Cajanus platycarpus Flavonoid 3'5' Hydroxylase_2 (CpF3'5'H_2) Confers Resistance to Helicoverpa armigera by Modulating Total Polyphenols and Flavonoids in Transgenic Tobacco.

Pod borer Helicoverpa armigera, a polyphagus herbivorous pest, tremendously incurs crop damage in economically important crops. This necessitates the identification and utility of novel genes for the control of the herbivore. The present study deals with the characterization of a flavonoid 3'5' hydroxylase_2 (F3'5'H_2) from a pigeonpea wild relative Cajanus platycarpus, possessing a robust chemical resistance response to H. armigera. Though F3'5'H_2 displayed a dynamic expression pattern in both C. platycarpus (Cp) and the cultivated pigeonpea, Cajanus cajan (Cc) during continued herbivory, CpF3'5'H_2 showed a 4.6-fold increase vis a vis 3-fold in CcF3'5'H_2. Despite similar gene copy numbers in the two Cajanus spp., interesting genic and promoter sequence changes highlighted the stress responsiveness of CpF3'5'H_2. The relevance of CpF3'5'H_2 in H. armigera resistance was further validated in CpF3'5'H_2-overexpressed transgenic tobacco based on reduced leaf damage and increased larval mortality through an in vitro bioassay. As exciting maiden clues, CpF3'5'H_2 deterred herbivory in transgenic tobacco by increasing total flavonoids, polyphenols and reactive oxygen species (ROS) scavenging capacity. To the best of our knowledge, this is a maiden attempt ascertaining the role of F3'5'H_2 gene in the management of H. armigera. These interesting leads suggest the potential of this pivotal branch-point gene in biotic stress management programs.

The plant specialized metabolite epicatechin- 3-gallate (EC3G) perturbs lipid metabolism and attenuates fat accumulation in pigeonpea pod borer, Helicoverpa armigera.

Control of pod borer Helicoverpa armigera, a notorious polyphagous pest requires paramount attention with focus on environment-friendly management approaches. Overproduction of catechins (epigallocatechin-EGC and epicatechin-3-gallate-EC3G) in the pod borer-resistant pigeonpea wild relative, Cajanus platycarpus during continued herbivory prodded us to assess their underlying molecular effect on H. armigera. Significant reduction in larval and pupal growth parameters was observed when reared on artificial diet incorporated with 100 ppm EC3G vis a vis 100 ppm EGC and EGC + EC3G. Comparative RNAseq analyses of larvae that fed on normal and EC3G-incorporated diet revealed 62 differentially expressed genes dominated by detoxification and lipid metabolism. While lipase and fatty acid-binding protein 2-like were up-regulated, delta9-FADS-like involved in fatty acid synthesis was downregulated, indicating effect of EC3G on fat metabolism. Validation of RNAseq data by qPCR; midgut glutathione-S-transferase and esterase assays depicted increased lipolysis and reduced lipogenesis in EC3G-fed larvae. Additionally, differential accumulation of stearic acid and oleic acid in EC3G-fed and control larvae/adults ascertained perturbation in lipogenesis. Supported by modelling, molecular docking and simulations, we demonstrate the possible involvement of the insect adipokinetic hormone receptor (AKHR) in the EC3G-mediated response. The study demonstrates plant specialized metabolite EC3G as a potential candidate for H. armigera control.

Bedside Chest Ultrasound in Postoperative Pediatric Cardiac Surgery Patients: Comparison With Bedside Chest Radiography.

OBJECTIVE: The primary objective was to study the degree of agreement between the chest ultrasound (CUS) studies and chest x-ray (CXR) studies in postoperative pediatric cardiac surgical patients regarding the diagnosis of thoracic abnormalities, and also to compare the diagnostic performance of CUS in reference to CXR for the detection of thoracic abnormalities. The secondary objective was to compare the necessity for interventions done on the basis of CUS and CXR findings in the postoperative setting. DESIGN: A prospective observational study. SETTING: At a postoperative pediatric cardiac surgical intensive care unit in a tertiary-care center. PARTICIPANTS: One hundred sixty patients between the age of 2 months to 18 years undergoing elective cardiac surgery for various congenital heart diseases. INTERVENTIONS: After obtaining permission from the institutional ethics committee, 160 pediatric cardiac surgical patients were studied prospectively in the postoperative period. On the day of surgery (postoperative day [POD] 0), bedside CXR was done in the immediate postoperative period. After bedside CXR, CUS examination was performed and then interpreted by the principal investigator. The CXR was interpreted by the surgical team. Provisional diagnosis was made by the principal investigator and surgical team. Any intervention required was decided based on CXR or CUS findings or both. The procedure was repeated in the morning of POD 1. MEASUREMENTS AND MAIN RESULTS: The degree of agreement between CUS studies and CXR studies in detecting abnormalities was evaluated by Cohen's kappa (k) statistics. The diagnostic performance of CUS was compared with that of CXR using sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy. Overall, kappa analysis (k) showed substantial agreement between the findings of the CUS and CXR studies (k = 0.749). The diagnostic performance of CUS, as compared with CXR, was found to have a sensitivity of 96.9%, specificity of 84.75%, PPV of 73.4%, NPV of 98.43%, and diagnostic accuracy of 88.44%. In 94 abnormal findings, the interventions were done based on CUS or CXR findings or both. Overall, there was a substantial agreement (k = 0.787) between CUS and CXR regarding the necessity for interventions. CONCLUSIONS: The degree of agreement between CUS and CXR studies was substantial for atelectasis, interstitial edema, and diaphragmatic weakness. The degree of agreement between CUS and CXR studies was almost perfect for pneumothorax and fair for pleural effusion. More CUS studies detected intrathoracic pathologies than CXR studies. The CUS also detected abnormalities earlier than CXR and was found to be useful for the early institution of intervention therapy in patients with interstitial edema and atelectasis. It would be reasonable to conclude that CUS may be considered in some instances as an alternative to CXR.

Recovery of Latent HIV-1 from Brain Tissue by Adoptive Cell Transfer in Virally Suppressed Humanized Mice.

Defining the latent human immunodeficiency virus type 1 (HIV-1) burden in the human brain during progressive infection is limited by sample access. Human hematopoietic stem cells (hu-HSCs)-reconstituted humanized mice provide an opportunity for this study. The model mimics, in measure, HIV-1 pathophysiology, transmission, treatment, and elimination in an infected human host. However, to date, brain HIV-1 latency in hu-HSC mice during suppressive antiretroviral therapy (ART) was not studied. To address this need, hu-HSC mice were administered long acting (LA) ART 14 days after HIV-1 infection was established. Animals were maintained under suppressive ART for 3 months, at which time HIV-1 infection was detected at low levels in brain tissue by droplet digital polymerase chain reaction (ddPCR) test on DNA. Notably, adoptive transfer of cells acquired from the hu-HSC mouse brains and placed into naive hu-HSC mice demonstrated viral recovery. These proof-of-concept results demonstrate replication-competent HIV-1 reservoir can be established in hu-HSC mouse brains that persists during long-term ART treatment. Hu-HSC mice-based mouse viral outgrowth assay (hu-MVOA) serves as a sensitive tool to interrogate latent HIV-1 brain reservoirs.

Humanized Mice for Infectious and Neurodegenerative disorders.

Humanized mice model human disease and as such are used commonly for research studies of infectious, degenerative and cancer disorders. Recent models also reflect hematopoiesis, natural immunity, neurobiology, and molecular pathways that influence disease pathobiology. A spectrum of immunodeficient mouse strains permit long-lived human progenitor cell engraftments. The presence of both innate and adaptive immunity enables high levels of human hematolymphoid reconstitution with cell susceptibility to a broad range of microbial infections. These mice also facilitate investigations of human pathobiology, natural disease processes and therapeutic efficacy in a broad spectrum of human disorders. However, a bridge between humans and mice requires a complete understanding of pathogen dose, co-morbidities, disease progression, environment, and genetics which can be mirrored in these mice. These must be considered for understanding of microbial susceptibility, prevention, and disease progression. With known common limitations for access to human tissues, evaluation of metabolic and physiological changes and limitations in large animal numbers, studies in mice prove important in planning human clinical trials. To these ends, this review serves to outline how humanized mice can be used in viral and pharmacologic research emphasizing both current and future studies of viral and neurodegenerative diseases. In all, humanized mouse provides cost-effective, high throughput studies of infection or degeneration in natural pathogen host cells, and the ability to test transmission and eradication of disease.

Ex-foliar application of glycine betaine and its impact on protein, carbohydrates and induction of ROS scavenging system during drought stress in flax (Linum usitatissimum).

Crop plants have an innate capacity to acclimatize and survive myriad stresses in field conditions. This acclimatization to stress enhances crop stand in field and productivity of plant. Inter alia field crops withstand drought stress (hydropenia) by inducing synthesis or accumulation of osmolytes such as (i) proline and other amino acids, (ii) glycine betaine (GB), (iii) soluble carbohydrates, and (iv) reactive oxygen species (ROS) scavenging system as intrinsic drought antagonizing molecules. Precise in vivo induction of osmolytes and their effect on ROS scavenging system in flax/linseed has not been elucidated. The investigation was carried out to identify a tolerant and susceptible cultivar of flax from a core collection of 53 core accessions and evaluate the role of compatible osmolytes in Linum usitatissimum under hydropenia. We screened eight morphometrically diverse flax genotypes in field under irrigated and un-irrigated condition and classified them as tolerant and susceptible genotypes. Further, we examined the effect of ex-foliar glycine betaine application - a signature molecule involved in drought tolerance, on selected tolerant and susceptible varieties. Our results showed stimulatory impact of glycine betaine on accumulation of ROS scavenging antioxidants, total soluble protein and on its own accumulation. While the ex-foliar application had no inhibitory effect on the growth of plants; accumulation of free proline, amino acids and carbohydrates are inhibited par se in flax. Our findings reveal, flax is a non-accumulator of glycine betaine and exogenous application of glycine betaine enhances its own levels during drought stress.

Adjuvant chemotherapy in uterine carcinosarcoma: Comparison of a doublet and a triplet chemotherapeutic regimen.

BACKGROUND: Uterine carcinosarcoma (UCS) is a rare and aggressive malignancy, and there are no existing standard guidelines for adjuvant therapy. Doublet chemotherapy regimens are most favored in adjuvant setting; however, given the early chances of distant recurrences, does a triple-drug adjuvant chemotherapy improve disease-free survival (DFS), remains to be seen. Our aim of the study is to compare and review different adjuvant regimens used in UCS. METHODS: Retrospective chart analysis included 37 optimally staged UCS patients. Each of them had either received paclitaxel plus carboplatin (PC) or paclitaxel, ifosfamide, and cisplatin (TIP). A toxicity analysis was charted as per common terminology criteria for adverse events (CTCAE) 4 criteria. A survival analysis was done by the Kaplan-Meier method, and log-rank test was used for comparison of two variables. RESULTS: Incidence of UCS was 4.1% and mean age (standard deviation) was 58.73 ± 6.3 (range 42 - 71) years. TIP and PC chemotherapies were given to 22 and 15 patients, respectively. Five-year DFS and overall survival for TIP versus PC were 38.2% versus 35.9% (P = 0.118) and 49% versus 50.3% (P = 0.306), respectively, and for Stage I, II versus Stage III was 78.8% versus 12.7%(P = 0.001) and 92.3% versus 34.2% (P = 0.002), respectively. However, in advanced disease (Stage III), there is a trend toward DFS advantage of triple-drug adjuvant regimen (Hazards ratio (HR) = 0.35, 95% confidence interval (CI) = 0.12-1.07). Grade 3 and 4 toxicities were seen in 54.5% patients of TIP chemotherapy group and in 13.3% patients of the PC chemotherapy (P = 0.012). CONCLUSION: Triple-drug adjuvant chemotherapy (TIP) confers no survival advantage over doublet chemotherapy (PC), and in turn, increases the grade 3/4 toxicity in the adjuvant setting of optimally staged UCS patients.

HIV-1-Associated Left Ventricular Cardiac Dysfunction in Humanized Mice.

The molecular cause(s) for early onset heart failure in people living with HIV-1 infection (PLWH) remains poorly defined. Herein, longitudinal echocardiography was used to assess whether NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice reconstituted with human hematopoietic stem cells (Hu-NSG mice) and infected with HIV-1ADA can recapitulate the salient features of this progressive human disease. Four weeks post infection, Hu-NSG mice of both sexes developed left ventricular (LV) diastolic dysfunction (DD), with 25% exhibiting grade III/IV restrictive DD with mitral regurgitation. Increases in global longitudinal and circumferential strains and declines in LV ejection fraction and fractional shortening were observed eight weeks post infection. After twelve weeks of infection, 33% of Hu-NSG mice exhibited LV dyskinesia and dyssynchrony. Histopathological analyses of hearts seventeen weeks post infection revealed coronary microvascular leakage, fibrosis and immune cell infiltration into the myocardium. These data show for the first time that HIV-1ADA-infected Hu-NSG mice can recapitulate key left ventricular cardiac deficits and pathophysiological changes reported in humans with progressive HIV-1 infection. The results also suggest that HIV-1 infected Hu-NSG mice may be a useful model to screen for pharmacological agents to blunt LV dysfunction and associated pathophysiologic causes reported in PLWH.

Amplification of Replication Competent HIV-1 by Adoptive Transfer of Human Cells From Infected Humanized Mice.

Detection of latent human immunodeficiency virus type 1 (HIV-1) in "putative" infectious reservoirs is required for determining treatment efficiency and for viral elimination strategies. Such tests require induction of replication competent provirus and quantitative testing of viral load for validation. Recently, humanized mice were employed in the development of such tests by employing a murine viral outgrowth assay (mVOA). Here blood cells were recovered from virus infected antiretroviral therapy suppressed patients. These cells were adoptively transferred to uninfected humanized mice where replication competent virus was recovered. Prior reports supported the notion that an mVOA assay provides greater sensitivity than cell culture-based quantitative VOA tests for detection of latent virus. In the current study, the mVOA assays was adapted using donor human hematopoietic stem cells-reconstituted mice to affirm research into HIV-1 elimination. We simulated an antiretroviral therapy (ART)-treated virus-infected human by maintaining the infected humanized mice under suppressive treatment. This was operative prior to human cell adoptive transfers. Replication-competent HIV-1 was easily detected in recipient animals from donors with undetectable virus in plasma. Moreover, when the assay was used to investigate viral presence in tissue reservoirs, quantitative endpoints were determined in "putative" viral reservoirs not possible in human sample analyses. We conclude that adoptive transfer of cells between humanized mice is a sensitive and specific assay system for detection of replication competent latent HIV-1.

Assessment of Pigeonpea (Cajanus cajan L.) transgenics expressing Bt ICPs, Cry2Aa and Cry1AcF under nethouse containment implicated an effective control against herbivory by Helicoverpa armigera (Hübner).

BACKGROUND: Pigeonpea is a source of quality proteins and the main constituent of a well-balanced diet for majority of Indian population. One of the major constraints in the production of pigeonpea is a polyphagous insect pest, Helicoverpa armigera. Non-availability of resistant sources in the germplasm and limitations in conventional breeding have been key factors for continued yield losses. Additionally, hazards of chemical fertilizers on the environment have prompted the scientific community to develop alternative strategies. Bacillus thuringiensis (Bt) insecticidal proteins (ICPs) have emerged as the most reliable source for the control of insect pests through transgenics. RESULTS: Transgenic pigeonpea plants harboring validated Bt ICPs, Cry2Aa and Cry1AcF were developed by a non-tissue culture based in planta transformation strategy and assessed for integration of Transfer-DNA (T-DNA) and efficacy against pod borer under in vitro conditions. For the first time this study demonstrates the successful evaluation of 19 transgenic pigeonpea events (11 with cry2Aa and 8 with cry1AcF) under soil and pot conditions in a nethouse containment. The stability in the performance was assessed stringently by deliberate H. armigera larval challenging. The trial identified ten promising events of both the genes that portrayed reduced damage to the herbivore. CONCLUSION: We present the first ever successful evaluation of pigeonpea transgenics with the ability to mitigate pod borer under nethouse conditions. The transgenics depicted molecular evidence for the stability of T-DNA integration, consistency in the expression of Cry proteins and resistance against H. armigera. These events can form a pool of useful transgenics to manage the devastating pod borer. © 2019 Society of Chemical Industry.

Creation of a long-acting rilpivirine prodrug nanoformulation.

Antiretroviral therapy requires lifelong daily dosing to attain viral suppression, restore immune function, and improve quality of life. As a treatment alternative, long-acting (LA) antiretrovirals can sustain therapeutic drug concentrations in blood for prolonged time periods. The success of recent clinical trials for LA parenteral cabotegravir and rilpivirine highlight the emergence of these new therapeutic options. Further optimization can improve dosing frequency, lower injection volumes, and facilitate drug-tissue distributions. To this end, we report the synthesis of a library of RPV prodrugs designed to sustain drug plasma concentrations and improved tissue biodistribution. The lead prodrug M3RPV was nanoformulated into the stable LA injectable NM3RPV. NM3RPV treatment led to RPV plasma concentrations above the protein-adjusted 90% inhibitory concentration for 25 weeks with substantial tissue depots after a single intramuscular injection in BALB/cJ mice. NM3RPV elicited 13- and 26-fold increases in the RPV apparent half-life and mean residence time compared to native drug formulation. Taken together, proof-of-concept is provided that nanoformulated RPV prodrugs can extend the apparent drug half-life and improve tissue biodistribution. These results warrant further development for human use.

Immune Activations and Viral Tissue Compartmentalization During Progressive HIV-1 Infection of Humanized Mice.

Human immunodeficiency virus type one (HIV-1) tissue compartments are established soon after viral infection. However, the timing in which virus gains a permanent foothold in tissue and the cellular factors that control early viral-immune events are incompletely understood. These are critical events in studies of HIV-1 pathogenesis and in the development of viral reservoirs after antiretroviral therapy. Moreover, factors affecting the permanence of viral-tissue interactions underlie barriers designed to eliminate HIV-1 infection. To this end we investigated the temporal and spatial viral and host factors during HIV-1 seeding of tissue compartments. Two humanized NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ mouse models were employed. In the first, immune deficient mice were reconstituted with human CD34+ cord blood hematopoietic stem cells (HSC) (hu-HSC) and in the second mice were transplanted with adult mature human peripheral lymphocytes (hu-PBL). Both, in measure, reflect relationships between immune activation and viral infection as seen in an infected human host. Following humanization both mice models were infected with HIV-1ADA at 104 50% tissue culture infective doses. Viral nucleic acids and protein and immune cell profiles were assayed in brain, lung, spleen, liver, kidney, lymph nodes, bone marrow, and gut from 3 to 42 days. Peripheral CD4+ T cell loss began at 3 days together with detection of HIV-1 RNA in both mouse models after initiation of HIV-1 infection. HIV-1 was observed in all tested tissues at days 3 and 14 in hu- PBL and HSC mice, respectively. Immune impairment was most prominent in hu-PBL mice. T cell maturation and inflammation factors were linked directly to viral tissue seeding in both mouse models. We conclude that early viral tissue compartmentalization provides a roadmap for investigations into HIV-1 elimination.

Modulating cellular autophagy for controlled antiretroviral drug release.

AIM: Pharmacologic agents that affect autophagy were tested for their abilities to enhance macrophage nanoformulated antiretroviral drug (ARV) depots and its slow release. METHODS: These agents included URMC-099, rapamycin, metformin, desmethylclomipramine, 2-hydroxy-ß-cyclodextrin (HBC) and clonidine. Each was administered with nanoformulated atazanavir (ATV) nanoparticles to human monocyte-derived macrophages. ARV retention, antiretroviral activity and nanocrystal autophagosomal formation were evaluated. RESULTS: URMC-099, HBC and clonidine retained ATV. HBC, URMC-099 and rapamycin improved intracellular ATV retention. URMC-099 proved superior among the group in affecting antiretroviral activities. CONCLUSION: Autophagy inducing agents, notably URMC-099, facilitate nanoformulated ARV depots and lead to sustained release and improved antiretroviral responses. As such, they may be considered for development as part of long acting antiretroviral treatment regimens.

Gemcitabine with carboplatin for advanced intrahepatic cholangiocarcinoma: A study from North India Cancer Centre.

BACKGROUND: Gemcitabine plus cisplatin has been established as a standard chemotherapy regimen for advanced biliary tract cancers (BTCs) based on the phase III UK ABC-02 study, which included all types of biliary cancers. There is very limited data regarding the effectiveness of known chemotherapeutic regimens especially in IHCC. METHODS: Records of 63 patients diagnosis of IHCC who received Gemcitabine and Carboplatin (G-C Regimen) chemotherapy as a first line were retrospectively reviewed. The primary aim of this study was to assess the response rate of gemcitabine carboplatin-based chemotherapy as a first line therapy in advanced intrahepatic cholangiocarcinoma (IHCC). The secondary objectives were to assess toxicity, progression free survival and overall survival. RESULTS: There were 38 men and 25 women in our study with a median age of 56.75 years (range 31-78 years). Of the 38+25= 63 patients, 21 patients (33.8%) progressed, 5 patients (8.06%) had complete response, 25 patients (40.3%) had partial response, 12 patients (19.3%) had stable disease. Overall response rate was 48.36% and tumor control rate was 67.6%. Progression free survival was 5.3 months and overall survival of 10.3 months was seen. The most common grade 3-4 toxicities were anemia, neutropenia, and thrombocytopenia. Most common nonhematological toxicity was fatigue. CONCLUSION: Gemcitabine in combination with carboplatin has activity against advanced IHCC. Our results are comparable with other gemcitabine carboplatin studies as well as gemcitabine cisplatin-based studies.

Autophagy facilitates macrophage depots of sustained-release nanoformulated antiretroviral drugs.

Long-acting anti-HIV products can substantively change the standard of care for patients with HIV/AIDS. To this end, hydrophobic antiretroviral drugs (ARVs) were recently developed for parenteral administration at monthly or longer intervals. While shorter-acting hydrophilic drugs can be made into nanocarrier-encased prodrugs, the nanocarrier encasement must be boosted to establish long-acting ARV depots. The mixed-lineage kinase 3 (MLK-3) inhibitor URMC-099 provides this function by affecting autophagy. Here, we have shown that URMC-099 facilitates ARV sequestration and its antiretroviral responses by promoting the nuclear translocation of the transcription factor EB (TFEB). In monocyte-derived macrophages, URMC-099 induction of autophagy led to retention of nanoparticles containing the antiretroviral protease inhibitor atazanavir. These nanoparticles were localized within macrophage autophagosomes, leading to a 4-fold enhancement of mitochondrial and cell vitality. In rodents, URMC-099 activation of autophagy led to 50-fold increases in the plasma drug concentration of the viral integrase inhibitor dolutegravir. These data paralleled URMC-099-mediated induction of autophagy and the previously reported antiretroviral responses in HIV-1-infected humanized mice. We conclude that pharmacologic induction of autophagy provides a means to extend the action of a long-acting, slow, effective release of antiretroviral therapy.

Pharmacodynamics of long-acting folic acid-receptor targeted ritonavir-boosted atazanavir nanoformulations.

Long-acting nanoformulated antiretroviral therapy (nanoART) that targets monocyte-macrophages could improve the drug's half-life and protein-binding capacities while facilitating cell and tissue depots. To this end, ART nanoparticles that target the folic acid (FA) receptor and permit cell-based drug depots were examined using pharmacokinetic and pharmacodynamic (PD) tests. FA receptor-targeted poloxamer 407 nanocrystals, containing ritonavir-boosted atazanavir (ATV/r), significantly increased drug bioavailability and PD by five and 100 times, respectively. Drug particles administered to human peripheral blood lymphocyte reconstituted NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ mice and infected with HIV-1ADA led to ATV/r drug concentrations that paralleled FA receptor beta staining in both the macrophage-rich parafollicular areas of spleen and lymph nodes. Drug levels were higher in these tissues than what could be achieved by either native drug or untargeted nanoART particles. The data also mirrored potent reductions in viral loads, tissue viral RNA and numbers of HIV-1p24+ cells in infected and treated animals. We conclude that FA-P407 coating of ART nanoparticles readily facilitates drug carriage and antiretroviral responses.

Dexmedetomidine as an Anesthetic Adjuvant in Patients Undergoing Transsphenoidal Resection of Pituitary Tumor.

BACKGROUND: Transnasal transsphenoidal (TNTS) resection of pituitary tumors involves wide fluctuation in hemodynamic parameter and causes hypertension and tachycardia due to intense noxious stimuli during various stages of surgery. None of routinely used anesthetic agents effectively blunts the undesirable hemodynamic responses, and therefore usually there is a need to use increased doses of anesthetic agents. Dexmedetomidine (DEX) an α-2 adrenergic receptor agonist, because its sympatholytic and antinociceptive properties may ensure optimal intraoperative hemodynamic stability during critical moments of surgical manipulation. In addition, DEX reduced the anesthetic requirement with rapid recovery at the end of surgery. The main aim of our study was to evaluate the effect of DEX on perioperative hemodynamics, anesthetic requirements, and recovery characteristics in patients undergoing TNTS resection of pituitary tumors. MATERIALS AND METHODS: Forty-six patients scheduled for elective TNTS resection of pituitary tumor were randomized to receive a continuous infusion of DEX (group D) or 0.9% saline (group C). Patients in both the groups were subjected to a standardized anesthesia comprising of induction with propofol, fentanyl, vecuronium, and positive pressure ventilation with O2/air (1:1)/isoflurane. The response entropy target range during maintenance of anesthesia was 40 to 60. The hemodynamic variables at various stages of surgery, intraoperative anesthetic, and analgesic and recovery characteristics were recorded. RESULTS: Total fentanyl consumption during the study period was significantly lower in group D compared with group C (4.7 and 7.7 µg/kg, respectively; P<0.01). End-tidal isoflurane concentration requirement was found to be significantly reduced in group D compared with group C throughout the surgical period. Fentanyl and end-tidal isoflurane concentration requirement was reduced in group D compared with group C by 40% and 33.3%, respectively. Heart rate and mean arterial pressure were significantly higher in the group C compared with group D after intubation, during various stages of surgery and immediately after extubation. The group D had excellent surgical conditions and lesser bleeding in comparison to group C. Emergence time and extubation time were significantly shorter in group D compared with group C. CONCLUSIONS: DEX as an anesthetic adjuvant improved hemodynamic stability and decreased anesthetic requirements in patients undergoing TNTS resection of pituitary tumor. In addition, DEX provided better surgical field exposure conditions and early recovery from anesthesia.

Associations between brain microstructures, metabolites, and cognitive deficits during chronic HIV-1 infection of humanized mice.

BACKGROUND: Host-species specificity of the human immunodeficiency virus (HIV) limits pathobiologic, diagnostic and therapeutic research investigations to humans and non-human primates. The emergence of humanized mice as a model for viral infection of the nervous system has overcome such restrictions enabling research for HIV-associated end organ disease including behavioral, cognitive and neuropathologic deficits reflective of neuroAIDS. Chronic HIV-1 infection of NOD/scid-IL-2Rgcnull mice transplanted with human CD34+ hematopoietic stem cells (CD34-NSG) leads to persistent viremia, profound CD4+ T lymphocyte loss and infection of human monocyte-macrophages in the meninges and perivascular spaces. Murine cells are not infected with virus. METHODS: Changes in mouse behavior were measured, starting at 8 weeks after viral infection. These were recorded coordinate with magnetic resonance spectroscopy metabolites including N-acetylaspartate (NAA), creatine and choline. Diffusion tensor magnetic resonance imaging (DTI) was recorded against multispectral immunohistochemical staining for neuronal markers that included microtubule associated protein-2 (MAP2), neurofilament (NF) and synaptophysin (SYN); for astrocyte glial fibrillary acidic protein (GFAP); and for microglial ionized calcium binding adaptor molecule 1 (Iba-1). Oligodendrocyte numbers and integrity were measured for myelin associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG) antigens. RESULTS: Behavioral abnormalities were readily observed in HIV-1 infected mice. Longitudinal open field activity tests demonstrated lack of habituation indicating potential for memory loss and persistent anxiety in HIV-1 infected mice compared to uninfected controls. End-point NAA and creatine in the cerebral cortex increased with decreased MAG. NAA and glutamate decreased with decreased SYN and MAG. Robust inflammation reflected GFAP and Iba-1 staining intensities. DTI metrics were coordinate with deregulation of NF, Iba-1, MOG and MAG levels in the whisker barrel and MAP2, NF, MAG, MOG and SYN in the corpus callosum. CONCLUSIONS: The findings are consistent with some of the clinical, biochemical and pathobiologic features of human HIV-1 nervous system infections. This model will prove useful towards investigating the mechanisms of HIV-1 induced neuropathology and in developing novel biomarkers and therapeutic strategies for disease.

Combinatorial assessments of brain tissue metabolomics and histopathology in rodent models of human immunodeficiency virus infection.

Metabolites are biomarkers for a broad range of central nervous system disorders serving as molecular drivers and byproducts of disease pathobiology. However, despite their importance, routine measures of brain tissue metabolomics are not readily available based on the requirements of rapid tissue preservation. They require preservation by microwave irradiation, rapid freezing or other methods designed to reduce post mortem metabolism. Our research on human immunodeficiency virus type one (HIV-1) infection has highlighted immediate needs to better link histology to neural metabolites. To this end, we investigated such needs in well-studied rodent models. First, the dynamics of brain metabolism during ex vivo tissue preparation was shown by proton magnetic resonance spectroscopy in normal mice. Second, tissue preservation methodologies were assessed using liquid chromatography tandem mass spectrometry and immunohistology to measure metabolites and neural antigens. Third, these methods were applied to two animal models. In the first, immunodeficient mice reconstituted with human peripheral blood lymphocytes then acutely infected with HIV-1. In the second, NOD scid IL2 receptor gamma chain knockout mice were humanized with CD34+ human hematopoietic stem cells and chronically infected with HIV-1. Replicate infected animals were treated with nanoformulated antiretroviral therapy (nanoART). Results from chronic infection showed that microgliosis was associated with increased myoinostitol, choline, phosphocholine concentrations and with decreased creatine concentrations. These changes were partially reversed with nanoART. Metabolite responses were contingent on the animal model. Taken together, these studies integrate brain metabolomics with histopathology towards uncovering putative biomarkers for neuroAIDS.

High quality RNA isolation from ployphenol-, polysaccharide- and protein-rich tissues of lentil (Lens culinaris).

Current RNA isolation methods have limitations in their ability to yield good quality and quantity of RNA from plants that have high content of phenols, polysaccharides and storage proteins. Existing methods also do not eliminate accompanying chromosomal DNA in RNA preparation that causes false positives in gene expression studies. Standard isolation technique was modified for rapid and quick extraction of RNA, and lentil tissue most appropriate to extract good quality RNA was determined. The concentration of the phenol blocker polyvinylpyrrolidone in the extraction buffer was determined, DNase I was added to eliminate chromosomal DNA and the timing of this step was optimized. RNA up to 568 µg of RNA from 1 g of tissue was isolated from four different tissues of lentil in less than half the time typically required by reported methods. The method avoids the use of toxic phenol-chloroform, hazardous guanidinium thiocyanate (GTC) and laborious CsCl ultracentrifugation. Absorbance A260/A280 ratio of 1.9 and A260/A230 ratio of 2.7 reveal RNA to be of high purity. Modified method yielded RNA that was free from contaminants and suitable for RT-PCR and cDNA library construction.