Accelerated microbial identification "directly" from positive blood cultures using MALDI-TOF MS: Local clinical laboratory challenges.

Rapid identification of microbial pathogens "directly" from positive blood cultures (PBCs) is critical for prompt initiation of empirical antibiotic therapy and clinical outcomes. Towards higher microbial identification rates, we modified a published initial serum separator tubes-based MALDI-TOF-MS protocol, for blood culture specimens received at a non-hospital based standalone diagnostic laboratory, Bangalore, India: (a) "Initial" protocol #1: From 28 PBCs, identification= 39% (Gram-negative= 43%: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa; Gram-positive: 36%: Enterococcus faecalis, Staphylococcus aureus, Staphylococcus haemolyticus); mis-identification= 14%; non-identification= 47%. (b) "Modified" protocol #2: Quality controls (ATCC colonies spiked in negative blood cultures) From 7 analysis, identification= 100% (Escherichia coli, Klebsiella pneumonia, Klebsiella oxytoca, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus); From 7 PBCs, identification= 57%; mis-identification= 14%; non-identification= 29%. Microbial preparations of highest quality and quantity for proteomic analysis and separate spectra matching reference databases for colonies and PBCs are needed for best clinical utility.

Myofibroblast progeny in wound biology and wound healing studies.

Fibroblasts and myofibroblasts play a myriad of important roles in human tissue function, especially in wound repair and healing. Among all cells, fibroblasts are group of cells that decide the status of wound as they maintain tissue homeostasis. Currently, the increase in the deleterious effects of chronic wound and their morbidity rate has necessitated the need to understand the influence of fibroblasts and myofibroblasts, which chiefly originate locally from tissue-resident fibroblasts to address the same. Wound pathophysiology is complex, herein, we have discussed fibroblast and myofibroblast heterogeneity in skin and different organs by understanding the phenotypical and functional properties of each of its sub-populations in the process of wound healing. Recent advancements in fibroblast activation, differentiation to myofibroblasts, proliferation and migration are discussed in detail. Fibroblasts and myofibroblasts are key players in wound healing and wound remodelling, respectively, and their significance in wound repair is discussed. An increased understanding of their biology during wound healing also gives an opportunity to explore more of fibroblast and myofibroblast focused therapies to treat chronic wounds which are clinical challenges. In this regard, in the current review, we have described the different methods for isolation of primary fibroblasts and myofibroblasts from both animal models and humans, and their characterization. Additionally, we have also provided details on possible molecular targets for better understanding of prognosis, diagnosis and treatment of chronic wounds. Information will help both researchers and clinicians in providing molecular insight that enable them for effective chronic wound management. The knowledge of intimate dialogue between the fibroblast, sub-populations like, myofibroblast and their microenvironment, will serve useful in determining novel, efficient and specific therapeutic targets to treat pathological wound conditions.

Cytotoxicity of obacunone and obacunone glucoside in human prostate cancer cells involves Akt-mediated programmed cell death.

Obacunone and obacunone glucoside (OG) are naturally occurring triterpenoids commonly found in citrus and other plants of the Rutaceae family. The current study reports the mechanism of cytotoxicity of citrus-derived obacunone and OG on human androgen-dependent prostate cancer LNCaP cells. Both limonoids exhibited time- and dose-dependent inhibition of cell proliferation, with more than 60% inhibition of cell viability at 100 µM, after 24 and 48 h. Analysis of fragmentation of DNA, activity of caspase-3, and cytosolic cytochrome-c in the cells treated with limonoids provided evidence for activation of programmed cell death by limonoids. Treatment of LNCaP cells with obacunone and OG resulted in dose-dependent changes in expression of proteins responsible for the induction of programmed cell death through the intrinsic pathway and down-regulation of Akt, a key molecule in cell signaling pathways. In addition, obacunone and OG also negatively regulated an inflammation-associated transcription factor, androgen receptor, and prostate-specific antigen, and activated proteins related to the cell cycle, confirming the ability of limonoids to induce cytotoxicity through multiple pathways. The results of this study provided, for the first time, an evidence of the cytotoxicity of obacunone and OG in androgen-dependent human prostate cancer cells.

5-Geranyloxy-7-methoxycoumarin inhibits colon cancer (SW480) cells growth by inducing apoptosis.

For the first time, three coumarins were isolated from the hexane extract of limes (Citrus aurantifolia) and purified by flash chromatography. The structures were identified by NMR (1D, 2D) and mass spectral analyses as 5-geranyloxy-7-methoxycoumarin, limettin, and isopimpinellin. These compounds inhibited human colon cancer (SW-480) cell proliferation, with 5-geranyloxy-7-methoxycoumarin showing the highest inhibition activity (67 %) at 25 µM. Suppression of SW480 cell proliferation by 5-geranyloxy-7-methoxycoumarin was associated with induction of apoptosis, as evidenced by annexin V staining and DNA fragmentation. In addition, 5-geranyloxy-7-methoxycoumarin arrested cells at the G0/G1 phase, and induction of apoptosis was demonstrated through the activation of tumour suppressor gene p53, caspase8/3, regulation of Bcl2, and inhibition of p38 MAPK phosphorylation. These findings suggest that 5-geranyloxy-7-methoxycoumarin has potential as a cancer preventive agent.

Inhibition of prostate cancer (LNCaP) cell proliferation by volatile components from Nagami kumquats.

Fresh Nagami kumquats (Fortunella margarita) were subjected to hydrodistillation using a Clevenger-type apparatus to obtain volatile oil. The chemical composition of the volatile oil was analyzed by GC-MS using Rtx-5 Sil MS and DB Wax columns. A total of 25 volatile compounds were identified by mass spectra, retention index, and comparison with known standards. The major identified compounds are d-limonene (41.64 %), ß-myrecene (16.54 %), linalyl propionate (9.55 %), and germacrene-D (5.93 %) from the Rtx-5 Sil MS column; d-limonene and ß-myrecene were also separated as major compounds on the DB wax column. The oil is rich in hydrocarbons (77.41 %) consisting of 60.05 % monoterpenes and 17.36 % sesquiterpenes. Interestingly, oxygenated hydrocarbons (17.6 %) were also found in kumquat volatile oil. Certain volatile compounds were also confirmed by positive chemical ionization and NMR spectra. Further, the volatile oil demonstrated good DPPH radical scavenging activity and antioxidant capacity. Kumquat volatile oil at 200 ppm concentration exhibited 55 %, 61 %, and 63.4 % inhibition of human prostate cancer (LNCaP) cell proliferation at 24, 48, and 72 h, respectively, by cell count assays. Significant increases in expression of bax/bcl2 and p53 proteins confirmed that volatile oil induces apoptosis. In addition, inhibition of inflammatory markers such as NF-κB and Cox-2 was observed. The cleavage of caspase-8 in the LNCaP cells treated with volatile oil demonstrated that apoptosis occurred through an extrinsic pathway. This is the first report of the identification and possible mechanisms of in vitro antiproliferative effects of kumquat volatile components on human prostate cancer (LNCaP) cells.

Antioxidant potentials of flaxseed by in vivo model.

The present study reports the antioxidant activity of flaxseed as measured by feeding weanling albino rats with 5.0% and 10.0% of flaxseed (constituting approximately 0.75 and 1.5 g kg(-)(1)) for 14 days followed by challenging animals with 2.0 g kg(-)(1) b.w. CCl(4) as toxin. Activity was assessed by measuring hepatic marker enzymes like catalase, superoxide dismutase (SOD), and peroxidase and comparing with those from the normal group and from a group receiving toxin without flaxseed. Treatment of CCl(4) at dose of 2.0 g kg(-)(1) b.w. decreased the activities of various antioxidant enzymes such as catalase, superoxide dismutase (SOD), and peroxidase by 35.6%, 47.76%, and 53.0%, respectively, compared to the control group, and the lipid peroxidation value increased nearly 1.2-fold compared to that of the group treated with toxin without flaxseed. Pretreatment of rats with 5.0% flaxseed followed by CCl(4) treatment caused restoration of catalase, SOD, and peroxidase by 39.7%, 181.42%, and 123.7%, respectively, as compared to control. The group treated with 10.0% flaxseed has shown the restoration of 95.02%, 182.31%, and 136.0% of catalase, SOD, and peroxidase. In the case of the group treated with toxin without flaxseed, the level of superoxide dismutase and the catalse value decreased 91.4% and 55.33%, respectively, in comparison with the control group. These results clearly indicate the beneficial effect of flaxseed components as an antioxidant as seen by restoration of hepatic enzymes, which were varied from normal to one due to toxicity induced by toxin (CCl(4)). Owing to this property, the flaxseed known for its functional properties can be further extended to exploit its possible application for various health benefits as nutraceuticals and food ingredient.

Genetically modified hairy roots of Withania somnifera Dunal: a potent source of rejuvenating principles.

Transgenic hairy roots were induced from Withania somnifera Dunal, by infecting leaf explants with Agrobacterium rhizogenes. Polymerase chain reaction for rol A gene and Southern blot confirmed the integration of T-DNA in the genome. Cultures were grown in Murashige and Skoog solid as well as in liquid medium. The antioxidant activity was assayed in roots grown in solid media and liquid media. Hairy roots grown in liquid media found to possess highly significant activity in 1,1-diphenyl-2-pecryl-hydrazyl radical, beta-carotene linoleic acid model system. The activity was 57.34%, 75.64%, and 93.41% in case DPPH model and 55.3%, 76.3%, and 90.5% in case of b-CLAMS in 25, 50, and 100 mg L(-1) concentration, respectively. In case of hydroxyl radical trapping and brain lipid peroxidation assay, the activity was more significant in hairy roots grown on solid medium in comparison with commercial formulation prepared using normal roots and standard withanaloids. Root extract grown in solid medium has shown 93.2% hydroxyl radical trapping activity at 100 mg L(-1) concentration, and 500 mg L(-1) has shown 83.6% in case of brain lipid peroxidation assay. High-performance liquid chromatography analysis demonstrated the presence of withanaloids in the hairy root extracts. The results of the study clearly indicate that there is enhancement of secondary metabolites in hairy roots, which is indicated through significant enhancement of the antioxidant activity, since these are the major constituents responsible for the activity. This is the first report on the presence of antioxidant principles in genetically modified roots of W. somnifera. These results of the present study may aid in utilization of the W. somnifera hairy roots for its rejuvenating principles.