Is preoperative neutrophil-to-lymphocyte ratio a red flag which can predict high-risk pathological characteristics in renal cell carcinoma?

INTRODUCTION: Renal cell carcinoma (RCC) is known to invoke both immunological and inflammatory responses. While the neutrophils mediate the tumor-induced inflammatory response, the lymphocytes bring about the various immunological events associated with it. The neutrophil-to-lymphocyte ratio (NLR) is a simple indicator of this dual response. We investigated the association between preoperative NLR and histopathological prognostic variables of RCC intending to find out whether it can be of value as a red flag capable of alerting the clinician as to the biological character of the tumor under consideration. METHODS: Preoperative NLR and clinicopathological variables, namely histological subtype, nuclear grade, staging, lymphovascular invasion, capsular invasion, tumor necrosis, renal sinus invasion, and sarcomatoid differentiation of 60 patients who underwent radical or partial nephrectomy, were analyzed to detect the association between the two. RESULTS: We found that mean preoperative NLR was significantly higher in clear-cell carcinomas (3.25 ± 0.29) when compared with nonclear-cell carcinomas (2.25 ± 0.63). There was a linear trend of NLR rise as the stage of the disease advanced. A significant rise in preoperative NLR was noted in tumors with various high-risk histopathological features such as tumor size, capsular invasion, tumor necrosis, and sarcomatoid differentiation. CONCLUSION: Preoperative measurement of NLR is a simple test which may provide an early clue of high-risk pathological features of renal cell cancer.

Role of Preoperative Renal Stone Complexity Assessment by Guy's Stone Score as a Predictor of Percutaneous Nephrolithotomy Outcomes.

INTRODUCTION: Various nephrolithometric scoring systems have recently emerged to predict the outcomes of percutaneous nephrolithotomy (PCNL). However, there is no consensus upon an ideal tool. The current study aimed to assess the correlation between Guy's stone score (GSS) and PCNL outcomes. METHOD: This was a hospital-based observational study of 2-year duration. All patients electively undergoing PCNL for renal stones during the study period were included. Based on the imaging findings, the stones were categorized as simple (GSS I & II) and complex (GSS III & IV). The association between GSS and duration of the procedure, the number of percutaneous tracts needed, stone-free rate (SFR), and the severity of complications based on modified Clavien classification and postoperative stay were assessed. RESULTS: Total number of the patients studied were 100 (n = 100), and most of the patients were in the category of GSS II (51%). Post-extrapolation of χ2 to Pearson's test, GSS demonstrated a significant association with duration of surgery, the number of percutaneous tracts needed, the severity of complications, and SFR. CONCLUSIONS: Preoperative assessment of stone complexity by using GSS effectively correlated with SFR as well as other PCNL outcomes. Hence, we recommend utilizing this predictive tool for standardized documentation, preoperative planning, and better patient counseling.

Renal salvage, an achievable goal in patients with emphysematous pyelonephritis: Outcomes of an algorithmic renal preserving strategy.

INTRODUCTION: Emergency nephrectomy has been the time-honored treatment of choice for emphysematous pyelonephritis (EPN), a fatal gas-forming necrotizing infection. Recent years have seen a shift toward nonextirpative approach aimed to achieve higher rates of renal salvage, limiting the indications for nephrectomy to severe grades of the disease. This study aimed at analyzing the role of initial renal preserving measures algorithmically applied across grades of EPN. MATERIALS AND METHODS: We prospectively analyzed the clinical data and outcome of 36 consecutive patients of EPN in 5 years' study period, treated by renal preserving measures, which include aggressive resuscitation, parenteral antibiotics, effective drainage of infected fluid/gas, and relieving the urinary tract obstruction. Huang-Tseng computed tomography-based classification system was used to categorize the patients as well as to employ suitable treatment modality. RESULTS: The mean age of the patients was 57.5 ± 12 years with female preponderance (2:1). Diabetes mellitus (97%) was the most common associated factor. Escherichia coli was (72%) the most frequent causative organism found. Urinary tract obstruction was seen in 27 patients (75%) attributable to ureteric calculi, renal papillary necrosis, ureteric stricture, and fungal bezoar in the descending order of frequency. Only 2 (6%) out of 36 patients managed according to our hospital renal salvage protocol required salvage nephrectomy. The overall survival rate was 94%. CONCLUSION: Our hospital-based algorithmic renal preserving strategy not only improved the survival but also decreased the need for nephrectomy.

Can new, improvised Visual Prostate Symptom Score replace the International Prostate Symptom Score? Indian perspective.

INTRODUCTION: Visual Prostate Symptom Score (VPSS) was introduced to overcome the drawbacks of the International Prostate Symptom Score (IPSS). However, this score also has potential for improvement. MATERIALS AND METHODS: The primary objective of this study was to evaluate the utility of VPSS in patients with benign enlarged prostate (BEP) after uroflowmetric validation of the stream component. The secondary objective was to improve VPSS by adding a new severity grading and to assess if the "new upgraded VPSS" can replace IPSS in terms of ease of completion without assistance and the time taken. RESULTS: Of 115 patients, 42.60% of them were of the age group between 61-70 years; mean ± standard deviation age was 64.75 years ± 8.042 (range 48-90 years). Nineteen (16.52%) patients, who had education level ≥10th grade completed IPSS without assistance. One hundred and eight (93.91%) patients completed VPSS without assistance (P = 0.000). None of those (0/6) with no formal education (but able to read and write) could complete the IPSS without assistance, whereas 66.67% completed the VPSS without assistance. Six minutes and two minutes was the average time taken to complete IPSS (4-10 min) and VPSS (1-3 min), respectively. Responses to different variables of VPSS were statistically significant (P < 0.001) compared to the IPSS. Correlation between the severity grading of the two scores was statistically significant (P < 0.001), with a statistically significant positive correlation between VPSS and IPSS (r = +0.582, P < 0.001). The new severity grading system developed on par with the IPSS, improvising the existing VPSS, showed statistically significant positive correlation to the IPSS (r = +0.587, P < 0.001). CONCLUSIONS: VPSS correlated well with IPSS. The "new improvised VPSS" developed by incorporating severity grading is a potential tool that can replace IPSS by overcoming its limitations.

Development of a Screening Platform to Identify Small Molecules That Modify ELP1 Pre-mRNA Splicing in Familial Dysautonomia.

Familial dysautonomia (FD) is an autonomic and sensory neuropathy caused by a mutation in the splice donor site of intron 20 of the ELP1 gene. Variable skipping of exon 20 leads to a tissue-specific reduction in the level of ELP1 protein. We have shown that the plant cytokinin kinetin is able to increase cellular ELP1 protein levels in vivo and in vitro through correction of ELP1 splicing. Studies in FD patients determined that kinetin is not a practical therapy due to low potency and rapid elimination. To identify molecules with improved potency and efficacy, we developed a cell-based luciferase splicing assay by inserting renilla (Rluc) and firefly (Fluc) luciferase reporters into our previously well-characterized ELP1 minigene construct. Evaluation of the Fluc/Rluc signal ratio enables a fast and accurate way to measure exon 20 inclusion. Further, we developed a secondary assay that measures ELP1 splicing in FD patient-derived fibroblasts. Here we demonstrate the quality and reproducibility of our screening method. Development and implementation of this screening platform has allowed us to efficiently screen for new compounds that robustly and specifically enhance ELP1 pre-mRNA splicing.

Specific correction of a splice defect in brain by nutritional supplementation.

Recent studies emphasize the importance of mRNA splicing in human genetic disease, as 20-30% of all disease-causing mutations are predicted to result in mRNA splicing defects. The plasticity of the mRNA splicing reaction has made these mutations attractive candidates for the development of therapeutics. Familial dysautonomia (FD) is a severe neurodegenerative disorder, and all patients have an intronic IVS20+6T>C splice site mutation in the IKBKAP gene, which results in tissue-specific skipping of exon 20 and a corresponding reduction in ikappaB kinase complex associated protein (IKAP) levels. We created transgenic mouse lines using a human IKBKAP bacterial artificial chromosome (BAC) into which we inserted the IKBKAP splice mutation (FD BAC) and have shown that the transgenic mice exhibit the same tissue-specific aberrant splicing patterns as seen in FD patients. We have previously demonstrated that the plant cytokinin kinetin can significantly improve the production of wild-type IKBKAP transcripts in FD lymphoblast cell lines by improving exon inclusion. In this study, we tested the ability of kinetin to alter IKBKAP splicing in the transgenic mice carrying the FD BAC and show that it corrects IKBKAP splicing in all major tissues assayed, including the brain. The amount of wild-type IKBKAP mRNA and IKAP protein was significantly higher in the kinetin-treated mice. These exciting results prove that treatment of FD, as well as other mechanistically related splicing disorders, with kinetin holds great promise as a potential therapeutic aimed at increasing normal protein levels, which may, in turn, slow disease progression.

Loss of mouse Ikbkap, a subunit of elongator, leads to transcriptional deficits and embryonic lethality that can be rescued by human IKBKAP.

Familial dysautonomia (FD), a devastating hereditary sensory and autonomic neuropathy, results from an intronic mutation in the IKBKAP gene that disrupts normal mRNA splicing and leads to tissue-specific reduction of IKBKAP protein (IKAP) in the nervous system. To better understand the roles of IKAP in vivo, an Ikbkap knockout mouse model was created. Results from our study show that ablating Ikbkap leads to embryonic lethality, with no homozygous Ikbkap knockout (Ikbkap(-)(/)(-)) embryos surviving beyond 12.5 days postcoitum. Morphological analyses of the Ikbkap(-)(/)(-) conceptus at different stages revealed abnormalities in both the visceral yolk sac and the embryo, including stunted extraembryonic blood vessel formation, delayed entry into midgastrulation, disoriented dorsal primitive neural alignment, and failure to establish the embryonic vascular system. Further, we demonstrate downregulation of several genes that are important for neurulation and vascular development in the Ikbkap(-)(/)(-) embryos and show that this correlates with a defect in transcriptional elongation-coupled histone acetylation. Finally, we show that the embryonic lethality resulting from Ikbkap ablation can be rescued by a human IKBKAP transgene. For the first time, we demonstrate that IKAP is crucial for both vascular and neural development during embryogenesis and that protein function is conserved between mouse and human.

A humanized IKBKAP transgenic mouse models a tissue-specific human splicing defect.

Familial dysautonomia (FD) is a severe hereditary sensory and autonomic neuropathy, and all patients with FD have a splice mutation in the IKBKAP gene. The FD splice mutation results in variable, tissue-specific skipping of exon 20 in IKBKAP mRNA, which leads to reduced IKAP protein levels. The development of therapies for FD will require suitable mouse models for preclinical studies. In this study, we report the generation and characterization of a mouse model carrying the complete human IKBKAP locus with the FD IVS20+6T-->C splice mutation. We show that the mutant IKBKAP transgene is misspliced in this model in a tissue-specific manner that replicates the pattern seen in FD patient tissues. Creation of this humanized mouse is the first step toward development of a complex phenotypic model of FD. These transgenic mice are an ideal model system for testing the effectiveness of therapeutic agents that target the missplicing defect. Last, these mice will permit direct studies of tissue-specific splicing and the identification of regulatory factors that play a role in complex gene expression.

Therapeutic potential and mechanism of kinetin as a treatment for the human splicing disease familial dysautonomia.

Mutations that affect the splicing of pre-mRNA are a major cause of human disease. Familial dysautonomia (FD) is a recessive neurodegenerative disease caused by a T to C transition at base pair 6 of IKBKAP intron 20. This mutation results in variable tissue-specific skipping of exon 20. Previously, we reported that the plant cytokinin kinetin dramatically increases exon 20 inclusion in RNA isolated from cultured FD cells. The goal of the current study was to investigate the nature of the FD splicing defect and the mechanism by which kinetin improves exon inclusion, as such knowledge will facilitate the development of future therapeutics aimed at regulating mRNA splicing. In this study, we demonstrate that treatment of FD lymphoblast cell lines with kinetin increases IKBKAP mRNA and IKAP protein to normal levels. Using a series of minigene constructs, we show that deletion of a region at the end of IKBKAP exon 20 disrupts the ability of kinetin to improve exon inclusion, pinpointing a kinetin responsive sequence element. We next performed a screen of endogenously expressed genes with multiple isoforms resulting from exon skipping events and show that kinetin's ability to improve exon inclusion is not limited to IKBKAP. Lastly, we highlight the potential of kinetin for the treatment of other human splicing disorders by showing correction of a splicing defect in neurofibromatosis.

Gene expression and specificity in the mature zone of the lobster olfactory organ.

The lobster olfactory organ is an important model for investigating many aspects of the olfactory system. To facilitate study of the molecular basis of olfaction in lobsters, we made a subtracted cDNA library from the mature zone of the olfactory organ of Homarus americanus, the American lobster. Sequencing of the 5'-end of 5,184 cDNA clones produced 2,389 distinct high-quality sequences consisting of 1,944 singlets and 445 contigs. Matches to known sequences corresponded with the types of cells present in the olfactory organ, including specific markers of olfactory sensory neurons, auxiliary cells, secretory cells of the aesthetasc tegumental gland, and epithelial cells. The wealth of neuronal mRNAs represented among the sequences reflected the preponderance of neurons in the tissue. The sequences identified candidate genes responsible for known functions and suggested new functions not previously recognized in the olfactory organ. A cDNA microarray was designed and tested by assessing mRNA abundance differences between two of the lobster's major chemosensory structures: the mature zone of the olfactory organ and the dactyl of the walking legs, a taste organ. The 115 differences detected again emphasized the abundance of neurons in the olfactory organ, especially a cluster of mRNAs encoding cytoskeletal-associated proteins and cell adhesion molecules such as 14-3-3zeta, actins, tubulins, trophinin, Fax, Yel077cp, suppressor of profilin 2, and gelsolin.

Transcriptional changes during neuronal death and replacement in the olfactory epithelium.

The olfactory epithelium has the unusual ability to replace its neurons. We forced replacement of mouse olfactory sensory neurons by bulbectomy. Microarray, bioinformatics, and in situ hybridization techniques detected a rapid shift in favor of pro-apoptotic proteins, a progressive immune response by macrophages and dendritic cells, and identified or predicted 439 mRNAs enriched in olfactory sensory neurons, including gene silencing factors and sperm flagellar proteins. Transcripts encoding cell cycle regulators, axonogenesis proteins, and transcription factors and signaling proteins that promote proliferation and differentiation were increased at 5--7 days after bulbectomy and were expressed by basal progenitor cells or immature neurons. The transcription factors included Nhlh 1, Hes 6, Lmyc 1, c-Myc, Mxd 4, Id 1, Nmyc 1, Cited 2, c-Myb, Mybl 1, Tead 2, Dp 1, Gata 2, Lmo 1, and Sox1 1. The data reveal significant similarities with embryonic neurogenesis and make several mechanistic predictions, including the roles of the transcription factors in the olfactory sensory neuron lineage.

Transcriptional changes during neuronal death and replacement in the olfactory epithelium.

The olfactory epithelium has the unusual ability to replace its neurons.We forced replacement of mouse olfactory sensory neurons by bulbectomy. Microarray, bioinformatics, and in situ hybridization techniques detected a rapid shift in favor of pro-apoptotic proteins, a progressive immune response by macrophages and dendritic cells, and identified or predicted 439 mRNAs enriched in olfactory sensory neurons, including gene silencing factors and sperm flagellar proteins. Transcripts encoding cell cycle regulators, axonogenesis proteins, and transcription factors and signaling proteins that promote proliferation and differentiation were increased at 5-7 days after bulbectomy and were expressed by basal progenitor cells or immature neurons. The transcription factors included Nhlhl, Hes6, Lmycl, c-Myc, Mxd4, Idl,Nmycl, Cited2, c-Myb, Mybll, Tead2, Dpl, Gata2, Lmol, and Soxll. The data reveal significant similarities with embryonic neurogenesis and make several mechanistic predictions, including the roles of the transcription factors in the olfactory sensory neuron lineage.

Fluorescence-based sensing system for copper using genetically engineered living yeast cells.

A whole cell-based optical sensing system for copper was developed based on Saccharomyces cerevisiae cells harboring plasmid pYEX-GFPuv. The basis of this system was the ability of the transcriptional activator protein Ace1 present in S. cerevisiae to control the expression of the reporter protein, GFPuv. When copper ions are present in the sample, the Ace1 protein activates the cup1 promoter located upstream from the gfpuv gene in plasmid pYEX-GFPuv, thus inducing the production of GFPuv. The concentration of copper ions in the sample can then be related to the GFPuv expressed in the yeast. The amount of GFPuv produced in the system was determined by monitoring the fluorescence emitted at 507 nm after excitation at 397 nm. This system can detect copper at concentrations as low as 5 x 10(-7) M, and is selective for copper over a variety of metal ions, with the exception of silver. The applicability of this sensing system to different analytical platforms and in real samples is demonstrated.

Luminescence-based whole-cell-sensing systems for cadmium and lead using genetically engineered bacteria.

Whole-cell-based sensing systems that respond to cadmium and lead ions have been designed and developed using genetically engineered bacteria. These systems take advantage of the ability of certain bacteria to survive in environments polluted with cadmium and lead ions. The bacteria used in this investigation have been genetically engineered to produce reporter proteins in response to the toxic ions. This was achieved by modifying a strain of Escherichia colito harbor plasmids pYSC1 and pYS2/pYSG1. In these dual-plasmid-based sensing systems, the expression of the reporters beta-galactosidase and red-shifted green fluorescent protein (rs-GFP) was controlled by CadC, the regulatory protein of the cad operon. Regulation of the expression of the reporter proteins is related to the amount of cadmium and lead ions employed to induce the bacteria. The bacterial sensing systems were found to respond to cadmium, lead, and zinc ions, and had no significant response to nickel, copper, manganese, and cobalt.