-pathogen interactions in a range of pathosystems have revealed an array of mechanisms by which plants reduce the efficiency of pathogenesis. While R-gene mediated resistance confers highly effective defense responses against pathogen invasion, quantitative resistance is associated with intermediate levels of resistance that reduces disease progress. To test the hypothesis that specific loci affect distinct stages of fungal pathogenesis, a set of maize introgression lines was used for mapping and characterization of quantitative trait loci (QTL) conditioning resistance to Setosphaeria turcica, the causal agent of northern leaf blight (NLB). To better understand the nature of quantitative resistance, the identified QTL were further tested for three secondary hypotheses: (1) that disease QTL differ by host developmental stage; (2) that their performance changes across environments; and (3) that they condition broad-spectrum resistance.

Results

Among a set of 82 introgression lines, seven lines were confirmed as more resistant or susceptible than B73. Two NLB QTL were validated in BC₄F₂ segregating populations and advanced introgression lines. These loci, designated qNLB1.02 and qNLB1.06, were investigated in detail by comparing the introgression lines with B73 for a series of macroscopic and microscopic disease components targeting different stages of NLB development. Repeated greenhouse and field trials revealed that qNLB1.06 _Tx303 (the Tx303 allele at bin 1.06) reduces the efficiency of fungal penetration, while qNLB1.02 _B73 (the B73 allele at bin 1.02) enhances the accumulation of callose and phenolics surrounding infection sites, reduces hyphal growth into the vascular bundle and impairs the subsequent necrotrophic colonization in the leaves. The QTL were equally effective in both juvenile and adult plants; qNLB1.06 _Tx303 showed greater effectiveness in the field than in the greenhouse. In addition to NLB resistance, qNLB1.02 _B73 was associated with resistance to Stewart's wilt and common rust, while qNLB1.06 _Tx303 conferred resistance to Stewart's wilt. The non-specific resistance may be attributed to pleiotropy or linkage.

Conclusions

Our research has led to successful identification of two reliably-expressed QTL that can potentially be utilized to protect maize from S. turcica in different environments. This approach to identifying and dissecting quantitative resistance in plants will facilitate the application of quantitative resistance in crop protection.     

iPathology cockpit diagnostic station: validation according to College of American Pathologists Pathology and Laboratory Quality Center recommendation at the Hospital Trust and University of Verona

Background

Validation of digital whole slide images is crucial to ensure that diagnostic performance is at least equivalent to that of glass slides and light microscopy. The College of American Pathologists Pathology and Laboratory Quality Center recently developed recommendations for internal digital pathology system validation. Following these guidelines we sought to validate the performance of a digital approach for routine diagnosis by using an iPad and digital control widescreen-assisted workstation through a pilot study.

Methods

From January 2014, 61 histopathological slides were scanned by ScanScope Digital Slides Scanner (Aperio, Vista, CA). Two independent pathologists performed diagnosis on virtual slides in front of a widescreen by using two computer devices (ImageScope viewing software) located to different Health Institutions (AOUI Verona) connected by local network and a remote image server using an iPad tablet (Aperio, Vista, CA), after uploading the Citrix receiver for iPad. Quality indicators related to image characters and work-flow of the e-health cockpit enterprise system were scored based on subjective (high vs poor) perception. The images were re-evaluated two weeks apart.

Results

The whole glass slides encountered 10 liver: hepatocarcinoma, 10 renal carcinoma, 10 gastric carcinoma and 10 prostate biopsies: adenocarcinoma, 5 excisional skin biopsies: melanoma, 5 lymph-nodes: lymphoma. 6 immuno- and 5 special stains were available for intra- and internet remote viewing. Scan times averaged two minutes and 54 seconds per slide (standard deviation 2 minutes 34 seconds). Megabytes ranged from 256 to 680 (mean 390) per slide storage. Reliance on glass slide, image quality (resolution and color fidelity), slide navigation time, simultaneous viewers in geographically remote locations were considered of high performance score. Side by side comparisons between diagnosis performed on tissue glass slides versus widescreen were excellent showing an almost perfect concordance (0.81, kappa index).

Conclusions

We validated our institutional digital pathology system for routine diagnostic facing with whole slide images in a cockpit enterprise digital system or iPad tablet. Computer widescreens are better for diagnosing scanned glass slide that iPad. For urgent requests, iPad may be used. Legal aspects have to be soon faced with to permit the clinical use of this technology in a manner that does not compromise patient care.

     

The ABC transporter-encoding gene AFR1 affects the resistance of Cryptococcus neoformans to microglia-mediated antifungal activity by delaying phagosomal maturation

The pathogenic yeast Cryptococcus neoformans has evolved several strategies to survive within phagocytes. Recently, it has been demonstrated that upregulation of the ATP binding cassette transporter-encoding gene antifungal resistance 1 ( AFR1 ) is important not only for determining the resistance of C. neoformans to fluconazole but also in influencing fungal virulence. In the present study, we showed that the fluconazole-resistant AFR1- overexpressing mutant strain was not sensitive to microglia-mediated anticryptococcal activity, as compared with the fluconazole-susceptible isogenic strains, the wild type and the afr1 Δ mutant. Interestingly, although the three strains were phagocytosed to a similar extent, reduced acidification and delayed maturation were observed in phagosomes containing the AFR1 -overexpressing strain with respect to the others. These findings provide the first evidence that upregulation of the AFR1 gene affects C. neoformans –microglia interplay, adding insights to the complexity of cryptococcal virulence and to its unexpected link with azole resistance.     

Role of pressor mechanisms from the NTS and CVLM in control of arterial pressure

In the present study, we investigated the effects of inhibition of the caudal ventrolateral medulla (CVLM) with the GABA(A) agonist muscimol combined with the blockade of glutamatergic mechanism in the nucleus of the solitary tract (NTS) with kynurenic acid (kyn) on mean arterial pressure (MAP), heart rate (HR), and regional vascular resistances. In male Holtzman rats anesthetized intravenously with urethane/chloralose, bilateral injections of muscimol (120 pmol) into the CVLM or bilateral injections of kyn (2.7 nmol) into the NTS alone increased MAP to 186 +/- 11 and to 142 +/- 6 mmHg, respectively, vs. control: 105 +/- 4 mmHg; HR to 407 +/- 15 and to 412 +/- 18 beats per minute (bpm), respectively, vs. control: 352 +/- 12 bpm; and renal, mesenteric and hindquarter vascular resistances. However, in rats with the CVLM bilaterally blocked by muscimol, additional injections of kyn into the NTS reduced MAP to 88 +/- 5 mmHg and mesenteric and hindquarter vascular resistances below control baseline levels. Moreover, in rats with the glutamatergic mechanisms of the NTS blocked by bilateral injections of kyn, additional injections of muscimol into the CVLM also reduced MAP to 92 +/- 2 mmHg and mesenteric and hindquarter vascular resistances below control baseline levels. Simultaneous blockade of NTS and CVLM did not modify the increase in HR but also abolished the increase in renal vascular resistance produced by each treatment alone. The results suggest that important pressor mechanisms arise from the NTS and CVLM to control vascular resistance and arterial pressure under the conditions of the present study.