Mitigation of Huanglongbing: Implications of a Biologically Enhanced Nutritional Program on Yield, Pathogen Localization, and Host Gene Expression Profiles.

Huanglongbing (HLB, citrus greening disease), the most destructive disease affecting citrus production, is primarily linked to the gram-negative, insect-vectored, phloem-inhabiting α-proteobacterium 'Candidatus Liberibacter asiaticus' (CLas). With no effective treatment available, management strategies have largely focused on the use of insecticides in addition to the destruction of infected trees, which are environmentally hazardous and cost-prohibitive for growers, respectively. A major limitation to combating HLB is the inability to isolate CLas in axenic culture, which hinders in vitro studies and creates a need for robust in situ CLas detection and visualization methods. The aim of this study was to investigate the efficacy of a nutritional program-based approach for HLB treatment, and to explore the effectiveness of an enhanced immunodetection method to detect CLas-infected tissues. To achieve this, four different biologically enhanced nutritional programs (bENPs; P1, P2, P3, and P4) were tested on CLas-infected citrus trees. Structured illumination microscopy preceded by a modified immunolabeling process and transmission electron microscopy were used to show treatment-dependent reduction of CLas cells in phloem tissues. No sieve pore plugging was seen in the leaves of P2 trees. This was accompanied by an 80% annual increase in fruit number per tree and 1,503 (611 upregulated and 892 downregulated) differentially expressed genes. These included an MLRQ subunit gene, UDP-glucose transferase, and genes associated with the alpha-amino linolenic acid metabolism pathway in P2 trees. Taken together, the results highlight a major role for bENPs as a viable, sustainable, and cost effective option for HLB management.

Cultural validation of the structured clinical interview for diagnostic and statistical manual of mental disorders in Indigenous Australians.

OBJECTIVE: This study determined the cultural appropriateness of the Structured Clinical Interview for the DSM-IV Axis I Disorders (SCID-I) as an acceptable tool for diagnosing mental illness among Indigenous people. METHODS: De-identified qualitative feedback from participants and psychologists regarding the cultural appropriateness of the SCID-I for Indigenous people using open-ended anonymous questionnaires was gathered. Aboriginal Medial Service staff and Indigenous Support Workers participated in a focus group. RESULTS: A total of 95.6% of participants felt comfortable during the 498 questionnaires completed. Psychologists also provided qualitative feedback for 502 (92.3%) interviews, of whom 40.4% established a good rapport with participants. Of the participants, 77.7% understood the SCID-I questions well, while 72.5% did not require any cultural allowances to reach a clinical diagnosis. CONCLUSION: When administered by a culturally safe trained psychologist, SCID-I is well tolerated in this group.

The unintended mitochondrial uncoupling effects of the FDA-approved anti-helminth drug nitazoxanide mitigates experimental parkinsonism in mice.

Mitochondria play a primary role in the pathophysiology of Parkinson's disease (PD), and small molecules that counteract the initial stages of disease may offer therapeutic benefit. In this regard, we have examined whether the off-target effects of the Food and Drug Administration (FDA)-approved anti-helminth drug nitazoxanide (NTZ) on mitochondrial respiration could possess any therapeutic potential for PD. Results indicate that MPP+-induced loss in oxygen consumption rate (OCR) and ATP production by mitochondria were ameliorated by NTZ in real time by virtue of its mild uncoupling effect. Pretreatment of cells with NTZ mitigated MPP+-induced loss in mitochondrial OCR and reactive oxygen species (ROS). Similarly, addition of NTZ to cells pretreated with MPP+ could reverse block in mitochondrial OCR and reactive oxygen species induced by MPP+ in real time. The observed effects of NTZ were found to be transient and reversible as removal of NTZ from incubation medium restored the mitochondrial respiration to that of controls. Apoptosis induced by MPP+ was ameliorated by NTZ in a dose-dependent manner. In vivo results demonstrated that oral administration of NTZ (50 mg/kg) in an acute MPTP mouse model of PD conferred significant protection against the loss of tyrosine hydroxylase (TH)-positive neurons of substantia nigra. Based on the above observations we believe that repurposing of NTZ for PD may offer therapeutic benefit.

Rational design of a ligand-controlled protein conformational switch.

Design of a regulatable multistate protein is a challenge for protein engineering. Here we design a protein with a unique topology, called uniRapR, whose conformation is controlled by the binding of a small molecule. We confirm switching and control ability of uniRapR in silico, in vitro, and in vivo. As a proof of concept, uniRapR is used as an artificial regulatory domain to control activity of kinases. By activating Src kinase using uniRapR in single cells and whole organism, we observe two unique phenotypes consistent with its role in metastasis. Activation of Src kinase leads to rapid induction of protrusion with polarized spreading in HeLa cells, and morphological changes with loss of cell-cell contacts in the epidermal tissue of zebrafish. The rational creation of uniRapR exemplifies the strength of computational protein design, and offers a powerful means for targeted activation of many pathways to study signaling in living organisms.

Tumebacillus lipolyticus sp. nov., isolated from river water.

An aerobic, endospore-forming, alkali-tolerant, Gram-stain-positive, non-motile, rod-shaped bacterium, designated strain NIO-S10T, was isolated from a surface water sample collected from the Godavari River, Kapileswarapuram, India. Colonies on nutrient agar were circular, 3-4 mm in diameter, creamish and raised after incubation for 36 h at 37 °C. Growth occurred at 20-40 °C, at pH 6-9 and in the presence of 0-2 % (w/v) NaCl. Strain NIO-S10T was positive for oxidase, caseinase, DNase, gelatinase, lipase and urease activities, and negative for catalase, aesculinase, amylase and cellulase activities. The fatty acids were dominated by branched and saturated fatty acids, with a high abundance of iso-C15 : 0, anteiso-C15 : 0 and C18 : 0.The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid, and MK-7 was the major menaquinone. The DNA G+C content of strain NIO-S10T was 54.4 mol%. A blast sequence similarity search based on 16S rRNA gene sequences indicated that Tumebacillus ginsengisoli Gsoil 1105T was the nearest phylogenetic neighbour to strain NIO-S10T, with a pairwise sequence similarity of 94.9 %. Phylogenetic analysis showed that strain NIO-S10T was clustered with members of the genus Tumebacillus and together with members of the genus Effusibacillus. Based on phenotypic characteristics and phylogenetic inference, strain NIO-S10T represents a novel species of the genus Tumebacillus, for which the name Tumebacillus lipolyticus sp. nov. is proposed. The type strain is NIO-S10T ( = KCTC 32289T = MTCC 12483T).

Quantitation of VEGFR2 (vascular endothelial growth factor receptor) inhibitors--review of assay methodologies and perspectives.

The introduction of small-molecule tyrosine kinase VEGFR2 (vascular endothelial growth factor receptor) inhibitors has added another dimension in the treatment of several oncology indications as they offer a unique mechanism. The VEGFR2 inhibitors have demonstrated superior benefits in treating certain types of cancer, such as renal cell carcinoma and hepatocellular carcinoma, as a monotherapy option. Many of the approved VEGFR2 inhibitors have also shown promise when used in combination with other anticancer agents. There are numerous bioanalytical methods published for the analysis of VEGFR2 inhibitors in preclinical and clinical samples. This review covers VEGFR2 inhibitors such as sunitinib, sorafenib, pazopanib and JI-101. In addition to providing a comprehensive review of the available methods for the above-mentioned VRGFR2 inhibitors, it also provides information on assays that can simultaneously measure multiple tyrosine kinase inhibitors, including VEGFR2 molecules. Based on the review, the published methodologies using LC/MS-MS or HPLC-UV are adequate for the quantification of the VEGFR2 inhibitors and can easily be established in a modern day bioanalytical laboratory. The availability of a plethora of assays for multiple tyrosine kinase inhibitors makes it easy to analyze a panel of compounds to support either therapeutic drug monitoring and/or clinical pharmacokinetics.

Transcriptional Responses of Different Brain Cell Types to Oxygen Decline.

Brain hypoxia is associated with a wide range of physiological and clinical conditions. Although oxygen is an essential constituent of maintaining brain functions, our understanding of how specific brain cell types globally respond and adapt to decreasing oxygen conditions is incomplete. In this study, we exposed mouse primary neurons, astrocytes, and microglia to normoxia and two hypoxic conditions and obtained genome-wide transcriptional profiles of the treated cells. Analysis of differentially expressed genes under conditions of reduced oxygen revealed a canonical hypoxic response shared among different brain cell types. In addition, we observed a higher sensitivity of neurons to oxygen decline, and dissected cell type-specific biological processes affected by hypoxia. Importantly, this study establishes novel gene modules associated with brain cells responding to oxygen deprivation and reveals a state of profound stress incurred by hypoxia.

Comparison of Opal self-ligating brackets with manually ligating brackets.

This study was conducted with aim to evaluate the efficiency of the newly introduced Opal self-ligating brackets (SLB). The chairside time saved, appliance efficiency, bracket bond failures, breakages and bracket staining in the Opal SLBs were compared with the conventional standard metal manually ligating brackets (MLBs) of MBT system. Seven patients were compared in each group. Standard light curing bonding methods were used in both the group. Chairside time saving, appliance efficiency and bracket bond failures were compared among the groups whereas staining was observed with the SLBs. Appliance efficiency was evaluated by PAR scores. Results showed significant chairside time being saved in SLBs, whereas the appliance efficiency was not significant. Bond failures were found only in SLBs as well as breakages along with staining. Henceforth, we could conclude that though SLBs had advantage of saving chairside time but also had disadvantage of losing more time with bond failures.

Targeting acute ischemic stroke with a calcium-sensitive opener of maxi-K potassium channels.

During ischemic stroke, neurons at risk are exposed to pathologically high levels of intracellular calcium (Ca++), initiating a fatal biochemical cascade. To protect these neurons, we have developed openers of large-conductance, Ca++-activated (maxi-K or BK) potassium channels, thereby augmenting an endogenous mechanism for regulating Ca++ entry and membrane potential. The novel fluoro-oxindoles BMS-204352 and racemic compound 1 are potent, effective and uniquely Ca++-sensitive openers of maxi-K channels. In rat models of permanent large-vessel stroke, BMS-204352 provided significant levels of cortical neuroprotection when administered two hours after the onset of occlusion, but had no effects on blood pressure or cerebral blood flow. This novel approach may restrict Ca++ entry in neurons at risk while having minimal side effects.

Extracellular-Signal-Regulated Kinase Inhibition Switches APP Processing from ß- to α-Secretase under Oxidative Stress: Modulation of ADAM10 by SIRT1/NF-κB Signaling.

The sequential cleavage of full-length amyloid precursor protein (APP) by secretases has been at the center of efforts for understanding the onset of Alzheimer's disease (AD). A decrease in α-secretase activity was observed during the progression of AD; however, the precise molecular mechanism involved in the downregulation of α-secretase under oxidative stress is not fully understood. In the present study, we have demonstrated that pharmacological inhibition of mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) by mitogen-activated protein kinase kinase-1 (MEK-1) inhibitor (PD98059) restored the expression of a disintegrin and metalloproteinase 10 (ADAM10) with a concomitant decrease in ß-site APP cleavage enzyme 1 (BACE1) under oxidative stress. Silent mating-type information regulation 2 homologue 1 (SIRT1) activation by resveratrol also mitigated alterations in secretase levels through MAPK/ERK signaling. Intracerebroventricular (ICV) administration of streptozotocin in rats showed amyloidogenic processing of APP and altered the SIRT1/ERK axis in the hippocampus. We also observed that the ADAM10 expression is controlled at the transcriptional level by oxidative stress. Using the luciferase reporter activity of ADAM10 promoter deletion constructs, we have identified the region 290 bp upstream of the transcription start site (TSS) possessing regulatory elements responsible for ADAM10 downregulation with hydrogen peroxide (H2O2) treatment. Further, bioinformatics analysis revealed the presence of putative nuclear factor kappa B (NF-κB) binding sites in the ADAM10 promoter region. Treatment of cortical neurons with the NF-κB inhibitor (Bay 11-7082) mitigated the transcriptional upregulation of ADAM10 by PD98059. Overall, our findings suggest that SIRT1/ERK/NF-κB axis contributes to the downregulation of ADAM10, resulting in the shift from nonamyloidogenic to amyloidogenic processing of APP under oxidative stress.

Spatial variations in dissolved inorganic nutrients in the groundwaters along the Indian coast and their export to adjacent coastal waters.

Submarine Groundwater Discharge (SGD) is one of the main external nutrient sources to the coastal waters. The concentrations of nutrients in groundwaters are a few folds higher than that of adjacent coastal waters; therefore, SGD enhances nutrients levels in the coastal waters and influences coastal biota. In order to examine the spatial and seasonal variability in nutrient concentrations and exchange to the coastal waters, groundwater samples were collected at ~ 90 locations along the Indian coast during the wet and dry seasons. This study revealed that dissolved inorganic nitrogen (DIN) and dissolved inorganic phosphates (DIP) and urea were found to be high during the dry than wet period. Higher concentrations of DIN and DIP were observed during both wet and dry periods in the groundwater along the east than the west coast of India. The State-wise mean amount of fertilizer used during Kharif (wet) and Rabi (dry) period in each Indian State showed significant correlation with mean concentrations of DIN and urea. The observed linear relationship of DIN with bacterial respiration and inverse relationship with DO saturation and ammonium in groundwater suggested that decomposition of organic matter and nitrification contributed to the DIN pool in the groundwater. The mean rate of SGD fluxes varied between 1.6 × 104 m3/day and 1.75 × 1011 m3/day in the Indian coastal region. The annual mean SGD flux of DIN and DIP was estimated to be 0.103 ± 0.02 and 0.021 ± 0.01 Tg (1 Tg = 1012 g) to the western coastal Bay of Bengal (east coast of India) and 0.06 ± 0.03 and 0.015 ± 0.01 Tg/y to the eastern coastal Arabian Sea (west coast of India) respectively. The estimated SGD flux of DIN and DIP to the Indian coastal waters amounted to 0.163 ± 0.04 and 0.036 ± 0.02 Tg/y respectively, and it is almost close to that of nutrients discharged by rivers (0.22 ± 0.05 and 0.11 ± 0.03 Tg/y respectively). Among the external sources of nitrogen and phosphorus, such as river discharge, atmospheric deposition, the contribution by SGD is highly significant in the Bay of Bengal (30 and 17% respectively) than in the case of Arabian Sea (24 and 25% respectively).

Clinical Validation of Rapid Gout Detection Method and Kit.

Gout is an inflammatory arthritis, which causes intense, acute pain due to the buildup of uric acid crystals in synovial fluid. The gold standard for gout diagnosis consists of synovial fluid analysis by polarized light microscopy, which is costly, time-intensive, and technique-dependent, therefore meriting a more efficient, inexpensive, and accessible method for diagnosis. We previously developed and validated a novel colorimetric gout detection method and device based on the reduction of silver nitrate by uric acid; here, we clinically validated our method and device using arthroscopically obtained synovial fluid samples from gout patients. We successfully identified uric acid crystals in clinical samples via our colorimetric method, visualized uric acid crystals in synovial fluid via handheld microscopy, and determined that silver nitrate stain did not interfere with the microscopic visualization of uric acid crystals necessary for diagnosis. We also developed and validated a method of processing turbid clinical samples for use in our device to prevent the obscuration of uric acid crystals by suspended material. Our method and device will clinically facilitate the immediate colorimetric diagnosis of gout and the subsequent bedside visualization of uric acid crystals in both ideal and turbid synovial fluid samples, allowing for a point-of-care diagnosis of gout.

Baicalin, an emerging multi-therapeutic agent: pharmacodynamics, pharmacokinetics, and considerations from drug development perspectives.

Baicalin was extensively researched for utility in a number of therapeutic areas owing to its anti-inflammatory, anti-oxidant, anti-bacterial, and anti-cancer properties. A number of preclinical studies, in vitro work, and mechanistic studies were performed to understand the absorption, distribution, metabolism, and excretion profiles of baicalin. The absorption of baicalin involved several complexities: the restriction to two distant sites; the conversion of baicalin to baicalein; the possible role of transporter(s); and enhanced absorption due to breakdown of conjugates by beta-glucuronidase. Limited distribution data suggest that baicalin reached several sites such as the brain, eye lens, thymus, etc. Hepatobiliary recycling also served as a distribution phase for sustained delivery of baicalin. Metabolism data suggest the rapid conversion of baicalin to baicalein, which was extensively subjected to Phase 2 metabolism, conjugates baicalein glucuronide/sulfate have been identified. Limited excretion data suggest involvement of renal and faecal routes--glucuronide and sulfate conjugates were excreted in urine and faeces (via biliary excretion). The published data on baicalin suggest imminent challenges for developing baicalin and/or during co-administration with other agents. These challenges are absorption related (transporter or changes in the microenvironment), metabolism related (CYP2B6 induction and/or CYP2E1 inhibition), and excretion/efflux related (competitive biliary pathway and/or OATP1B1 transport).

Drug-drug interaction studies in preclinical species: should metabolite(s) kinetics be studied?

Drug-drug interaction studies are important building blocks in drug development to understand the perceived risk of a purported interaction due to the differing clinical pharmacology attributes of the co-administered drugs. Two case studies are presented that justify the importance of evaluating the metabolite kinetics data along with the parent in a preclinical model. Atorvastatin and verapamil have interesting clinical pharmacology attributes in that both agents are substrates and/or inhibitors of the dual cytochrome P450 (CYP) 3A4 and P-glycoprotein (Pgp) efflux transporter interplay. As articulated by the two case studies, the presence of metabolite kinetic data (i.e., norverapamil) provided unequivocal evidence in order to tease out the actual pathway responsible for the interaction between atorvastatin and verapamil. Therefore, consideration for metabolite kinetics, wherever feasible, appears to be prudent in defining the interaction liability between the two agents in a preclinical model.

Optimization of USSP duration for enhanced corrosion resistance of AA7075.

This investigation was carried out following our earlier work on the effect of ultrasonic shot peening (USSP) on corrosion resistance of the 7075 aluminium alloy in 3.5 wt% NaCl solution to optimize the duration of USSP. The samples not treated with USSP and different samples treated with USSP were subjected to potentiodynamic polarization and electrochemical impedance spectroscopy. Among the specimens USSP treated from 5 to 30 s, the one USSP treated for 15 s (USSP 15) was found to exhibit highest corrosion potential (Ecorr) and lowest corrosion current density (icorr). Corrosion products were characterized by Scanning Electron Microscopy (SEM) and X-ray Photoelectron Spectroscopy (XPS). Scanning Kelvin Probe Force Microscopy (SKPFM) was used to measure the surface free potential. The enhanced corrosion resistance of the USSP 15 sample was found to be due to combined effect of surface nanostructure of the matrix, homogeneity and refinement of second phase precipitates. There was enhancement in formation of adherent passive layer in the USSP15 specimen.

Interspecies scaling of a camptothecin analogue: human predictions for intravenous topotecan using animal data.

As a class, camptothecin analogues via market entry of topotecan and irinotecan, have shown promise for the treatment of various solid tumours. Topotecan, in particular, was chosen as the substrate for allometric scaling and prediction of human parameter values for both total clearance (CL) and volume of distribution (V(ss)). The availability of published data in mouse, rat, dog, and monkey paved the way for interspecies scaling via allometry. Although it appeared that at a minimum mouse, rat, and dog would reasonably fit in a three-species allometry scale-up, the inclusion of monkey data enabled a better prediction of the human parameter values for total topotecan-e.g., CL: allometric equation: 1.5234W(0.7865); predicted value = 43.04 l h(-1): observed CL = 24-53 l h(-1); V(ss): allometric equation: 1.1939W(1.0208); predicted value = 91.29 litres: observed V(ss) = 66-146 litres. The proximity of the allometric exponent values of CL (0.7885) and V(ss) (1.0208) to the suggested values of 0.75 and 1.00 was not only encouraging, but also confirmed the applicability of interspecies scaling approach for topotecan. The data suggest that allometric scaling approaches with suitable correction factors could potentially be used to predict the human pharmacokinetics of novel CPT analogues prospectively.

Carbamylated Hemoglobin can Differentiate Acute Kidney Injury from Chronic Kidney Disease.

Carbamylated hemoglobin (CarHb) was found to have a potential role in the differentiation of patients with acute kidney injury (AKI) from chronic kidney disease (CKD). This study was aimed at the evaluation of the diagnostic performance and usefulness of CarHb in the differentiation of AKI from CKD. Forty patients with renal disease and twenty age- and sex-matched healthy controls were included in the study. Urea, creatinine, Hb, and CarHb were measured in all the subjects. Patients with AKI and CKD were found to have significantly increased levels of CarHb when compared to controls (P < 0.05 for both groups). Patients with CKD had significantly increased levels of CarHb when compared to patients with AKI (P < 0.05). CarHb showed significant positive correlation with urea in patients with renal disease (r = 0.776, P < 0.0001). Significant area under curve (AUC = 0.840, P < 0.0001) was obtained for CarHb and a cut-off value of 98.33 µg VH/g Hb resulted with the best combination of 85% sensitivity and 75% specificity. CarHb may provide clinical utility since patients with AKI and CKD have similar clinical presentation usually. A cut-off value of 98.33 µg VH/g Hb has been found to be useful to differentiate AKI from CKDs.

Differential expression of leaf proteins in four cultivars of peanut (Arachis hypogaea L.) under water stress.

Drought is a major constraint to the productivity of many crops affecting various physiological and biochemical processes. Seventy percent of the peanuts are grown in semiarid tropics that are frequently prone to drought stress. So, we analyzed its effect in 4 cultivars of peanut, with different degrees of drought tolerance, under 10 and 20 days of water stress using two-dimensional gel electrophoresis and mass spectrometry. A total of 189 differentially expressed protein spots were identified in the leaf proteome of all the 4 cultivars using PD Quest Basic software; 74 in ICGV 91114, 41 in ICGS 76, 44 in J 11 and 30 in JL 24. Of these, 30 protein spots were subjected to in-gel trypsin digestion followed by MALDI-TOF that are functionally categorized into 5 groups: molecular chaperones, signal transducers, photosynthetic proteins, defense proteins and detoxification proteins. Of these, 12 proteins were sequenced. Late embryogenesis abundant protein, calcium ion binding protein, sucrose synthase isoform-1, 17.3 kDa heat shock protein and structural maintenance of chromosome proteins were overexpressed only in the 15 and 20 days stressed plants of ICGV 91114 cultivar while cytosolic ascorbate peroxidase was expressed with varying levels in the 10 and 20 days stressed plants of all the 4 cultivars. Signaling protein like 14-3-3 and defense proteins like alpha-methyl-mannoside-specific lectin and mannose/glucose-binding lectins were differentially expressed in the 4 cultivars. Photosynthetic protein like Rubisco was down-regulated in the stressed plants of all 4 cultivars while Photosystem-I reaction center subunit-II of chloroplast precursor protein was overexpressed in only 20 days stressed plants of ICGV 91114, ICGS 76 and J11 cultivars. These differentially expressed proteins could potentially be used as protein markers for screening the peanut germplasm and further crop improvement.