Cytotoxic effect of Rotheca serrata on cancer cell lines MCF-7 and neuroblastoma SH-SY5Y.

INTRODUCTION: Rotheca serrata (Lamiaceae), a highly medicinal plant is used as an antidote for snakebite and the plant possesses medicinal properties like hepatoprotective, antitussive, antioxidant, anticancer, neuro-protective, used in rheumatoid arthritis and is also a α-glucoside inhibitor. AIM OF THE STUDY: This work aimed to study the anticancerous effect of Rotheca serrata (root and leaf) on cancer cell lines MCF-7 (breast cancer cell line) and Neuroblastoma SH-SY5Y. MATERIALS AND METHODS: This investigation was a preliminary one which supported the retrospective and safe use of plants as described in Ayurveda. Dulbecco's Modified Eagle Medium with High Glucose (DMEM-HG) for culturing MCF-7- Human Breast cancer cell line and Minimum essential Medium (MEM)+F12 medium for culturing SH-SY5Y- Homo sapiens bone marrow neuroblast were used. MTT assay measured the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability. RESULTS: The results indicated that the Methanolic extract of Rotheca serrata (root and leaf) showed high anticancer activity. Different concentrations of plant extracts (25, 50, 100, 200, 400 µg/ml) were used to study the anticancerous activity, amongst which the significant results were obtained for 400 µg/ml concentration (both root & leaf). Effective anticancer activity against MCF - 7 breast cancer cells was shown in methanoilc extracts and were expressed as IC 50 values; in root (IC 50 value = 61.8259 ± 7.428 µg/ml) and in leaf (IC 50 value = 78.1497 ± 6.316 µg/ml). The MTT assay in case of neuroblastoma (SH-SY5Y) cell lines revealed that 400 µg/ml concentration of leaf methanolic extract showed effective inhibition of cancer cells with IC 50 value 37.8462 ± 2.957 µg/ml as compared to IC 50 value of root methanolic extract which was 57.0895 ± 2.351 µg/ml. CONCLUSION: R. serrata possess anticancer activity against breast cancer cell line (MCF-7) and neuroblastoma (SH-SY 5Y) cell lines. This study may to design plant-based drugs without side effects. Dosage compensation for specific type of cancer needs to be monitored in patients with 1st stage.

Tumor grading model employing geometric analysis of histopathological images with characteristic nuclei dictionary.

Histopathological study has been shown to improve diagnosis of various disease classifications effectively as any disease condition is correlated to characteristic set of changes in the tissue structure. This study aims at developing an automated neural network system for grading brain tumors (Glioblastoma Multiforme) from histopathological images within the Whole Slide Images (WSI) of hematoxylin and eosin (H&E) stains with significant accuracy. Hematoxylin channels are extracted from the histopathological image patches using color de-convolution. Cell nuclei are precisely segmented using three level Otsu thresholding. From each segmented image, nuclei boundaries are extracted to extract nucleus level features based on their shape and size. Geometric features including ellipse eccentricities, nucleus perimeter, area, and polygon edge counts are extracted using geometric algorithms to define the nuclei boundaries of the segmented image. These features are collected for a large number of nuclei and the nuclei are clustered using the K-Means algorithm in order to create a dictionary. One of the major contributions involves the creation of dictionary of a fixed number of representative cell nuclei to speed up patch level classification. This optimal dictionary is used for clustering extracted cell nuclei and a fixed length histogram of counts on different types of nuclei is obtained. The proposed system has been tested with a total of 239600 TCGA patches of GBM and 206000 patches of LGG collected from GDC data portal and it showed good diagnosis performance with auto-classification accuracy of 97.2% compared to other state-of-art methods. Our results on segmentation and classification are encouraging, with better attainment with regard to precision and accuracy in contrast with previous models. The auto grading proposed system will act as a potential guide for pathologists to make more accurate decisions.

Long-term neurodevelopment outcome of caffeine versus aminophylline therapy for apnea of prematurity.

OBJECTIVE: Methylxanthines are the most commonly prescribed drug in neonatal setups. However, Clinicians show indecision in choosing the right agent for Apnea of Prematurity in most of the developing countries. Present study aimed to compare rate of mortality and survival with normal neurodevelopment outcome at 18 to 24 months of corrected age, between Caffeine- and Aminophylline-treated infants for apnea of prematurity. METHODS: 240 infants were randomly allocated to caffeine and aminophylline for apnea of prematurity during February 2012 to January 2015. Long-term neurodevelopmental assessment was done only from children who had attained corrected age of 18 to 24 months during April 2014 to February 2016. Cognitive, language and motor deficits were assessed by Bayley Scale of infant and toddler development (BSID - III). Postnatal characteristics such as hearing and visual impairments during NICU stay were noted and same were followed up. RESULTS: Infants allocated to caffeine group showed 83% less risk of getting cognitive impairment (RR 0.16; CI 95% range 0.02 to 1.36), 50% less risk of developing motor deficits (RR 0.50; CI 95% range 0.12 to 1.95) and 24% less risk of developing language problems (RR 0.76; CI 95% range 0.36 to 1.58). However in all the neurodevelopment domains the difference between groups was not statistically significant. Risk of mortality in caffeine group was 9% less over aminophylline group which was statistically non-significant (RR - 0.92; CI 95% range - 0.45 to 1.84; p = 0.81). Physical growth parameters were found to be similar in both the groups. Risk of developing visual abnormality and hearing impairments was also statistically non-significant between the groups. CONCLUSION: Caffeine and aminophylline showed similar effects in reducing the rate of mortality and improving the survival without neurodevelopment delays; though the clinical significance of caffeine over aminophylline cannot be undermined.

Comparative Efficacy and Safety of Caffeine and Aminophylline for Apnea of Prematurity in Preterm (≤34 weeks) Neonates: A Randomized Controlled Trial.

OBJECTIVE: To compare the efficacy and safety of standard doses of Caffeine and Aminophylline for Apnea of prematurity. STUDY DESIGN: Randomized controlled trial. SETTING: Tertiary-care referral centre and a teaching institution in Southern India. Trial was conducted from February 2012 to January 2015. PARTICIPANTS: 240 preterm (≤34 wk) neonates with apnea of prematurity. INTERVENTIONS: Neonates randomized into two groups: Caffeine group received loading dose of caffeine citrate (20 mg/kg) followed by 5 mg/kg/day maintenance dose every 24 hour. Aminophylline group received loading dose of Aminophylline - 5 mg/kg and maintenance dose of 1.5 mg/kg 8-hourly. OUTCOME MEASURES: Difference in apneic spells, associated respiratory morbidity, and acute adverse events were assessed. Association of efficacy with therapeutic drug levels was also evaluated. RESULTS: Infants on aminophylline experienced less apnea spells in 4-7 days of therapy (P=0.03). Mean apnea rate and isolated desaturations were similar in 1-3, 4-7 and 8-14 days of therapy. No difference was noted in duration of Neonatal Intensive Care Unit stay and hospital stay. Mean heart rate was significantly high in Aminophylline group (P<0.001). Risk of developing tachycardia was less (RR 0.30; 95% CI range 0.15 to 0.60; P<0.001) in Caffeine- over Aminophylline-treated infants. CONCLUSION: Aminophylline is as effective as caffeine for prevention of apneic spells in preterm neonates; however, dosage optimization needs to be done to reduce toxicity.

Pfeiffer Syndrome with Extreme Proptosis, Hypothyroidism and Tail like Appendage.

Pfeiffer syndrome is a rare genetic disorder with combination of bicoronal craniosynostosis, broad thumbs, broad great toes, ankylosis of elbow and partial variable syndactyly of the hands and feet. Since the disorder was reported by Pfeiffer in 1964, new associations have been added on. Authors report a newborn with features of Pfeiffer syndrome type 3 with hypothyroidism, tail like appendage and extremely anteriorly placed anus as new associations.

Mechanisms of Adipocytokine-Mediated Trastuzumab Resistance in HER2-Positive Breast Cancer Cell Lines.

Acquired resistance to trastuzumab is a clinical problem in the treatment of HER2-over-expressing metastatic breast cancer. Importantly, an earlier report suggested that high body mass index was associated with reduced overall survival and reduced time to progression in patients with early stage or metastatic HER2-positive breast cancer treated with trastuzumab. Adipocyte-secreted factors may stimulate growth of HER2-positive cancers, blocking the growth inhibitory action of trastuzumab. Leptin and growth differentiation factor 15 (GDF15) are two adipocytokines that have been reported to stimulate HER2-PI3K signaling. We previously showed that cells with acquired trastuzumab resistance express increased levels of GDF15, and that GDF15 knockdown restores sensitivity to trastuzumab. The objective of the current study was to identify potential molecular mechanisms by which adipocytes stimulate resistance to trastuzumab in HER2-over-expressing breast cancer cell lines. Cells were cultured in complete media or conditioned media from differentiated adipocytes (CM). Cell viability of trastuzumab-treated cells was examined under anchorage-dependent and -independent conditions. Phosphorylation of Akt was assessed by Western blotting, and response to trastuzumab was reassessed upon treatment with the PI3K inhibitor LY294002 or after transfection with kinase-dead Akt. We report that CM significantly reduced trastuzumab-mediated growth inhibition of HER2-positive cells, and stimulated rapid phosphorylation of Akt. Pharmacologic or genetic inhibition of PI3K overcame CM-mediated trastuzumab resistance. Leptin and GDF15 were both measured in CM, but only GDF15 conferred resistance to trastuzumab. Leptin, on the other hand, abrogated sensitivity to lapatinib but not trastuzumab. Our observations suggest that adipocyte-secreted factors such as GDF15 stimulate PI3K signaling, resulting in reduced response to trastuzumab. The utility of adipocytokines as predictors of drug resistance and approaches to mitigate the cancer-promoting effects of adipocyte-secreted factors should be further examined. Our work supports additional investigation into GDF15 as a potential biomarker of trastuzumab resistance, and development of approaches to therapeutically target GDF15 in HER2-positive breast cancers that have progressed on trastuzumab.

Prevalence, clinical and biochemical profile of subclinical hypothyroidism in normal population in Mumbai.

BACKGROUND: Subclinical Hypothyroidism (ScHt) affects 3-15% of the adult population. It's clinical and biochemical profile is not well defined, especially in Indian scenario. Our study aimed at screening normal population to define normative ranges of thyroid hormones and Serum thyroid stimulating hormone (S.TSH) and prevalence of ScHt and thyroid autoimmunity. MATERIALS AND METHODS: Two-hundred thirty-seven normal subjects without family history of thyroid disease were evaluated for symptoms and laboratory tests for thyroid dysfunction and autoimmunity. RESULTS: The thyroid function tests were as follows: EUTHYROID GROUP: MEAN VALUES WERE: T3: 1.79 ± 0.42 ng/mL, T4: 10.23 ± 2.25 µg/dL, FT3: 1.88 ± 0.19 pg/mL, FT4: 1.12 ± 0.21 ng/dL, S.TSH: 2.22 ± 1.06 µlu/mL. 10.2% of euthyroid subjects had antimicrosomal antibodies (AMA) +ve (mean titer 1:918) and 23.6% were anti-thyroid peroxidase autoantibody (anti-TPO) +ve (mean titer 15.06 Au/mL). The euthyroid outlier range for S.TSH was 0.3-4.6 µlu/mL. The values were comparable in both the sexes. Those with S.TSH ≥ 5 µlu/mL were defined to have ScHt. SCHT GROUP: Prevalence of ScHt was 11.3% (M:F ratio 1:3.7). 74% belonged to 35-54 years age group and prevalence increased with age (post-menopausal females: prevalence 20%). S.TSH was 9.8 ± 7.22 µlu/mL, mean S.AMA was 1:5079 (40.7% positivity) and mean S.anti-TPO was 260 Au/mL (47.6% positivity). Majority were agoitrous (74%), and stage I goiter was seen in 26% of this population. Symptom score of 5-8 was seen in 55% ScHt subjects versus 35% normal subjects. CONCLUSION: Mean S.TSH in our population was 2.22 µlu/mL (euthyroid outliers: 0.3-4.6 µlu/mL); hence, S.TSH above 4.6 µlu/mL should be considered as abnormal. The prevalence of thyroid autoimmunity increases after age of 35 years. ScHt presents mainly in agoitrous form and with positive antibodies, suggesting autoimmunity as the cause.

Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion.

Identification of novel molecular markers and therapeutic targets may improve survival rates for patients with ovarian cancer. In the current study, immunohistochemical (IHC) analysis of two human ovarian tumor tissue arrays showed high staining for GDF15 in a majority of tissues. Exogenous stimulation of ovarian cancer cell lines with recombinant human GDF15 (rhGDF15) or stable over-expression of a GDF15 expression plasmid promoted anchorage-independent growth, increased invasion, and up-regulation of matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF). MMP inhibition suppressed GDF15-mediated invasion. In addition, IHC analysis of human ovarian tumor tissue arrays indicated that GDF15 expression correlated significantly with high MMP2 and MMP9 expression. Exogenous and endogenous GDF15 over-expression stimulated phosphorylation of p38, Erk1/2, and Akt. Pharmacologic inhibition of p38, MEK, or PI3K suppressed GDF15-stimulated growth. Further, proliferation, growth, and invasion of GDF15 stable clones were blocked by rapamycin. IHC analysis demonstrated significant correlation between GDF15 expression and phosphorylation of mTOR. Finally, knockdown of endogenous GDF15 or neutralization of secreted GDF15 suppressed invasion and growth of a GDF15-over-expressing ovarian cancer cell line. These data indicate that GDF15 over-expression, which occurred in a majority of human ovarian cancers, promoted rapamycin-sensitive invasion and growth of ovarian cancer cells. Inhibition of mTOR may be an effective therapeutic strategy for ovarian cancers that over-express GDF15. Future studies should examine GDF15 as a novel molecular target for blocking ovarian cancer progression.

Catalytic deoxyribozyme-modified nanoparticles for RNAi-independent gene regulation.

DNAzymes are catalytic oligonucleotides with important applications in gene regulation, DNA computing, responsive soft materials, and ultrasensitive metal-ion sensing. The most significant challenge for using DNAzymes in vivo pertains to nontoxic delivery and maintaining function inside cells. We synthesized multivalent deoxyribozyme "10-23" gold nanoparticle (DzNP) conjugates, varying DNA density, linker length, enzyme orientation, and linker composition in order to study the role of the steric environment and gold surface chemistry on catalysis. DNAzyme catalytic efficiency was modulated by steric packing and proximity of the active loop to the gold surface. Importantly, the 10-23 DNAzyme was asymmetrically sensitive to the gold surface and when anchored through the 5' terminus was inhibited 32-fold. This property was used to generate DNAzymes whose catalytic activity is triggered by thiol displacement reactions or by photoexcitation at λ = 532 nm. Importantly, cell studies revealed that DzNPs are less susceptible to nuclease degradation, readily enter mammalian cells, and catalytically down-regulate GDF15 gene expression levels in breast cancer cells, thus addressing some of the key limitations in the adoption of DNAzymes for in vivo work.

Growth differentiation factor 15 (GDF15)-mediated HER2 phosphorylation reduces trastuzumab sensitivity of HER2-overexpressing breast cancer cells.

Resistance to the anti-HER2 monoclonal antibody trastuzumab is a major problem in the treatment of HER2-overexpressing metastatic breast cancer. Growth differentiation factor 15 (GDF15), which is structurally similar to TGF beta, has been reported to stimulate phosphorylation of HER2. We tested the hypothesis that GDF15-mediated phosphorylation of HER2 reduces the sensitivity of HER2-overexpressing breast cancer cell lines to trastuzumab. Gene microarray analysis, real-time PCR, and ELISA were used to assess GDF15 expression. Growth inhibition and proliferation assays in response to pharmacologic inhibitors of HER2, TGF beta receptor, or Src were performed on cells stimulated with recombinant human GDF15 or stable GDF15 transfectants. Western blotting was performed to determine effects of GDF15 on HER2 signaling. Cells were infected with lentiviral GDF15 shRNA plasmid to determine effects of GDF15 knockdown on cell survival in response to trastuzumab. Cells with acquired or primary trastuzumab resistance showed increased GDF15 expression. Exposure of trastuzumab-sensitive cells to recombinant human GDF15 or stable transfection of a GDF15 expression plasmid inhibited trastuzumab-mediated growth inhibition. HER2 tyrosine kinase inhibition abrogated GDF15-mediated Akt and Erk1/2 phosphorylation and blocked GDF15-mediated trastuzumab resistance. Pharmacologic inhibition of TGF beta receptor blocked GDF15-mediated phosphorylation of Src. Further, TGF beta receptor inhibition or Src inhibition blocked GDF15-mediated trastuzumab resistance. Finally, lentiviral GDF15 shRNA increased trastuzumab sensitivity in cells with acquired or primary trastuzumab resistance. These results support GDF15-mediated activation of TGF beta receptor-Src-HER2 signaling crosstalk as a novel mechanism of trastuzumab resistance.

Endometrioid carcinoma of the upper urinary tract.

Herein, we report a second case of endometrioid carcinoma of the upper urinary tract presenting 17 years after hysterectomy for high grade adenocarcinoma of ovary. A 51-year-old nullipara presented to us with a complaint of hematuria. After complete work up, she underwent right radical nephro-ureterectomy with bladder cuff excision. The histology showed endometrioid carcinoma of upper urinary tract without any evidence of endometriosis.

Antioxidant activity and free radical scavenging reactions of hydroxybenzyl alcohols. Biochemical and pulse radiolysis studies.

Comparative studies on free radical scavenging by isomers of hydroxybenzyl alcohols (HBAs) were carried out to understand the molecular mechanisms involved in the antioxidant action. Using rat liver mitochondria as model systems, we have examined the radioprotective and antioxidant effects of hydroxybenzyl alcohols. Apart from their ability to scavenge free radicals and ferric reducing power, HBAs have shown good protection against radiation and oxidative stress. Using peroxyl radicals as initiator of reactive oxygen species (ROS), studies were carried out to evaluate antioxidant properties of HBAs against rat liver mitochondrial membrane components such as lipid and protein. Our results show that HBAs are potent inhibitor of lipid peroxidation and protein oxidation thus suggesting their role as free radical scavengers. In the presence of HBAs, restoration of depleted activity of Mn-SOD has also been investigated. In the presence of 2-hydroxybenzyl alcohol (2-HBA) complete restoration in the activity of Mn-SOD was observed on exposure to 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). Free radical scavenging ability of HBAs were found to be comparable to alpha tocopherol. Pulse radiolysis technique has been used to study the reactions of HBAs with various biologically relevant reactive species such as hydroxyl radical (OH) and trichloromethyl peroxyl radical (CCl3O2). HBAs could scavenge OH radical giving initially OH-adducts which in turn decays to phenoxyl radicals. Reactions of phenoxyl radicals of HBAs with ascorbic acid have been also studied. Redox potential of HBAs has been evaluated with cyclic voltammetry. Studies clearly suggest a structural reactivity correlation between radical scavenging and antioxidant properties of these isomers of HBA. Among the 3 isomers of HBAs, 4-HBA and 2-HBA are found to have better radical scavenging and antioxidant properties than 3-HBA.

Cerebral aspergilloma presenting as atypical meningioma in an immunologically competent patient: a case report.

A 36-year-old man presented with history suggestive of intracranial space occupying lesion. Computed tomography of brain revealed a large lobulated, extra axial, hyperdense lesion in the right fronto-temporal region extending up to to the right frontal and ethmoidal sinuses, eroding the bone, enhancing homogeneously with contrast, which was suggestive of atypical meningioma. He had no predisposing factors that could lead to the suspicion of opportunistic infection. Craniotomy and total excision of the lesion was done. Histopathological study revealed aspergilloma.

Biological significance of singlet oxygen.

The biological significance of singlet oxygen (1O2), an electronically excited species of oxygen, has been realized only in the last two decades. This was mainly due to the lack of proper methodology to generate this reactive oxygen species (ROS) in pure form and its reactions with biological molecules. Recent studies, using newly developed detection methods, show that 1O2 being generated in many biological systems, can significantly and quite often adversely alter several crucial biomolecules including DNA, proteins and lipids with undesirable consequences including cytotoxicity and/or disesase development. The reactions of 1O2 with the biological molecules are rather specific, as compared to other ROS. There are various compounds, mainly derived from natural sources that offer protection against damage induced by 1O2. Among the antioxidants carotenoids are the most effective singlet oxygen quenchers followed by tocopherols and others. The same reactive species if generated specifically in diseased states such as cancer can lead to the cure of the disease, and this principle is utilized in the newly developing modality of cancer treatment namely photodynamic therapy. Singlet oxygen, in low concentrations can also act as signaling molecule with several biological implications. This review clearly brings out the biological significance of 1O2.

A new phenotype for sbcB mutations in Escherichia coli: RecA-dependent increase in plasmid-borne gene expression.

A new chromosomal mutation (cpeA), that causes increased expression of plasmid-borne genes in Escherichia coli, was identified and mapped to the sbcB locus. The effect of the mutation on plasmid transcription was non-specific with respect to the various promoters that were studied, but was more pronounced for an IncW low-copy-number plasmid than for ColE1- or p15A-based, high-copy-number plasmids. The mutant phenotype was observed even in recB+C+D+ strains, but not in recA mutants. The increased-expression phenotype was also observed in sbcB15 but not in xonA1 (another sbcB allele) mutants, suggesting that the expression of this phenotype is mediated by genes of the so-called RecF pathway family. Consistent with this interpretation was the observation that the cpeA mutant phenotype was less pronounced in recF, recJ and recO mutants. The increased-expression phenotype was also correlated with increased recovery of plasmid DNA from the cpeA/sbcB mutant strains, but there was no evidence for the occurrence of linear plasmid multimers in these strains.

Molecular cloning of an osmoregulatory locus in Escherichia coli: increased proU gene dosage results in enhanced osmotolerance.

The proU locus in Escherichia coli encodes an important osmoregulatory function which mediates the growth-promoting effect of L-proline and glycine betaine in high-osmolarity media. This locus was cloned, in contiguity with a closely linked Tn10 insertion, onto a multicopy plasmid directly from the E. coli chromosome. For a given level of osmotic stress, the magnitude of osmoresponsive induction of a single-copy proU::lac fusion was reduced in strains with multiple copies of the proU+ genes; in comparison with haploid proU+ strains, strains with the multicopy proU+ plasmids also exhibited enhanced osmotolerance in media supplemented with 1 mM L-proline or glycine betaine. Experiments involving subcloning, Tn1000 mutagenesis, and interplasmid complementation in a deletion mutant provided evidence for the presence at this locus of two cistrons, both of which are necessary for the expression of ProU function. We propose the designations proU for the gene originally identified by the proU224::Mu d1(lac Ap) insertion and proV for the gene upstream (that is, counterclockwise) of proU.