Low-Dose Near-Infrared Light-Activated Mitochondria-Targeting Photosensitizers for PDT Cancer Therapy.

Phthalocyanines (Pcs) are promising candidates for photodynamic therapy (PDT) due to their absorption in the phototherapeutic window. However, the highly aromatic Pc core leads to undesired aggregation and decreased reactive oxygen species (ROS) production. Therefore, short PEG chain functionalized A3B type asymmetric Pc photosensitizers (PSs) were designed in order to decrease aggregation and increase the aqueous solubility. Here we report the synthesis, characterization, optical properties, cellular localization, and cytotoxicity of three novel Pc-based agents (LC31, MLC31, and DMLC31Pt). The stepwise functionalization of the peripheral moieties has a strong effect on the distribution coefficient (logP), cellular uptake, and localization, as well as photocytotoxicity. Additional experiments have revealed that the presence of the malonic ester moiety in the reported agent series is indispensable in order to induce photocytotoxicity. The best-performing agent, MLC31, showed mitochondrial targeting and an impressive phototoxic index (p.i.) of 748 in the cisplatin-resistant A2780/CP70 cell line, after a low-dose irradiation of 6.95 J/cm2. This is the result of a high photocytotoxicity (IC50 = 157 nM) upon irradiation with near-infrared (NIR) light, and virtually no toxicity in the dark (IC50 = 117 µM). Photocytotoxicity was subsequently determined under hypoxic conditions. Additionally, a preliminarily pathway investigation of the mitochondrial membrane potential (MMP) disruption and induction of apoptosis by MLC31 was carried out. Our results underline how agent design involving both hydrophilic and lipophilic peripheral groups may serve as an effective way to improve the PDT efficiency of highly aromatic PSs for NIR light-mediated cancer therapy.

Fate-mapping of erythropoietin-producing cells in mouse models of hypoxaemia and renal tissue remodelling reveals repeated recruitment and persistent functionality.

AIM: Fibroblast-like renal erythropoietin (Epo) producing (REP) cells of the corticomedullary border region "sense" a decrease in blood oxygen content following anaemia or hypoxaemia. Burst-like transcription of Epo during tissue hypoxia is transient and is lost during fibrotic tissue remodelling, as observed in chronic kidney disease. The reason for this loss of Epo expression is under debate. Therefore, we tested the hypothesis that REP cell migration, loss and/or differentiation may cause Epo inhibition. METHODS: Using a reporter mouse that allows permanent labelling of active REP cells at any given time point, we analysed the spatiotemporal fate of REP cells following their initial hypoxic recruitment in models of hypoxaemia and renal tissue remodelling. RESULTS: In long-term tracing experiments, tagged REP reporter cells neither died, proliferated, migrated nor transdifferentiated into myofibroblasts. Approximately 60% of tagged cells re-expressed Epo upon a second hypoxic stimulus. In an unilateral model of tissue remodelling, tagged cells proliferated and ceased to produce Epo before a detectable increase in myofibroblast markers. Treatment with a hypoxia-inducible factor (HIF) stabilizing agent (FG-4592/roxadustat) re-induced Epo expression in the previously active REP cells of the damaged kidney to a similar extent as in the contralateral healthy kidney. CONCLUSIONS: Rather than cell death or differentiation, these results suggest cell-intrinsic transient inhibition of Epo transcription: following long-term dormancy, REP cells can repeatedly be recruited by tissue hypoxia, and during myofibrotic tissue remodelling, dormant REP cells are efficiently rescued by a pharmaceutic HIF stabilizer, demonstrating persistent REP cell functionality even during phases of Epo suppression.

Patient-Reported Outcomes in Middle Ear and Active Transcutaneous Bone Conduction Hearing Implants.

OBJECTIVE: This study used questionnaires to examine the patient-reported satisfaction with 2 hearing implant devices to determine the level of overall satisfaction with the devices, which, if any, factors predicted good or poor perceived outcomes, or whether there were any specific aspects of the devices where dissatisfaction was apparent. METHODS: A post-treatment questionnaire survey of 39 adult patients who had received a Vibrant Soundbridge (VSB) or Bonebridge (BB) hearing implant, with at least 3 months of follow-up, was conducted using the Glasgow Benefit Inventory (GBI) and Hearing Device Satisfaction Scale (HDSS). Satisfaction scores were compared to pre- and post-operative audiologic outcomes. The correlation between GBI and HDSS scores was also examined. RESULTS: A total of 28 of the 39 patients (72%) responded: 13 with a BB and 15 with a VSB at a mean of 13 months after implantation. The overall mean total GBI score was 30, with no significant differences across the groups. The responders generally reported that they were "satisfied" across most domains of the HDSS. In the study, 25 of the 28 responders were largely satisfied with their devices but 3 respondents were not. Two were known non-users, while one used the device but did not gain the benefit expected. It is instructive to note that all of these dissatisfied recipients were close to the manufacturer recommended limits for implantation of their respective devices at the time of surgery. Certain themes were identified within the patients' responses, indicating common aspects where satisfaction was poorer. CONCLUSION: This series of 28 implant recipients demonstrates high levels of satisfaction with implantable hearing devices across 2 different validated questionnaires. Implant teams could exercise caution and manage patient expectations if the patients are close to the recommended limits of a particular device.

Distal and proximal hypoxia response elements cooperate to regulate organ-specific erythropoietin gene expression.

While it is well-established that distal hypoxia response elements (HREs) regulate hypoxia-inducible factor (HIF) target genes such as erythropoietin (Epo), an interplay between multiple distal and proximal (promoter) HREs has not been described so far. Hepatic Epo expression is regulated by a HRE located downstream of the EPO gene, but this 3' HRE is dispensable for renal EPO gene expression. We previously identified a 5' HRE and could show that both HREs direct exogenous reporter gene expression. Here, we show that whereas in hepatic cells the 3' but not the 5' HRE is required, in neuronal cells both the 5' and 3' HREs contribute to endogenous Epo induction. Moreover, two novel putative HREs were identified in the EPO promoter. In hepatoma cells HIF interacted mainly with the distal 3' HRE, but in neuronal cells HIF most strongly bound the promoter, to a lesser extent the 3' HRE, and not at all the 5' HRE. Interestingly, mutation of either of the two distal HREs abrogated HIF binding to the 3' and promoter HREs. These results suggest that a canonical functional HRE can recruit multiple, not necessarily HIF, transcription factors to mediate HIF binding to different distant HREs in an organ-specific manner.

Device profile of the Bonebridge bone conduction implant system in hearing loss: an overview of its safety and efficacy.

INTRODUCTION: The Bonebridge is an active transcutaneous semi-implantable bone conduction hearing device suitable for several types of hearing loss. It has unique benefits over some more established technologies. It consists of an internal active implant and an external sound processor. It was first launched in 2012, with a newer model released in late 2019. AREAS COVERED: The structure and features of the device are described. Indications, audiological criteria, and contraindications to implantation are discussed. The planning and procedure of implantation surgery are also described. Research outlining the outcomes of implant use and risk of adverse events is highlighted. EXPERT OPINION: The evidence included in this article demonstrates the successful audiological outcomes and patient satisfaction with Bonebridge implantation. The rate of adverse events following surgery is low and compares well with other devices which may be considered for Bonebridge candidates. The device should be considered as an option for suitable candidates and in many cases may be the better option available, given the low incidence of skin complications and the absence of a skin penetrating abutment. Future advances are likely to affect sound processor technology, connectivity, and possibly further reduction in implant size and gain.

Oxygen-dependent bond formation with FIH regulates the activity of the client protein OTUB1.

Protein:protein interactions are the basis of molecular communication and are usually of transient non-covalent nature, while covalent interactions other than ubiquitination are rare. For cellular adaptations, the cellular oxygen and peroxide sensor factor inhibiting HIF (FIH) confers oxygen and oxidant stress sensitivity to the hypoxia inducible factor (HIF) by asparagine hydroxylation. We investigated whether FIH contributes to hypoxia adaptation also through other mechanisms and identified a hypoxia sensitive, likely covalent, bond formation by FIH with several client proteins, including the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1). Biochemical analyses were consistent with a co-translational amide bond formation between FIH and OTUB1, occurring within mammalian and bacterial cells but not between separately purified proteins. Bond formation is catalysed by FIH and highly dependent on oxygen availability in the cellular microenvironment. Within cells, a heterotrimeric complex is formed, consisting of two FIH and one covalently linked OTUB1. Complexation of OTUB1 by FIH regulates OTUB1 deubiquitinase activity. Our findings reveal an alternative mechanism for hypoxia adaptation with remarkably high oxygen sensitivity, mediated through covalent protein-protein interactions catalysed by an asparagine modifying dioxygenase.

Quality of Life Outcomes following Treatment of Hypopharyngeal Cancer.

Quality of life (QoL) is an important consideration in the management of individuals with head and neck cancer. The poor prognosis and significant impact of treatment modalities on function of the salivary glands, larynx and pharynx combine to make hypopharyngeal carcinoma a particularly challenging condition to treat. The impact of diagnosis and treatment on health related QoL is substantial. There is increased understanding that organ preservation does not necessarily correlate with function preservation as was previously expected. The impact on QoL, of chemoradiotherapy (CRT) or surgery, must be taken into account when managing individuals and deciding on treatment. Several QoL tools have been developed to understand the subjective consequences of functional impairment. The number and quality of studies specifically for hypopharyngeal carcinoma are low. The effects on QoL differ for surgery and CRT, as one would expect, but there are no demonstrable significant differences in most domains. Those treated with CRT show higher levels of dry mouth and sticky saliva, while those patients who have undergone surgery report greater levels of sensory disturbance. Significant differences were not noted in speech outcomes or global (general) health scores. The psychological morbidity and lack of good coping strategies are thought to play an important role in the high suicide rates of these patients (12-fold higher than the average population in the USA). Large, long-term, longitudinal studies of patients surviving treatment, answering both general and disease-specific questionnaires are required to direct clinicians towards the least morbid treatment strategies. The ability to cope and the availability of emotional support probably have a greater impact on subjective QoL than the functional outcomes of treatment.

Generation of renal Epo-producing cell lines by conditional gene tagging reveals rapid HIF-2 driven Epo kinetics, cell autonomous feedback regulation, and a telocyte phenotype.

Erythropoietin (Epo) is essential for erythropoiesis and is mainly produced by the fetal liver and the adult kidney following hypoxic stimulation. Epo regulation is commonly studied in hepatoma cell lines, but differences in Epo regulation between kidney and liver limit the understanding of Epo dysregulation in polycythaemia and anaemia. To overcome this limitation, we have generated a novel transgenic mouse model expressing Cre recombinase specifically in the active fraction of renal Epo-producing (REP) cells. Crossing with reporter mice confirmed the inducible and highly specific tagging of REP cells, located in the corticomedullary border region where there is a steep drop in oxygen bioavailability. A novel method was developed to selectively grow primary REP cells in culture and to generate immortalized clonal cell lines, called fibroblastoid atypical interstitial kidney (FAIK) cells. FAIK cells show very early hypoxia-inducible factor (HIF)-2α induction, which precedes Epo transcription. Epo induction in FAIK cells reverses rapidly despite ongoing hypoxia, suggesting a cell autonomous feedback mechanism. In contrast, HIF stabilizing drugs resulted in chronic Epo induction in FAIK cells. RNA sequencing of three FAIK cell lines derived from independent kidneys revealed a high degree of overlap and suggests that REP cells represent a unique cell type with properties of pericytes, fibroblasts, and neurons, known as telocytes. These novel cell lines may be helpful to investigate myofibroblast differentiation in chronic kidney disease and to elucidate the molecular mechanisms of HIF stabilizing drugs currently in phase III studies to treat anemia in end-stage kidney disease.

Self-Evaluated Quality of Life and Functional Outcomes After Microsurgery, Stereotactic Radiation or Observation-Only for Vestibular Schwannoma of the Adult Patient: A Systematic Review.

OBJECTIVE: To evaluate the impact of different management options on health-related quality of life (HRQoL) in vestibular schwannoma patients. DATA SOURCES: A systematic search of the Cochrane Database, Database of Abstracts of Reviews of Effectiveness, and the Ovid Medline & EMBASE was performed. English and German language studies published between 1980 and 2015 were considered. STUDY SELECTION: This is a systematic review of HRQoL of patients managed for vestibular schwannoma. Studies in which HRQoL after one management option were evaluated or compared with other managements or with control populations using validated or reliable questionnaires, were included. DATA EXTRACTION: The included studies were independently evaluated by two reviewers. The quality of studies was assessed and graded as per Oxford Centre of Evidence Based Medicine System. RESULTS: Ten prospective and 29 retrospective studies were identified: microsurgery initially exerted a negative effect on HRQoL but this tended to improve with follow up. Radiotherapy had a less negative effect but with minimal change over follow up. A significant limitation was that studies did not present results stratified by tumor size. Many patients will need active treatment despite the potential for negative effects on their QoL. The concept of a minimal clinically important difference has been introduced into this field and was compared with five studies. CONCLUSION: A number of prospective studies are available but none yet with a disease-specific questionnaire. Heterogeneity and the methodological weaknesses of the included studies constitute the principle limitation of this review. The introduction of the minimal clinically important difference should improve the relevance of studies and allow a sensitive comparison of treatments.

Hypoxia ameliorates intestinal inflammation through NLRP3/mTOR downregulation and autophagy activation.

Hypoxia regulates autophagy and nucleotide-binding oligomerization domain receptor, pyrin domain containing (NLRP)3, two innate immune mechanisms linked by mutual regulation and associated to IBD. Here we show that hypoxia ameliorates inflammation during the development of colitis by modulating autophagy and mammalian target of rapamycin (mTOR)/NLRP3 pathway. Hypoxia significantly reduces tumor necrosis factor α, interleukin (IL)-6 and NLRP3 expression, and increases the turnover of the autophagy protein p62 in colon biopsies of Crohn's disease patients, and in samples from dextran sulfate sodium-treated mice and Il-10 -/- mice. In vitro, NF-κB signaling and NLRP3 expression are reduced through hypoxia-induced autophagy. We also identify NLRP3 as a novel binding partner of mTOR. Dimethyloxalylglycine-mediated hydroxylase inhibition ameliorates colitis in mice, downregulates NLRP3 and promotes autophagy. We suggest that hypoxia counteracts inflammation through the downregulation of the binding of mTOR and NLRP3 and activation of autophagy.Hypoxia and HIF-1α activation are protective in mouse models of colitis, and the latter regulates autophagy. Here Cosin-Roger et al. show that hypoxia ameliorates intestinal inflammation in Crohn's patients and murine colitis models by inhibiting mTOR/NLRP3 pathway and promoting autophagy.

FIH Regulates Cellular Metabolism through Hydroxylation of the Deubiquitinase OTUB1.

The asparagine hydroxylase, factor inhibiting HIF (FIH), confers oxygen-dependence upon the hypoxia-inducible factor (HIF), a master regulator of the cellular adaptive response to hypoxia. Studies investigating whether asparagine hydroxylation is a general regulatory oxygen-dependent modification have identified multiple non-HIF targets for FIH. However, the functional consequences of this outside of the HIF pathway remain unclear. Here, we demonstrate that the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1) is a substrate for hydroxylation by FIH on N22. Mutation of N22 leads to a profound change in the interaction of OTUB1 with proteins important in cellular metabolism. Furthermore, in cultured cells, overexpression of N22A mutant OTUB1 impairs cellular metabolic processes when compared to wild type. Based on these data, we hypothesize that OTUB1 is a target for functional hydroxylation by FIH. Additionally, we propose that our results provide new insight into the regulation of cellular energy metabolism during hypoxic stress and the potential for targeting hydroxylases for therapeutic benefit.

Hypoxia attenuates the proinflammatory response in colon cancer cells by regulating IκB.

Two main features common to all solid tumors are tissue hypoxia and inflammation, both of which cause tumor progression, metastasis, therapy resistance and increased mortality. Chronic inflammation is associated with increased cancer risk, as demonstrated for inflammatory bowel disease patients developing colon cancer. However, the interplay between hypoxia and inflammation on the molecular level remains to be elucidated. We found that MC-38 mouse colon cancer cells contain functional hypoxic (HIF-1α) and inflammatory (p65/RelA) signaling pathways. In contrast to cells of the myeloid lineage, HIF-1α levels remained unaffected in MC-38 cells treated with LPS, and hypoxia failed to induce NF-κB. A similar regulation of canonical HIF and NF-κB target genes confirmed these results. RNA deep sequencing of HIF-1α and p65/RelA knock-down cells revealed that a surprisingly large fraction of HIF target genes required p65/RelA for hypoxic regulation and a number of p65/RelA target genes required HIF-1α for proinflammatory regulation, respectively. Hypoxia attenuated the inflammatory response to LPS by inhibiting nuclear translocation of p65/RelA independently of HIF-1α, which was associated with enhanced IκBα levels and decreased IKKß phosphorylation. These data demonstrate that the interaction between hypoxic and inflammatory signaling pathways needs to be considered when designing cancer therapies targeting HIF or NF-κB.

Hypoxia enhances lipid uptake in macrophages: role of the scavenger receptors Lox1, SRA, and CD36.

OBJECTIVES: The core of advanced atherosclerotic plaques turns hypoxic as the arterial wall thickens and oxygen diffusion capacity becomes impaired. Macrophage-derived foam cells play a pivotal role in atherosclerotic plaque formation by expressing scavenger receptors that regulate lipid uptake. However, the role of hypoxia in scavenger receptor regulation remains incompletely understood. METHODS AND RESULTS: Using RT-qPCR, flow cytometry and immunoblotting, we found that mRNA and protein expression levels of the scavenger receptor A (SRA) and the cluster of differentiation 36 (CD36) were upregulated by oxidized low-density lipoprotein (oxLDL), but decreased following exposure of macrophages to hypoxia. In contrast, lectin-like oxLDL receptor (Lox-1) mRNA and protein levels were upregulated under hypoxic conditions. Flow cytometry confirmed the increased lipid content in macrophages after exposure to 0.2% oxygen and the hypoxia-mimetic dimethyloxalylglycine (DMOG). Antibody-mediated blocking of Lox-1 receptor decreased the hypoxic induction of oxLDL uptake and lipid content. RNAi-mediated knock-down of hypoxia-inducible factor (HIF)-1α in macrophages attenuated the hypoxic induction of Lox-1. CONCLUSIONS: Hypoxia increases lipid uptake into macrophages and differentially regulates the expression of oxLDL receptors. Lox-1 plays a major role in hypoxia-induced foam cell formation which is, at least in part, mediated by HIF-1α.

Assessing the threshold for vestibular schwannoma resection and the behavior of residual tumor.

OBJECTIVES: In vestibular schwannoma surgery postoperative facial nerve function is generally described as being related to tumor size, with 20 mm extrameatal diameter suggested as the threshold at which facial nerve function deteriorates. Near-total resection is accepted if the risk to nerve function becomes unacceptably high. We sought to review outcomes of vestibular schwannoma resection and follow residual tumor fragments. Second, we pooled data to quantify the risk of regrowth and need for subsequent intervention. METHODS: Prospectively collected data on all patients undergoing microsurgical removal of vestibular schwannoma by the senior author over the past 15 years. RESULTS: Approximately 247 patients were identified, the spread of tumors was 74 intracanalicular, 120 15 mm or lesser, 57 between 16 and 30 mm, and 6 greater than 30 mm. Overall, 91.5% recovered to House-Brackmann Grade 1 or 2, with no difference between the groups with 15 mm or lesser and greater than 15 mm (p > 0.05); 26 had incomplete resection, and 3 showed regrowth. Pooled data: 193 near total resections with 12% regrowing; 181 subtotal resections with 30% regrowing. Of 108 growing fragments, 78% had intervention. CONCLUSION: The present series suggests that the threshold for preservation of good facial nerve function may be moved to a tumor of 30-mm extrameatal diameter. Although historically tumor fragments were felt to have a low growth potential, the cumulative data would suggest that a significant proportion of fragments do grow and may need intervention.

To drain or not to drain - management of pediatric deep neck abscesses: a case-control study.

UNLABELLED: Optimal management of deep neck abscesses has been the subject of debate for more than a century: surgical drainage has been the mainstay of treatment, but recently many centres have reported successful non-operative management in selected cases. OBJECTIVES: Our objective was to review the management of deep neck abscesses in our institution and to identify characteristics that would predict successful non-operative management. METHODS: A retrospective chart review from January 2001 to August 2010 was performed. Children up to age fifteen years with a CT-confirmed diagnosis of retropharyngeal or parapharyngeal abscess were included. A case-control study of small deep space neck abscesses (≤ 25 mm maximal diameter) was performed, comparing antibiotic treatment alone with antibiotics plus abscess drainage. RESULTS: 54 children met the inclusion criteria, of whom half had abscesses ≤ 25 mm diameter. Younger children within the group with smaller abscesses were more likely to need surgical drainage (p<0.05). Of 13 children requiring operative management, ten underwent a period of antibiotic treatment and observation prior to surgery, eight (80%) had fever beyond 48 h compared with three (23%) in the non-surgical group (p<0.01). 27 children had an abscess > 25 mm diameter on CT scan, four (15%) of whom responded quickly to antibiotics and were managed non-operatively, while the rest underwent surgery. There were no significant differences between the surgical and non-surgical group characteristics with larger abscesses. CONCLUSION: High dose intravenous antibiotics are an effective treatment for deep space neck abscesses and may obviate the need for surgical drainage, particularly in smaller abscesses. Children who do not respond quickly to antibiotics are more likely to require surgery to achieve resolution. Children with larger abscesses may respond to antibiotic therapy alone but should be closely observed. A trial of high dose intravenous antibiotics in stable children with close observation is warranted as first line treatment, especially for small deep space neck abscesses.

Do high-speed drills generate high-frequency noise in mastoid surgery?

BACKGROUND: The introduction of increasingly high speed drills for mastoid surgery has heightened the concern that cochlea damage may occur in both the operated and nonoperated ear. It has been observed clinically that this damage could be associated with frequencies above 8,000 Hz and that, to observe these changes, high-frequency audiometry should be performed. Previous studies have investigated noise transmission to the cochlea at frequencies below 4,000 Hz only. There having been, until recently, limitations to the equipment available to measure higher frequencies. OBJECTIVE: To define the characteristics of noise transmitted to the cochlea during drilling of temporal bone, specifically in the higher frequency ranges up to 20,000 Hz. METHODS: Cleaned temporal bones were fitted with 3 mutually perpendicular accelerometers, capable of measuring frequencies in the range 500 to 20,000 Hz. The system was calibrated using a Kamplex Audio Traveller AA220 pure tone audiometer, and accelerometer outputs were recorded on a personal computer at a sampling frequency of 102.4 kHz per channel. The magnitude of the noise transmitted to the cochlea was determined for a range of burrs. RESULTS: Maximum transmission of sound was 108 dBA at 4,000 Hz using a 6.5-mm burr on the cortical mastoid bone. The average results showed that the sound transmission tailed off at the higher frequencies dropping to 84 dBA at 8,000 Hz and 40 dBA at 16,000 Hz. CONCLUSION: The high-frequency hearing reduction noted in patients after mastoid surgery was shown not to be due to excessive high-frequency noise generated by drilling.

Substrate preference and phosphatidylinositol monophosphate inhibition of the catalytic domain of the Per-Arnt-Sim domain kinase PASKIN.

The Per-Arnt-Sim (PAS) domain serine/threonine kinase PASKIN, or PAS kinase, links energy flux and protein synthesis in yeast, regulates glycogen synthesis and protein translation in mammals, and might be involved in insulin regulation in the pancreas. According to the current model, binding of a putative ligand to the PAS domain disinhibits the kinase domain, leading to PASKIN autophosphorylation and increased kinase activity. To date, only synthetic but no endogenous PASKIN ligands have been reported. In the present study, we identified a number of novel PASKIN kinase targets, including ribosomal protein S6. Together with our previous identification of eukaryotic elongation factor 1A1, this suggests a role for PASKIN in the regulation of mammalian protein translation. When searching for endogenous PASKIN ligands, we found that various phospholipids can bind PASKIN and stimulate its autophosphorylation. Interestingly, the strongest binding and autophosphorylation was achieved with monophosphorylated phosphatidylinositols. However, stimulated PASKIN autophosphorylation did not correlate with ribosomal protein S6 and eukaryotic elongation factor 1A1 target phosphorylation. Although autophosphorylation was enhanced by monophosphorylated phosphatidylinositols, di- and tri-phosphorylated phosphatidylinositols inhibited autophosphorylation. By contrast, target phosphorylation was always inhibited, with the highest efficiency for di- and tri-phosphorylated phosphatidylinositols. Because phosphatidylinositol monophosphates were found to interact with the kinase rather than with the PAS domain, these data suggest a multiligand regulation of PASKIN activity, including a still unknown PAS domain binding/activating ligand and kinase domain binding modulatory phosphatidylinositol phosphates.

Vitamin C is dispensable for oxygen sensing in vivo.

Prolyl-4-hydroxylation is necessary for proper structural assembly of collagens and oxygen-dependent protein stability of hypoxia-inducible transcription factors (HIFs). In vitro function of HIF prolyl-4-hydroxylase domain (PHD) enzymes requires oxygen and 2-oxoglutarate as cosubstrates with iron(II) and vitamin C serving as cofactors. Although vitamin C deficiency is known to cause the collagen-disassembly disease scurvy, it is unclear whether cellular oxygen sensing is similarly affected. Here, we report that vitamin C-deprived Gulo(-/-) knockout mice show normal HIF-dependent gene expression. The systemic response of Gulo(-/-) animals to inspiratory hypoxia, as measured by plasma erythropoietin levels, was similar to that of animals supplemented with vitamin C. Hypoxic HIF induction was also essentially normal under serum- and vitamin C-free cell-culture conditions, suggesting that vitamin C is not required for oxygen sensing in vivo. Glutathione was found to fully substitute for vitamin C requirement of all 3 PHD isoforms in vitro. Consistently, glutathione also reduced HIF-1α protein levels, transactivation activity, and endogenous target gene expression in cells exposed to CoCl(2). A Cys201Ser mutation in PHD2 increased basal hydroxylation rates and conferred resistance to oxidative damage in vitro, suggesting that this surface-accessible PHD2 cysteine residue is a target of antioxidative protection by vitamin C and glutathione.

15-Year review of laryngeal and oral dysplasias and progression to invasive carcinoma.

The objective of this study is to identify a management or follow-up strategy for patients with laryngeal and oral dysplasia. A chart review of all patients with laryngeal and oral dysplasia over a 15-year period was performed. All patients were followed for a minimum period of 5 years from initial diagnosis of oropharyngeal or laryngeal dysplasia. If invasive carcinoma was demonstrated on subsequent biopsies, the exact time of this was recorded and Kaplan-Meier survival curves were plotted. In the laryngeal cohort, 45 patients were identified, 15 (33%) developed carcinoma, 7 of 30 patients (23%) with mild or moderate dysplasia, compared with 8 of 15 (53%) with severe dysplasia or CIS (P = 0.01). Thirteen of the carcinomas (87%) developed within 36 months of original biopsy. In the oral cohort, 32 patients were identified, 17 (53%) developed carcinoma, 1 of 9 patients (11%) with mild dysplasia, compared with 8 of 12 (67%) with severe dysplasia and 8 of 10 (80%) with CIS (P < 0.001). Fifteen of the 17 patients (88%) developed carcinoma within 36 months of original biopsy. In conclusion, although numbers are small, our results show that mild and moderate laryngeal dysplasia behaves differently to severe dysplasia and CIS. Mild oral dysplasia also behaves differently to severe dysplasia or CIS. In general, progress to malignancy happens within a 3-year period. Severe dysplasia or CIS should be managed aggressively.