To evaluate the effects of near-infrared (NIR) laser irradiation of microspheres (MS) containing hollow gold nanospheres (HAuNS) and paclitaxel (PTX) administered intra-arterially in an animal model.
Materials and Methods
For the ex-vivo experiments, VX2 tumor-bearing rabbits underwent hepatic artery (HA) administration of MS-HAuNS or MS. The animals were killed, the liver tumors were subjected to NIR irradiation, and temperature changes were estimated with magnetic resonance imaging. For the in-vivo study, VX2 tumor-bearing rabbits were randomized to 3 groups: MS-HAuNS-PTX-plus-NIR, MS-HAuNS-PTX, and saline-plus-NIR. Laser irradiation was delivered at 1 hour and at 3 days after HA administration of saline or MS-HAuNS-PTX. Animals were euthanized and tumors were analyzed for necrosis and apoptosis. Plasma samples were collected from the MS-HAuNS-PTX-plus-NIR animals for PTX analysis.
Ex-vivo experiments showed intratumoral heating in animals that received MS-HAuNS but no temperature change in animals that received MS. Animals treated with MS-HAuNS-PTX-plus-NIR showed a transient increase in plasma PTX levels after each NIR irradiation and significantly greater tumor necrosis than did those that received MS-HAuNS-PTX or saline-plus-NIR (44.9% vs. 13.8% or 23.7%, respectively; P < .0001). The mean apoptotic index in the MS-HAuNS-PTX-NIR group (5.01 ± 1.66) was significantly higher than that in the MS-HAuNS-PTX (2.99 ± 0.97) or saline-plus-NIR (1.96 ± 0.40) groups (P = .0013).
NIR laser irradiation after MS-HAuNS-PTX administration results in intratumoral heating and increases the efficacy of treatment. Further studies are required to evaluate the optimal laser settings to maximize therapeutic efficacy.
Elevated blood pressure (BP) is associated with increased cardiovascular risks manifested by ischemic heart disease and stroke. Studies of cardiothoracic surgeons and neurosurgeons suggest surgery induces a hemodynamic stress malresponse. However, it is unclear whether these occur in orthopaedic surgeons.
We measured the BP of surgeons during hallux valgus surgery, TKA, and THA with the: (1) trainee assisting the trainer, (2) the trainer assisting the trainee, (3) the trainee operating independently, and (4) compared the intraoperative changes in BP and heart rate of orthopaedic surgeons with those of a clinic day and during an exercise tolerance test.
We used an ambulatory BP monitor to measure the BP and heart rate of three consultants and their respective trainees during hallux valgus surgery, TKA, or THA. We noted if there were any differences in the stress response of the lead surgeon in comparison to when the same individual was assisting a trainee, and vice versa. Additionally, we recorded the trainee’s BP and heart rate when they were operating independently. The intraoperative changes in BP and heart rate of orthopaedic surgeons were compared with those measured during a clinic day and during an exercise tolerance test.
When the trainer was leading the operation, their mean arterial pressure gradually increased to 105 (range, 102–109) until implant placement. However, when the trainee was operating and the trainer assisting, the trainer’s BP peaked (mean, 101; range, 95–111) at the beginning of the procedure and slowly declined as it progressed. The trainee’s BP remained elevated throughout. The highest peaks for trainees were noted during independent operating. All of the surgeons had higher average BP readings (mean, 100; range, 95–108) and heart rate (mean, 86; range, 57–117) on days when they did surgery compared with baseline.
The elective operations studied induced a hypertensive response. The response was more marked in trainees than in trainers, particularly if the trainee was operating independently.
The nucleocapsid (N) protein of Chandipura virus (CHPV) plays a crucial role in viral life cycle, besides being an important structural component of the virion through proper organization of its interactions with other viral proteins. In a recent study, the authors had mapped the associations among CHPV proteins and shown that N protein interacts with four of the viral proteins: N, phosphoprotein (P), matrix protein (M), and glycoprotein (G). The present study aimed to distinguish the regions of CHPV N protein responsible for its interactions with other viral proteins. In this direction, we have generated the structure of CHPV N protein by homology modeling using SWISS-MODEL workspace and Accelrys Discovery Studio client 2.55 and mapped the domains of N protein using PiSQRD. The interactions of N protein fragments with other proteins were determined by ZDOCK rigid-body docking method and validated by yeast two-hybrid and ELISA. The study revealed a unique binding site, comprising of amino acids 1–30 at the N terminus of the nucleocapsid protein (N1) that is instrumental in its interactions with N, P, M, and G proteins. It was also observed that N2 associates with N and G proteins while N3 interacts with N, P, and M proteins.
This paper describes a rare finding of an inguinal ovary in an adult woman who presented with pelvic pain. Inguinal ovary may occur if the gubernaculum fails to attach to the uterus or if the canal of Nuck remains open during fetal development.
Electronic supplementary material
The online version of this article (doi:10.1186/2193-1801-2-545) contains supplementary material, which is available to authorized users.
Hippophae salicifolia D. Don (Seabuckthorn) grows in stressful environment of high altitude under conditions of low temperature and low availability of water. We have studied gender based differences in physiochemical response to cold stress in male and female plants of Seabuckthorn. After 24 h of cold stress about 32 and 66 % higher electrolyte leakage (EL) was recorded in male and female plants respectively. Relative water content (RWC) at the end of 24 h stress was higher in male plants (~64 %) compared to female plants (~60 %). Proline content in leaf samples of cold stressed male and female plants also increased upon cold stress. After 24 h about 2.7 fold higher amount of proline was assessed in male and female in comparison to control plants. Similarly, about two fold increase in the specific activities of superoxide dismutase (SOD) and catalase was also observed upon cold stress in male and female plants. These findings have important inferences for community of molecular biologists exploring seabuckthorn genome for agronomically important genes.
Seabuckthorn; Hippophae salicifolia; Cold stress; Dioecy; Physiochemical response
Photothermal ablation (PTA) is an emerging technique that uses near-infrared laser light-generated heat to destroy tumor cells. However, complete tumor eradication by PTA therapy alone is difficult because heterogeneous heat distribution can lead to sub-lethal thermal dose in some areas of the tumor. Successful PTA therapy requires selective delivery of photothermal conducting nanoparticles to mediate effective PTA of tumor cells, and the ability to combine PTA with other therapy modalities. Here, we synthesized multifunctional doxorubicin (DOX)-loaded hollow gold nanospheres (DOX@HAuNS) that target EphB4, a member of the Eph family of receptor tyrosine kinases overexpressed on the cell membrane of multiple tumors and angiogenic blood vessels. Increased uptake of targeted nanoparticles T-DOX@HAuNS was observed in three EphB4-positive tumors both in vitro and in vivo. In vivo release of DOX from DOX@HAuNS, triggered by near-infrared laser, was confirmed by dual radiotracer technique. Treatment with T-DOX@HAuNS followed by near-infrared laser irradiation resulted in significantly decreased tumor growth when compared to treatments with non-targeted DOX@HAuNS plus laser or HAuNS plus laser. The tumors in six of the eight mice treated with T-DOX@HAuNS plus laser regressed completely with only residual scar tissue by 22 days following injection, and none of the treatment groups experienced a loss in body weight. Together, our findings demonstrate that concerted chemo-photothermal therapy with a single nanodevice capable of mediating simultaneous PTA and local drug release may have promise as a new anticancer therapy.
Hollow Gold Nanospheres; EphB4 receptors; Targeting; Doxorubicin; Multimodal Therapy
Histone modifications occur in precise patterns, with several modifications known to affect the binding of proteins. These interactions affect the chromatin structure, gene regulation, and cell cycle events. The dual modifications on the H3 tail, serine10 phosphorylation, and lysine14 acetylation (H3Ser10PLys14Ac) are reported to be crucial for interaction with 14-3-3ζ. However, the mechanism by which H3Ser10P along with neighboring site-specific acetylation(s) is targeted by its regulatory proteins, including kinase and phosphatase, is not fully understood. We carried out molecular modeling studies to understand the interaction of 14-3-3ζ, and its regulatory proteins, mitogen-activated protein kinase phosphatase-1 (MKP1), and mitogen- and stress-activated protein kinase-1 (MSK1) with phosphorylated H3Ser10 alone or in combination with acetylated H3Lys9 and Lys14. In silico molecular association studies suggested that acetylated Lys14 and phosphorylated Ser10 of H3 shows the highest binding affinity towards 14-3-3ζ. In addition, acetylation of H3Lys9 along with Ser10PLys14Ac favors the interaction of the phosphatase, MKP1, for dephosphorylation of H3Ser10P. Further, MAP kinase, MSK1 phosphorylates the unmodified H3Ser10 containing N-terminal tail with maximum affinity compared to the N-terminal tail with H3Lys9AcLys14Ac. The data clearly suggest that opposing enzymatic activity of MSK1 and MKP1 corroborates with non-acetylated and acetylated, H3Lys9Lys14, respectively. Our in silico data highlights that site-specific phosphorylation (H3Ser10P) and acetylation (H3Lys9 and H3Lys14) of H3 are essential for the interaction with their regulatory proteins (MKP1, MSK1, and 14-3-3ζ) and plays a major role in the regulation of chromatin structure.
modeling; histone H3 modifications; 14-3-3ζ; MSK1; MKP1
Activation of nuclear factor-kappaB (NF-κB) has been implicated in chronic inflammatory disorders and is a key regulator of genes involved in response to infection, inflammation and stress. Interstitial cystitis and painful bladder syndrome are common inflammatory disorders of the urinary bladder characterized by frequent urination and bladder pain. The role of NF-κB activation in bladder inflammation has not been well defined.
Material and Methods
Female transgenic NF-κB-Luciferase (Luc) Tag mice were used to perform serial, noninvasive in vivo and ex vivo molecular imaging of NF-κB activation in the whole body after administration of arsenic trioxide (5mg/kg), lipopolysaccharide (2mg/kg), or cyclophosphamide (200mg/kg) to initiate acute transient inflammation in the bladder. Pretreatment with dexamethasone (10mg/kg) was used to modulate the cyclophosphamide-induced NF-κB-dependent luminescence in vivo.
Treatment of NF-κB-Luc mice with chemicals increased luminescence in a time- and organ- specific manner both in vivo and ex vivo. The highest levels of bladder NF-κB-dependent luminescence were observed 4 h after cyclophosphamide administration. Pretreatment with dexamethasone 1 h before injection of cyclophosphamide significantly down-regulated cyclophosphamide-induce bladder NF-κB-dependent luminescence, ameliorated the grossly evident pathologic features of acute inflammation, and diminished cellular immunostaining for NF-κB in the bladder.
NF-κB activity may play an important role in the patho-physiology of bladder inflammation and NF-κB-Luc mice can serve as a useful model for screening potential candidate drugs for treatment of cystitis associated with aberrant NF-κB activity. Such screening may significantly aid the development of therapeutic strategies to manage inflammatory disorders in the urinary bladder.
urinary bladder; cystitis; NF-kappaB; molecular imaging; inflammation
The calcium (Ca2+) transporters, like Ca2+ channels, Ca2+ ATPases, and Ca2+ exchangers, are instrumental for signaling and transport. However, the mechanism by which they orchestrate the accumulation of Ca2+ in grain filling has not yet been investigated. Hence the present study was designed to identify the potential calcium transporter genes that may be responsible for the spatial accumulation of calcium during grain filling. In silico expression analyses were performed to identify Ca2+ transporters that predominantly express during the different developmental stages of Oryza sativa. A total of 13 unique calcium transporters (7 from massively parallel signature sequencing [MPSS] data analysis, and 9 from microarray analysis) were identified. Analysis of variance (ANOVA) revealed differential expression of the transporters across tissues, and principal component analysis (PCA) exhibited their seed-specific distinctive expression profile. Interestingly, Ca2+ exchanger genes are highly expressed in the initial stages, whereas some Ca2+ ATPase genes are highly expressed throughout seed development. Furthermore, analysis of the cis-elements located in the promoter region of the subset of 13 genes suggested that Dof proteins play essential roles in regulating the expression of Ca2+ transporter genes during rice seed development. Based on these results, we developed a hypothetical model explaining the transport and tissue specific distribution of calcium in developing cereal seeds. The model may be extrapolated to understand the mechanism behind the exceptionally high level of calcium accumulation seen in grains like finger millet.
Protection of cells from oxidative insult may be possible through direct scavenging of reactive oxygen species, or through stimulation of intracellular antioxidant defense mechanisms by induction of antioxidant gene expression. In this study we investigated the cytoprotective effect of chamomile and elucidated the underlying mechanisms.
The cytoprotective effect of chamomile was examined on H2O2-induced cellular stress in RAW 264.7 murine macrophages.
RAW 264.7 murine macrophages treated with chamomile were protected from cell death caused by H2O2. Treatment with 50 μM H2O2 for 6 h caused significant increase in cellular stress accompanied by cell death in RAW 264.7 macrophages. Pretreatment with chamomile at 10-20 μg/mL for 16 h followed by H2O2 treatment protected the macrophages against cell death. Chamomile exposure significantly increased the expression of antioxidant enzymes viz. heme oxygenase-1 (HO-1), peroxiredoxin-1 (Prx-1), and thioredoxin-1 (Trx-1) in a dose-dependent manner, compared with their respective controls. Chamomile increased nuclear translocation of Nrf2 with increased phosphorylated Nrf2 levels, and binding to the antioxidant response element in the nucleus.
These molecular findings for the first time provide insights into the mechanisms underlying the induction of phase 2 enzymes through the Keap1-Nrf2 signaling pathway by chamomile, and provide evidence that chamomile possesses antioxidant and cytoprotective properties.
chamomile; macrophages; phase II enzymes; oxidative stress; antioxidant defense; inflammation
To evaluate the importance of morphology in quantifying expression after in vivo gene transfer and to compare gene expression after intra-arterial (IA) and intra-tumoral (IT) delivery of adenovirus expressing a SSTR2-based reporter gene in a large animal tumor model.
Materials and Methods
Tumor directed IA or IT delivery of adenovirus containing a human somatostatin receptor type 2A (Ad-CMV-HA-SSTR2A) gene chimera or control adenovirus (Ad-CMV-GFP) was performed in VX2 tumors growing in both rabbit thighs. Three days later, 111In-octreotide was administered intravenously after CT imaging using a clinical scanner. 111In-octreotide uptake in tumors was evaluated the following day using a clinical gamma-camera. Gene expression was normalized to tumor weight with and without necrosis. This procedure was repeated on nine additional rabbits to investigate longitudinal gene expression both 5 days and 2 weeks after adenovirus delivery. CT images were used to evaluate tumor morphology and excised tissue samples were analyzed to determine 111In-octreotide biodistribution ex vivo.
VX2 tumors infected with Ad-CMV-HA-SSTR2 had greater 111In-octreotide uptake than with control virus (P<0.05). Intra-arterial and intra-tumoral routes resulted in similar levels of gene expression. Longitudinally, expression appeared to wane at 2 weeks versus 5 days after delivery. Areas of necrosis did not demonstrate significant uptake ex vivo. Morphology identified areas of necrosis on contrast enhanced CT and upon excluding necrosis, in vivo biodistribution analysis resulted in greater percent injected dose per gram (P<0.01) and corresponded better with ex vivo biodistribution(r = 0.72, P<0.01, Coefficient of the x-variable = .72) at 2 weeks than without excluding necrosis (P<0.01).
Tumor specificity and high transgene expression can be achieved in tumors via both tumor directed intra-arterial and intra-tumoral delivery in a large animal tumor model. Using clinical machines, morphologic imaging contributes to functional imaging for quantifying SSTR2-based reporter expression in vivo.
Background & objectives:
HIV/AIDS patients may have renal involvement also, however, Indian data are sparse. The present study was done to find the spectrum of renal diseases in HIV/AIDS patients in north India.
In this prospective pilot study, HIV positive patients aged >18 yr were screened for renal involvement [serum creatinine >1.5 mg% and/or significant proteinuria (>500 mg /day)]. Patients who were positive on screening were followed up prospectively and underwent kidney biopsy if indicated.
A total of 526 patients were screened, of these, 91 (17.3%) were found to have renal involvement. Group A (Treatment naïve) comprised 392 patients who were not on antiretroviral treatment (ART) and group B (patients on ART) comprised 134 patients. More patients (74/392, 18.9%) in group A had renal involvement as compared to patients in group B (17/134, 12.7%). Of the 91 patients with renal involvement, 26 were followed up and underwent kidney biopsy. Thirteen patients had only proteinuria and another 13 had renal dysfunction with or without proteinuria. Most common histological diagnosis was mesangioproliferative glomerulonephritis (mes PGN) (10/26). Two patients had collapsing FSGS (focal segmental glomerulosclerosis) and three patients had immune complex glomerulonephritis. Seven patients had acute kidney injury, whom six totally recovered from their renal function. All patients with mesPGN tolerated angiotensin converting enzyme (ACE) inhibitors well. There was mixed response of collapsing FSGS to steroids. Both patients with MPGN (membranoproliferative glomerulonephritis) did well on low dose of steroid and ART.
Interpretation & conclusions:
Renal involvement was found to be common in HIV positive patients (17.3%). A low occurrence of renal involvement found in patients already on ART suggests some renoprotective effect of ART. Our preliminary results showed that collapsing FSGS was not rare in Indian HIV positive population, but classical HIV associated nephropathy was not seen. Longitudinal studies with robust study design and large sample size need to be done to confirm the findings.
ART; human immunodeficiency virus (HIV); nephropathy; renal disease
Among patients with Alzheimer’s disease who have had a response to antipsychotic medication for psychosis or agitation–aggression, the risk of a recurrence of symptoms after discontinuation of the medication has not been established.
Patients with Alzheimer’s disease and psychosis or agitation–aggression received open-label treatment with risperidone for 16 weeks. Those who had a response to risperidone therapy were then randomly assigned, in a double-blind fashion, to one of three regimens: continued risperidone therapy for 32 weeks (group 1), risperidone therapy for 16 weeks followed by placebo for 16 weeks (group 2), or placebo for 32 weeks (group 3). The primary outcome was the time to relapse of psychosis or agitation.
A total of 180 patients received open-label risperidone (mean dose, 0.97 mg daily). The severity of psychosis and agitation were reduced, although there was a mild increase in extrapyramidal signs; 112 patients met the criteria for response to treatment, of whom 110 underwent randomization. In the first 16 weeks after randomization, the rate of relapse was higher in the group that received placebo than in the groups that received risperidone (60% [24 of 40 patients in group 3] vs. 33% [23 of 70 in groups 1 and 2]; P = 0.004; hazard ratio with placebo, 1.94; 95% confidence interval [CI], 1.09 to 3.45; P = 0.02). During the next 16 weeks, the rate of relapse was higher in the group that was switched from risperidone to placebo than in the group that continued to receive risperidone (48% [13 of 27 patients in group 2] vs. 15% [2 of 13 in group 1]; P = 0.02; hazard ratio, 4.88; 95% CI, 1.08 to 21.98; P = 0.02). The rates of adverse events and death after randomization did not differ significantly among the groups, although comparisons were based on small numbers of patients, especially during the final 16 weeks.
In patients with Alzheimer’s disease who had psychosis or agitation that had responded to risperidone therapy for 4 to 8 months, discontinuation of risperidone was associated with an increased risk of relapse.
Research studies on the effects of discontinuing antipsychotic medications in patients with dementia have not identified specific target symptoms or response to antipsychotics prior to discontinuation. The Antipsychotic Discontinuation in Alzheimer Disease (ADAD) trial addresses these issues in a randomized, double-blind, placebo-controlled, multicenter risperidone treatment and discontinuation trial. In Phase A, AD patients with psychosis or agitation receive open treatment with risperidone for 16 weeks. Responders are randomized, double-blind, to one of three arms in Phase B: 1) continuation risperidone for the next 32 weeks, 2) risperidone for the next 16 weeks followed by placebo for 16 weeks, or 3) placebo for the next 32 weeks.
Several design features provide unique strengths to this trial: identification of target symptoms and systematic open antipsychotic treatment with only responders randomized in the discontinuation trial, use of a single antipsychotic medication, two clinically relevant time-points for discontinuation to evaluate the impact of duration of treatment on relapse, exclusion of patients at increased risk of stroke, assessment of several affected symptom domains, and state-of-the-art approaches to assess relapse and handle dropout.
This study will provide clinically relevant data on the likelihood and time to relapse, and predictors of relapse, in patients switched from risperidone to placebo after response to risperidone treatment. Given the warnings about antipsychotic use in patients with dementia, studies of this type are essential to determine the optimal duration of treatment that confers the greatest benefit to risk ratio and to improve evidence-based treatment strategies.
agitation; Alzheimer disease; antipsychotic; discontinuation trial; psychosis; risperidone
Identification of ethylene-regulated and ripening-related genes from banana (Musa acuminata Var. Harichaal) fruits using DDRT-PCR led to the isolation of differentially expressed partial cDNA of pectin methylesterase inhibitor (MaPMEI) gene. Its full-length cDNA sequence consisted of a 567 bp ORF, encoding a protein of 189 aa with deduced molecular mass 19.6 kDa. Expression pattern of MaPMEI gene revealed that upon ethylene treatment, this gene is up-regulated initially giving maximum expression in post-climacteric stage then decreases slightly in later stages of ripening. 1-MCP, a known ethylene perception inhibitor, inhibits both fruit ripening as well as the transcript level of this gene. Also, the transcripts of MaPMEI gene were not detected during the short time ethylene treatment suggesting this gene appears to be not directly induced by ethylene. Interestingly, MaPMEI gene showed fruit specific expression that indicates its possible role in the regulations of PMEs in fruits. In silico analysis revealed a predicted signal peptide sequence necessary for localization of MaPMEI in the cell wall. Furthermore, the four Cys residues involved in disulfide bridges are conserved in MaPMEI similar to other PMEIs and invertase inhibitors. Phylogenetic analysis further suggests that the MaPMEI identified in this study is more closely related to PMEIs than to invertase inhibitors.
Banana; Fruit ripening; Pectin methylesterase inhibitor; Ethylene responsive expression; DDRT-PCR
In India, there are a large number of end-stage renal disease (ESRD) patients waiting for renal transplant. Deceased donor organ transplantation (DDOT) is the possible solution to bridge the disparity between organ supply and demand. The concept of expanded criteria donors (ECDs) was developed to combat the huge discrepancy between demand and organ availability. However, ECD kidneys have a higher propensity for delayed graft function (DGF), and therefore worse long-term survival. We present our experience of deceased donor renal transplantation.
We report single centre experience on DDOT including ECDs vis-à-vis patient/graft survival, graft function in terms of serum creatinine (SCr), rejection episodes, and delayed graft function in 44 DDOT
Materials and Methods:
Between August 1998 and April 2011, 44 renal transplants from 35 deceased donors were performed, of which 37.2% were expanded criteria donors. Results were analyzed in terms of age of donor, terminal SCr, graft ischemia time, graft function, post-transplant complications, and graft and patient survival. All recipients received sequential triple drug immunosuppression and induction with rabbit antithymocyte globulin (rATG). The induction is commenced by giving first dose of rATG intraoperatively (dose 1.5 mg/kg) and subsequent rATG infusions were administered daily for a minimum of 5 and maximum of 7 doses depending on initial graft function.
We have been able to achieve a mean cold ischemia time of 6.25 ± 2.55 h due to the coordinated team efforts. Delayed graft function occurred in 34% patients and 31.8% had prolonged drainage. There were no urinary leaks. Seven (16%) patients had biopsy-proven rejection episodes, all of which were reversed with treatment. Two patients underwent graft nephrectomy. One of these was due to hyperacute rejection and another due to anastomotic hemorrhage. One-year graft survival was 92.4% and the patient survival was 83.8%.
Deceased donor renal transplants have satisfactory graft function and patient survival despite the high incidence of delayed graft function. Retrieving kidneys from marginal donors can add to the donor pool.
Cold ischemia time; deceased donor; delayed graft function; end-stage renal disease
Kartagener’s syndrome is a very rare congenital malformation comprising of a classic triad of sinusitis, situs inversus and bronchiectasis. Primary ciliary dyskinesia is a genetic disorder with manifestations present from early life and this distinguishes it from acquired mucociliary disorders. Approximately one half of patients with primary ciliary dyskinesia have situs inversus and, thus are having Kartagener syndrome. We present a case of 12 year old boy with sinusitis, situs inversus and bronchiectasis. The correct diagnosis of this rare congenital autosomal recessive disorder in early life is important in the overall prognosis of the syndrome, as many of the complications can be prevented if timely management is instituted, as was done in this in this case.
Bronchiectasis; primary ciliary dyskinesia; Kartagener’s syndrome; sinusitis; situs inversus
Our containment trials have established cold tolerance in Nicotiana tabacum osmotin (Nt Osm) transgenic tomato (Solanum lycopersicum L. cv. Pusa Ruby). Though, the stress tolerance mechanisms have been studied at physio-biochemical levels, molecular mechanisms underlying the tolerant response are still not well studied. Therefore, quantitative transcript expression of Osmotin and other stress responsive genes (CBF1, P5CS and APX) was studied in response to cold (4°C; 2 and 24 h) treatment in the transgenic and wild type tomato plants. The expression analysis revealed differential transcript regulation in the transgenic and wild type plants on the cold exposure. In general, the genes were either earlier induced or the extent of fold change in transcript expression over the respective untreated controls was higher in transgenic than in the wild type plants on cold exposure. The transcript expression data also supported the metabolite analysis on free Proline and ascorbate content. The results thus suggest that constitutive over expression of the Osmotin modulate transcript abundance and functional expression products of the other stress responsive genes thereby, imparting cold tolerance in the transgenic tomato plants.
Electronic supplementary material
The online version of this article (doi:10.1186/2193-1801-2-117) contains supplementary material, which is available to authorized users.
Osmotin; Transgenic tomato; qRT PCR; Transcript expression; Free Proline; Ascorbate; Cold stress
We sought to determine whether PI3K pathway mutation or activation state and rapamycin-induced feedback-loop activation of Akt is associated with rapamycin sensitivity or resistance.
Cancer cell lines were tested for rapamycin-sensitivity, Akt phosphorylation and mTOR target inhibition. Mice injected with breast or neuroendocrine cancer cells and patients with neuroendocrine tumor (NET) were treated with rapalogs, and Akt phosphorylation was assessed.
31 cell lines were rapamycin-sensitive (RS) and 12 were relatively rapamycin-resistant (RR; IC50>100 nM). Cells with PIK3CA and/or PTEN mutations were more likely to be RS (p=0.0123). Akt phosphorylation (S473 and T308) was significantly higher in RS cells (p<0.0001). Rapamycin led to a significantly greater pathway inhibition and greater increase in p-Akt T308 (p<0.0001) and p-Akt S473 (p=0.0009) in RS cells. Rapamycin and everolimus significantly increased Akt phosphorylation but inhibited growth in an in vivo NET model (BON). In patients with NETs treated with everolimus and octreotide, progression-free survival correlated with p-Akt T308 in pretreatment (R=0.4762, p=0.0533) and on-treatment tumor biopsies (R=0.6041, p=0.0102). Patients who had a documented partial response were more likely to have an increase in p-Akt T308 with treatment compared to non-responders (p=0.0146).
PIK3CA/PTEN genomic aberrations and high p-Akt levels are associated with rapamycin sensitivity in vitro. Rapamycin-mediated Akt activation is greater in RS cells, with a similar observation in patients with clinical responses on exploratory biomarker analysis; thus feedback-loop activation of Akt is not a marker of resistance but rather may function as an indicator of rapamycin activity.
Akt; mTOR; rapamycin sensitivity; RPPA; everolimus; pharmacodynamic markers
Acetylation of the tumor suppressor gene p53 at the carboxy-terminal lysine (Lys) residues enhances its transcriptional activity associated with cell cycle arrest and apoptosis. Histone deacetylases (HDACs), a family of evolutionarily conserved enzymes, counterbalance the acetylation of lysine residues on histone and non-histone proteins. In this study, we demonstrate that green tea polyphenols (GTPs) and their major constituent, (−) epigallocatechin-3-gallate (EGCG), activate p53 through acetylation at the Lys373 and Lys382 residues by inhibiting class I HDACs in LNCaP human prostate cancer cells. Treatment of cells with GTPs (2.5–10 µg/ml) and EGCG (5–20 µM) resulted in dose- and time-dependent inhibition of class I HDACs (HDAC1, 2, 3 and 8), albeit at varying levels. Discontinuation of treatment with GTP/EGCG resulted in the loss of p53 acetylation at both the sites in these cells. GTP/EGCG treatment also resulted in increased expression of p21/waf1 and Bax at the protein and message levels in these cells. The increased GTP/EGCG-mediated p53 acetylation enhanced its binding on the promoters of p21/waf1 and Bax, which was associated with increased accumulation of cells in the G0/G1 phase of the cell cycle and induction of apoptosis. Our findings indicate that GTP/EGCG causes acetylation of p53 by inhibiting class I HDACs, a function that is likely to be part of the mechanisms that control the physiological activity of p53.
prostate cancer; green tea polyphenols; epigallocatechin-3-gallate; p53 acetylation; histone deacetylases
Signal transducer and activator of transcription (Stat)-3 and nuclear factor-kappa B (NF-κB) are important signaling pathways constitutively activated during inflammation. We previously reported that high-fat diet (HFD) intake induces oxidative stress in the prostate through elevated expression of NADPH oxidase subunits causing NF-κB activation. We sought to determine whether Stat-3 is involved in the activation of NF-κB in the prostate as a result of HFD feeding, leading to inflammation.
C57BL/6 mice were either fed with regular diet (RD) or HFD for 4 and 8 weeks. Plasma cytokine levels were determined by multiplex analysis. Western blotting was performed to determine the expression of NF-κB, Stat-3, Akt, PDK1, PKCε and their phosphorylated forms along with pathologic evaluation of the prostate. Immunoprecipitation and electrophoretic mobility shift assay were conducted to study the association between Stat-3 and NF-κB.
C57BL/6 mice fed with HFD showed a significant increase in the plasma levels of IL-1β, IL-6, IL-17 and TNFα after 4 and 8 weeks of feeding, compared with RD controls. HFD feeding elevated the intraprostatic expression of IL-6 and caused activation of PKCε and Akt, the upstream kinase regulating Stat-3 and NF-κB. Nuclear extracts from the prostates of mice fed with HFD exhibited constitutively activated levels of Stat-3 and NF-κB/p65. Increased association between the activated forms of Stat-3 and NF-κB/p65 was observed in the nucleus as a result of HFD feeding, a finding that was accompanied by morphologic evidence of increased intraprostatic inflammation.
Our findings suggest that HFD activates Stat-3 and NF-κB/p65 in the prostate, and their interaction is associated with increased inflammation in the prostate.
inflammation; high-fat diet; prostate; Stat-3; NF-κB
Green tea polyphenols (GTPs) reactivate epigenetically silenced genes in cancer cells and trigger cell cycle arrest and apoptosis; however, the mechanisms whereby these effects occur are not well understood. We investigated the molecular mechanisms underlying the antiproliferative effects of GTP, which may be similar to those of histone deacetylase (HDAC) inhibitors. Exposure of human prostate cancer LNCaP cells (harboring wild-type p53) and PC-3 cells (lacking p53) with 10–80 μg/ml of GTP for 24 h resulted in dose-dependent inhibition of class I HDAC enzyme activity and its protein expression. GTP treatment causes an accumulation of acetylated histone H3 in total cellular chromatin, resulting in increased accessibility to bind with the promoter sequences of p21/waf1 and Bax, consistent with the effects elicited by an HDAC inhibitor, trichostatin A. GTP treatment also resulted in increased expression of p21/waf1 and Bax at the protein and message levels in these cells. Furthermore, treatment of cells with proteasome inhibitor, MG132 together with GTP prevented degradation of class I HDACs, compared with cells treated with GTP alone, indicating increased proteasomal degradation of class I HDACs by GTP. These alterations were consistent with G0–G1 phase cell cycle arrest and induction of apoptosis in both cell lines. Our findings provide new insight into the mechanisms of GTP action in human prostate cancer cells irrespective of their p53 status and suggest a novel approach to prevention and/or therapy of prostate cancer achieved via HDAC inhibition.
Despite advances in diagnosis, surgery, and antimicrobial therapy, mortality rates associated with complicated intra-abdominal infections remain exceedingly high.
The 2013 update of the World Society of Emergency Surgery (WSES) guidelines for the management of intra-abdominal infections contains evidence-based recommendations for management of patients with intra-abdominal infections.