Response of pipeline Nepalese maize hybrids to different doses of chemical fertilizers under varying plant densities

Low plant population and inadequate fertilizer application are constraints for maize production among farmers in Nepal. In order to identify the integrated effects of plant density and fertilizer dose for production of hybrid maize, series of on station experiments were carried out at Rampur, Chitwan, Nepal using strip plot design with three replications during winter seasons in two consecutive years 2016/17 and 2017/18. Two promising Nepalese hybrids namely RML-95/RML-96 and RML-86/RML-96 were taken as vertical factor whereas five densities viz. 55,555 ha-1(D1), 66,666 ha-1 (D2), 77,519 ha-1 (D3), 87,719 ha-1 (D4) and 98,039 ha-1 (D5) in combination with two doses of NPK fertilizers i.e. recommended and rational as horizontal factor. Recommended dose of NPK for maize was 120:60:40 kg ha-1 and the rational doses for above mentioned densities were calculated based on bench mark of fertilizer recommendation for 53,333 plants ha-1. Derived rational doses of NPK for D1, D2, D3, D4 and D5 were 125:63:42, 150:75:50, 174:87:58, 197:99:66, 221:110:74 kg ha-1 respectively. Genotypic variation of two tested hybrids for growth, yield attributes and grain yield were found statistically similar. However, planting the hybrids in various densities and fertilizer doses significantly influenced grain yield ranging from 5.63 to 7.91 t ha-1. Significantly higher grain yield (7.91 t ha-1) and benefit cost ratio (2.21) were observed when plant population was maintained at 77,519 ha-1 with fertilizer dose 174:87:58 NPK kg ha-1.  Applying fertilizer as rational dose enhanced the yield up to the density of 77519 plants ha-1 then the yield gradually declined as the density increased. Whereas recommended dose of fertilizer with increasing plant density from 55,555 to 87,719 ha-1 had statistically similar yield and declined more at 98039 ha-1.  The present recommended dose of fertilizer application is inadequate for higher plant density to increase the grain yield of hybrid maiz

Kinetic Isotope Effects and Hydrogen/Deuterium Exchange Reveal Large Conformational Changes During the Catalysis of the Clostridium acetobutylicum Spore Photoproduct Lyas

Spore photoproduct lyase (SPL) catalyzes the direct reversal of a thymine dimer 5-thyminyl-5,6-dihydrothymine (i.e. the spore photoproduct (SP)) to two thymine residues in germinating endospores. Previous studies suggest that SPL from the bacterium Bacillus subtilis (Bs) harbors an unprecedented radical-transfer pathway starting with cysteine 141 proceeding through tyrosine 99. However, in SPL from the bacterium Clostridium acetobutylicum (Ca), the cysteine (at position 74) and the tyrosine are located on the opposite sides of a substrate-binding pocket that has to collapse to bring the two residues into proximity, enabling the C→Y radical passage as implied in SPL(Bs) . To test this hypothesis, we adopted hydrogen/deuterium exchange mass spectrometry (HDX-MS) to show that C74(Ca) is located at a highly flexible region. The repair of dinucleotide SP TpT by SPL(Ca) is eight-fold to 10-fold slower than that by SPL(Bs) ; the process also generates a large portion of the aborted product TpTSO2- . SPL(Ca) exhibits apparent (D V) kinetic isotope effects (KIEs) of ~6 and abnormally large competitive (D V/K) KIEs (~20), both of which are much larger than the KIEs observed for SPL(Bs) . All these observations indicate that SPL(Ca) possesses a flexible active site and readily undergoes conformational changes during catalysis

Epitope mapping of Japanese encephalitis virus neutralizing antibodies by native mass spectrometry and hydrogen/deuterium exchange

Japanese encephalitis virus (JEV) remains a global public health concern due to its epidemiological distribution and the existence of multiple strains. Neutralizing antibodies against this infection have shown efficacy in in vivo studies. Thus, elucidation of the epitopes of neutralizing antibodies can aid in the design and development of effective vaccines against different strains of JEV. Here, we describe a combination of native mass spectrometry (native-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) to complete screening of eight mouse monoclonal antibodies (MAbs) against JEV E-DIII to identify epitope regions. Native-MS was used as a first pass to identify the antibodies that formed a complex with the target antigen, and it revealed that seven of the eight monoclonal antibodies underwent binding. Native mass spectra of a MAb (JEV-27) known to be non-binding showed broad native-MS peaks and poor signal, suggesting the protein is a mixture or that there are impurities in the sample. We followed native-MS with HDX-MS to locate the binding sites for several of the complex-forming antibodies. This combination of two mass spectrometry-based approaches should be generally applicable and particularly suitable for screening of antigen-antibody and other protein-protein interactions when other traditional approaches give unclear results or are difficult, unavailable, or need to be validated

FINE NEEDLE ASPIRATION CYTOLOGY OF BREAST LUMP IN T.U. TEACHING HOSPITAL

This is a retrospective study of fine needle aspiration cytology of breast lesions performed between May 1998 and April 2000. During this period, a total of 2001 fine needle aspirations were done in the Tribhuvan Universtiy Teaching Hospital (TUTH), of which 470 (23.48 %) were of the breast. Fibrocystic disease found to be the most frequent and was diagnosed in 183 cases (38.9%). Fibroadenoma was the third common pathological condition, numbering 61 (12.9%). Malignant conditions of the breast were 72 cases (15.3%). During these two years, histological diagnosis was available in 154 cases. Histological examination revealed benign conditions in 74 cases (48%), malignancy in 59 cases (38.3%) and inflammatory & lactational changes in 21 cases (13.7%). Out of these 154 cases, 71 cases had also undergone fine needle aspiration cytology. The 36 cases diagnosed as benign cytologically, 34 cases were also benign histologically, whereas 2 cases turned out to be malignant. Out of 37 cases that were histologically malignant, on cytology 35 cases were diagnosed as malignant. The sensitivity and specificity of cytopathological diagnosis for breast lesions was 100% and 94.6% respectively. Inflammatory lesions correlated well cytologically and histolgically. FNAC is a safe and rapid diagnostic method for evaluation of various lesions. However, there are possibilities of false negative and false positive results because of wide range of appearance of breast lesions. Key Words: Breast lumps, Fine Needle Aspiration Cytology, Benign Lesions, Malignant Lesions, Histopathology and Correlation

Global Analysis of Protein Folding Thermodynamics for Disease State Characterization and Biomarker Discovery

<p>Protein biomarkers can facilitate the diagnosis of many diseases such as cancer and they can be important for the development of effective therapeutic interventions. Current large-scale biomarker discovery and disease state characterization studies have largely focused on the global analysis of gene and protein expression levels, which are not directly tied to function. Moreover, functionally significant proteins with similar expression levels go undetected in the current paradigm of using gene and protein expression level analyses for protein biomarker discovery. Protein-ligand interactions play an important role in biological processes. A number of diseases such as cancer are reported to have altered protein interaction networks. Current understanding of biophysical properties and consequences of altered protein interaction network in disease state is limited due to the lack of reproducible and high-throughput methods to make such measurements. Thermodynamic stability measurements can report on a wide range of biologically significant phenomena (e.g., point mutations, post-translational modifications, and new or altered binding interactions with cellular ligands) associated with proteins in different disease states. Investigated here is the use of thermodynamic stability measurements to probe the altered interaction networks and functions of proteins in disease states. This thesis outlines the development and application of mass spectrometry based methods for making proteome-wide thermodynamic measurements of protein stability in multifactorial complex diseases such as cancer. Initial work involved the development of SILAC-SPROX and SILAC-PP approaches for thermodynamic stability measurements in proof-of-concept studies with two test ligands, CsA and a non-hydrolyzable adenosine triphosphate (ATP) analogue, adenylyl imidodiphosphate (AMP-PNP). In these proof-of-principle studies, known direct binding target of CsA, cyclophilin A, was successfully identified and quantified. Similarly a number of known and previously unknown ATP binding proteins were also detected and quantified using these SILAC-based energetics approaches. </p><p>Subsequent studies in this thesis involved thermodynamic stability measurements of proteins in the breast cancer cell line models to differentiate disease states. Using the SILAC-SPROX, ~800 proteins were assayed for changes in their protein folding behavior in three different cell line models of breast cancer including the MCF-10A, MCF-7, and MDA-MB-231 cell lines. Approximately, 10-12% of the assayed proteins in the comparative analyses performed here exhibited differential stability in cell lysates prepared from the different cell lines. Thermodynamic profiling differences of 28 proteins identified with SILAC-SPROX strategy in MCF-10A versus MCF-7 cell line comparison were also confirmed with SILAC-PP technique. The thermodynamic analyses performed here enabled the non-tumorigenic MCF-10A breast cell line to be differentiated from the MCF-7 and MDA-MB-231 breast cancer cell lines. Differentiation of the less invasive MCF-7 breast cancer cell line from the more highly invasive MDA-MB-231 breast cancer cell line was also possible using thermodynamic stability measurements. The differentially stabilized protein hits in these studies encompassed those with a wide range of functions and protein expression levels, and they included a significant fraction (~45%) with similar expression levels in the cell line comparisons. These proteins created novel molecular signatures to differentiate the cancer cell lines studied here. Our results suggest that protein folding and stability measurements complement the current paradigm of expression level analyses for biomarker discovery and help elucidate the molecular basis of disease.</p>Dissertatio

Effect of Dexmedetomidine on Intraoperative Haemodynamics and Postoperative Analgesia in Laparoscopic Cholecystectomy

Background and objectives:Dexmedetomidine is an α2 agonist with sympatholytic, anxiolytic, sedative and analgesic effect used as adjunct during surgeries for its haemodynamic stabilizing effect and analgesic effect. Primary aims of the study were to evaluate the haemodynamic effect of intravenous dexmedetomidine and the duration and quality of analgesia in laparoscopic cholecystectomy. Secondary aims were sedation levels and occurrence of side effects.Materials and methods:Eighty four patients, American society of Anaesthesiologists physical status I and II, aged 18-60 years of either gender undergoing laparoscopic cholecystectomy in general anaesthesia were randomly allocated into two equal groups. Group C patient received Normal Saline and Group S patient received dexmedetomidine loading dose infusion of 1µg/kg over 10 minutes before induction and maintained with 0.4µg/kg/hr till the removal of gall bladder. Induction with propofol and fentanyl was done. Standard monitoring including Heart Rate, Mean arterial pressure, oxygen saturation were monitored perioperatively. Postoperative analgesia requirement and sedation score were assessed.Results: In Group S, the haemodynamic responses were significantly attenuated. During postoperative period, 24 hours analgesic requirement of diclofenac sodium was 141.43mg in group S as compared to 217.50mg in group C(p&lt;0.001). Side effects were treatable. Sedation was better in Group S.Conclusion:Dexmedetomidine effectively attenuates haemodynamic stress response during laparoscopic surgery with reduction in postoperative analgesic requirements.</p

Global Analysis of Protein Folding Thermodynamics for Disease State Characterization

Current methods for the large-scale characterization of disease states generally rely on the analysis of gene and/or protein expression levels. These existing methods fail to detect proteins with disease-related functions and unaltered expression levels. Here we describe the large-scale use of thermodynamic measurements of protein folding and stability for the characterization of disease states. Using the Stable Isotope Labeling with Amino Acids in Cell Culture and Stability of Proteins from Rates of Oxidation (SILAC-SPROX) technique, we assayed ∼800 proteins for protein folding and stability changes in three different cell culture models of breast cancer including the MCF-10A, MCF-7, and MDA-MB-231 cell lines. The thermodynamic stability profiles generated here created distinct molecular markers to differentiate the three cell lines, and a significant fraction (∼45%) of the differentially stabilized proteins did not have altered expression levels. Thus, the differential thermodynamic profiling strategy reported here created novel molecular signatures of breast cancer and provided additional insight into the molecular basis of the disease. Our results establish the utility of protein folding and stability measurements for the study of disease processes, and they suggest that such measurements may be useful for biomarker discovery in disease

Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry

Copyright © 2020 American Chemical Society. Although knowledge of the coordination chemistry and metal-withholding function of the innate immune protein human calprotectin (hCP) has broadened in recent years, understanding of its Ca2+-binding properties in solution remains incomplete. In particular, the molecular basis by which Ca2+ binding affects structure and enhances the functional properties of this remarkable transition-metal-sequestering protein has remained enigmatic. To achieve a molecular picture of how Ca2+ binding triggers hCP oligomerization, increases protease stability, and enhances antimicrobial activity, we implemented a new integrated mass spectrometry (MS)-based approach that can be readily generalized to study other protein-metal and protein-ligand interactions. Three MS-based methods (hydrogen/deuterium exchange MS kinetics; protein-ligand interactions in solution by MS, titration, and H/D exchange (PLIMSTEX); and native MS) provided a comprehensive analysis of Ca2+ binding and oligomerization to hCP without modifying the protein in any way. Integration of these methods allowed us to (i) observe the four regions of hCP that serve as Ca2+-binding sites, (ii) determine the binding stoichiometry to be four Ca2+ per CP heterodimer and eight Ca2+ per CP heterotetramer, (iii) establish the protein-to-Ca2+ molar ratio that causes the dimer-to-tetramer transition, and (iv) calculate the binding affinities associated with the four Ca2+-binding sites per heterodimer. These quantitative results support a model in which hCP exists in its heterodimeric form and is at most half-bound to Ca2+ in the cytoplasm of resting cells. With release into the extracellular space, hCP encounters elevated Ca2+ concentrations and binds more Ca2+ ions, forming a heterotetramer that is poised to compete with microbial pathogens for essential metal nutrients

Near-atomic structure of a giant virus

Although the nucleocytoplasmic large DNA viruses (NCLDVs) are one of the largest group of viruses that infect many eukaryotic hosts, the near-atomic resolution structures of these viruses have remained unknown. Here we describe a 3.5 Å resolution icosahedrally averaged capsid structure of Paramecium bursaria chlorella virus 1 (PBCV-1). This structure consists of 5040 copies of the major capsid protein, 60 copies of the penton protein and 1800 minor capsid proteins of which there are 13 different types. The minor capsid proteins form a hexagonal network below the outer capsid shell, stabilizing the capsid by binding neighboring capsomers together. The size of the viral capsid is determined by a tape-measure, minor capsid protein of which there are 60 copies in the virion. Homologs of the tape-measure protein and some of the other minor capsid proteins exist in other NCLDVs. Thus, a similar capsid assembly pathway might be used by other NCLDVs