Validation of physician certified verbal autopsy using conventional autopsy: a large study of adult non-external causes of death in a metropolitan area in Brazil.

BACKGROUND: Reliable mortality data are essential for the development of public health policies. In Brazil, although there is a well-consolidated universal system for mortality data, the quality of information on causes of death (CoD) is not even among Brazilian regions, with a high proportion of ill-defined CoD. Verbal autopsy (VA) is an alternative to improve mortality data. This study aimed to evaluate the performance of an adapted and reduced version of VA in identifying the underlying causes of non-forensic deaths, in São Paulo, Brazil. This is the first time that a version of the questionnaire has been validated considering the autopsy as the gold standard. METHODS: The performance of a physician-certified verbal autopsy (PCVA) was evaluated considering conventional autopsy (macroscopy plus microscopy) as gold standard, based on a sample of 2060 decedents that were sent to the Post-Mortem Verification Service (SVOC-USP). All CoD, from the underlying to the immediate, were listed by both parties, and ICD-10 attributed by a senior coder. For each cause, sensitivity and chance corrected concordance (CCC) were computed considering first the underlying causes attributed by the pathologist and PCVA, and then any CoD listed in the death certificate given by PCVA. Cause specific mortality fraction accuracy (CSMF-accuracy) and chance corrected CSMF-accuracy were computed to evaluate the PCVA performance at the populational level. RESULTS: There was substantial variability of the sensitivities and CCC across the causes. Well-known chronic diseases with accurate diagnoses that had been informed by physicians to family members, such as various cancers, had sensitivities above 40% or 50%. However, PCVA was not effective in attributing Pneumonia, Cardiomyopathy and Leukemia/Lymphoma as underlying CoD. At populational level, the PCVA estimated cause specific mortality fractions (CSMF) may be considered close to the fractions pointed by the gold standard. The CSMF-accuracy was 0.81 and the chance corrected CSMF-accuracy was 0.49. CONCLUSIONS: The PCVA was efficient in attributing some causes individually and proved effective in estimating the CSMF, which indicates that the method is useful to establish public health priorities.

The link between cutaneous inflammation and cognitive impairment.

Cognitive impairment is a symptom of neurological disorders, including dementia and Alzheimer's disease; and mild cognitive impairment can be a precursor of both disorders. Aged humans and animal models with other systemic disorders, such as cardiovascular diseases and diabetes, display a higher incidence of cognitive decline. Epidemiological studies have shown that the incidence of cognitive impairment also is higher in subjects with certain inflammatory skin disorders, including psoriasis and chronic eczematous dermatitis. Chronologically aged individuals exhibit increased cutaneous inflammation and elevated circulating cytokine levels, linked to alterations in epidermal function, which itself can induce cutaneous inflammation. Conversely, strategies that improve epidermal function can lower cytokine levels in both the skin and circulation. Thus, it seems likely that epidermal dysfunction could contribute, at least in part, to the development of chronic low-grade inflammation, also termed 'inflammaging', in the elderly. The evidence of cognitive impairment in patients with inflammatory dermatoses suggests a link between cutaneous inflammation and cognitive impairment. Because of the pathogenic role of epidermal dysfunction in ageing-associated cutaneous inflammation, improvements in epidermal function could be an alternative approach for mitigation of the ageing-associated decline in cognitive function.

Macroevolutionary diversification rates show time dependency.

For centuries, biologists have been captivated by the vast disparity in species richness between different groups of organisms. Variation in diversity is widely attributed to differences between groups in how fast they speciate or go extinct. Such macroevolutionary rates have been estimated for thousands of groups and have been correlated with an incredible variety of organismal traits. Here we analyze a large collection of phylogenetic trees and fossil time series and describe a hidden generality among these seemingly idiosyncratic results: speciation and extinction rates follow a scaling law in which both depend on the age of the group in which they are measured, with the fastest rates in the youngest clades. Using a series of simulations and sensitivity analyses, we demonstrate that the time dependency is unlikely to be a result of simple statistical artifacts. As such, this time scaling is likely a genuine feature of the tree of life, hinting that the dynamics of biodiversity over deep time may be driven in part by surprisingly simple and general principles.

CERKL is upregulated in cutaneous squamous cell carcinoma and maintains cellular sphingolipids and resistance to oxidative stress.

BACKGROUND: Ceramide kinase-like protein (CERKL) was originally described in retinal tissue. CERKL has been shown to protect cells from oxidative stress, and mutations in CERKL underlie the inherited disease retinitis pigmentosa. CERKL expression maintains cellular sphingolipids via an unknown mechanism. OBJECTIVES: To determine whether CERKL is expressed in epidermis and cutaneous squamous cell carcinoma (cSCC) and whether CERKL expression affects cSCC sphingolipid metabolism and susceptibility to oxidative stress. METHODS: CERKL expression was determined by RNA-Seq, quantitative polymerase chain reaction and immunohistochemistry. CERKL was knocked down in cSCC cells using small interfering RNA. Sphingolipid content was analysed by liquid chromatography-mass spectrometry. Oxidative stress was induced by treatment with H2 O2 , and apoptosis was measured using flow cytometry to determine annexin V binding. RESULTS: CERKL mRNA and protein are highly expressed in actinic keratosis and cSCC in comparison with normal epidermis. CERKL is also expressed in metabolically active epithelial cells in normal hair bulbs and sebaceous glands. CERKL knockdown in cultured cSCC cells reduces cellular sphingolipid content and enhances susceptibility to oxidative stress. CONCLUSIONS: These findings suggest that CERKL may be important in cSCC progression and could lead to novel strategies for prevention and treatment of cSCC.

CERKL is Upregulated in Cutaneous Squamous Cell Carcinoma and Maintains Cellular Sphingolipids and Resistance to Oxidative Stress.

BACKGROUND: Ceramide Kinase-Like Protein (CERKL) was originally described in retinal tissue. CERKL has been shown to protect cells from oxidative stress, and mutations in CERKL underlie the inherited disease, retinitis pigmentosa. CERKL expression maintains cellular sphingolipids via an unknown mechanism. OBJECTIVES: To determine whether CERKL is expressed in epidermis and cutaneous squamous cell carcinoma (cSCC) and whether CERKL expression affects cSCC sphingolipid metabolism and susceptibility to oxidative stress. METHODS: CERKL expression was determined by RNA-Seq, qPCR and immunohistochemistry. CERKL was knocked down in cSCC cells using siRNA. Sphingolipid content was analyzed by liquid chromatography-mass spectrometry (LC-MS). Oxidative stress was induced by treatment with H2 O2 , and apoptosis was measured using flow cytometry to determine annexin v binding. RESULTS: CERKL mRNA and protein are highly expressed in actinic keratosis and cSCC in comparison with normal epidermis. CERKL also is expressed in metabolically active epithelial cells in normal hair bulbs and sebaceous glands. CERKL knockdown in cultured cSCC cells reduces cellular sphingolipid content and enhances susceptibility to oxidative stress. CONCLUSIONS: These findings suggest that CERKL may be important in cSCC progression and could lead to novel strategies for prevention and treatment of cSCC.

Comparison of Fundus Biomicroscopy Examination of the Optic Nerve Head with and without Mydriasis.

BACKGROUND: Notwithstanding the significant advances in automated imaging techniques in the past 2 decades, subjective evaluation of the optic disc still remains an important part of glaucoma propaedeutic. In places with limited resources and a high demand for ophthalmic care, anatomical evaluation of glaucoma cases often relies solely on slit-lamp-based fundus biomicroscopic examination, which is frequently performed without mydriasis. OBJECTIVE: The aim of this study was to compare metrics related to fundus biomicroscopy examination of the optic nerve head and peripapillary retinal nerve fiber layer (pRNFL) with and without mydriasis. MATERIAL AND METHODS: Healthy individuals, patients with early glaucoma, and glaucoma suspects were prospectively enrolled. Patients were examined before and after mydriasis by three glaucoma specialists, who estimated patients' vertical cup-to-disc ratio (CDR) and evaluated the presence of glaucomatous signs: laminar dot sign, disc hemorrhage, disc saucering, disc notching, peripapillary atrophy, localized pRNFL defect, and loss of the ISNT pattern. Main outcome measures were the intra-observer comparison, the inter-observer agreement, and the abilities to identify glaucomatous signs before and after mydriasis. RESULTS: Thirty patients (60 eyes) were enrolled (mean age, 62.3 ± 11.6 years). Considering the evaluation of the three examiners, the mean vertical CDR increased from 0.41 to 0.44 (p = 0.02), and the median of the coefficient of variation of the measures was reduced from 0.24 to 0.11 (p = 0.01) after mydriasis. Regarding the inter-observer agreement evaluation, the kappa coefficient values ranged from 0.64 to 0.72 before mydriasis and from 0.71 to 0.77 after mydriasis. Dot sign and disc notching were better identified through fundoscopic examination with mydriasis compared to the nonmydriatic examination (p < 0.01). CONCLUSION: Our results suggest that fundus biomicroscopy should be performed with mydriasis whenever possible, as it yells a better intra- and inter-observer agreement and improves the detection of glaucomatous signs. Moreover, examiners seem to underestimate CDR values without mydriasis. Further investigation is warranted to validate these findings by general ophthalmologists and in different populations.

Topical applications of an emollient reduce circulating pro-inflammatory cytokine levels in chronically aged humans: a pilot clinical study.

BACKGROUND: While increased levels of circulating inflammatory cytokines in chronologically aged humans have been linked to the development of ageing-associated chronic disorders (e.g., cardiovascular disease, type II diabetes, osteoporosis and Alzheimer's disease), approaches that reduce circulating cytokines are not yet available. In chronologically aged mice, we recently demonstrated that epidermal dysfunction largely accounts for age-associated elevations in circulating cytokine levels, and that improving epidermal function reduced circulating cytokine levels. OBJECTIVE: We performed a pilot study to determine whether improving epidermal function reduces circulating pro-inflammatory cytokine levels in aged humans. METHODS: Thirty-three aged humans were topically treated twice-daily for 30 days, with ≈ 3 mL of an emollient, previously shown to improve epidermal function, while untreated, aged humans and a cohort of young volunteers served as controls. Changes in epidermal function and levels of three key, age-related, plasma cytokines (IL-1ß, IL-6 and TNFα) were measured at baseline and after treatment, using Luminex 200™ system. RESULTS: We also found significantly higher baseline levels of IL-1ß, IL-6 and TNFα in aged vs. young humans (P < 0.001), as previously reported. Topical applications of the barrier repair emollient significantly enhanced epidermal permeability barrier function (P < 0.01) and stratum corneum hydration (P < 0.05). In parallel, circulating levels of IL-1ß and IL-6 normalized, while TNFα levels declined substantially. CONCLUSION: The results of this preliminary study suggest that a larger clinical trial should be performed to confirm whether improving epidermal function also can reduce circulating pro-inflammatory cytokine levels in aged humans, while also possibly attenuating the downstream development of chronic inflammatory disorders in the aged humans.

Validation of clinical risk score for colorectal liver metastases resected in a contemporary multicenter cohort.

BACKGROUND: Recent advances in care for colorectal liver metastases (CRLM) have lengthened 5-year survival. In this new era, prognostic tools such as the clinical risk score (CRS) for colorectal liver metastases require reevaluation. METHODS: Patients undergoing resection for CRLM between 2008 and 2012 at 4 specialty hepatobiliary centers in Canada (N = 740) were stratified by CRS and analyzed in Kaplan-Meier survival curves. Primary outcome of overall survival (OS) and secondary outcome of recurrence-free survival (RFS). Multivariate Cox regression compared CRS to patient factors. RESULTS: Median OS not reached (>60 months), median RFS 16 months. Original CRS strata was a significant (p < 0.001) predictor of both OS (5-year OS: 0; 75%, 1; 71%, 2; 57%, 3; 57%, 4; 46%) and RFS (5-year RFS: 0; 39%, 1; 33%, 2; 21%, 3; 21%, 4; 8%). The presence of extrahepatic colorectal metastatic disease increased recurrence risk (RFS hazard ratio of 1.32 (1.06-1.65)), and the use of intraoperative portal pedicle clamping reduced recurrence risk (RFS hazard ratio of 0.78 (0.61-0.99)). CONCLUSIONS: The CRS remains a relevant tool for predicting long-term outcomes for patients undergoing resection of CRLM. Additional factors such as the presence of extrahepatic colorectal metastatic disease and the use of intraoperative portal pedicle clamping may improve the prognostic power of the CRS.

Immature mosquitoes in bamboo internodes in municipal parks, city of são paulo, Brazil.

We conducted an inventory of the mosquito fauna of the internodes of bamboo plants grown in municipal parks in the city of São Paulo, Brazil. During October 2010 to July 2013, a total of 8,541 immature stages belonging to 21 mosquito species were collected from bamboos over 61 municipal parks. Of these, Aedes albopictus was the most abundant species followed by Ae. aegypti found in broken bamboos in most parks. The former species was 7.2 times more abundant than the latter when both species shared the same habitat. Other species collected from bamboos included Culex quinquefasciatus and Haemagogus leucocelaenus. In bamboos with perforated internodes, species of the genus Wyeomyia were the most prevalent. Differences were also observed in species composition and abundance of mosquitoes collected in transversely broken bamboos and those collected from perforated bamboo internodes. Constant surveillance of these breeding sites is crucial due to the epidemiological importance of the species found. Furthermore, these breeding sites may help maintain some native wild mosquito populations along with a variety of other invertebrates found in these urban green areas.

Mutations in ATP2C1, encoding a calcium pump, cause Hailey-Hailey disease.

Hailey-Hailey disease (HHD, MIM 16960) is inherited in an autosomal dominant manner and characterized by persistent blisters and erosions of the skin. Impaired intercellular adhesion and epidermal blistering also occur in individuals with pemphigus (which is due to autoantibodies directed against desmosomal proteins) and in patients with Darier disease (DD, MIM 124200), which is caused by mutations in a gene encoding a sarco/endoplasmic reticulum (ER)-Golgi calcium pump. We report here the identification of mutations in ATP2C1, encoding the human homologue of an ATP-powered pump that sequesters calcium into the Golgi in yeast, in 21 HHD kindreds. Regulation of cytoplasmic calcium is impaired in cultured keratinocytes from HHD patients, and the normal epidermal calcium gradient is attenuated in vivo in HHD patients. Our findings not only provide an understanding of the molecular basis of HHD, but also underscore the importance of calcium control to the functioning of stratified squamous epithelia.

Diagnosing preperimetric glaucoma with spectral domain optical coherence tomography.

PURPOSE: To evaluate the diagnostic accuracy of spectral domain optical coherence tomography (SD-OCT) for detection of preperimetric glaucoma and compare it with the performance of confocal scanning laser ophthalmoscopy (CSLO). DESIGN: Cohort study. PARTICIPANTS: A cohort of 134 eyes of 88 glaucoma suspects based on the appearance of the optic disc. METHODS: Patients were recruited from the Diagnostic Innovations in Glaucoma Study (DIGS). All eyes underwent retinal nerve fiber layer (RNFL) imaging with Spectralis SD-OCT (Heidelberg Engineering, Carlsbad, CA) and topographic imaging with Heidelberg Retinal Tomograph III (HRT-III) (Heidelberg Engineering) CSLO within 6 months of each other. All patients had normal visual fields at the time of imaging and were classified on the basis of history of documented stereophotographic evidence of progressive glaucomatous change in the appearance of the optic nerve occurring before the imaging sessions. MAIN OUTCOME MEASURES: Areas under the receiver operating characteristic curves (AUCs) were calculated to summarize diagnostic accuracies of the SD-OCT and CSLO. Likelihood ratios (LRs) were reported using the diagnostic categorization provided by each instrument after comparison to its normative database. RESULTS: Forty-eight eyes of 42 patients had evidence of progressive glaucomatous change and were included in the preperimetric glaucoma group. Eighty-six eyes of 46 patients without any evidence of progressive glaucomatous change followed untreated for an average of 14.0 ± 3.6 years were included in the control group. The parameter with the largest AUC obtained with the SD-OCT was the temporal superior RNFL thickness (0.88 ± 0.03), followed by global RNFL thickness (0.86 ± 0.03) and temporal inferior RNFL thickness (0.81 ± 0.04). The parameter with the largest AUC obtained with the CSLO was rim area (0.72 ± 0.05), followed by rim volume (0.71 ± 0.05) and linear cup-to-disk ratio (0.66 ± 0.05). Temporal superior RNFL average thickness measured by SD-OCT performed significantly better than rim area measurements from CSLO (0.88 vs. 0.72; P=0.008). Outside normal limits results for SD-OCT parameters were associated with strongly positive LRs. CONCLUSIONS: The RNFL assessment with SD-OCT performed well in detecting preperimetric glaucomatous damage in a cohort of glaucoma suspects and had a better performance than CSLO.

Comparison of the diagnostic accuracies of the Spectralis, Cirrus, and RTVue optical coherence tomography devices in glaucoma.

PURPOSE: To compare the diagnostic accuracies of retinal nerve fiber layer (RNFL) thickness measurements obtained with the Spectralis (Heidelberg Engineering, Dossenheim, Germany), Cirrus (Carl Zeiss Meditec, Dublin, CA), and RTVue (Optovue Inc., Fremont, CA) devices for the detection of glaucoma. DESIGN: Diagnostic, case-control study. PARTICIPANTS: A total of 233 (107 healthy, 126 glaucomatous) of 149 participants from the longitudinal Diagnostic Innovations in Glaucoma Study (DIGS) and from the African Descent and Glaucoma Evaluation Study (ADAGES). METHODS: All participants underwent RNFL thickness imaging with the Spectralis, Cirrus, and RTVue devices in the same visit. Receiver operating characteristic (ROC) curves adjusted for age and race were obtained for quadrants (superior, nasal, inferior, temporal) and global RNFL thickness for all instruments. Areas under ROC (AUC) and sensitivities at fixed specificities (80% and 95%) were calculated and compared. MAIN OUTCOME MEASURES: Comparison of diagnostic accuracy using AUCs and sensitivities at fixed specificities of 80% and 95%. RESULTS: The RNFL thickness parameter with the largest AUCs was the superior quadrant for the Spectralis (0.88) and the global RNFL thickness for the Cirrus (0.88) and the RTVue (0.87). The pairwise comparison among the ROC curves showed no statistical difference for all parameters except for the nasal quadrant, which had significantly larger AUC in the Spectralis and RTVue compared with the Cirrus (P<0.03 for both comparisons). There were no significant differences in sensitivities among the best parameters from each instrument (P>0.05). The superior quadrant thickness measured with the Spectralis had sensitivity of 81.9% at a fixed specificity of 80% and 70% at a fixed specificity of 95%. The global thickness measured by the Cirrus had a sensitivity of 80.3% at a fixed specificity of 80% and 65.6% at a fixed specificity of 95%. For the RTVue, the global thickness had a sensitivity of 77.9% at a fixed specificity of 80% and 62.1% at a fixed specificity of 95%. CONCLUSIONS: Although the spectral-domain optic coherence tomography (SD-OCT) instruments have different resolution and acquisition rates, their ability to detect glaucoma was similar.

Endoplasmic reticulum Ca2+ depletion activates XBP1 and controls terminal differentiation in keratinocytes and epidermis.

BACKGROUND: Endoplasmic reticulum (ER) Ca(2+) depletion, previously shown to signal pathological stress responses, has more recently been found also to trigger homeostatic physiological processes such as differentiation. In keratinocytes and epidermis, terminal differentiation and barrier repair require physiological apoptosis and differentiation, as evidenced by protein synthesis, caspase 14 expression, lipid secretion and stratum corneum (SC) formation. OBJECTIVES: To investigate the role of Ca(2+) depletion-induced ER stress in keratinocyte differentiation and barrier repair in vivo and in cell culture. METHODS: The SERCA2 Ca(2+) pump inhibitor thapsigargin (TG) was used to deplete ER calcium both in cultured keratinocytes and in mice. Levels of the ER stress factor XBP1, loricrin, caspase 14, lipid synthesis and intracellular Ca(2+) were compared after both TG treatment and barrier abrogation. RESULTS: We showed that these components of terminal differentiation and barrier repair were signalled by physiological ER stress, via release of stratum granulosum (SG) ER Ca(2+) stores. We first found that keratinocyte and epidermal ER Ca(2+) depletion activated the ER-stress-induced transcription factor XBP1. Next, we demonstrated that external barrier perturbation resulted in both intracellular Ca(2+) emptying and XBP1 activation. Finally, we showed that TG treatment of intact skin did not perturb the permeability barrier, yet stimulated and mimicked the physiological processes of barrier recovery. CONCLUSIONS: This report is the first to quantify and localize ER Ca(2+) loss after barrier perturbation and show that homeostatic processes that restore barrier function in vivo can be reproduced solely by releasing ER Ca(2+), via induction of physiological ER stress.

Estimation of the post-mortem interval: Effect of storage conditions on the determination of vitreous humour [K+].

Obtaining a reliable estimate of the post-mortem interval (PMI) has been a long-running challenge in forensic medicine. Several more or less successful techniques for making such estimates have been developed, but in recent years important advances have been made thanks to the detailed study of the relationship between the PMI and the analytes - in particular K+ - of the vitreous humour (VH). The extraction and pre-treatment of VH samples has been standardized, the influence of certain environmental factors on analytical results has been quantified, and some of the circumstances under which techniques become unreliable have been identified. The present work examines how the conditions to which VH samples are subject in routine practice may alter the results of their analysis. Exposure to light and ambient temperature was found to alter the values returned in determinations of VH [K+], [Na+] and [Cl-], while exposure to several freezing/thawing cycles (even with final heating) led to no significant modifications in determinations of VH [K+] and [Na+]. It is recommended that if analysis has to be delayed, VH should be frozen for storage in a refrigerator before bringing to room temperature for processing. It is also recommended that samples not be exposed to ambient light and temperature.

Heavy metal contamination in Peru: implications on children's health.

Cerro de Pasco, Peru, has been excessively contaminated with heavy metals due to high mining activities in the region. We investigated the presence of chronic exposure to heavy metals in children living in Cerro de Pasco and its effect on health. Heavy metal concentrations were determined in hair samples collected from 78 children living in a region exposed to an open-pit mine (Paragsha region) and from other 16 children unexposed to mine activities (Carhuamayo region). Children exposed to the mine showed statistically significant higher concentration of aluminum, antimony, arsenic, cadmium, chromium, iron, lead, tin and thallium (p < 0.05) than control children. Hair samples collected from the same children in two occasions (2016 and 2018) showed that the exposure is chronic with higher levels of heavy metals observed in 2018. The concentration of heavy metals was higher in hair tip than in hair root samples. Heavy metals are associated with substantial higher risk of nosebleed (odds ratio, OR = 15.40), chronic colic (OR = 7.30), dermatologic alterations (OR = 6.16), mood alterations (OR = 7.07), presence of white lines on nails (OR = 12.10), reduced visual camp (OR = 3.97) and other symptoms (OR = 5.12). Chronic heavy metal exposure implies various negative consequences on children's health. Preventive measures are crucial to protect children's health.

Reemergence of Yellow Fever in Brazil: The Role of Distinct Landscape Fragmentation Thresholds.

Yellow Fever Virus (YFV) reemergence in Brazil was followed by human suffering and the loss of biodiversity of neotropical simians on the Atlantic coast. The underlying mechanisms were investigated with special focus on distinct landscape fragmentation thresholds in the affected municipalities. An ecological study in epidemiology is employed to assess the statistical relationship between events of YFV and forest fragmentation in municipal landscapes. Negative binomial regression model showed that highly fragmented forest cover was associated with an 85% increase of events of YFV in humans and simians (RR = 1.85, CI 95% = 1.24-2.75, p=0.003) adjusted by vaccine coverage, population size, and municipality area. Intermediate levels of forest cover combined with higher levels of forest edge densities contribute to the YFV dispersion and the exponential growth of YF cases. Strategies for forest conservation are necessary for the control and prevention of YF and other zoonotic diseases that can spillover from the fragmented forest remains to populated cities of the Brazilian Atlantic coast.

The epidermal Ca(2+) gradient: Measurement using the phasor representation of fluorescent lifetime imaging.

Ionic gradients are found across a variety of tissues and organs. In this report, we apply the phasor representation of fluorescence lifetime imaging data to the quantitative study of ionic concentrations in tissues, overcoming technical problems of tissue thickness, concentration artifacts of ion-sensitive dyes, and calibration across inhomogeneous tissue. We used epidermis as a model system, as Ca(2+) gradients in this organ have been shown previously to control essential biologic processes of differentiation and formation of the epidermal permeability barrier. The approach described here allowed much better localization of Ca(2+) stores than those used in previous studies, and revealed that the bulk of free Ca(2+) measured in the epidermis comes from intracellular Ca(2+) stores such as the Golgi and the endoplasmic reticulum, with extracellular Ca(2+) making a relatively small contribution to the epidermal Ca(2+) gradient. Due to the high spatial resolution of two-photon microscopy, we were able to measure a marked heterogeneity in average calcium concentrations from cell to cell in the basal keratinocytes. This finding, not reported in previous studies, calls into question the long-held hypothesis that keratinocytes increase intracellular Ca(2+), cease proliferation, and differentiate passively in response to changes in extracellular Ca(2+). The experimental results obtained using this approach illustrate the power of the experimental and analytical techniques outlined in this report. Our approach can be used in mechanistic studies to address the formation, maintenance, and function of the epidermal Ca(2+) gradient, and it should be broadly applicable to the study of other tissues with ionic gradients.

Ascorbic acid concentration is reduced in the secondary aqueous humour of glaucomatous patients.

BACKGROUND: We aimed to evaluate the ascorbic acid concentration in secondary aqueous humour (AH) from glaucomatous patients and to compare it with primary AH from primary open-angle glaucoma patients and non-glaucomatous patients. METHODS: Primary AH samples were prospectively obtained from clinically uncontrolled primary open-angle glaucoma patients and senile cataract patients (controls) prior to trabeculectomy and cataract surgery. Secondary AH samples were obtained from eyes with previous intraocular surgery, prior to trabeculectomy or cataract surgery. AH (0.1 mL) was aspirated by inserting a 26-gauge needle into the anterior chamber just before surgery and then immediately stored at -80 degrees C. The ascorbic acid concentration was determined in a masked fashion by high-pressure liquid chromatography. RESULTS: A total of 18 patients with senile cataract, 16 glaucomatous patients with primary AH (no previous intraocular surgery) and 11 glaucomatous patients with secondary AH (previous intraocular surgery) were included. There was no difference in mean age between groups (P = 0.15). The mean +/- standard deviation concentration of ascorbic acid in the secondary AH from glaucomatous patients (504 +/- 213 micromol/L [95% confidence interval {CI}, 383-624]) was significantly lower than the concentration of ascorbic acid found in the primary aqueous of primary open-angle glaucoma (919 +/- 427 micromol/L [95% CI, 709-1128]) and control patients (1049 +/- 433 micromol/L [95% CI, 848-1249]; P < 0.01, Kruskal-Wallis test). CONCLUSIONS: The ascorbic acid concentration in secondary AH of glaucomatous patients was approximately twofold lower in comparison with primary AH of glaucomatous and cataract patients. The implications of a reduced concentration of ascorbic acid in the secondary AH deserve further investigation.