Subcellular localization of p27 and prostate cancer recurrence: automated digital microscopy analysis of tissue microarrays

Previous investigations have linked decreased nuclear expression of the cell cycle inhibitor p27 with poor outcome in prostate cancer. However, these reports are inconsistent regarding the magnitude of that association and its independence from other predictors. Moreover, cytoplasmic translocation of p27 has been proposed as a negative prognostic sign. Given the cost and accuracy limitations of manual scoring, particularly of tissue microarrays, we determined if laser-based fluorescence microscopy could provide automated analysis of p27 in both nuclear and cytoplasmic locations and, thus, clarify its significance as a prognostic biomarker. We constructed tissue microarrays covering 202 recurrent cases (rising prostate-specific antigen) and 202 matched controls without recurrence. Quadruplicate tumor samples encompassed 5 slides and 1616 cancer histospots. Cases and controls matched on age, Gleason grade, stage, and hospital. We immunolabeled epithelial cytoplasm with Alexafluor 647, p27 with Alexafluor 488, and nuclei with 4c6-diamidino-2-phenylindole·2HCl. Slides were scanned on an iCys laser scanning cytometer (CompuCyte Corp, Cambridge, MA). Nuclear crowding required a stereological approach--random arrays of circles (phantoms) were layered on images and the content of each phantom was analyzed in scatter plots. Both nuclear and cytoplasmic p27 were significantly lower in cases versus controls (P = .014 and P = .004, respectively). Regression models controlling for matching variables plus prostate-specific antigen showed strong linear trends for increased risk of recurrence with lower p27 in both nucleus and cytoplasm (highest versus lowest quartile; odds ratio, 0.35; P = .006). Manual scoring identified an inverse association between p27 expression and tumor grade but no independent association with recurrence. In conclusion, we developed an automated method for subcellular scoring of p27 without the need to segment individual cells. Our method identified a strong relationship, independent of tumor grade, stage, and prostate-specific antigen, between p27 expression--regardless of subcellular location--and prostate cancer recurrence. This relationship was not observed with manual scoring.     

A prospective comparison of laparoscopic and robotic radical nephrectomy for T1-2N0M0 renal cell carcinoma

Objectives  We prospectively evaluated the safety, feasibility, and efficiency of robotic radical nephrectomy (RRN) for localized renal tumors (T1-2N0M0) and compared this with laparoscopic radical nephrectomy (LRN). Materials and methods  Between October 2006 to August 2007, a prospective data analysis of 15 cases of renal cell carcinoma (RCC) stage T1-2N0M0, undergoing RRN was done. These patients were compared with a contemporary cohort of 15 patients of RCC with clinical stage T1-2N0M0, undergoing LRN. To keep comparison robust, all cases were performed by a single surgeon. Demographic, intra-operative, post-operative outcomes, pathological characteristics and follow-up data of the two groups were recorded and analyzed statistically. Results  Patients in group A (RRN) experienced significantly (P = 0.001) long operating time than group B (LRN). However, mean estimated blood loss, intra-operative and post-operative complications, blood transfusion rate, analgesic requirement, hospital stay and convalescence were comparable in two groups (P < 0.05). There was one conversion to open surgery in group A, and none in group B. The mean follow-up was comparable in two groups (8.3 and 9.1 months, respectively, in group A and B, P = 0.09). There were no local, port-site or distal recurrences in either group. Conclusions  Robotic radical nephrectomy is a safe, feasible and effective for performing radical nephrectomy for localized RCC. Both groups (RRN and LRN) had comparable intra-operative, peri-operative, post-operative and oncological outcomes except for longer operating time with increased cost for RRN. In this comparative study, there were no outstanding benefits of RRN observed over LRN for localized RCC.     

Epileptic Spike Recognition in Electroencephalogram Using Deterministic Finite Automata

This Paper presents an automated method of Epileptic Spike detection in Electroencephalogram (EEG) using Deterministic Finite Automata (DFA). It takes pre-recorded single channel EEG data file as input and finds the occurrences of Epileptic Spikes data in it. The EEG signal was recorded at 256 Hz in two minutes separate data files using the Visual Lab-M software (ADLink Technology Inc., Taiwan). It was preprocessed for removal of baseline shift and band pass filtered using an infinite impulse response (IIR) Butterworth filter. A system, whose functionality was modeled with DFA, was designed. The system was tested with 10 EEG signal data files. The recognition rate of Epileptic Spike as on average was 95.68%. This system does not require any human intrusion. Also it does not need any short of training. The result shows that the application of DFA can be useful in detection of different characteristics present in EEG signals. This approach could be extended to a continuous data processing system. Certificate of Originality This is to certify that the article submitted for publication in ‘Journal of Medical Systems’ has not been published, nor is being considered for publication, elsewhere. All experimental procedures on rats were performed in compliance with “Committee for the purpose of control and supervision of experiments on animals (CPCSEA)”, India.     

Microfluidic immunoassays as rapid saliva-based clinical diagnostics

At present, point-of-care (POC) diagnostics typically provide a binary indication of health status (e.g., home pregnancy test strip). Before anticipatory use of diagnostics for assessment of complex diseases becomes widespread, development of sophisticated bioassays capable of quantitatively measuring disease biomarkers is necessary. Successful translation of new bioassays into clinical settings demands the ability to monitor both the onset and progression of disease. Here we report on a clinical POC diagnostic that enables rapid quantitation of an oral disease biomarker in human saliva by using a monolithic disposable cartridge designed to operate in a compact analytical instrument. Our microfluidic method facilitates hands-free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. Using 20 microl of saliva, we demonstrate rapid (<10 min) measurement of the collagen-cleaving enzyme matrix metalloproteinase-8 (MMP-8) in saliva from healthy and periodontally diseased subjects. In addition to physiologically measurable indicators of periodontal disease, conventional measurements of salivary MMP-8 were used to validate the microfluidic assays described in this proof-of-principle study. The microchip-based POC diagnostic demonstrated is applicable to rapid, reliable measurement of proteinaceous disease biomarkers in biological fluids.     

Efficacy of Iliac Crest vs. Medpor in Orbital Floor Reconstruction

Aim  

The present study is to compare the effectiveness of iliac crest graft and medpor implant, for repairing traumatic orbital floor defects.     

Caffeine augments Alprazolam induced cytotoxicity in human cell lines

Combined effects of alprazolam (Alp), a member of benzodiazepine group of drugs and caffeine on human cell lines, HeLa and THP1 were investigated in this study. Alp mediated cytotoxicity was enhanced while caffeine was present. The cell death was confirmed by observing morphological changes, LDH assay and membrane anisotropic study. Also such combined effects induced elevated level of ROS and depletion of GSH. The mechanism of cell death induced by simultaneous treatment of Alp and caffeine was associated with the calcium-mediated activation of μ-calpain, release of lysosomal protease cathepsin B, activation of PARP and cleavage of caspase 3. Our results indicate that, Alp alone induces apoptosis in human cells but in the presence of caffeine it augments necrosis in a well-regulated pathway. Thus our observations strongly suggest that, alprazolam and caffeine together produce severe cytotoxicity in human cell lines.     

Reducing out-of-pocket expenditures to reduce poverty: a disaggregated analysis at rural-urban and state level in India

Out-of-pocket (OOP) expenditure on health care has significantimplications for poverty in many developing countries. Thispaper aims to assess the differential impact of OOP expenditureand its components, such as expenditure on inpatient care, outpatientcare and on drugs, across different income quintiles, betweendeveloped and less developed regions in India. It also attemptsto measure poverty at disaggregated rural-urban and state levels. Based on Consumer Expenditure Survey (CES) data from the NationalSample Survey (NSS), conducted in 1999–2000, the shareof households’ expenditure on health services and drugswas calculated. The number of individuals below the state-specificrural and urban poverty line in 17 major states, with and withoutnetting out OOP expenditure, was determined. This also enabledthe calculation of the poverty gap or poverty deepening in eachregion. Estimates show that OOP expenditure is about 5% of total householdexpenditure (ranging from about 2% in Assam to almost 7% inKerala) with a higher proportion being recorded in rural areasand affluent states. Purchase of drugs constitutes 70% of thetotal OOP expenditure. Approximately 32.5 million persons fellbelow the poverty line in 1999–2000 through OOP payments,implying that the overall poverty increase after accountingfor OOP expenditure is 3.2% (as against a rise of 2.2% shownin earlier literature). Also, the poverty headcount increaseand poverty deepening is much higher in poorer states and ruralareas compared with affluent states and urban areas, exceptin the case of Maharashtra. High OOP payment share in totalhealth expenditures did not always imply a high poverty headcount;state-specific economic and social factors played a role. The paper argues for better methods of capturing drugs expenditurein household surveys and recommends that special attention bepaid to expenditures on drugs, in particular for the poor. Targetedpolicies in just five poor states to reduce OOP expenditurecould help to prevent almost 60% of the poverty headcount increasethrough OOP payments.     

First digital flexible ureterorenoscope: initial experience

BACKGROUND: Flexible ureterorenoscopy is a minimally invasive procedure that is rapidly gaining popularity as a treatment for patients with upper urinary tract lithiasis. One of the main drawbacks is the limited visualization imposed by fiberoptic technology in an endoscope, often only 7 to 8F and the fragility of fiber-based optics in a flexible endoscope. MATERIALS AND METHODS: We report our initial experience with digital flexible URS and holmium laser lithotripsy using digital image capture in three patients. Two patients had upper tract calculi (1 cm lower pole calculus and 8 mm caliceal diverticular stone, respectively), and one patient underwent diagnostic URS. RESULTS: After basket capture and intrarenal relocation of the lower pole stone, complete stone destruction was achieved. Similarly, the caliceal diverticular stone was destroyed after holmium laser incision of the caliceal diverticulum. Remarkable improvement in image quality was observed throughout the procedures in comparison with current fiberoptic image acquisition. Minor image interference was noted during fluoroscopic screening, which increased slightly during laser activation. CONCLUSIONS: The first digital flexible ureterorenoscope provided excellent image quality and zoom capability, with a substantial improvement compared with fiberoptic technology. It does not require a separate light source and is an important step for the continued evolution of flexible URS as a therapeutic modality for upper urinary tract pathology.     

Inactivation of human mutL homolog 1 and mutS homolog 2 genes in head and neck squamous cell carcinoma tumors and leukoplakia samples by promoter hypermethylation and its relation with microsatellite instability phenotype

BACKGROUND: A subset of head and neck squamous cell carcinoma (HNSCC) exhibits a microsatellite instability (MIN) phenotype. The authors correlated alterations in the mismatch-repair genes human mutL homolog 1 (hMLH1) and human mutS homolog 2 (hMSH2) in primary head and neck squamous cell carcinoma (HNSCC) tumors and in samples of leukoplakia with the MIN phenotype. METHODS: One hundred twenty-three paired HNSCC normal and tumor tissues and 27 leukoplakia samples were examined for hypermethylation of hMLH1 and hMSH2 promoters. The hypermethylation status of the tissues was confirmed by expression studies. Sixty-three of 123 randomly selected tumors and all 27 leukplakia samples were genotyped with 8 microsatellite markers to determine MIN. RESULTS: Fifty percent of HNSCC tumors and 63% of leukoplakia samples harbored hypermethylation at either or both hMLH1 and hMSH2 promoters. Normal tissues adjacent to methylation-positive tumors also demonstrated hypermethylation of both promoters at a high frequency (25%). A positive correlation between tobacco habit and promoter hypermethylation was observed (P = .001). A correlation was observed between MIN and the frequency of promoter hypermethylation in the leukoplakia samples, but no such trend was observed in the HNSCC tumors. It is noteworthy that patients who had a high frequency of MIN-positive tumors exhibited hypermethylation in both the affected tissues and the adjacent normal tissues (P = .007). Patients with a tobacco habit who had promoter hypermethylation at both the affected tissues and the adjacent normal tissues had tumors that mostly were MIN positive (P = .047). CONCLUSIONS: The current results suggested that tobacco-addicted individuals are more susceptible to promoter hypermethylation of hMLH1 and hMSH2 and that, if such hypermethylation occurs in the normal squamous epithelium of the head and neck region, then those tissues are likely to develop into tumors that involve the MIN pathway.