Uncovering and engineering the mechanical properties of the adhesion GPCR ADGRG1 GAIN domain.

Key cellular functions depend on the transduction of extracellular mechanical signals by specialized membrane receptors including adhesion G-protein coupled receptors (aGPCRs). While recently solved structures support aGPCR activation through shedding of the extracellular GAIN domain, the molecular mechanisms underpinning receptor mechanosensing remain poorly understood. When probed using single-molecule atomic force spectroscopy and molecular simulations, ADGRG1 GAIN dissociated from its tethered agonist at forces significantly higher than other reported signaling mechanoreceptors. Strong mechanical resistance was achieved through specific structural deformations and force propagation pathways under mechanical load. ADGRG1 GAIN variants computationally designed to lock the alpha and beta subdomains and rewire mechanically-induced structural deformations were found to modulate the GPS-Stachel rupture forces. Our study provides unprecedented insights into the molecular underpinnings of GAIN mechanical stability and paves the way for engineering mechanosensors, better understanding aGPCR function, and informing drug-discovery efforts targeting this important receptor class.

Stapes surgery in osteogenesis imperfecta: retrospective analysis of 18 operated ears.

PURPOSE: To evaluate short and long term results of stapes surgery in patients with osteogenesis imperfecta (OI), METHODS: Retrospective case series of 18 primary stapes surgeries performed on 11 hearing-impaired OI patients with evidence of stapes fixation, in a Tertiary referral center. We analysed pre-operative and post-operative hearing results at 1 month and at least 1 year RESULTS: The main operative findings were stapes fixation, thickened footplate and fragile or fractured stapes crura. No revision surgery was necessary. Hearing improvement was achieved in 94% of the cases. We obtained an air-bone gap closure to within 10 dB in 46% of the cases and to within 15 dB in 92% of the cases at 1-year follow-up. The mean hearing gain in air conduction (at 0.5, 1, 2 and 4 kHz) was 18.4 dB at 1 month and 22.4 dB at 1 year. CONCLUSION: Stapes surgery in OI gives good results with few complications in our series. A hearing gain is often obtained in spite of the sensorineural hearing loss caused by the natural progression of the disease.

Clinical and cytological study of the oral mucosa of smoking and non-smoking qat chewers in Yemen.

OBJECTIVE: The study was conducted to investigate the role of qat and smoking habits on the prevalence of visible and cytological abnormalities in the oral mucosa among Yemenites. METHODS: We recruited 30 non-smoking and 30 smoking Yemenites chewing qat unilaterally for at least 5 years. We inspected oral cavities for the presence of lesions and took brush biopsies from the buccal mucosa/gingiva of the chewing/non-chewing region. RESULTS: All visible oral lesions were flat and homogeneous, and cytological changes were detected frequently. Among both non-smokers and smokers, white lesions and cytological changes were detected in 77% of all cases. On the chewing area, the proportion with white lesions ranged--depending on anatomical area and smoking status--between 47 and 93% and was significantly more frequent than on the non-chewing side (range 3-47%). The proportion of regions with changes was similar in non-smokers and smokers. Kappa statistics for "interobserver" agreement between visual inspection and cytological specimens of brush biopsies was at best fair (≤0.25). CONCLUSIONS: The high prevalence of visible lesions and cytological abnormalities among qat chewers was independent of smoking status. CLINICAL RELEVANCE: The moderate level of agreement between visual inspection and exfoliative cytology demonstrates the still challenging clinical management of chronic qat chewers, though brush biopsies including adjuvant techniques like DNA cytometry may support the clinical decision-making process in future.

Heregulin regulates the actin cytoskeleton and promotes invasive properties in breast cancer cell lines.

The metastatic process requires changes in tumor cell adhesion properties, cell motility and remodeling of the extracellular matrix. The erbB2 proto-oncogene is overexpressed in approximately 30% of breast cancers and is a major prognostic parameter when present in invasive disease. A ligand for the erbB2 receptor has not yet been identified but it can be activated by heterodimerization with heregulin (HRG)-stimulated erbB3 and erbB4 receptors. The HRGs are a family of polypeptide growth factors that have been shown to play a role in embryogenesis, tumor formation, growth and differentiation of breast cancer cells. The erbB3 and erbB4 receptors are involved in transregulation of erbB2 signaling. The work presented here suggests biological roles for HRG including regulation of the actin cytoskeleton and induction of motility and invasion in breast cancer cells. HRG-expressing breast cancer cell lines are characterized by low erbB receptor levels and a high invasive and metastatic index, while those which overexpress erbB2 demonstrate minimal invasive potential in vitro and are non-tumorigenic in vivo. Treatment of the highly tumorigenic and metastatic HRG-expressing breast cancer cell line MDA-MB-231 with an HRG-neutralizing antibody significantly inhibited proliferation in culture and motility in the Boyden chamber assay. Addition of exogenous HRG to non-invasive erbB2 overexpressing cells (SKBr-3) at low concentrations induced formation of pseudopodia, enhanced phagocytic activity and increased chemomigration and invasion in the Boyden chamber assay. The specificity of the chemomigration response to HRG is demonstrated by inhibition with the anti-HRG neutralizing antibody. These results suggest that either HRG can act as an autocrine or paracrine ligand to promote the invasive behavior of breast cancer cells in vitro or thus may enhance the metastatic process in vivo.

Advances in infection control: ventilator-associated pneumonia.

Mechanically ventilated patients are 6-21 times more likely to develop nosocomial pneumonia. It is estimated that between 6% and 52% of ventilated patients develop ventilator-associated pneumonia (VAP) with attributable mortality of 27-51%. Certain high risk organisms carry higher mortality (e.g., Pseudomonas aeruginosa and Acinetobacter spp.). Aspiration of colonized orodigestive secretions is the commonly recognized route of infection, whereas inhalation of contaminated aerosol hematogenous spread and direct infection are less common. Gram-negative pathogens are responsible for 40-60% of VAP, whereas gram-positive pathogens cause 15-20%, and it is commonly polymicrobial. Diagnosis remains difficult, and studies showed that early appropriate treatment can improve patient outcome. Better understanding of the pathogenesis and risk factors is important for implementing more effective infection control measures. Clinical trials evaluating outcome will help in assessing current and future preventive and therapeutic measures.

Nebulized glyceryl trinitrate exerts acute bronchodilator effects in patients with acute bronchial asthma.

Glyceryl trinitrate (GTN) is a potent smooth muscle relaxant and vasodilator. There are conflicting reports regarding its efficacy as a bronchodilator. The aim of this study was to examine whether nebulized GTN has bronchodilating effects in patients with acute bronchial asthma. We studied 18 patients (five female, 13 male) who were admitted to the hospital with acute severe asthma on two occasions, administering either 6 mg nebulized GTN or placebo (saline) in a double-blind, randomized, crossover fashion. Bronchial response was assessed by measurement of peak expiratory flow (PEF), forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC). A systematic effect of this dose of GTN was demonstrated by a mean increase in heart rate of 38.1% (SEM=7.6%) after GTN administration from supine to erect posture, compared with 10.2% (SEM=1.8%) after placebo (P<0.005). Systolic blood pressure decreased by 8.7% (SEM=1.1%) after GTN, compared with 4.0% (SEM=2.1%) after placebo (P<0.05). Diastolic blood pressure did not change significantly. Baseline PEF, FEV1 and FVC did not differ on the two experimental days; however, acute bronchodilating effects were seen: PEF (l/min); 368 (21) pre-GTN, 411 (22) post-GTN and 384 (23) post-placebo (P<0.001). FEV1 (l); 2.12 (0.13) pre-GTN, 2.46 (0.15) post-GTN and 2.25 (0.16) post-placebo (P<0.001). FVC (l); 3.31 (0.17) pre-GTN, 3.75 (0.2) post-GTN and 3.54 (0.2) post-placebo (P<0.001). In conclusions, nebulized GTN has bronchodilating effects in patients with acute bronchial asthma. The exact mechanism of bronchodilation is not known, but it may be due to local effect on bronchial smooth muscles through nitric oxide or by systemic vasodilatation which leads to a decrease in pulmonary artery pressure and pulmonary vascular resistance, or an increase in systemic catecholamine release.

NRG-3 in human breast cancers: activation of multiple erbB family proteins.

Ligands of the EGF/Heregulin family control the growth of epithelial cells by binding to receptors of the erbB family. By searching a large database of cDNA sequences at Human Genome Sciences Inc. we have identified a new encoded protein sequence containing all the conserved elements of the EGF/Heregulin family. The same sequence has recently been independently identified as NRG-3. The EGF-like domain of NRG-3 was generated as a recombinant protein in E. coli and used to test the specificity of receptor binding. In human breast cancer cells and in 32D cells transfected by erbB family members, NRG-3 activated multiple erbB family members. These include EGF receptor (erbB1) and erbB4 when expressed individually and erbB2 and erbB3 when expressed together. Recombinant NRG-3 altered the growth of human breast cancer cells growing in vitro. NRG-3 was expressed in cell lines derived from breast cancer. These results indicate that NRG-3 is a potential regulator of normal and malignant breast epithelial cells in vivo.

The significance of heregulin in breast cancer tumor progression and drug resistance.

The erbB-2 receptor plays an important role in the prognosis of breast cancer and is expressed at high levels in nearly 30% of tumors in breast cancer patients. While evidence accumulates to support the relationship between erbB-2 overexpression and poor overall survival in human breast cancer, understanding of the biological consequence(s) of erbB-2 overexpression remains elusive. The discovery of heregulin has allowed us to identify a number of related but distinct biological endpoints which appear responsive to signal transduction through the erbB-2/4 receptor. These endpoints of growth, invasiveness, and differentiation have clear implications for the emergence, maintenance, and/or control of malignancy, and represent established endpoints in the assessment of malignant progression in human breast cancer. Preliminary studies in vitro have shown that heregulin induces a biphasic growth effect on cells with erbB-2 overexpression. Interestingly, we observed that expression of heregulin correlates with a more aggressive/invasive, vimentin-positive phenotype in breast cancer cells lines. Therefore, we have postulated that heregulin is involved in breast cancer tumor progression. We have shown that heregulin induces in vitro chemoinvasion and chemotaxis of breast cancer cells as well as growth in an anchorage dependent and independent manner. Interestingly, a heregulin neutralizing antibody inhibits chemotaxis and results in cell growth inhibition and blockade of the invasive phenotype. Strikingly, genetically engineered cells which constitutively express heregulin demonstrate critical phenotypic changes that are associated with a more aggressive phenotype. Specifically, these cells are no longer dependent on estrogen for growth and are resistant to tamoxifen in vitro and in vivo, and moreover these cells metastasize to lymph nodes in athymic nude mice. These tumors appear to have lost bcl-2 expression as compared with the control tumors. In addition, presumably by activation/regulation of topoisomerase II, the heregulin-transfected cells become exquisitely sensitive to doxorubicin and VP-16. Clearly, mechanistic aspects of the erbB-2/4 and heregulin interaction need to be understood from a therapeutic standpoint which could provide additional insights into synergistic treatments for certain patients, or improve treatment regimens for a large number of women. The study of heregulin and its co-expression with erbB-2/4 receptor and the assessment of its involvement in the progression from the in situ stage of breast tumors to the invasive one will additionally increase the relevance of heregulin as a prognostic/diagnostic factor. We believe that our studies provide new insights into breast cancer diagnosis, prognosis, and treatment.

Interaction between erbB-receptors and heregulin in breast cancer tumor progression and drug resistance.

The type I growth factor receptor family is increasingly recognized as important in the development and maintenance of breast cancer. The family currently consists of four closely related members: the epidermal growth factor receptor (EGF-R/erbB-1), erbB-2, erbB-3 and erbB-4. Putative ligands which bind directly to or indirectly activate erbB-2/3/4 have been characterized recently. This still growing family of EGF-related growth factors includes gp30, its homolog heregulin (HRG), the rat homolog neu differentiation factor (NDF), glial growth factors (GLIA), ARIA and a 50 kDa factor from COLO 16 cells. The understanding of the function, biology and interactions of these growth factor receptors and their ligands will have far-reaching implications for the prognosis and treatment of breast cancer. This review focuses on advances and future directions for further investigations intended to clarify the mechanism and significance of erbB/ligand interactions in breast cancer.

Dynamic patterns of medial preoptic mu-opiate receptor regulation by gonadal steroid hormones.

The density of mu-opiate receptors located in the medial preoptic area (MPOA) of the rat hypothalamus is cyclical and sexually dimorphic. The hormonal regulation of MPOA mu-receptors was examined in ovariectomized rats treated with a variety of hormone regimens. In experiment 1, animals received acute estradiol (E2), progesterone (P), or prolactin (PRL), or E2 followed in 48 h by either P or vehicle by subcutaneous injection. Brains were removed 3 h after the final injection. In experiment 2, animals were implanted with empty or E2-filled Silastic capsules, and received either P or vehicle by injection 48 h later, at which time E2 capsules were removed. One group received E2 implants which remained in place following sham removal surgery. Brains and trunk blood for radioimmunoassay of E2 and P were collected 3, 27 or 51 h after the final injection. Frozen brain sections were prepared, incubated in [3H]D-Ala2,MePhe4,Gly-ol5-enkephalin, which selectively labels mu-receptors, and analyzed using quantitative receptor autoradiography. P treatment significantly increased MPOA mu-receptors, but only 27 h after E2 priming. Neither shorter P exposure, nor E2, P or PRL alone affected MPOA mu-receptor density. Following this delayed E2,P-induced increase, mu-receptor density subsequently decreased in the presence of absence of E2. The results suggest that E2,P treatment produces a gradual and transient increase of MPOA mu-receptor density. The subsequent decrease of receptor density is independent of the presence of E2 and may be related to receptor turnover. The time course of this effect is consistent with that of the estrus cycle. Such hormone-induced regulation of MPOA mu-receptor density could influence the physiologic effects of opiates on gonadotropin secretion and reproductive behavior in cycling females.