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Joubert Syndrome (JBTS) is a neurodevelopmental ciliopathy defined by a highly specific midbrain-hindbrain malformation, variably associated with additional neurological features. JBTS displays prominent genetic heterogeneity with >40 causative genes that encode proteins localising to the primary cilium, a sensory organelle that is essential for transduction of signalling pathways during neurodevelopment, among other vital functions. JBTS proteins localise to distinct ciliary subcompartments, suggesting diverse functions in cilium biology. Currently, there is no unifying pathomechanism to explain how dysfunction of such diverse primary cilia-related proteins results in such a highly specific brain abnormality. To identify the shared consequence of JBTS gene dysfunction, we carried out transcriptomic analysis using zebrafish mutants for the JBTS-causative genes cc2d2aw38, cep290fh297, inpp5ezh506, talpid3i264 and togaram1zh510 and the Bardet-Biedl syndrome-causative gene bbs1k742. We identified no commonly dysregulated signalling pathways in these mutants and yet all mutants displayed an enrichment of altered gene sets related to central nervous system function. We found that JBTS mutants have altered primary cilia throughout the brain but do not display abnormal brain morphology. Nonetheless, behavioural analyses revealed reduced locomotion and loss of postural control which, together with the transcriptomic results, hint at underlying abnormalities in neuronal activity and/or neuronal circuit function. These zebrafish models therefore offer the unique opportunity to study the role of primary cilia in neuronal function beyond early patterning, proliferation and differentiation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Chemokines are proteins important for a range of biological processes from cell-directed migration (chemotaxis) to cell activation and differentiation. Chemokine C-C ligand 5 (CCL5) is an important pro-inflammatory chemokine attracting immune cells towards inflammatory sites through interaction with its receptors CCR1/3/5. Recombinant production of large quantities of CCL5 in Escherichia coli is challenging due to formation of inclusion bodies which necessitates refolding, often leading to low recovery of biologically active protein. To combat this, we have developed a method for CCL5 production that utilises the purification of SUMO tagged CCL5 from E. coli SHuffle cells avoiding the need to reform disulfide bonds through inclusion body purification and yields high quantities of CCL5 (~ 25 mg/L). We demonstrated that the CCL5 produced was fully functional by assessing well-established cellular changes triggered by CCL5 binding to CCR5, including receptor phosphorylation and internalisation, intracellular signalling leading to calcium flux, as well as cell migration. Overall, we demonstrate that the use of solubility tags, SHuffle cells and low pH dialysis constitutes an approach that increases purification yields of active CCL5 with low endotoxin contamination for biological studies., (© 2024. The Author(s).)

Objective: Investigate the accuracy of ChatGPT in the manner of medical questions related to otolaryngology., Methods: A ChatGPT session was opened within which 93 questions were asked related to otolaryngology topics. Questions were drawn from all major domains within otolaryngology and based upon key action statements (KAS) from clinical practice guidelines (CPGs). Twenty-one "patient-level" questions were also asked of the program. Answers were graded as either "correct," "partially correct," "incorrect," or "non-answer.", Results: Correct answers were given at a rate of 45.5% (71.4% correct in patient-level, 37.3% CPG); partially correct answers at 31.8% (28.6% patient-level, 32.8% CPG); incorrect at 21.6% (0% patient-level, 28.4% CPG); and 1.1% non-answers (% patient-level, 1.5% CPG). There was no difference in the rate of correct answers between CPGs published before or after the period of data collection cited by ChatGPT. CPG-based questions were less likely to be correct than patient-level questions (p = 0.003)., Conclusion: Publicly available artificial intelligence software has become increasingly popular with consumers for everything from story-telling to data collection. In this study, we examined the accuracy of ChatGPT responses to questions related to otolaryngology over 7 domains and 21 published CPGs. Physicians and patients should understand the limitations of this software as it applies to otolaryngology, and programmers in future iterations should consider giving greater weight to information published by well-established journals and written by national content experts., Level of Evidence: N/A Laryngoscope, 134:4011-4015, 2024., (© 2024 The Authors. The Laryngoscope published by Wiley Periodicals LLC on behalf of The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives: Posterior glottic diastasis (PGD) is an underappreciated etiology of dysphonia in patients with prior airway reconstruction or prolonged intubation. In endoscopic posterior cricoid reduction (ePCR), cricoid is removed to minimize the posterior glottic gap. Dynamic voice computed tomography (DVCT) permits visualization of the posterior glottis, estimating the amount of cricoid to be removed. Posterior glottic gaps in patients undergoing ePCR were compared to non-dysphonic patients to describe pediatric PGD and establish surgical parameters for ePCR., Methods: DVCTs performed in non-dysphonic patients and dysphonic patients undergoing ePCR from 2014 to 2023 were reviewed. EPCR operative reports were queried. Pre- and postoperative Pediatric Voice Handicap Index (pVHI) and Consensus Auditory-Perceptual Evaluation of Voice (CAPE-V) scores and aerodynamic measures were reviewed., Results: Seventeen pediatric patients who underwent ePCR and 19 non-dysphonic patients were included. Posterior glottic gaps were significantly larger in the dysphonic group (median 2.4 mm [IQR: 2.0, 2.8] vs. 1.3 mm [IQR: 1.1, 1.7], p < 0.001). Mean width of the cricoid removed was 1.6 mm (SD 0.4 mm). Mean (SD) pre- and postoperative pVHI scores were 55.5 (19.9) and 34.6 (16.0; p < 0.001). Mean (SD) pre- and postoperative CAPE-V scores were 52.7 (15.4) and 36.5 (20.4; p < 0.001), respectively., Conclusions: Children in this cohort tolerated an average 1.3 mm posterior glottic gap without dysphonia. Dysphonic patients with PGD had a median 2.4 mm gap and underwent cricoid reduction by 1.6 mm. All ePCR patients demonstrated improvement in dysphonia. Results seek to optimize the management of pediatric PGD and present a safe and effective amount of cricoid to remove during ePCR., Level of Evidence: 4 Laryngoscope, 134:5139-5144, 2024., (© 2024 The American Laryngological, Rhinological and Otological Society, Inc.)

Prostate cancer is the most common cancer in men in the UK with over 50 000 new cases diagnosed each year and although therapeutic advances in surgery, anti-androgens, radio- and chemotherapy have increased survival rates, there still remains a need for new treatments to combat the most aggressive forms of the disease. Gene therapy offers promise as an alternative approach but is reliant on selective targeting to the cancer cell surface. Herein we describe the novel construction of a prostate specific membrane antigen (PSMA) binding bioconjugate-polyplex, based on a glutamate-urea peptide scaffold using 'click' chemistry, which we demonstrate is capable of targeted delivery of a GFP gene to PSMA overexpressing prostate cancer cells, and therefore may have potential future application as part of a prostate cancer gene delivery therapy., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

Nucleoside analogues are established treatments for cancer and viral infection. Gemcitabine is a commonly employed nucleoside analogue displaying anticancer properties against a range of tumor types but is rapidly inactivated in vivo . Efforts to bolster its pharmaceutical profile include investigating prodrug forms. Herein, we explore the synthesis of a novel glucose-gemcitabine glycoconjugate, targeting uptake via glucose transport. We select a redox-reactive disulfide linker for conjugation of gemcitabine (through N 4-cytosine) with glucose. Evaluation of this glycoconjugate reveals increased toxicity against androgen insensitive PC3 prostate cancer cells compared to LNCaP (which have lower levels of glucose transporter GLUT1). These preliminary results suggest that glycoconjugation of nucleosides may be an effective approach to targeting cells which display increased uptake and metabolism of glucose., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Objective: Cochlear implantation of prelingually deaf infants provides auditory input sufficient to develop spoken language; however, outcomes remain variable. Inability to participate in speech perception testing limits testing device efficacy in young listeners. In postlingually implanted adults (aCI), speech perception correlates with spectral resolution an ability that relies independently on frequency resolution (FR) and spectral modulation sensitivity (SMS). The correlation of spectral resolution to speech perception is unknown in prelingually implanted children (cCI). In this study, FR and SMS were measured using a spectral ripple discrimination (SRD) task and were correlated with vowel and consonant identification. It was hypothesized that prelingually deaf cCI would show immature SMS relative to postlingually deaf aCI and that FR would correlate with speech identification., Study Design: Cross-sectional study., Setting: In-person, booth testing., Methods: SRD was used to determine the highest spectral ripple density perceived at various modulation depths. FR and SMS were derived from spectral modulation transfer functions. Vowel and consonant identification was measured; SRD performance and speech identification were analyzed for correlation., Results: Fifteen prelingually implanted cCI and 13 postlingually implanted aCI were included. FR and SMS were similar between cCI and aCI. Better FR was associated with better speech identification for most measures., Conclusion: Prelingually implanted cCI demonstrated adult-like FR and SMS; additionally, FR correlated with speech identification. FR may be a measure of CI efficacy in young listeners., (© 2023 American Academy of Otolaryngology-Head and Neck Surgery Foundation.)

Objectives: Spectral resolution correlates with speech understanding in post-lingually deafened adults with cochlear implants (CIs) and is proposed as a non-linguistic measure of device efficacy in implanted infants. However, spectral resolution develops gradually through adolescence regardless of hearing status. Spectral resolution relies on two different factors that mature at markedly different rates: Resolution of ripple peaks (frequency resolution) matures during infancy whereas sensitivity to across-spectrum intensity modulation (spectral modulation sensitivity) matures by age 12. Investigation of spectral resolution as a clinical measure for implanted infants requires understanding how each factor develops and constrains speech understanding with a CI. This study addresses the limitations of the present literature. First, the paucity of relevant data requires replication and generalization across measures of spectral resolution. Second, criticism that previously used measures of spectral resolution may reflect non-spectral cues needs to be addressed. Third, rigorous behavioral measurement of spectral resolution in individual infants is limited by attrition. To address these limitations, we measured discrimination of spectrally modulated, or rippled, sounds at two modulation depths in normal hearing (NH) infants and adults. Non-spectral cues were limited by constructing stimuli with spectral envelopes that change in phase across time. Pilot testing suggested that dynamic spectral envelope stimuli appeared to hold infants' attention and lengthen habituation time relative to previously used static ripple stimuli. A post-hoc condition was added to ensure that the stimulus noise carrier was not obscuring age differences in spectral resolution. The degree of improvement in discrimination at higher ripple depth represents spectral frequency resolution independent of the overall threshold. It was hypothesized that adults would have better thresholds than infants but both groups would show similar effects of modulation depth., Design: Participants were 53 6- to 7-month-old infants and 23 adults with NH with no risk factors for hearing loss who passed bilateral otoacoustic emissions screening. Stimuli were created from complexes with 33- or 100-tones per octave, amplitude-modulated across frequency and time with constant 5 Hz envelope phase-drift and spectral ripple density from 1 to 20 ripples per octave (RPO). An observer-based, single-interval procedure measured the highest RPO (1 to 19) a listener could discriminate from a 20 RPO stimulus. Age-group and stimulus pure-tone complex were between-subjects variables whereas modulation depth (10 or 20 dB) was within-subjects. Linear-mixed model analysis was used to test for the significance of the main effects and interactions., Results: All adults and 94% of infants provided ripple density thresholds at both modulation depths. The upper range of threshold approached 17 RPO with the 100-tones/octave carrier and 20 dB depth condition. As expected, mean threshold was significantly better with the 100-tones/octave compared with the 33-tones/octave complex, better in adults than in infants, and better at 20 dB than 10 dB modulation depth. None of the interactions reached significance, suggesting that the effect of modulation depth on the threshold was not different for infants or adults., Conclusions: Spectral ripple discrimination can be measured in infants with minimal listener attrition using dynamic ripple stimuli. Results are consistent with previous findings that spectral resolution is immature in infancy due to immature spectral modulation sensitivity rather than frequency resolution., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Objectives/hypothesis: A tracheal cartilaginous sleeve (TCS) is a rare anomaly characterized by anterior fusion of tracheal cartilages. TCS is associated with syndromic craniosynostoses including Apert, Crouzon and Pfeiffer syndromes and FGFR2, FGFR3, and TWIST1 variants. This study presents a 30-year review of patients with syndromic craniosynostosis and TCS and describes diagnostic methods, genetic variants, surgical interventions, and long-term outcomes., Study Design: Retrospective, single-institution review., Methods: This review included patients with syndromic craniosynostosis and TCS treated at Seattle Children's Hospital from 1990 to 2020. Tracheostomy, genetic variants, and additional surgery were primary measures. Fisher's exact test compared need for tracheostomy in patients with proposed high-risk (FGFR2 p.W290 or FGFR2 p.C342) versus low-risk genetic variants., Results: Thirty patients with TCS were identified. Average age at diagnosis was 12 months (range 2-weeks to 7.9-years; standard deviation 19.8 months). Syndromes included Pfeiffer (37%), Apert (37%), and Crouzon (26%). Severe obstructive sleep apnea was present in 76% of patients. Tracheostomy was performed in 17 patients (57%); five were successfully decannulated. Additional interventions included adenotonsillectomy (57%), nasal (20%), laryngeal (17%), and craniofacial skeletal surgery (87%). All patients with Pfeiffer syndrome and FGFR2 p.W290C variants and 83% of patients with FGFR2 p.C342 variants required tracheostomy, differing from other variants (P = .02, odds ratio 33, 95% confidence interval 1.56-697.96). One patient (3%) died., Conclusion: TCS contributes to multilevel airway obstruction in patients with syndromic craniosynostosis. Genetic testing in patients with FGFR2-related syndromic craniosynostoses may identify those at risk of TCS and facilitate early intervention. A better understanding of this patient population may foster individualized airway management strategies and improve outcomes., Level of Evidence: 4 Laryngoscope, 132:215-221, 2022., (© 2021 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives: Cochlear implant depth of insertion affects audiologic outcomes and can be measured in adults using plain films obtained in the "cochlear view." The objective of this study was to assess interrater and intrarater reliability of measuring depth of insertion using cochlear view radiography., Study Design: Prospective, observational., Setting: Tertiary referral pediatric hospital., Subjects and Methods: Patients aged 11 months to 20 years (median, 4 years; interquartile range [IQR], 1-8 years) undergoing cochlear implantation at our institution were studied over 1 year. Children underwent cochlear view imaging on postoperative day 1. Films were deidentified and 1 image per ear was selected. Two cochlear implant surgeons and 2 radiologists evaluated each image and determined angular depth of insertion. Images were re-reviewed 6 weeks later by all raters. Inter- and intrarater reliability were calculated with intraclass correlation coefficients (ICCs)., Results: Fifty-seven ears were imaged from 42 children. Forty-nine ears (86%) had successful cochlear view x-rays. Median angular depth of insertion was 381° (minimum, 272°; maximum, 450°; IQR, 360°-395°) during the first round of measurement. Measurements of the same images reviewed 6 weeks later showed median depth of insertion of 382° (minimum, 272°; maximum, 449°; IQR, 360°-397°). Interrater and intrarater reliability ICCs ranged between 0.81 and 0.96, indicating excellent reliability., Conclusions: Postoperative cochlear view radiography is a reliable tool for measurement of cochlear implant depth of insertion in infants and children. Further studies are needed to determine reliability of intraoperatively obtained cochlear view radiographs in this population.

COVID-19 is a rapidly growing global pandemic caused by a novel coronavirus. With no vaccine or definitive treatment, public health authorities have recommended a strategy of "social distancing," reducing individual interaction, canceling elective procedures, and limiting nonessential services. Health care providers must determine what procedures are considered "elective," balancing risk of treatment delays with that of coronavirus exposure to patient, family, and providers. Given critical periods for language development and the long-term impact of auditory deprivation, some audiologic and otologic services should be considered essential. In this article, we describe the experience of a quaternary referral pediatric hospital in Seattle, the epicenter of COVID-19 in the United States, and share strategies for risk minimization employed by Seattle Children's Hospital. We hope that this work can be a reference for other centers continuing care for children who are deaf and hard of hearing during the COVID-19 and future resource-limiting crises.

Spectral ripple discrimination tasks are commonly used to probe spectral resolution in cochlear implant (CI), normal-hearing (NH), and hearing-impaired individuals. In addition, these tasks have also been used to examine spectral resolution development in NH and CI children. In this work, stimulus sine-wave carrier density was identified as a critical variable in an example spectral ripple-based task, the Spectro-Temporally Modulated Ripple (SMR) Test, and it was demonstrated that previous uses of it in NH listeners sometimes used values insufficient to represent relevant ripple densities. Insufficient carry densities produced spectral under-sampling that both eliminated ripple cues at high ripple densities and introduced unintended structured interference between the carriers and intended ripples at particular ripple densities. It was found that this effect produced non-monotonic psychometric functions for NH listeners that would cause systematic underestimation of thresholds with adaptive techniques. Studies of spectral ripple detection in CI users probe a density regime below where this source of aliasing occurs, as CI signal processing limits dense ripple representation. While these analyses and experiments focused on the SMR Test, any task in which discrete pure-tone carriers spanning frequency space are modulated to approximate a desired pattern must be designed with the consideration of the described spectral aliasing effect.

Background: Phospholipases D1 and D2 (PLD1/2) are implicated in tumorigenesis through their generation of the signalling lipid phosphatidic acid and its downstream effects. Inhibition of PLD1 blocks prostate cell growth and colony formation. Here a role for PLD2 in prostate cancer (PCa), the major cancer of men in the western world, is examined., Methods: PLD2 expression was analysed by immunohistochemistry and western blotting. The effects of PLD2 inhibition on PCa cell viability and cell motility were measured using MTS, colony forming and wound-healing assays., Results: PLD2 protein is expressed about equally in luminal and basal prostate epithelial cells. In cells from different Gleason-scored PCa tissue PLD2 protein expression is generally higher than in non-tumorigenic cells and increases in PCa tissue scored Gleason 6-8. PLD2 protein is detected in the cytosol and nucleus and had a punctate appearance. In BPH tissue stromal cells as well as basal and luminal cells express PLD2. PLD2 protein co-expresses with chromogranin A in castrate-resistant PCa tissue. PLD2 inhibition reduces PCa cell viability, colony forming ability and directional cell movement., Conclusions: PLD2 expression correlates with increasing Gleason score to GS8. PLD2 inhibition has the potential to reduce PCa progression.

Choosing an appropriate cell model(s) is the first decision to be made before starting a new project or programme of study. Here, we address the rationale that can be behind this decision and we summarize the current cell models that are used to study prostate cancer. Researchers face the challenge of choosing a model that recapitulates the complexity and heterogeneity of prostate cancer. The use of primary prostate epithelial cells cultured from patient tissue is discussed, and the necessity for close clinical-academic collaboration in order to do this is highlighted. Finally, a novel quantitative phase imaging technique is described, along with the potential for cell characterization to not only include gene expression and protein markers but also morphological features, cell behaviour and kinetic activity.

Background: Phospholipases D1 and D2 (PLD1/2) hydrolyse cell membrane glycerophospholipids to generate phosphatidic acid, a signalling lipid, which regulates cell growth and cancer progression through effects on mTOR and PKB/Akt. PLD expression and/or activity is raised in breast, colorectal, gastric, kidney and thyroid carcinomas but its role in prostate cancer (PCa), the major cancer of men in the western world, is unclear., Methods: PLD1 protein expression in cultured PNT2C2, PNT1A, P4E6, LNCaP, PC3, PC3M, VCaP, 22RV1 cell lines and patient-derived PCa cells was analysed by western blotting. PLD1 protein localisation in normal, benign prostatic hyperplasia (BPH), and castrate-resistant prostate cancer (CRPC) tissue sections and in a PCa tissue microarray (TMA) was examined by immunohistochemistry. PLD activity in PCa tissue was assayed using an Amplex Red method. The effect of PLD inhibitors on PCa cell viability was measured using MTS and colony forming assays., Results: PLD1 protein expression was low in the luminal prostate cell lines (LNCaP, VCaP, 22RV1) compared with basal lines (PC3 and PC3M). PLD1 protein expression was elevated in BPH biopsy tissue relative to normal and PCa samples. In normal and BPH tissue, PLD1 was predominantly detected in basal cells as well in some stromal cells, rather than in luminal cells. In PCa tissue, luminal cells expressed PLD1. In a PCa TMA, the mean peroxidase intensity per DAB-stained Gleason 6 and 7 tissue section was significantly higher than in sections graded Gleason 9. In CRPC tissue, PLD1 was expressed prominently in the stromal compartment, in luminal cells in occasional glands and in an expanding population of cells that co-expressed chromogranin A and neurone-specific enolase. Levels of PLD activity in normal and PCa tissue samples were similar. A specific PLD1 inhibitor markedly reduced the survival of both prostate cell lines and patient-derived PCa cells compared with two dual PLD1/PLD2 inhibitors. Short-term exposure of PCa cells to the same specific PLD1 inhibitor significantly reduced colony formation., Conclusions: A new specific inhibitor of PLD1, which is well tolerated in mice, reduces PCa cell survival and thus has potential as a novel therapeutic agent to reduce prostate cancer progression. Increased PLD1 expression may contribute to the hyperplasia characteristic of BPH and in the progression of castrate-resistant PCa, where an expanding population of neuroendocrine-like cells express PLD1.

Background: Achieving locoregional control in high-risk patients with head and neck cancer who are poor candidates for standard continuous-course (chemo) radiotherapy due to advanced age, comorbidities, or very advanced disease is challenging. At our Institution, we have significant experience with a regimen of split-course, accelerated, hypofractionated radiotherapy (SCAHRT) for these patients., Patients and Methods: The SCAHRT regimen consisted of 60-72 Gy in 20-24 fractions separated by several weeks mid-course to allow for toxicity recovery and disease reassessment. It was used for patients with advanced age, significant co-morbidities, anticipated intolerance to definitive (chemo)radiation, and those with oligometastatic disease. Disease-free and overall survival rates were calculated using Kaplan-Meier analysis., Results: Fifty-eight out of 65 patients (89%) completed both courses of treatment. Patients without metastatic or recurrent disease were evaluated for treatment response and survival (n=39). Among this group, total tumor response was 91%, and median locoregional failure-free survival and overall survival were 25.7 and 8.9 months, respectively., Conclusion: In high-risk patients unable to tolerate continuous-course definitive (chemo)radiation, SCAHRT is a safe, well-tolerated and effective method of achieving durable locoregional disease control. In properly selected patients, this regimen is preferable to purely palliative approaches., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Aim: The purpose of the present study was to identify variables associated with high risk of failure in patients with locally advanced squamous cell carcinoma of the oral cavity (SCC-OC)., Patients and Methods: This retrospective study included 191 patients with stage III-IVb SCC-OC treated with post-operative radiotherapy (RT) or chemoradiotherapy (CRT) between 1995 and 2013. Disease-free (DFS) and overall survival (OS) were analyzed; variables associated with inferior DFS were identified., Results: Seventy-five patients (39%) recurred. DFS and five-year OS were 52% and 54%, respectively. Poorly differentiated tumors (p=0.03), recurrent tumors (p=0.02) and high nodal ratio (p=0.02) were associated with an increased risk of recurrence. CRT was associated with improved DFS in patients with positive margins and/or extracapsular extension (p=0.021)., Conclusion: Tumors that are recurrent, high grade, or have high nodal ratio are at risk of recurrence. Presence of these disease features should be taken into consideration for better risk stratification., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: Tumor human papillomavirus (HPV) status has emerged as one of the most powerful prognostic factors for disease control and survival in patients with oropharyngeal squamous cell carcinoma (OPSCC). We reviewed our experience in patients with OPSCC and known tumor HPV status treated with definitive chemoradiotherapy (CRT)., Methods: Patients with stage III-IVb OPSCC and known tumor HPV status treated with CRT between 2006 and 2011 were identified from an IRB approved registry for this retrospective review. Outcomes were estimated using the Kaplan-Meier method and compared between HPV-positive and negative patients using the log-rank test., Results: Of the 121 pts (89% male, 93% Caucasian) included in this study, median age was 57 (range: 40-73) and median follow-up was 21 months (range: 6-63). Ninety-seven (80%) patients were HPV-positive and 24 (20%) were HPV-negative. Primary site was base of tongue (55%), tonsil (44%), and oropharyngeal wall (2%). Two year rates of locoregional recurrence (3% vs. 26%; p = 0.002), disease free survival (93% vs. 64%; p = 0.001) and overall survival (94% vs 73%; p = 0.002) were superior in HPV-positive patients, while rates of distant recurrence were similar (3% vs. 5%; p = 0.98). While acute toxicities were similar between both groups, patients with HPV-positive disease were more likely to resume a normal diet (90% vs. 65%; p = 0.017) at last follow up. Also, no HPV-positive patient required a feeding tube beyond 6 months after treatment, compared with 24% of HPV-negative patients., Conclusions: Definitive CRT produces excellent rates of disease control with minimal late toxicity for patients with HPV-positive OPSCC. Studies of OPSCC should account for tumor HPV status when identifying factors prognostic for outcome.

The efficient and expeditious syntheses of both enantiomers of the amphibian alkaloid cis-225H have been achieved. Utilizing a common cis-2,5-disubstituted pyrrolidine building block derived from (+)-2-tropinone, the enantioselective syntheses have established the absolute configuration of these alkaloids as (+)-(2R,5S) and (-)-(5S,2R).

Background: The extended trochanteric osteotomy was introduced as a safe and effective exposure technique for revision hip surgery; however, intraoperative iatrogenic femoral fractures have been reported. This study examined the effects of the extended trochanteric osteotomy on the torsional strength of the femur with use of cadaver bones. We hypothesized that repair of the osteotomy fragment would restore the torsional strength to that of an intact femur and that an osteotomized femur containing a well-fixed stem would have the same torsional strength as an intact femur with a stem., Methods: Fifty-eight cadaveric human femora were divided into five groups, according to the repair technique, to examine the effects of the extended trochanteric osteotomy: intact, osteotomy, repaired osteotomy, implant, and implant-repaired osteotomy. Osteotomy fragments were reattached with use of three double-looped 18-gauge wires. A femoral stem was cemented into the last two groups. Specimens were tested mechanically in rotation until failure. Rotational properties were compared with one-way analysis of variance followed by post hoc pairwise comparisons. Linear regression analysis was performed for bone mineral density and torsional strength., Results: Torque to failure was reduced by 73% for the specimens in the osteotomy group compared with the intact group (p < 0.0001). Repair of the osteotomy did not improve torque to failure (p > 0.99). Femora in the implant-repaired osteotomy group displayed significantly improved torque-to-failure values compared with the specimens in the osteotomy and repaired osteotomy groups (p < 0.0001). However, the strength of the femora in the implant-repaired osteotomy group remained significantly less than that of the specimens in the implant group (p < 0.007). A significant linear relationship was observed between bone mineral density and torque to failure for femora in the intact (p < 0.006), osteotomy (p < 0.002), and repaired osteotomy (p < 0.001) groups., Conclusions: The extended trochanteric osteotomy reduces torsional strength by 73% even when the osteotomy fragment is repaired. Bone mineral density directly affects absolute femoral strength in this model.

The orientation and dynamics of two 40-nm thick films of 4-n-pentyl-4'-cyanobiphenyl (5CB), a nematic liquid crystal, have been studied using step-scan Fourier transform infrared spectroscopy (FTIR). The films are confined in nanocavities bounded by an interdigitated electrode array (IDA) patterned on a zinc selenide (ZnSe) substrate. The effects of the ZnSe surface morphology (specifically, two variations of nanometer-scale corrugations obtained by mechanical polishing) on the initial ordering and reorientation dynamics of the electric-field-induced Freedericksz transition are presented here. The interaction of the 5CB with ZnSe surfaces bearing a spicular corrugation induces a homeotropic (surface normal) alignment of the film confined in the cavity. Alternately, when ZnSe is polished to generate fine grooves along the surface, a planar alignment is promoted in the liquid crystalline film. Time-resolved FTIR studies that enable the direct measurement of the rate constants for the electric-field-induced orientation and thermal relaxation reveal that the dynamic transitions of the two film structures are significantly different. These measurements quantitatively demonstrate the strong effects of surface morphology on the anchoring, order, and dynamics of liquid crystalline thin films.

Purpose: Long-term clinical studies have now shown that tight control of blood pressure in type 2 diabetes reduces the risk of diabetes-related death and common diabetic complications, including diabetic retinopathy. However, the mechanisms by which hypertension enhances diabetic microvascular disease, especially diabetic retinopathy, are poorly understood. We developed an experimental model of hypertension in diabetic rats and studied the early ultrastructural changes in retinal capillaries under these conditions., Methods: Hypertension was induced in diabetic BioBreeding (BB) rats by unilateral nephrectomy, weekly subcutaneous mineralocorticoid and 0.9% oral saline. Serial blood pressures and ultrastructural features of retinal capillaries were recorded in four groups: normotensive Wistar rats, normotensive diabetic rats, hypertensive Wistar rats and hypertensive diabetic rats., Results: A significant and sustained increase in systolic blood pressure occurred in both groups of nephrectomised rats. There was a significant increase in the number of caveolae (i) in both pericytes and endothelial cells in animals with hypertension and diabetes together compared with all other groups and (ii) in pericytes in animals with diabetes alone. The number of direct contacts between pericytes and endothelial cells was reduced in diabetic and hypertensive diabetic animals. Hypertension and diabetes had an interactive effect in producing retinal capillary basement membrane thickening., Conclusions: In the BB rat hypertension and diabetes have an interactive effect in increasing the number of caveolae in both endothelial cells and pericytes. We speculate that this may be a reflection of changes in calcium and nitric oxide metabolism in these animals.

The role of cGMP as a second messenger for renin secretion is contentious. This was investigated using a superfused collagenase-dispersed rat kidney cortex cell preparation devoid of indirect influences on renin secretion. Nitroprusside, atriopeptin II and 8-Br-cGMP all increased renin release but the dose-response relationships were biphasic. At low dose ranges there was a positive correlation between increasing drug concentration and renin secretion, but at high drug concentrations, a negative correlation was apparent. Methylene blue, a guanylate cyclase inhibitor, also suppressed baseline renin release at 10(-5) and 10(-6) M, but stimulated release at 10(-3) M. Using mid-range drug concentrations, the cGMP specific phosphodiesterase inhibitor MB22948 potentiated renin release in response to nitroprusside and 8-Br-cGMP. Inhibition of guanylate cyclase with either methylene blue or LY83583 attenuated renin release in response to nitroprusside, but, as expected, had no effect on 8-Br-cGMP induced release. We conclude that, under physiological conditions, cGMP is a stimulatory second messenger for renin release. This activity is mimicked at low dose ranges by 8-Br-cGMP, nitroprusside and atriopeptin II. In response to high doses of these drugs an unknown inhibitory pathway is activated and this opposes, in a dose-related manner, the stimulatory actions of cGMP for renin release.

Systolic blood pressure in the central ear artery of eight rabbits increased by 21 mmHg (1 mmHg = 133.32 Pa) over 40 days following renal artery clipping and contralateral nephrectomy (one-kidney, one-clip). Plasma active and acid-activatable (pH 2.8) renin did not change significantly. Similar data were obtained from a group of 12 rabbits following renal artery clipping alone (two-kidney, one-clip) except that blood pressure in this group increased for 26 days but then declined until 40 days. Two animals with one-kidney, one-clip hypertension and three rabbits with two-kidney, one-clip hypertension had large increases in plasma active and inactive renin levels, which followed a more exaggerated rise in blood pressure than in the previous two groups. Forty days after unilateral renal artery clipping, the unclipped kidney was removed in 10 animals with two-kidney, one-clip hypertension. A further increase in blood pressure (+29%) occurred in seven of the animals but no change in plasma active or inactive renin. Results were compared with two groups of control animals, a unilateral nephrectomy group and a laparotomy group. None of the surgical procedures used produced a consistent pattern of change in the relative amounts of active and inactive renin in plasma. No marked changes in sodium, potassium, or water balance occurred in any group of animals.

1. Beta-adrenoceptor linked regulation of plasma active and inactive renin was investigated in sheep with indwelling artery, vein and bladder catheters. Concurrent changes in renal function were also monitored. 2. The beta-adrenoceptor agonist isoprenaline (bolus intravenous (i.v.) dose 0.5 mg/kg, 0.05 microgram/kg per min) increased plasma active renin concentration within 15 min and reached a plateau increase of 306%. Initially plasma inactive renin remained unchanged but after 105 min it decreased. 3. The non-selective beta-adrenoceptor antagonist propranolol (bolus i.v. dose 4 mg/kg, 40 micrograms/kg per min) did not significantly alter basal plasma active renin but selectively reduced inactive renin. The absence of any change in active renin may reflect the relatively low level of stress-induced sympathetic activation in these conscious animals compared to equivalent studies carried out on human subjects. 4. Using the same dose schedules, propranolol completely blocked the isoprenaline-induced increases in plasma active renin secretion even though it had no effect on the basal active renin levels. 5. The selective beta 1-adrenoceptor antagonist atenolol (bolus i.v. dose 5 mg/kg) decreased basal plasma active renin by 24% (+ 135 min) but reduced plasma inactive renin by 75% compared to control. 6. Therefore, both a beta-adrenoceptor agonist (isoprenaline) and two beta-adrenoceptor antagonists (propranolol and atenolol) decreased plasma inactive renin concentration in conscious sheep. 7. These results are discussed in relation to the accompanying changes in arterial blood pressure and aspects of renal function.

Secretion control for active and inactive (acid-activatable) renin was investigated using a rabbit kidney cortex slice preparation. In complete Krebs-Ringer bicarbonate buffer (pH 7.4) the calcium-channel-blocking drugs verapamil (50 microM) and flunarizine (50 microM) had no effect on active renin release but increased inactive renin secretion. In Ca-free or Ca-depleted (+EGTA) buffers secretion of both active and inactive renin was increased. Neither verapamil nor flunarizine under these conditions had any additional effect on active renin secretion but inactive renin release was stimulated by verapamil and inhibited by flunarizine. Although having no effect in the presence of Ca++ ions, the ionophore A23187 (17 microM) selectively abolished the secretion of inactive renin in the absence of calcium. The mechanisms regulating the secretion of the two forms of renin are clearly not identical. Differential release of active and inactive renin by the kidney may contribute to the overall control of the renin-angiotensin system.

1. Dietary sodium depletion and subsequent repletion was studied in rabbits. Potassium intake was maintained constant. 2. during sodium depletion and repletion blood pressure, packed cell volume, food consumption and body weight remained at control values. 3. Decreased sodium excretion was observed in both urine and faeces during sodium depletion and the physiological control of these changes is discussed in relation to the renin-angiotensin-aldosterone system. 4. Potassium excretion during sodium depletion initially fell as a result of reduced urine volume and gradually returned to normal. Urine potassium concentration remained constant. 5. Faecal excretion of potassium rose by 63% during sodium depletion and there was a rise from a control value of 17-25% in the proportion of total potassium excretion accounted for by the faecal component. 6. Water consumption and urine volume both decreased in the initial phase of sodium depletion and then returned to control levels. 7. It is important to consider both urinary and faecal excretion of sodium and potassium when calculating balance status for either ion. Faecal excretion, as well as kidney function, shows important physiological adaptations.

1. The role of Ca2+ in the control of renin release was investigated using a collagenase-dispersed rat kidney cortex cell preparation. 2. Superfusion with a series of low [Ca2+] buffers in either ascending or descending order of concentration increased renin release. Exposure to 0.06 mmol/l Ca2+ increased release by 120% (P less than 0.001) when presented as the first buffer in ascending order of concentration and by 79% (P less than 0.001) when presented as the fourth and last in a series of descending order. 3. The Ca2+ entry blocking drug diltiazem in a range of concentrations increased renin release and at 10(-5) mol/l diltiazem the mean stimulation was 35% (P less than 0.01). 4. 8-(N.N-Diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) reduces the release of Ca2+ from intracellular stores and, studied over a range of concentrations, this compound increased renin release. At 10(-5) mol/l TMB-8 the mean increase was 44% (P less than 0.001). 5. None of these experimental manipulations, low [Ca2+], diltiazem or TMB-8, had any effect on the release of adenosine 3':5'-cyclic monophosphate into the cell superfusate, indicating that a decrease in intracellular [Ca2+] increases renin release by a mechanism which is independent of changes in adenosine 3':5'-cyclic monophosphate production. 6. Effects of low [Ca2+], diltiazem and TMB-8 on renin secretion were all shown to be reversible when superfusion with control buffer was resumed.

Hyponatremia developing some days after transsphenoidal pituitary adenectomy is a treacherous complication of uncertain cause. Of 19 patients monitored in a pilot study at the Wessex Neurological Centre, plasma sodium fell below 125 mmol/liter in three patients at times ranging from 6 to 9 days postoperatively. One patient had evidence of inappropriate secretion of arginine vasopressin (AVP), and the other two probably had steroid insufficiency despite apparently adequate steroid cover. In a more detailed study, the fluid and sodium balance of a further 16 patients was monitored for 7 to 11 days following transsphenoidal surgery together with plasma cortisol, renin, and AVP concentrations. No patient became severely hyponatremic. Three developed partial diabetes insipidus. Two patients with Cushing's disease had evidence of postoperative corticosteroid insufficiency despite normal steroid protection. An inappropriately low plasma cortisol concentration was recorded in both. Plasma AVP concentrations did not show a delayed surge postoperatively. Delayed hyponatremia appears to occur most often in patients with hypoadrenalism, as glucocorticoid cover is decreased. It results from water retention combined with natriuresis, and is reversed by glucocorticoid treatment.

Excitation-secretion coupling for active and inactive renin was investigated by manipulating the ionic composition of media in which rabbit kidney cortex slices were incubated. Alterations in [Ca++] and [K+] produced qualitatively similar, but quantitatively distinct, changes in the release of the two forms of renin. Under control conditions, the slices secreted 15-20% of total renin in the inactive form and exposure to Ca++-free or low [K+] media increased this to 30-40%, a similar proportion to that stored within the kidney. Decreasing [Na+] stimulated release of active renin but reduced inactive renin. There appeared to be antagonism between the effects of Na+ and Ca++ ions in respect of active renin but synergism for release of the inactive form. It is suggested that the characteristics of a single renin release mechanism in the rat kidney resemble those for secretion of inactive, rather than active, renin in other species.

The response of plasma levels of active and inactive renin to haemorrhage was investigated in sheep with indwelling artery and vein catheters. In conscious animals, loss of 10% of estimated blood volume over a 5 min period increased plasma active renin by a mean of 59%, a surprisingly small change. Plasma inactive renin also increased, but only by 86%. Maximum increases in both forms of renin occurred within 1 h of the haemorrhage. The effects of an equivalent blood loss were investigated in pentobarbitone-anaesthetized sheep maintained in an upright posture using padded slings. Anaesthesia per se had no effect on plasma active or inactive renin. In anaesthetized sheep, 3 h after haemorrhage, plasma active renin had increased by 403% and inactive renin by 299% above control values, but a plateau (maximum) response was not reached during this time. In both conscious and anaesthetized animals the haemorrhage-induced increases in active and inactive renin occurred in parallel. It appears that haemorrhage of this intensity is a comparatively mild stimulus to increase plasma renin concentration in conscious sheep but is much more effective in anaesthetized animals. This may be linked to anaesthetic-induced increases in prostaglandin synthesis within the kidney.

The effect of tachyphylaxis to angiotensin II amide on the pressor potency of angiotensin I, II and III and noradrenaline was investigated in conscious rabbits with indwelling cannulae. No significant difference was observed between the reduction in responses to angiotensin II (58 +/- 4%) and angiotensin III (42 +/- 6%) but angiotensin I (15 +/- 5 %) and noradernaline (17 +/- 10%) responses were less markedly inhibited. If all angiotensin I pressor activity was mediated through its conversion to angiotensin II one would expect an equivalent degree of cross-tachyphylaxis to occur with the two peptides. Our results suggest that angiotensin I may have significant inherent pressor activity of its own, independent of conversion. This compound is already known to have activity at other target tissues. The absence of a significant difference in the degree of tachyphylaxis with angiotensin II and III may indicate they have substantially common pathways as pressor agents.

Angiotensin I is known to have direct agonist activity at some target tissues, independent of its conversion to angiotensin II. Aspects of its possible direct role as a pressor agent were investigated in conscious rabbits. Phentolamine (3 mg/kg i.v.), a dose which did not affect baseline blood pressure, reduced the pressor response to angiotensin II by 17% (P less than 0.05) but did not alter the sensitivity to angiotensin I. Noradrenaline activity was reduced by 56%. Propranolol (2 mg/kg i.v.), a dose which did not affect baseline blood pressure but which substantially reduced the depressor response to isoprenaline, had no effect on the pressor activity of either angiotensin I or II. Noradrenaline potency was reduced by 32%. Hexamethonium (20 mg/kg i.v.), caused a potentiation of the response to angiotensin I and II and noradrenaline. This was probably a non-specific action associated with the decrease in baseline blood pressure. No differential effect of ganglion blockade on the two angiotensins was noted. The dissociation of the effects of phentolamine on angiotensins I and II provides further evidence that pressor actions of these two compounds act through partially different mechanisms.

Release of active and inactive renin by rabbit kidney cortex slices was investigated. Inactive renin was estimated as the increase in renin activity after acidification (pH 2.8) of slice supernatant solutions. For kidney slices incubated in complete Krebs bicarbonate buffer, the Na-K-ATPase inhibitor ouabain (100 microM) reduced the secretion of both active (-19.2%) and inactive (-78.9%) forms of renin. In low Na buffers ([Na+] = 23 mM) active renin release was increased and inactive renin was suppressed. Both of these changes were abolished by addition of ouabain (100 microM). The reduction in inactive renin secretion produced by ouabain in complete Krebs buffer did not occur in low [Na+] buffers. In zero Ca2+ buffers containing EGTA (5 mM), secretion of both active and inactive renin was increased but these changes were abolished by addition of ouabain (100 microM). Incubating kidney slices in low Na+, zero Ca2+ media revealed differences between the secretion control mechanisms for the two forms of renin. The separate stimulatory effects of low Na+ and low Ca2+ were not additive for the release of active renin and inclusion of ouabain resulted in similar secretion rates to those under control conditions. For inactive renin secretion, in the absence of Ca2+ release mechanisms still respond to reduction in Na+ with decreased secretion. Conversely, in low Na+ buffers, removal of Ca2+ still promotes inactive renin secretion. These changes were abolished by the addition of ouabain (100 microM). Slices did not change in weight during incubation in media which did not contain ouabain. Addition of this inhibitor to control buffers and low Na buffers did result in an increase in weight. This correlated with the presence of Ca2+ in the buffer and did not appear to be related to [Na+]. These studies again show that the mechanisms regulating the secretion of active and inactive renin are not identical and support the hypothesis that Na+ have differing roles to play in the regulation of these two forms of renin.

1. Active and inactive renin release by rabbit kidney cortex slices was investigated. Inactive renin was estimated as the increase in renin activity after acidification (pH 2 . 8) of slice supernatant solutions. 2. Active renin release was increased when incubation medium [Na+] was reduced. This relationship was linear (r2 = 0 . 96) over the range [Na+] = 23-133 mM. 3. For the same range of [Na+] inactive renin secretion decreased when medium [Na+] was reduced (r2 = 0 . 92). Therefore, the proportion of total renin which was in the inactive form decreased linearly as [Na+] was reduced (r2 = 0 . 97). 4. Chloride ions did not appear to be important in altering the secretion of either active or inactive renin. 5. Adding furosemide to the incubation medium in concentrations up to 40 micrograms/ml. did not change secretion of either form of renin. The action of furosemide on secretion of active and inactive renin in vivo is therefore secondary to altered renal function. 6. Regulation of the relative amount of active and inactive renin in plasma could be entirely an intrarenal event. It is not essential to invoke a plasma activating enzyme for inactive renin in order to explain changes in plasma levels of the two forms of renin. 7. This paper supports the hypothesis that release of inactive renin by the kidney is controlled by a sodium-sensitive mechanism.

Regulation of plasma levels of active and inactive renin was investigated using sheep with indwelling artery, vein and bladder catheters. Control and experimental studies were carried out in the same animals on different days. Volume depletion during any single experiment was limited to a maximum of 50 ml. Despite large changes in sodium and water excretion, the diuretic furosemide at two dose levels, 1 and 10 mg/kg, failed to alter plasma levels of either active or inactive renin in conscious sheep. Induction of pentobarbitone anaesthesia in the sheep did not, per se, alter either plasma active or inactive renin. Furosemide (10 mg/kg) in anaesthetized animals produced a similar diuresis and natriuresis response to conscious sheep, but plasma active renin increased by 270% and inactive renin decreased to zero. In conscious sheep given an infusion of papaverine, furosemide also produced an increase in plasma active renin and a concurrent decrease in the inactive form. In both anaesthetized animals and in conscious sheep infused with papaverine, furosemide-induced intrarenal vasodilation, as evidenced by changes in clearance of p-aminohippuric acid, was much reduced in comparison to the conscious animals. This may be significant in relation to the control of renin secretion. It appears that the macula densa sodium receptor, which is considered to regulate renin release, will only function after it has been primed by other intra- or extrarenal factors. This is discussed, particularly in relation to the possible role played by the prostaglandin system.

Regulation of plasma active and inactive renin was investigated using conscious sheep with indwelling artery, vein and bladder catheters. Control and experimental studies were carried out in the same animals on different days. The calcium antagonist drug verapamil was given as an initial bolus injection (0.5 mg/kg) followed by a continuous infusion (0.1 mg/kg per h) over a 2.5 h period. Plasma active and inactive renin changed in parallel. Both were significantly increased within 15 min of the initial drug dose and both attained a peak increase after 45 min. Thereafter, the two forms of renin returned to basal levels despite continued infusion of the drug. Effective renal plasma flow (CPAH) was also transiently increased by verapamil and followed a similar time course to changes in plasma active and inactive renin concentration. Arterial blood pressure, however, remained suppressed by verapamil for the duration of the study. Verapamil did not alter urine flow or sodium and potassium excretion rates. These results are discussed in relation to the possible link between intrarenal haemodynamics and renin secretion in conscious and in anaesthetized animals and also in relation to the concept that variation in the relative amounts of active and inactive renin secreted in differing physiological situations represents a mechanism for regulating the renin-angiotensin system.

1. The effect of the beta-adrenoceptor agonist isoprenaline on the secretion of active and inactive renin was investigated in two preparations. 2. In ten urethane-anaesthetized rabbits isoprenaline, given as a renal artery infusion, had relatively minor effects on renal sodium excretion (increased) and systemic arterial blood pressure (decreased). Urine volume, potassium excretion, creatinine clearance and serum electrolytes were all unchanged. Plasma active and inactive renin both increased immediately and returned to basal values after ceasing the isoprenaline infusion. 3. No significant changes in either plasma renin activity or renal function were observed in a group of ten control animals. 4. The magnitude of the isoprenaline-induced changes in plasma active renin was similar to that in a previous study of frusemide diuresis, but the time course was quite different. Inactive renin disappeared from plasma during frusemide diuresis. 5. Renin release by rabbit kidney cortex slices was also studied. Isoprenaline, added to the incubation medium, caused a dose-related increase in active renin secretion, but inactive renin release remained unchanged. This is in marked contrast to a previous study where reducing [Na+] increased active renin and inhibited inactive renin output. 6. These data support our previous suggestion that activation of inactive renin is regulated by a sodium-sensitive intrarenal mechanism.