In situ hybridization detection of homeobox genes reveals distinct expression patterns in oral squamous cell carcinomas

Aims: To analyse the expression of three homeobox genes (HOXA7, PITX1 and PRRX1) in oral squanous cell carcinomas (OSCC) and the relationship of such expression to certain distinct histopathological features of OSCC and in comparison to adjacent non-neoplastic epithelium (NT). Methods and results: Digoxigenin-labelled riboprobes that are specific for each homeobox gene were generated and in situ hybridization was carried out on frozen sections. In NT samples, HOXA7 and PITX1 transcripts were found more frequently in all epithelial layers, while PRRX1 was expressed in the basal layer. With OSCC samples, expression of the three genes was associated with all histological features. However, the HOXA7 and PITX1 signals were more intense in sheets and nests and PRRX1 in small nests and isolated cells. Conclusion: HOXA7, PIXT1 and PRRX1 homeobox genes have different patterns of expression in OSCC depending on its histological features.

Identification of intracellular and extracellular domains mediating signal transduction in the inhibitory glycine receptor chloride channel

Fast synaptic neurotransmission is mediated by transmitter-activated conformational changes in ligand-gated ion channel receptors, culminating in opening of the integral ion channel pore. Human hereditary hyperekplexia, or startle disease, is caused by mutations in both the intracellular or extracellular loops flanking the pore-lining M2 domain of the glycine receptor alpha 1 subunit. These flanking domains are designated the M1-M2 loop and the M2-M3 loop respectively. We show that four startle disease mutations and six additional alanine substitution mutations distributed throughout both loops result in uncoupling of the ligand binding sites from the channel activation gate. We therefore conclude that the M1-M2 and M2-M3 loops act in parallel to activate the channel. Their locations strongly suggest that they act as hinges governing allosteric control of the M2 domain. As the members of the ligand-gated ion channel superfamily share a common structure, this signal transduction model may apply to all members of this superfamily.

Immunohistochemical detection of polyductin and co-localization with liver progenitor cell markers during normal and abnormal development of the intrahepatic biliary system and in adult hepatobiliary carcinomas

The longest open reading frame of PKHD1 (polycystic kidney and hepatic disease 1), the autosomal recessive polycystic kidney disease (ARPKD) gene, encodes a single-pass, integral membrane protein named polyductin or fibrocystin. A fusion protein comprising its intracellular C-terminus, FP2, was previously used to raise a polyclonal antiserum shown to detect polyductin in several human tissues, including liver. In the current study, we aimed to investigate by immunohistochemistry the detailed polyductin localization pattern in normal (ductal plate [DP], remodelling ductal plate [RDP], remodelled bile ducts) and abnormal development of the primitive intrahepatic biliary system, known as ductal plate malformation (DPM). This work also included the characterization of polyductin expression profile in various histological forms of neonatal and infantile cholestasis, and in cholangiocellular carcinoma (CCC) and hepatocellular carcinoma (HCC). We detected polyductin expression in the intrahepatic biliary system during the DP and the RDP stages as well as in DPM. No specific staining was found at the stage of remodelled bile ducts. Polyductin was also detected in liver biopsies with neonatal cholestasis, including mainly biliary atresia and neonatal hepatitis with ductular reaction as well as congenital hepatic fibrosis. In addition, polyductin was present in CCC, whereas it was absent in HCC. Polyductin was also co-localized in some DP cells together with oval stem cell markers. These results represent the first systematic study of polyductin expression in human pathologies associated with abnormal development of intrahepatic biliary tree, and support the following conclusions: (i) polyductin expression mirrors developmental properties of the primitive intrahepatic biliary system; (ii) polyductin is re-expressed in pathological conditions associated with DPM and (iii) polyductin might be a potential marker to distinguish CCC from HCC.

Analysis of Different Staining Techniques for C4d Detection in Renal Allograft Biopsies

Capillary C4d deposition has been recognized as a marker of antibody-mediated rejection (AMR). Although the detection of capillary C4d by means of immunofluorescence (IF) in cryostat sections is well established, frozen tissue is not always available, thus limiting the diagnosis of AMR. The aim of the present study was to analyze different techniques for C4d staining and the prevalence of C4d in renal allograft biopsies. Detection of C4d was carried out using IF or immunohistochemistry (IHC) on frozen and paraffin sections of renal allograft biopsies available from the same patients. Biopsies obtained from 20 patients were classified into 3 groups: no rejection, acute rejection, and chronic allograft nephropathy (CAN). The capillary C4d deposition prevalence in frozen-IF, considered the gold standard technique for C4d detection, was 45% (9/20 cases). Compared with frozen-IF, the frozen-IHC technique presented an 85% concordance rate (17/20 cases; r =.70; P <.001; sensitivity = 77.8%; specificity = 90.9%). The paraffin-IF technique showed similar results, with an 80% concordance rate (16/20 cases; r =.64; P <.005; sensitivity = 55.6%; specificity = 100%), whereas C4d detection occurred in only 65% of paraffin-IHC cases (13/20; r =.30; not significant; sensitivity = 66.7%; specificity = 63.6%). No capillary C4d deposition was detected in cases without evidence of rejection. However, 4/7 cases (57%) of acute rejection were C4d positive. In the CAN group, 5111 cases (45%) were C4d positive. In conclusion, these results demonstrated that frozen-IHC and paraffin-IF can be considered alternative techniques to frozen-IF for C4d detection. The paraffin-IHC technique displayed the lowest concordance rate for C4d detection.

LOSS OF CD40 ENDOGENOUS S-NITROSYLATION DURING INFLAMMATORY RESPONSE IN ENDOTOXEMIC MICE AND PATIENTS WITH SEPSIS

Signal transduction through the surface molecule CD40 is critical for cellular activation in immunoinflammatory states such as sepsis. The mechanisms regulating this pathway are not completely understood. Because CD40 displays potentially regulatory cysteine residues and CD40 is probably exposed to NO in the inflammatory milieu, we hypothesized that S-nitrosylation, the interaction of NO with cysteines residues, acts as a post-translational modification on CD40, coregulating the signaling activity and, therefore, the level of cellular activation. As assessed by the biotin switch and the reduction/chemiluminescence S-nitrosylation detection techniques, CD40 was found to be S-nitrosylated endogenously and upon exposure to NO donors in both human and murine macrophages. S-nitrosylation of CD40 was associated with milder activation by its ligand (CD40L), leading to reduced in vitro cytokine (IL-1 beta, IL-12, and TNF-alpha) production, which was reversed in the presence of inhibitors of NO synthesis. S-nitrosylated CD40 was found in resting RAW 246.7 macrophages and BALB/c mice peritoneal macrophages, turning into the denitrosylated state upon in vitro or systemic exposure, respectively, to LPS. Moreover, monocytes from patients with sepsis displayed denitrosylated CD40 in contrast to the CD40 S-nitrosylation measured in healthy individuals. Finally, in an attempt to explain how S-nitrosylation regulates CD40 activation, we demonstrate that NO affects the redistribution of CD40 on the cell surface, which is a requirement for optimal signal transduction. Our results support a novel post-translational regulatory mechanism in which the CD40 signal may be, at least in part, dependent on cellular activation-induced receptor denitrosylation.

PCR-mediated detection of acidophilic, bioleaching-associated bacteria

The detection of acidophilic microorganisms from mining environments by culture methods is time consuming and unreliable. Several PCR approaches were developed to amplify small-subunit rRNA sequences from the DNA of six bacterial phylotypes associated with acidic mining environments, permitting the detection of the target DNA at concentrations as low as 10 fg.

Observer agreement of spine stenosis on magnetic resonance imaging analysis of patients with cervical spine myelopathy

Objectives: The purpose of this study was to measure the intraobserver and interobserver reliability of magnetic resonance detection of cervical spondylotic myelopathy with and without operational guidelines. Methods: Seven radiologists examined images from 10 patients with cord signal abnormalities and clinical signs of myelopathy. Radiologist examined films twice, with and without operational guidelines designed to define stenotic changes, while blinded to the clinical findings of the patients. Analyses included a Fleiss kappa assessment of intraobserver and interobserver reliability. Results: Results demonstrated high percentage of agreement and strong intraobserver reliability and variable Fleiss kappa, values for interobserver assessment. Operational guidelines did not improve the intraobserver or interobserver agreement. Conclusion: Although the percentage of agreement was high in some cases, the kappa agreement was low-most likely a result of the base rate problem of a kappa analysis. Sample bias toward severe degenerative changes resulted in highly prevalent selections and kappa adjusted values. Nonetheless, the results do suggest that substantial intraobserver kappa agreement and a wide range of interobserver kappa agreement exists among trained radiologists during detection of stenotic changes associated with cervical spondylotic myelopathy.

Renal Cell Carcinoma: T1 and T2 Signal Intensity Characteristics of Papillary and Clear Cell Types Correlated with Pathology

OBJECTIVE. The objective of our study was to describe the T1 and T2 signal intensity characteristics of papillary renal cell carcinoma (RCC) and clear cell RCC with pathologic correlation. MATERIALS AND METHODS. Of 539 RCCs, 49 tumors (21 papillary RCCs and 28 clear cell RCCs) in 45 patients were examined with MRI. Two radiologists retrospectively and independently assessed each tumor`s T1 and T2 signal intensity qualitatively and quantitatively (i.e., the signal intensity [SI] ratio [tumor SI/renal cortex SI]). Of the 49 tumors, 37 (76%) were assessed for pathology features including tumor architecture and the presence of hemosiderin, ferritin, necrosis, and fibrosis. MRI findings and pathology features were correlated. Statistical methods included summary statistics and Wilcoxon`s rank sum test for signal intensity, contingency tables for assessing reader agreement, concordance rate between the two readers with 95% CIs, and Fisher`s exact test for independence, all stratified by RCC type. RESULTS. Papillary RCCs and clear cell RCCs had a similar appearance and signal intensity ratio on T1-weighted images. On T2-weighted images, most papillary RCCs were hypointense (reader 1, 13/21; reader 2, 14/21), with an average mean signal intensity ratio for both readers of 0.67 +/- 0.2, and none was hyperintense, whereas most clear cell RCCs were hyperintense (reader 1, 21/28; reader 2, 17/28), with an average mean signal intensity ratio for both readers of 1.41 +/- 0.4 (p < 0.05). A tumor T2 signal intensity ratio of <= 0.66 had a specificity of 100% and sensitivity of 54% for papillary RCC. Most T2 hypointense tumors exhibited predominant papillary architecture; most T2 hyperintense tumors had a predominant nested architecture (p < 0.05). CONCLUSION. On T2-weighted images, most papillary RCCs are hypointense and clear cell RCCs, hyperintense. The T2 hypointense appearance of papillary RCCs correlated with a predominant papillary architecture at pathology.

Infrared seeded parametric four-wave mixing for sensitive detection of molecules

We have developed a sensitive resonant four-wave mixing technique based on two-photon parametric four-wave mixing with the addition of a phase matched ''seeder'' field. Generation of the seeder field via the same four-wave mixing process in a high pressure cell enables automatic phase matching to be achieved in a low pressure sample cell. This arrangement facilitates sensitive detection of complex molecular spectra by simply tuning the pump laser. We demonstrate the technique with the detection of nitric oxide down to concentrations more than 4 orders of magnitude below the capability of parametric four-wave mixing alone, with an estimated detection threshold of 10(12) molecules/cm(3).

The gaps-in-noise test: Gap detection thresholds in normal-hearing young adults

The aim of this study was to establish parameters for the gaps-in-noise test in normal-hearing young adults. One hundred subjects (50 males and 50 females) received an audiological evaluation to rule out hearing loss and auditory processing disorder. The gaps-in-noise test was then conducted on all subjects. The mean gap detection threshold was 4.19 ms. A psychometric function by gap duration was constructed, revealing that the percentage of correct responses was less than or equal to 5% for a gap duration of 2 ms, 10-30% for a gap duration of 3 ms, 60-70% for a gap duration of 4 ms, and over 96% for gap durations of 5 ms or longer. The results suggest that the data obtained can be applied as reference values for future testing. In the subjects evaluated, the gaps-in-noise test proved to be consistent with low variability.

Substrate-bound carbohydrates stimulate signal transduction and neurite outgrowth in an olfactory neuron cell line

SUBPOPULATIONS of olfactory receptor neurons, which are dispersed throughout the olfactory neuroepithelium, express specific cell surface carbohydrates and project to discrete regions of the olfactory bulb. Cell surface carbohydrates such as N-acetyl-lactosamine have been postulated to mediate sorting and selective fasciculation of discrete axon subpopulations during development of the olfactory pathway. Substrate-bound N-acetyl-lactosamine promotes neurite outgrowth by both clonal olfactory receptor neuron cell lines and olfactory receptor neurons in vitro, indicating that cell surface carbohydrates may be ligands for receptor-mediated stimulation of axon growth in vivo. In the present study, the role of transmembrane signaling in N-acetyl-lactosamine-stimulated neurite outgrowth was examined in the clonal olfactory neuron cell line 4.4.2. Substrate-bound N-acetyl-lactosamine stimulated neurite outgrowth which was specifically inhibited by antagonists to N- and L-type calcium channels and to tyrosine kinase phosphorylation. These results indicate that N-acetyl-lactosamine can evoke transmembrane receptor-mediated responses capable of influencing neurite outgrowth.

Detection of nosocomial malnutrition is improved in Amazon region by a standard clinical nutrition education program

Background: In Brazil hospital malnutrition is highly prevalent. physician awareness of malnutrition is low, and nutrition therapy is underprescribed. One alternative to approach this problem is to educate health care providers in clinical nutrition. The present study aims to evaluate the effect of an intensive education course given to health care professionals and students on the diagnosis ability concerning to hospital malnutrition. Materials and methods: An intervention study based on a clinical nutrition educational program, offered to medical and nursing students and professionals, was held in a hospital of the Amazon region. Participants were evaluated through improvement of diagnostic ability, according to agreement of malnutrition diagnosis using Subjective Global Assessment before and after the workshop, as compared to independent evaluations (Kappa Index, k). To evaluate the impact of the educational intervention on the hospital malnutrition diagnosis, medical records were reviewed for documentation of parameters associated with nutritional status of in-patients. The SPSS statistical software package was used for data analysis. Results: A total of 165 participants concluded the program. The majority (76.4%) were medical and nursing students. Malnutrition diagnosis improved after the course (before k = 0.5; after k = 0.64; p < 0.05). A reduction of false negatives from 50% to 33.3% was observed. During the course, concern of nutritional diagnosis was increased W = 17.57; p < 0.001) and even after the course, improvement on the height measurement was detected chi(2) 12.87;p < 0.001). Conclusions: Clinical nutrition education improved the ability of diagnosing malnutrition; however the primary impact was on medical and nursing students. To sustain diagnostic capacity a clinical nutrition program should be part of health professional curricula and be coupled with continuing education for health care providers.

The rms1 mutant of pea has elevated indole-3-acetic acid levels and reduced root-sap zeatin riboside content but increased branching controlled by graft-transmissible signal(s)

Rms1 is one of the series of five ramosus loci in pea (Pisum sativum L.) in which recessive mutant alleles confer increased branching at basal and aerial vegetative nodes. Shoots of the nonallelic rms1 and rms2 mutants are phenotypically similar in most respects. However, we found an up to 40-fold difference in root-sap zeatin riboside ([9R]Z) concentration between rms1 and rms2 plants. Compared with wild-type (WT) plants, the concentration of [9R]Z in rms1 root sap was very low and the concentration in rms2 root sap was slightly elevated. To our knowledge, the rms1 mutant is therefore the second ramosus mutant (rms4 being the first) to be characterized with low root-sap [9R]Z content. Like rms2, the apical bud and upper nodes of rms1 plants contain elevated indole-3-acetic acid levels compared with WT shoots. Therefore, the rms1 mutant demonstrates that high shoot auxin levels and low root-sap cytokinin levels are not necessarily correlated with increased apical dominance in pea. A graft-transmissible basis of action has been demonstrated for both mutants from reciprocal grafts between mutant and WT plants. Branching was also largely inhibited in rms1 shoots when grafted to rms2 rootstocks, but was not inhibited in rms2 shoots grafted to rms1 rootstocks. These grafting results are discussed, along with the conclusion that hormone-like signals other than auxin and cytokinin are also involved.

SUITABILITY OF A RAPID DNA ISOLATION AND AMPLIFICATION FOR DETECTION OF TRYPANOSOMA CRUZI IN TRIATOMA INFESTANS DRY FECAL SPOTS COLLECTED ON FILTER PAPER

A rapid DNA extraction was used for T. cruzi detection in triatomines dry fecal spots collected on filter paper and analyzed by PCR. Fifty T infestans were fed on experimentally infected Balb/C mice with high T. cruzi parasitemia and divided into five groups of len triatomines, and 100 triatomines were infected with lower parasitemia and divided into five groups of 20 triatomines, One dry fecal spot was analyzed per group on days 1, 2, 3, 4 and 5 post feeding. Amplification targeted T. cruzi TCZ sequence and resulted positive from day 4 after bugs feeding in the two models (high and lower parasitemia). The rapid DNA isolation and PCR proposed are suitable for detection of T. cruzi DNA in in filter paper and should be considered in field research.