Anticariogenic and phytochemical evaluation of Eucalyptus globules Labill.

In the present study, in vitro anticariogenic potential of ethyl acetate, hexane and methanol and aqueous extracts of plant leaves of Eucalyptus globules Labill. were evaluated by using four cariogenic bacteria, Lactobacillus acidophilus, Lactobacillus casei, Staphylococcus aureus and Streptococcus mutans. Agar well diffusion method and minimum inhibitory concentration (MIC) were used for this purpose. The ethyl acetate extracted fraction of plant leaves showed good inhibitory effects against all selected bacteria. In Eucalyptus globules, hexane and ethyl acetate extracts found highly effective against, Lactobacillus acidophilus with MIC value of 0.031 and 0.062 mg/mL, respectively. Qualitative phytochemical investigation of above extracts showed the presence of alkaloids, phenolic compounds, steroids, cardiac glycosides and terpenes. Based on the MIC value and bioautography, ethyl acetate of plant leaf was selected for further study. Further investigation on the structure elucidation of the bioactive compound using IR, GC-MS and NMR techniques revealed the presence of alpha-farnesene, a sesquiterpene. Eucalyptus globules plant leaf extracts have great potential as anticariogenic agents that may be useful in the treatment of oral disease.     

A hydrolytic ��-glutamyl transpeptidase from thermo-acidophilic archaeon Picrophilus torridus: binding pocket mutagenesis and transpeptidation

??-Glutamyl transpeptidase of a thermo-acidophilic archaeon Picrophilus torridus was cloned and expressed using E. coli Rosetta-pET 51b(+) expression system. The enzyme was expressed at 37 °C/200 rpm with ??-GT production of 1.99 U/mg protein after 3 h of IPTG induction. It was improved nearby 10-fold corresponding to 18.92 U/mg protein in the presence of 2 % hexadecane. The enzyme was purified by Ni²⁺-NTA with a purification fold of 3.6 and recovery of 61 %. It was synthesized as a precursor heterodimeric protein of 47 kDa with two subunits of 30 kDa and 17 kDa, respectively, as revealed by SDS-PAGE and western blot. The enzyme possesses hydrolase activity with optima at pH 7.0 and 55 °C. It was thermostable with a t _1/2 of 1 h at 50 °C and 30 min at 60 °C, and retained 100 % activity at 45 °C even after 24 h. It was inhibited by azaserine and DON and PMSF. Pt??-GT shared 37 % sequence identity and 53 % homology with an extremophile ??-GT from Thermoplasma acidophilum. Functional residues identified by in silico approaches were further validated by site-directed mutagenesis where Tyr327 mutated by Asn327 introduced significant transpeptidase activity.     

Neuromuscular Electrical Stimulation of the Quadriceps in Patients with Non-Small Cell Lung Cancer Receiving Palliative Chemotherapy: A Randomized Phase II Study

Background

A reduced exercise capacity is associated with increased morbidity and mortality in patients with advanced non-small cell lung cancer (NSCLC). Therapeutic exercise can be beneficial and neuromuscular electrical stimulation (NMES) of the quadriceps muscles may represent a practical approach. The primary aim of this study was to determine the acceptability of NMES of the quadriceps to patients with NSCLC used alongside palliative chemotherapy. Secondary aims explored aspects of safety and efficacy of NMES in this setting.

Methods

Patients with advanced NSCLC due to receive first-line palliative chemotherapy were randomized to usual care with or without NMES. They were asked to undertake 30 minute sessions of NMES, ideally daily, but as a minimum, three times weekly. For NMES to be considered acceptable, it was predetermined that ≥80% of patients should achieve this minimum level of adherence. Qualitative interviews were held with a subset of patients to explore factors influencing adherence. Safety was assessed according to the Common Terminology Criteria for Adverse Events. Quadriceps muscle strength, thigh lean mass, and physical activity level were assessed at baseline and after three cycles of chemotherapy.

Results

49 patients (28 male, median (IQR) age 69 (64−75) years) participated. Of 30 randomized to NMES, 18 were eligible for the primary endpoint, of whom 9 (50% [90% CI, 29 to 71]) met the minimum level of adherence. Adherence was enhanced by incorporating sessions into a daily routine and hindered by undesirable effects of chemotherapy. There were no serious adverse events related to NMES, nor significant differences in quadriceps muscle strength, thigh lean mass or physical activity level between groups.

Conclusions

NMES is not acceptable in this setting, nor was there a suggestion of benefit. The need remains to explore NMES in patients with cancer in other settings.

Trial Registration

Current Controlled Trials ISRCTN 42944026 www.controlled-trials.com/ISRCTN42944026     

Phytochemical Diversity of Murraya koenigii (L.) Spreng. from Western Himalaya

Murraya koenigii (L.) Spreng. (Rutaceae), commonly known as ‘curry leaf tree’, is a popular spice and condiment of India. To explore the diversity of the essential‐oil yield and aroma profile of curry leaf, growing wild in foot and mid hills of north India, 58 populations were collected during spring season. M. koenigii populations were found to grow up to an altitude of 1487 m in north India. Comparative results showed considerable variations in the essential‐oil yield and composition. The essential‐oil yield varied from 0.14 to 0.80% in shade‐dried leaves of different populations of M. koenigii. Analysis of the essential oils by GC and GC/MS, and the subsequent classification by statistical analysis resulted in four clusters with significant variations in their terpenoid composition. Major components of the essential oils of investigated populations were α‐pinene ( 2 ; 4.5–71.5%), sabinene ( 3 ; <0.05–66.1%), (E)‐caryophyllene ( 11 ; 1.6–18.0%), β‐pinene ( 4 ; <0.05–13.6%), terpinen‐4‐ol ( 9 ; 0.0–8.4%), γ‐terpinene ( 8 ; 0.2–7.4%), limonene ( 7 ; 1.1–5.5%), α‐terpinene ( 6 ; 0.0–4.5%), (E)‐nerolidol ( 14 ; 0.0–4.1%), α‐humulene ( 12 ; 0.6–3.5%), α‐thujene ( 1 ; 0.0–2.5%), β‐elemene ( 10 ; 0.2–2.4%), β‐selinene ( 13 ; 0.2–2.3%), and myrcene ( 5 ; 0.5–2.1%). Comparison of the present results with those in earlier reports revealed new chemotypes of M. koenigii in investigated populations from Western Himalaya. The present study documents M. koenigii populations having higher amounts of sabinene ( 3 ; up to 66.1%) for the first time.     

Construction of an amperometric TG biosensor based on AuPPy nanocomposite and poly (indole-5-carboxylic acid) modified Au electrode

A method is described for construction of an amperometric triglyceride (TG) biosensor based on covalent co-immobilization of lipase, glycerol kinase and glycerol-3-phosphate oxidase onto gold polypyrrole nanocomposite decorated poly indole-5-carboxylic acid electrodeposited on the surface of a gold electrode. The enzyme electrode was characterized by transmission electron microscopy, scanning electron microscopy, electrochemical impedance studies, Fourier transform infrared spectroscopy and cyclic voltammetry. Biosensor showed optimum response within 4 s at pH 6.5 and 35 °C, when polarized at +0.1 V against Ag/AgCl. There was a linear relationship between sensor response and triolein concentration in the range 50–700 mg/dl. Biosensor was employed for determination of TG in serum. Detection limit of the biosensor was 20 mg/dl. Biosensor was evaluated with 91–95 % recovery of added triolein in sera and 4.14 and 5.85 % within and between batch coefficients of variation, respectively. There was a good correlation (r = 0.99) between sera TG values by standard method (Enzymic colorimetric) and the present method. The biosensor was unaffected by a number of serum substances at their physiological concentration. Biosensor lost 50 % of its initial activity after its 100 uses over 7 months, when stored at 4 °C.     

In silico Platform for Prediction of N-, O- and C-Glycosites in Eukaryotic Protein Sequences

Glycosylation is one of the most abundant and an important post-translational modification of proteins. Glycosylated proteins (glycoproteins) are involved in various cellular biological functions like protein folding, cell-cell interactions, cell recognition and host-pathogen interactions. A large number of eukaryotic glycoproteins also have therapeutic and potential technology applications. Therefore, characterization and analysis of glycosites (glycosylated residues) in these proteins is of great interest to biologists. In order to cater these needs a number of in silico tools have been developed over the years, however, a need to get even better prediction tools remains. Therefore, in this study we have developed a new webserver GlycoEP for more accurate prediction of N-linked, O-linked and C-linked glycosites in eukaryotic glycoproteins using two larger datasets, namely, standard and advanced datasets. In case of standard datasets no two glycosylated proteins are more similar than 40%; advanced datasets are highly non-redundant where no two glycosites’ patterns (as defined in methods) have more than 60% similarity. Further, based on our results with several algorihtms developed using different machine-learning techniques, we found Support Vector Machine (SVM) as optimum tool to develop glycosite prediction models. Accordingly, using our more stringent and non-redundant advanced datasets, the SVM based models developed in this study achieved a prediction accuracy of 84.26%, 86.87% and 91.43% with corresponding MCC of 0.54, 0.20 and 0.78, for N-, O- and C-linked glycosites, respectively. The best performing models trained on advanced datasets were then implemented as a user-friendly web server GlycoEP (http://www.imtech.res.in/raghava/glycoep/). Additionally, this server provides prediction models developed on standard datasets and allows users to scan sequons in input protein sequences.     

MicroRNA Profiling in Prostate Cancer - The Diagnostic Potential of Urinary miR-205 and miR-214

Prostate cancer (PCa) is the most common type of cancer in men in the United States, which disproportionately affects African American descents. While metastasis is the most common cause of death among PCa patients, no specific markers have been assigned to severity and ethnic biasness of the disease. MicroRNAs represent a promising new class of biomarkers owing to their inherent stability and resilience. In the present study, we investigated potential miRNAs that can be used as biomarkers and/or therapeutic targets and can provide insight into the severity and ethnic biasness of PCa. PCR array was performed in FFPE PCa tissues (5 Caucasian American and 5 African American) and selected differentially expressed miRNAs were validated by qRT-PCR, in 40 (15 CA and 25 AA) paired PCa and adjacent normal tissues. Significantly deregulated miRNAs were also analyzed in urine samples to explore their potential as non-invasive biomarker for PCa. Out of 8 miRNAs selected for validation from PCR array data, miR-205 (p<0.0001), mir-214 (p<0.0001), miR-221(p<0.001) and miR-99b (p<0.0001) were significantly downregulated in PCa tissues. ROC curve shows that all four miRNAs successfully discriminated between PCa and adjacent normal tissues. MiR-99b showed significant down regulation (p<0.01) in AA PCa tissues as compared to CA PCa tissues and might be related to the aggressiveness associated with AA population. In urine, miR-205 (p<0.05) and miR-214 (p<0.05) were significantly downregulated in PCa patients and can discriminate PCa patients from healthy individuals with 89% sensitivity and 80% specificity. In conclusion, present study showed that miR-205 and miR-214 are downregulated in PCa and may serve as potential non-invasive molecular biomarker for PCa.     

NO and ROS implications in the organization of root system architecture

Over the past decades the role of nitric oxide (NO) and reactive oxygen species (ROS) in signaling and cellular responses to stress has witnessed an exponential trend line. Despite advances in the subject, our knowledge of the role of NO and ROS as regulators of stress and plant growth and their implication in signaling pathways is still partial. The crosstalk between NO and ROS during root formation offers new domains to be explored, as it regulates several plant functions. Previous findings indicate that plants utilize these signaling molecules for regulating physiological responses and development. Depending upon cellular concentration, NO either can stimulate or impede root system architecture (RSA) by modulating enzymes through post-translational modifications. Similarly, the ROS signaling molecule network, in association with other hormonal signaling pathways, control the RSA. The spatial regulation of ROS controls cell growth and ROS determine primary root and act in concert with NO to promote lateral root primordia. NO and ROS are two central messenger molecules which act differentially to upregulate or downregulate the expression of genes pertaining to auxin synthesis and to the configuration of root architecture. The investigation concerning the contribution of donors and inhibitors of NO and ROS can further aid in deciphering their role in root development. With this background, this review provides comprehensive details about the effect and function of NO and ROS in the development of RSA.     

Execution of programmed cell death by singlet oxygen generated inside the chloroplasts of Arabidopsis thaliana

Protoplasma - Absorption of excess excitation energy induces overproduction of singlet oxygen (1O2) in plants. The major sources of singlet oxygen production are chlorophyll and its intermediates...