Uncovering the neural control of laryngeal activity and subglottic pressure in anaesthetized rats: insights from mesencephalic regions.

To assess the possible interactions between the dorsolateral periaqueductal gray matter (dlPAG) and the different domains of the nucleus ambiguus (nA), we have examined the pattern of double-staining c-Fos/FoxP2 protein immunoreactivity (c-Fos-ir/FoxP2-ir) and tyrosine hydroxylase (TH) throughout the rostrocaudal extent of nA in spontaneously breathing anaesthetised male Sprague-Dawley rats during dlPAG electrical stimulation. Activation of the dlPAG elicited a selective increase in c-Fos-ir with an ipsilateral predominance in the somatas of the loose (p < 0.05) and compact formation (p < 0.01) within the nA and confirmed the expression of FoxP2 bilaterally in all the domains within the nA. A second group of experiments was made to examine the importance of the dlPAG in modulating the laryngeal response evoked after electrical or chemical (glutamate) dlPAG stimulations. Both electrical and chemical stimulations evoked a significant decrease in laryngeal resistance (subglottal pressure) (p < 0.001) accompanied with an increase in respiratory rate together with a pressor and tachycardic response. The results of our study contribute to new data on the role of the mesencephalic neuronal circuits in the control mechanisms of subglottic pressure and laryngeal activity.

Impact of a peer-to-peer escape room activity in the learning of Human Physiology of medical students from the university of Málaga.

Escape room's popularity has raised over the past years among young adults. It creates a distended competitive environment, where participants collaborate to achieve a common objective through teamwork. We decided to apply this format as a teaching method for medical students at the University of Malaga, Spain. A peer-to-peer physiological cardiorespiratory escape room was designed by intern undergraduate students, collaborating within the Department of Human Physiology. This activity integrated the contents of the Human Physiology syllabus, which were organized into four stages that culminated in a final medical case. Intern students oversaw the design, promotion, preparation and execution of the activity, and were in charge of conducting the evaluation and follow up. The escape room was done in mid-December, after all theoretical and practical contents had been delivered, for four consecutive years, improving from each year's experience. The target group for this activity were second year medical students, who were asked to team up freely in groups of four to six students before the start of the activity. The students in each group cooperated with each other while trying to solve the different puzzles and questions in each stage of the escape room. After the activity, the results of the final evaluation exam of these participants were compared against non-participants, who served as a control group. Qualitative feedback was also received from the participants via a special survey that was designed for this task. Results between 2020 and 2023 (three last activities) show that the final mark of the participants was significantly higher than in non-participants (6.39 ± 0.14 vs. 5.04 ± 0.2; p < 0.0007). The global exam mark also increased in the participants (5.43 ± 0.10 vs. 4.44 ± 0.15; p < 0.0007). A significant difference was observed in the performance in cardiovascular (p < 0.0007) and respiratory-related questions (p < 0.0007), which was substantial in the participants. The qualitative feedback received from the participants was mainly positive, indicating an overall acceptance of the format by the students. We conclude that escape room format with a peer-to-peer structure is an efficient teaching tool for medical students performed by medical students in the field of Human Physiology.

Impact of the glutamatergic neurotransmission within the A5 region on the cardiorespiratory response evoked from the midbrain dlPAG.

Stimulation of the dorsolateral periaqueductal grey matter (dlPAG) in rats evokes an active defensive behaviour together with a cardiorespiratory response characterised by tachypnoea, tachycardia and hypertension. The dlPAG neurons involved in these responses are excitatory, presumably glutamatergic, due to the presence of vesicular glutamate transporter VGLUT2 within their axon terminals. Previously, our group described a functional interaction between dlPAG and the pontine A5 region. Accordingly, in the present work, in order to characterize the role of glutamate within this interaction, experiments were carried out in spontaneously breathing anaesthetized rats (sodium pentobarbitone 60 mg/kg i.p., suplemented with 20 mg/kg i.p.). The cardiorespiratory response evoked by electrical stimulation of the dlPAG (1 ms pulses, 20-50 µA, given at 100 Hz, during 5 s) was analysed before and after the microinjection, within the A5 region, of either kynurenic acid (non-specific glutamate receptor antagonist; 5-10 nmol), DAP-5 (NMDA antagonist; 1 pmol), CNQX (non-NMDA antagonist; 1 pmol) or MCPG (metabotropic antagonist; 0,1 nmol). Kynurenic acid decreased the intensity of both the tachypnoea (p < 0,001) and tachycardia (p < 0,001) induced by dl-PAG stimulation. Blockade of no-NMDA receptors reduced the increase of respiratory frequency, heart rate and pressor response to dl-PAG stimulation (p < 0,01, p < 0,001, p < 0,05 respectively). Blockade of either NMDA or metabotropic receptors reduced the dlPAG-evoked tachycardia and pressor response (p < 0,01; p < 0,05 respectively). These results suggest a neuromodulatory role for A5 region via glutamate neurotransmission of the dlPAG-evoked cardiorespiratory response, confirming the role of the ventrolateral pons in the neuronal circuits involved in respiratory and heart rate control.

Glutamate receptors of the A5 region modulate cardiovascular responses evoked from the dorsomedial hypothalamic nucleus and perifornical area.

To assess the possible function of glutamate in the interaction between the dorsomedial hypothalamic nucleus-perifornical area (DMH-PeF) and the A5 pontine region (A5), cardiovascular and respiratory changes were studied in response to electrical stimulation of the DMH-PeF (1 ms pulses, 30-50 µA given at 100 Hz for 5 s) before and after the microinjection of kynurenic acid (non-specific glutamate receptor antagonist; 50 nl, 5 nmol), MK-801 (NMDA receptor antagonist; 50 nl, 50 nmol), CNQX (non-NMDA receptor antagonist; 50 nl, 50 nmol) or MCPG (metabotropic glutamate receptor antagonist; 50 nl, 5 nmol) within the A5 region. DMH-PeF electrical stimulation elicited a pressor (p < 0.001) and tachycardic response (p < 0.001) which was accompanied by an inspiratory facilitation characterised by an increase in respiratory rate (p < 0.001) due to a decrease in expiratory time (p < 0.01). Kynurenic acid within the A5 region decreased the tachycardia (p < 0.001) and the intensity of the blood pressure response (p < 0.001) to DMH-PeF stimulation. After the microinjection of MK-801 and CNQX into the A5 region, the magnitude of the tachycardia and the pressor response were decreased (p < 0.05 and p < 0.01; p < 0.001 and p < 0.05, respectively). After MCPG microinjection into the A5 region, a decrease in the tachycardia (p < 0.001) with no changes in the pressor response was observed during DMH-PeF stimulation. The respiratory response elicited by DMH-PeF stimulation was not changed after the microinjection of kynurenic acid, MK-801, CNQX or MCPG within the A5 region. These results suggest that A5 region glutamate receptors play a role in the cardiovascular response elicited from the DMH-PeF. The possible mechanisms involved in these interactions are discussed.

Genetic variability of hepatitis B virus in Uruguay: D/F, A/F genotype recombinants.

Hepatitis B virus (HBV) infection is a serious global health problem. Approximately 2 billion people worldwide have been infected, and approximately 350 million individuals currently suffer from HBV-induced chronic liver infection, which causes 600,000 deaths annually from chronic hepatitis, cirrhosis and hepatocellular carcinoma. HBV is classified in eight genotypes (A-H), and two more have been proposed (I-J). In this paper, complete genome sequences of nine Uruguayan HBV are reported. Five samples belong to genotype F1b and one to genotype A2. Three HBV recombinants were detected: A1/F1b, A2/F1b and D3/F1b. The following mutations were detected: a G1896A substitution, a 33-nucleotide deletion from position 2896 to 2928 in the Pre-S1 region involving Pre-S1 residues 3-13, a 33-nt deletion in the Pre-S1 region involving nt 2913-2945 and Pre-S1 residues 9-19. More F genotypes strains than expected were detected in this study, supporting the hypothesis that there are more people of indigenous origin than declared in our population. Also, one third of the samples analyzed were recombinants. This cannot be explained by the low HBV prevalence in Uruguay, but a high HBV infection rate in drug addicts and dialysis patients could act in favor of multiple-genotype HBV infections that could lead to recombination.

Neurons of the A5 region are required for the tachycardia evoked by electrical stimulation of the hypothalamic defence area in anaesthetized rats.

In order to assess the possible interactions between the pontine A5 region and the hypothalamic defence area (HDA), we have examined the pattern of double staining for c-Fos protein immunoreactivity (c-Fos-ir) and tyrosine hydroxylase, throughout the rostrocaudal extent of the A5 region in spontaneously breathing anaesthetized male Sprague-Dawley rats during electrical stimulation of the HDA. Activation of the HDA elicited a selective increase in c-Fos-ir with an ipsilateral predominance in catecholaminergic and non-catecholaminergic A5 somata (P < 0.001 in both cases). A second group of experiments was done to examine the importance of the A5 region in modulating the cardiorespiratory response evoked from the HDA. Cardiorespiratory changes were analysed in response to electrical stimulation of the HDA before and after ipsilateral microinjection of muscimol within the A5 region. Stimulation of the HDA evoked an inspiratory facilitatory response, consisting of an increase in respiratory rate (P < 0.001) due to a decrease in expiratory time (P < 0.01). The respiratory response was accompanied by a pressor response (P < 0.001) and tachycardia (P < 0.001). After muscimol microinjection within the A5 region, pressor and heart rate responses to HDA stimulation were reduced (P < 0.01 and P < 0.001, respectively). The respiratory response persisted unchanged. Finally, to confirm functional interactions between the HDA and the A5 region, extracellular recordings of putative A5 neurones were obtained during HDA stimulation. Seventy-five A5 cells were recorded, 35 of which were affected by the HDA (47%). These results indicate that neurones of the A5 region participate in the cardiovascular response evoked from the HDA. The possible mechanisms involved in these interactions are discussed.

Parabrachial complex glutamate receptors modulate the cardiorespiratory response evoked from hypothalamic defense area.

To characterize the possible role of glutamate in the interaction between Hypothalamic Defense Area (HDA) and Parabrachial complex (PBc) nuclei, cardiorespiratory changes were analyzed in response to electrical stimulation of the HDA (1 ms pulses, 30-50 µA given at 100 Hz for 5s) before and after the microinjection of the nonspecific glutamate receptor antagonist kynurenic acid (50 nl, 5 nmol), NMDA receptor antagonist MK-801 (50 nl, 50 nmol), non-NMDA receptor antagonist CNQX (50 nl, 50 nmol) or metabotropic glutamate receptor antagonist MCPG (50 nl, 5 nmol) within the PBc. HDA stimulation evoked an inspiratory facilitatory response, consisting of an increase in respiratory rate (p<0.001) due to a decrease in expiratory time (p<0.01). The respiratory response was accompanied by a pressor (p<0.001) and a tachycardic response (p<0.001). Kynurenic acid within the lateral parabrachial region (lPB) abolished the tachycardia (p<0.001) and decreased the magnitude of blood pressure response (p<0.001) to HDA stimulation. Similarly, the magnitude of the tachycardia and the pressor response was decreased after the microinjection of MK-801 (p<0.01 and p<0.001, respectively) and CNQX (p<0.05 in both cases) into the lPB. Kynurenic acid microinjection in this region produced an inhibition of the tachypnea (p<0.001) to HDA stimulation but the respiratory response persisted unchanged after MK-801 or CNQX microinjection into the lPB. Kynurenic acid within the medial parabrachial region (mPB) abolished the tachycardia (p<0.01) and decreased the magnitude of the pressor response (p<0.001) to HDA stimulation. MK-801 and CNQX microinjection in this region decreased the magnitude of the tachycardia (p<0.05, in both cases) and pressor response (p<0.05, in both cases). The respiratory response evoked by HDA stimulation was not changed after the microinjection of kynurenic acid, MK-801 or CNQX within the mPB. No changes were observed in the cardiorespiratory response evoked to HDA stimulation after MCPG microinjection within lPB and mPB. These results indicate that glutamate PBc receptors are involved in the cardiorespiratory response evoked from the HDA. The possible mechanisms involved in these interactions are discussed.

Snail nuclear expression parallels higher malignancy potential in neuroendocrine lung tumors.

INTRODUCTION: The aim of our study was to determine the integrity of the cell-cell adhesion E-cadherin-beta-catenin complex in neuroendocrine lung tumors (NELTs) and the possible involvement of Snail in its deregulation. METHODS: The studied series consisted of formalin-fixed-paraffin-embedded tissue samples from 70 patients diagnosed with NELT (2000-2006) including tumors of low malignancy potential (3 tumorlets, 33 typical carcinoids), intermediate malignancy potential (3 atypical carcinoids) and tumors of high malignancy potential (10 large cell neuroendocrine carcinomas-LCNEC and 21 small cell carcinoma-SCLC). E-cadherin, beta-catenin and Snail expression were immunohistochemically evaluated and mRNA levels were assessed by Q-RT-PCR for E-cadherin and Snail. RESULTS: Nuclear Snail signal was high in 46% tumors with the strongest level observed in high malignancy tumors. Furthermore, Snail levels correlated with tumor size, lymph node involvement and tobacco consumption. E-cadherin expression was downregulated in 24% cases and it was absent from the membrane in 31%, all of them cases of high malignancy potential. High E-cadherin levels and a membrane pattern were associated with tumor-free lymph node patients and inversely proportional to Snail protein expression. beta-catenin levels were weak in 43% and absent from the membrane in 59% cases. Interestingly, among high malignancy potential tumors, beta-catenin levels were significantly higher in LCNEC than in SCLC. The integrity of the E-cadherin-beta-catenin complex was retained in 37% cases, most of them carcinoid tumors, and correlated with low Snail levels, low malignancy potential and free lymph nodes. CONCLUSION: Snail nuclear expression and loss of integrity of cell adhesion complex E-cadherin/beta-catenin parallels higher malignancy potential in NELTs.

Distinctive clinicopathological associations of amplification of the cortactin gene at 11q13 in head and neck squamous cell carcinomas.

Amplification of the 11q13 region is a prevalent genetic alteration in head and neck squamous cell carcinoma (HNSCC). We investigated the clinical significance of cortactin (CTTN) and cyclin D1 (CCND1) amplification in both malignant transformation and tumour progression. CTTN and CCND1 amplification was analysed by differential and real-time PCR in a prospective series of laryngeal/pharyngeal carcinomas and archival premalignant tissues. CTTN mRNA and protein expression were respectively determined by real-time RT-PCR and immunohistochemistry, and correlated with gene status. Molecular alterations were associated with clinicopathological parameters and disease outcome. CTTN and CCND1 amplifications were respectively found in 75 (37%) and 90 (45%) tumours. Both correlated with advanced disease; however, only CTTN amplification was associated with recurrence and reduced disease-specific survival (p = 0.0022). Strikingly, CTTN amplification differentially influenced survival depending on tumour site (p = 0.0001 larynx versus p = 0.68 pharynx) and was an independent predictor of reduced survival in the larynx (p = 0.04). CCND1 amplification was detected in early tumourigenesis and increased with the severity of dysplasia. Importantly, CTTN amplification was only found in high-grade dysplasias that progressed to invasive carcinoma. CTTN gene status strongly correlated with mRNA and protein expression. Furthermore, CTTN overexpression correlated significantly with reduced disease-specific survival (p = 0.018). Taken together, these data indicate that CTTN may serve as a valuable biomarker to identify patients with laryngeal tumours at high risk of recurrence and poor outcome.

Human DESC1 serine protease confers tumorigenic properties to MDCK cells and it is upregulated in tumours of different origin.

Proteolysis of the extracellular matrix components plays a crucial role in the regulation of the cellular and physiological processes, and different pathologies have been associated with the loss or gain of function of proteolytic enzymes. DESC1 (differentially expressed in squamous cell carcinoma gene 1), a member of the TTSP (type II transmembrane serine protease) family of serine proteases, is an epithelial-specific enzyme that has been found downregulated in squamous cell carcinoma of the head and neck region. We describe new properties of DESC1 suggesting that this protease may be involved in the progression of some type of tumours. Thus, this enzyme hydrolyses some extracellular matrix components, such as fibronectin, gelatin or fibrinogen. Moreover, Madin-Darby canine kidney (MDCK) cells expressing exogenous human DESC1 acquire properties associated with tumour growth such as enhanced motility and an increase of tubular forms in a 3D collagen lattice following HGF treatment. Finally, we generated polyclonal anti-DESC1 antibodies and immunohistochemical analysis in tissues different from head and neck region indicated that this protease was overexpressed in tumours of diverse origins. Taken together, our results suggest that DESC1 could be considered as a potential therapeutic target in some type of tumours.

A5 region modulation of the cardiorespiratory responses evoked from parabrachial cell bodies in the anaesthetised rat.

We have examined the importance of the A5 region modulating cardiorespiratory responses evoked from the parabrachial complex (PB) in spontaneously breathing rats. Cardiorespiratory changes were analyzed in response to electrical stimulation and glutamate microinjections into the PB (10-20 nl, 1-2 nmol) before and after ipsilateral microinjection of muscimol (50 nl, 0.25 nmol) or lidocaine (50 nl, 0.5 nmol) within the A5 region. Stimulation of medial parabrachial and Kölliker-Fuse nuclei (mPB-KF) evoked a decrease in respiratory rate (P<0.001) with a rise in blood pressure (P<0.001) and heart rate (P<0.05). After muscimol or lidocaine microinjections within the A5 region, the pressor and heart rate responses to mPB-KF stimulation were reduced (P<0.05, both cases). Muscimol within the A5 region altered the respiratory response to glutamate stimulation of mPB-KF, evoking an increase in respiratory rate (P<0.05). Lidocaine abolished the respiratory response to mPB-KF stimulation. Stimulation of the lateral parabrachial nuclei (lPB) caused an increase in respiratory rate (P<0.001) with a rise in blood pressure (P<0.001) and heart rate (P<0.05). Muscimol or lidocaine microinjections within A5 region decreased heart rate (P<0.05) and pressor responses (P<0.05) evoked from lPB. The increase of respiratory rate persisted unchanged. To confirm functional interactions between A5 and PB, extracellular recordings of putative A5 neurones were obtained during PB stimulation. Eighty-three A5 cells were recorded, 35 were activated from the mPB-KF (42%). The results indicate that neurones of the A5 region participate in the cardiorespiratory response evoked from the different regions of the PB complex. The possible mechanisms involved in these interactions are discussed.

Determination of triazines in water samples by high-performance liquid chromatography with diode-array detection.

Triazines are widely used herbicides that can be detected in the environment at trace level. A preconcentration step is necessary to determinate them before analysis. In this study, carbonaceous and polymeric adsorbents are compared with C18 for the solid-phase extraction of simazine, atrazine, and propazine in water samples in order to quantitate their levels by high-performance liquid chromatography using photodiode-array detection.

Variability of genetic alterations in different sites of head and neck cancer.

OBJECTIVE: Tumors arising from different sites of the head and neck area have different clinical behavior. However, most of the studies on genetic alterations in head and neck squamous cell carcinomas do not make a distinction between the sites within this area. The objective of this study is to compare the genetic alterations in three different sites of the head and neck (larynx, oropharynx, and hypopharynx). STUDY DESIGN: Prospective study. METHODS: Thirty-eight laryngeal, 29 oropharyngeal, and 37 hypopharyngeal carcinomas were studied. DNA from tumor and healthy tissue was evaluated for amplification of the oncogenes at 11q13 region (CCND1, FGF3, FGF4 and EMS1) and of the oncogenes MYC and ERBB1; for integration of the human papillomavirus (HPV) types 6b and 16; for loss of heterozygosity (LOH) at p53 and NAT2; and for the cellular DNA content. RESULTS: FGF3 and FGF4 showed a significantly higher frequency of amplification in hypopharyngeal tumors (P =.006 and P =.0002, respectively). CCND1 amplification had a nearly statistically significant (P =.072) higher frequency of amplification in hypopharyngeal tumors. Aneuploid tumors were found in a significantly lower proportion in the larynx (P =.03) compared with the other sites. For the other genetic alterations, no significant differences among the three sites were found. CONCLUSIONS: These results suggest that cancers originating from different sites in the head and neck may have different tumor biology. Therefore, they should be considered as different entities.

P53 allelic loss and outcome in patients with squamous cell carcinoma of the head and neck.

Although loss of heterozygosity (LOH) on the p53 locus is frequent in squamous cell carcinomas of the head and neck (SCCHN), controversy still remains regarding the prognostic significance of such an event. Eighty consecutive SCCHN were studied. DNA was extracted from matched sets of normal and tumour tissue and used for polymerase chain reaction amplification of microsatellite markers located in the p53 locus. LOH at the p53 locus was found in 39 (70%) of the 56 informative cases. No relationship was found between p53 LOH and age, site, T stage, N stage, disease stage, and histological differentiation. Recurrence occurred in 53% of the cases with LOH and in 58% of the cases without it (P = 0.28). Moreover, no statistically significant association was found between p53 LOH and disease-specific survival (log-rank P = 0.98). These data suggest that although LOH at the p53 locus is common in SCCHN, this finding is of little clinical significance.

Glucocorticoids antagonize AP-1 by inhibiting the Activation/phosphorylation of JNK without affecting its subcellular distribution.

The immunosuppressive and antiinflammatory actions of glucocorticoid hormones are mediated by their transrepression of activating protein-1 (AP-1) and nuclear factor-kappa B (NFkappaB) transcription factors. Inhibition of the c-Jun NH(2)-terminal kinase (JNK) signaling pathway, the main mediator of AP-1 activation, has been described in extracts of hormone-treated cells. Here, we show by confocal laser microscopy, enzymatic assays, and immunoblotting that the synthetic glucocorticoid dexamethasone inhibited tumor necrosis factor alpha (TNF-alpha)-induced phosphorylation and activation of JNK in the cytoplasm and nucleus of intact HeLa cells. As a result, c-Jun NH(2)-terminal domain phosphorylation and induction were impaired. Dexamethasone did not block the TNF-alpha-induced JNK nuclear translocation, but rather induced, per se, nuclear accumulation of the enzyme. Consistently with previous findings, a glucocorticoid receptor mutant (GRdim), which is deficient in dimerization, DNA binding, and transactivation, but retains AP-1 transrepressing activity, was as efficient as wild-type GR in mediating the same effects of dexamethasone on JNK in transfected Cos-7 cells. Our results show that glucocorticoids antagonize the TNF-alpha-induced activation of AP-1 by causing the accumulation of inactive JNK without affecting its subcellular distribution.