Reverse phase protein arrays enable glioblastoma molecular subtyping

In the present study we investigated the phosphorylation status of the 12 most important signaling cascades in glioblastomas. More than 60 tumor and control biopsies from tumor center and periphery (based on neuronavigation) were subjected to selective protein expression analysis using reverse-phase protein arrays (RPPA) incubated with antibodies against posttranslationally modified cancer pathway proteins. The ratio between phosphorylated (or modified) and non-phosphorylated protein was assessed. All samples were histopathologically validated and proteomic profiles correlated with clinical and survival data. By RPPA, we identified three distinct activation patterns within glioblastoma defined by the ratios of pCREB1/CREB1, NOTCH-ICD/NOTCH1, and pGSK3β/GSK3β, respectively. These subclasses demonstrated distinct overall survival patterns in a cohort of patients from a single-institution and in an analysis of publicly available data. In particular, a high pGSK3β/GSK3β-ratio was associated with a poor survival. Wnt-activation/GSK3β-inhibition in U373 and U251 cell lines halted glioma cell proliferation and migration. Gene expression analysis was used as an internal quality control of baseline proteomic data. The protein expression and phosphorylation had a higher resolution, resulting in a better class-subdivision than mRNA based stratification data. Patients with different proteomic profiles from multiple biopsies showed a worse overall survival. The CREB1-, NOTCH1-, GSK3β-phosphorylation status correlated with glioma grades. RPPA represent a fast and reliable tool to supplement morphological diagnosis with pathway-specific information in individual tumors. These data can be exploited for molecular stratification and possible combinatorial treatment planning. Further, our results may optimize current glioma grading algorithms.     

Comparing the cost,glycaemic control and medication adherence of utilizing patients’ own medicines (POMs) versus usual dispensing among diabetic patients in an outpatient setting

BackgroundMillions worth of unused drugs particularly those indicated for chronic diseases such as diabetes were returned and disposed leading to substantial wastage. Use of patients’ own medications (POMs) in the inpatient setting has reduced wastage and saved cost. The impact of utilizing POMs in the outpatient setting has hitherto not been determined.PurposeThis study aims to compare the cost, medication adherence and glycaemic control of utilizing POMs versus usual dispensing.MethodsProspective randomized controlled study was conducted among diabetic patients that required monthly medication refill in the Outpatient Pharmacy in 2017. Patients who consented were equally divided into POMs and control groups. Both groups brought excess medications from home at week-0 and week-12. Patients in the POMs group brought excess medications monthly and sufficient amount of drugs were added until the next refill date. Drugs were dispensed as usual in the control group. Total cost consisting of the cost of drugs, staff and building was calculated. Glycosylated haemoglobin (HbA1c) was measured at baseline and week-12. Adherence was measured based on pill counting.ResultsThirty patients aged 56.77 ± 14.67 years with 13.37 ± 7.36 years of diabetes participated. Baseline characteristics were similar between the groups. POMs minimized the total cost by 38.96% which translated to a cost saving of USD 42.76 ± 6.98, significantly different versus USD 0.02 ± 0.52 in the control group, p = 0.025. Mean HbA1c reduced significantly (−0.79%, p = 0.016) in the POMs group but not significant in the control group (−0.11%, p = 0.740). Medication adherence improved significantly in both groups at week-12 (p < 0.010). Nevertheless, patients in the POMs group were more adherent, 87.20% vs. 66.32%, p = 0.034.ConclusionUtilizing POMs resulted in cost saving, improved adherence and better glycaemic control. Use of POMs should be practiced in the outpatient pharmacy to reduce wastage and cost.Graphical abstract      

Role of type-II pathway in apoptotic cell death induction by photosensitized CDRI-97/78 under ambient exposure of UV-B

Novel trioxane 97/78, developed by Central Drug Research Institute (CDRI), Lucknow has shown promising antimalarial activity. Clinical experience of anti-malarial drugs registered the occurrence of phototoxicity in patients exposed with sunlight subsequent to medication. Photodegradation study has identified one photo-product up to 4 h under UV-B/Sunlight by LC–MS/MS. UV-B irradiated 97/78 compound produced ¹O₂ via type-II dependent reaction mechanism, corroborated by its specific quencher. 2′-dGuO degradation and % tail development in photochemical as well as comet test, advocated the genotoxic potential of 97/78. The photocytotoxicity assays (MTT and NRU) on HaCaT cell line revealed the considerable decline in cell viability by 97/78. Cell cycle and Annexin V/PI double stain along with AO/EB demonstrated the G2/M phase arrest and apoptosis. Significant caspase-3 activity was measured in photoexcited 97/78 by colorimetric assay. Fluorescence stain with AO/JC-1 confirmed the lysosomal disruption and mitochondrial membrane destabilization by UV-B irradiated 97/78. Gene expression by RT-PCR showed significant upregulation of p21 and pro-apoptotic Bax, but no change observed in Bcl-2. In conclusion, the study highlights ROS mediated DNA damage, lysosomal and mitochondrial destabilization via upregulation of Bax and activation of caspase-3 which further leads to apoptosis.     

Marginal impaction in complex posterior wall acetabular fractures: role of allograft and mid-term results

European Journal of Orthopaedic Surgery & Traumatology - To evaluate the functional and radiological outcome of complex posterior wall acetabular fractures with marginal impaction treated with...     

Taenia solium taeniosis/cysticercosis in Asia: epidemiology,impact and issues

Several reports of patients with cysticercosis from many countries in Asia such as India, China, Indonesia, Thailand, Korea, Taiwan and Nepal are a clear indicator of the wide prevalence of Taenia solium cysticercosis and taeniosis in these and other Asian countries. However, epidemiological data from community based studies are sparse and available only for a few countries in Asia. Cysticercosis is the cause of epilepsy in up to 50% of Indian patients presenting with partial seizures. It is also a major cause of epilepsy in Bali (Indonesia), Vietnam and possibly China and Nepal. Seroprevalence studies indicate high rates of exposure to the parasite in several countries (Vietnam, China, Korea and Bali (Indonesia)) with rates ranging from 0.02 to 12.6%. Rates of taeniosis, as determined by stool examination for ova, have also been reported to range between 0.1 and 6% in the community in India, Vietnam, China, and Bali (Indonesia). An astonishingly high rate of taeniosis of 50% was reported from an area in Nepal populated by pig rearing farmers. In addition to poor sanitation, unhealthy pig rearing practices, low hygienic standards, unusual customs such as consumption of raw pork is an additional factor contributing to the spread of the disease in some communities of Asia. Undoubtedly, cysticercosis is a major public health problem in several Asian countries effecting several million people by not only causing neurological morbidity but also imposing economic hardship on impoverished populations. However, there are wide variations in the prevalence rates in different regions and different socio-economic groups in the same country. It is important to press for the recognition of cysticercosis as one of the major public health problems in Asia that needs to be tackled vigorously by the governments and public health authorities of the region.     

Does Parsonnet scoring model predict mortality following adult cardiac surgery in India?

Aims and Objectives:

To validate the Parsonnet scoring model to predict mortality following adult cardiac surgery in Indian scenario.

Materials and Methods:

A total of 889 consecutive patients undergoing adult cardiac surgery between January 2010 and April 2011 were included in the study. The Parsonnet score was determined for each patient and its predictive ability for in-hospital mortality was evaluated. The validation of Parsonnet score was performed for the total data and separately for the sub-groups coronary artery bypass grafting (CABG), valve surgery and combined procedures (CABG with valve surgery). The model calibration was performed using Hosmer–Lemeshow goodness of fit test and receiver operating characteristics (ROC) analysis for discrimination. Independent predictors of mortality were assessed from the variables used in the Parsonnet score by multivariate regression analysis.

Results:

The overall mortality was 6.3% (56 patients), 7.1% (34 patients) for CABG, 4.3% (16 patients) for valve surgery and 16.2% (6 patients) for combined procedures. The Hosmer–Lemeshow statistic was <0.05 for the total data and also within the sub-groups suggesting that the predicted outcome using Parsonnet score did not match the observed outcome. The area under the ROC curve for the total data was 0.699 (95% confidence interval 0.62–0.77) and when tested separately, it was 0.73 (0.64–0.81) for CABG, 0.79 (0.63–0.92) for valve surgery (good discriminatory ability) and only 0.55 (0.26–0.83) for combined procedures. The independent predictors of mortality determined for the total data were low ejection fraction (odds ratio [OR] - 1.7), preoperative intra-aortic balloon pump (OR - 10.7), combined procedures (OR - 5.1), dialysis dependency (OR - 23.4), and re-operation (OR - 9.4).

Conclusions:

The Parsonnet score yielded a good predictive value for valve surgeries, moderate predictive value for the total data and for CABG and poor predictive value for combined procedures.     

Reactive nitrogen species regulate autophagy through ATM-AMPK-TSC2–mediated suppression of mTORC1

Reactive intermediates such as reactive nitrogen species play essential roles in the cell as signaling molecules but, in excess, constitute a major source of cellular damage. We found that nitrosative stress induced by steady-state nitric oxide (NO) caused rapid activation of an ATM damage-response pathway leading to downstream signaling by this stress kinase to LKB1 and AMPK kinases, and activation of the TSC tumor suppressor. As a result, in an ATM-, LKB1-, TSC-dependent fashion, mTORC1 was repressed, as evidenced by decreased phosphorylation of S6K, 4E-BP1, and ULK1, direct targets of the mTORC1 kinase. Decreased ULK1 phosphorylation by mTORC1 at S757 and activation of AMPK to phosphorylate ULK1 at S317 in response to nitrosative stress resulted in increased autophagy: the LC3-II/LC3-I ratio increased as did GFP-LC3 puncta and acidic vesicles; p62 levels decreased in a lysosome-dependent manner, confirming an NO-induced increase in autophagic flux. Induction of autophagy by NO correlated with loss of cell viability, suggesting that, in this setting, autophagy was functioning primarily as a cytotoxic response to excess nitrosative stress. These data identify a nitrosative-stress signaling pathway that engages ATM and the LKB1 and TSC2 tumor suppressors to repress mTORC1 and regulate autophagy. As cancer cells are particularly sensitive to nitrosative stress, these data open another path for therapies capitalizing on the ability of reactive nitrogen species to induce autophagy-mediated cell death.Autophagy is a self-digestion process by which a eukaryotic cell degrades and recycles aggregate-prone proteins, macromolecules, and organelles. During autophagy, cytoplasmic contents are sequestered in double-membrane bound vesicles called autophagosomes and delivered to lysosomes for degradation, thereby allowing cells to eliminate and recycle the contents (1–3). Autophagy participates in both prosurvival (recycling of cellular building blocks) and prodeath (excess catalysis) pathways. A comprehensive understanding of signaling pathways that regulate autophagy holds great promise for new therapeutic opportunities by opening the possibility to compromise prosurvival autophagic pathways that enable tumor cells to evade therapy, or by promoting prodeath autophagic pathways to kill cancer cells.The classical pathway regulating autophagy in mammalian cells involves the serine/threonine kinase, mammalian target of rapamycin (mTOR). Active mTOR kinase in the mTORC1 complex phosphorylates and inhibits ULK1, a key proautophagy adapter involved in nucleation of the autophagophore membrane. Inactivation of mTORC1, either pharmacologically with rapamycin or via activation of the tuberous sclerosis complex (TSC) tumor suppressor, leads to downstream dephosphorylation events, including loss of ULK1 phosphorylation at S757. The TSC1/2 heterodimer is itself regulated by upstream kinases, including the AMP-activated protein kinase (AMPK), which regulates several metabolic processes and activates the TSC to repress mTORC1 under conditions of energy stress (4–6). AMPK also directly regulates autophagy by phosphorylating and activating ULK1 at S317 (7).Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes extending from its role as a chemical messenger and antibacterial agent to an integral component of the cardiovascular system and immune response to pathogens (8). NO has also been documented to play both promotional and inhibitory roles in cancer etiology. NO is produced endogenously by tumor cells of various histogenetic origins and has been associated with tumor proliferation and resistance to anticancer drugs. Although cancer cells may maintain endogenous NO at elevated levels that facilitate tumor growth, this strategy carries a survival liability, as high levels of NO can cause cytostasis or cytotoxicity (9). Thus, cancer cells may be more sensitive to NO-induced nitrosative stress, which could potentially provide a therapeutic avenue for modulating nitrosative stress to induce cell death. However, understanding at the molecular level regarding how NO participates in regulation of either prosurvival or prodeath autophagic pathways is limited.Recently, a novel signaling pathway involving activation of ATM to suppress mTORC1 in response to oxidative damage was identified (10). In the study reported here, we demonstrate that NO engages this pathway to suppress mTORC1 and promote autophagy. In response to nitrosative stress induced by steady-state exposure to NO, ATM is activated, signaling to AMPK via LKB1 to activate the TSC tumor suppressor and suppress mTORC1. Concomitant with suppression of mTORC1, autophagy is induced, accompanied by loss of cell viability. Our data provide strong evidence that NO regulates autophagy, with implications both for understanding the physiological role of NO-induced signaling and the development of therapies that can modulate nitrosative stress to kill cancer cells.     

Synaptophysin Positive Glomus Tumor of Trachea Simulating Typical Carcinoid: A Potential trap

Glomus tumors (GTs) are uncommon benign tumors, accounting for <2% of all soft tissue tumors and usually occur within the dermis or subcutis of distal extremities. Primary tracheal GT is rare, and to date less than 40 tracheal GTs have been reported. Tracheal GT usually present as a polypoidal mass. They express smooth muscle markers, and are negative for cytokeratin (CK) and neuroendocrine markers on immunohistochemistry (IHC). We here report a case of tracheal GT showing diffuse strong aberrant immunoexpression for synaptophysin, initially construed as carcinoid. Focal synaptophysin expression has been described in few gastric GTs, and a nasal GT. Diligent histomorphological examination and careful selection of IHC panel helps in clinching the diagnosis. Complete resection is the standard treatment of modality.     

A validated stability-indicating UPLC method for desloratadine and its impurities in pharmaceutical dosage forms

A novel stability-indicating gradient reverse phase ultra-performance liquid chromatographic (RP-UPLC) method was developed for the determination of purity of desloratadine in presence of its impurities and forced degradation products. The method was developed using Waters Aquity BEH C18 column with mobile phase containing a gradient mixture of solvents A and B. The eluted compounds were monitored at 280 nm. The run time was 8 min within which desloratadine and its five impurities were well separated. Desloratadine was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. Desloratadine was found to degrade significantly in oxidative and thermal stress conditions and stable in acid, base, hydrolytic and photolytic degradation conditions. The degradation products were well resolved from main peak and its impurities, thus proved the stability-indicating power of the method. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. This method was also suitable for the assay determination of desloratadine in pharmaceutical dosage forms.