Proteomic analysis reveals a role of ADP-glucose pyrophosphorylase in the asynchronous filling of rice superior and inferior spikelets.

The poor grain filling of inferior spikelets (IS) situated on the lower secondary rachis branch leads to a remarkable decrease in rice yield and quality. The AGPase small subunit 2 (AGPS2) encodes a small subunit of adenosine diphosphate-glucose pyrophosphorylase (AGPase) enzyme, which plays an important role in sucrose-starch conversion and starch biosynthesis in the grain filling of rice. In the present study, qPCR analysis showed low expression abundance of AGPS2 in IS, compared to the superior spikelets (SS), which was consistent with the lower grain weight of IS. To evaluate the molecular mechanism of AGPS2, we first identified the AGPS2 interaction network through Co-immunoprecipitation (Co-IP). In total, 29 proteins of AGPS2 interaction network were characterized by LC-MS/MS. Bioinformatics analysis revealed that, the characterized proteins in the interaction network are likely to be related to starch synthesis, sugar conversion, energy pathway, and folding/modification, and most of them were involved in the grain filling of rice. The sequent Co-IP analysis showed that AGPS2 can bind to starch branching enzyme (SBE), pullulanase (PUL) and starch debranching enzyme (DBE) and assemble into starch synthesizing protein complex (SSPC). In addition, the 14-3-3 protein GF14e was also found to interact with AGPS2. Further analysis by qPCR showed that the expression of GF14e was much higher on IS than on SS. The qPCR results also showed that the expression of GF14e was relatively stable in SS, but changed significantly in IS under alternate wetting and moderate soil drying (WMD), which is consistent with the AGPS2 expression pattern. Our present work provides direct molecular evidence for the different expression patterns of AGPS2 in SS and IS, which could be greatly helpful for the molecular amelioration of the poor grain filling of IS in rice.

Identification of phenylcarbamoylazinane-1,3,4-oxadiazole amides as lipoxygenase inhibitors with expression analysis and in silico studies.

In search for new anti-inflammatory agents that inhibit the enzymes of arachidonic acid pathway as the drug targets, the present article describes the screening of 1,3,4-oxadiazole analogues against lipoxygenase (LOX) enzyme. The work is based on the synthesis of new N-alkyl/aralky/aryl derivatives (6a-o) of 2-(4-phenyl-5-(1-phenylcarbamoylpiperidine)-4H-1,3,4-oxadiazol-3-ylthio)acetamide which were obtained by the reaction of 1,3,4-oxadiazole (3) with various electrophiles (5a-o), in KOH. The synthesized analogues showed potent to moderate inhibitory activity against the soybean 15-LOX enzyme; especially 6g, 6b, 6a and 6l displayed the potent inhibitory potential with IC50 values 7.15 ± 0.26, 9.32 ± 0.42, 15.83 ± 0.45 & 18.37 ± 0.53 µM, respectively, while excellent to moderate inhibitory profiles with IC50 values in the range of 26.13-98.21 µM were observed from the compounds 6k, 6m, 6j, 6o, 6h, 6f, 6n and 6c. Most of the active compounds exhibited considerable cell viability against blood mononuclear cells (MNCs) at 0.25 mM by MTT assay except 6f, 6h, 6k and 6m which showed around 50% cell viability. Flow cytometry studies of the selected compounds 6a, 6j and 6n revealed that these caused 79.5-88.51% early apoptotic changes in MNCs compared with 4.26% for control quercetin at their respective IC50 values. The relative expression of 5-LOX gene was monitored in MNCs after treatment with these three molecules and all down-regulated the enzyme activity. In silico ADME and molecular docking studies further supported these studies of oxadiazole derivatives and considered it as potential 'lead' compounds in drug discovery and development.

Exploring phenylcarbamoylazinane-1,2,4-triazole thioethers as lipoxygenase inhibitors supported with in vitro, in silico and cytotoxic studies.

Searching small molecules as an auspicious approach to develop new anti-inflammatory drugs is a challenge for the researchers especially by modifying active pharmacophoric groups in the targeted molecules. In the current work, a series of new S-alkyl/aralky derivatives (8a-h; 9a-h) of 2-(4-ethyl/phenyl-5-(1-phenylcarbamoylpiperidine)-4H-1,2,4-triazol-3-ylthio)ether were synthesized and assessed for their inhibitory action against the 15-lipoxygenase from soybean (15-sLOX). The basic precursor ethyl piperidine-4-carboxylate (a) was consecutively changed into phenylcarbamoyl derivative (1), hydrazide (2), semicarbazides (3/4) and N-ethyl/phenyl-5-(1-phenylcarbamoylpiperidine)-1,2,4-triazoles (5/6), which further in association with electrophiles (7a-h) promoted to the final products (8a-h; 9a-h). The synthesized derivatives were characterized by FT-IR, 1H-, 13C NMR spectroscopy, EI-MS, and HR-EI-MS spectrometry. Amongst these, 8a, 8c, and 9c, expressed potent inhibitory profiles against the 15-sLOX enzyme with IC50 values of 12.52 ± 0.35 to 35.64 ± 0.29 µM, followed by the compounds 9b, 9g, 9d, 9a, 8b, 8e, 8d, 8g, 8h, 8f and 9h with IC50 values in the range of 43.78 ± 0.43 to 108.65 ± 0.38 µM. All compounds exhibited variable cellular viability levels by MTT assay. Flow cytometric data demonstrated that 8f, 8g, 8h have maximal lymphocyte cellular viability and all compounds affected cells in the late apoptosis phase. In silico ADMET studies supported the drug-likeness of most of the molecules. These studies were supported by molecular docking against 15-sLOX, human 5-LOX (5-hLOX) and human 15-LOX (5-hLOX); that inhibitors of 15-sLOX docked-in the active pocket of either 5-hLOX or 15-hLOX and docking score remained constant for all three enzymes within a narrow range (-6.8 to -9.7) as did it for standard quercetin (-8.4 to -9.0). The most dominant bonding interactions were π-π, π-anion, and π-alkyl type along with the hydrogen bonding. The data collected altogether demonstrates the better possibility of some of these compounds as good LOX inhibitors in search for 'lead' as anti-inflammatory agents in the process of drug discovery and development.

Design and syntheses of 7-nitro-2-aryl-4H-benzo[d][1,3]oxazin-4-ones as potent anticancer and antioxidant agents.

A group of new nitro substituted benzoxazinones (3a-k) were synthesized from easily available 4-nitroanthranilic acid. All the synthesized compounds were characterized by FT-IR, 1H NMR, 13C NMR, mass spectrometry and elemental analysis. Anti-proliferative and pro-apoptotic potential of all the synthesized compounds (3a-k) was evaluated by MTT and Hoechst 33258 staining assay respectively whereas their antioxidant properties were determined via DPPH free radical scavenging assay. The most active compounds (3a, 3c and 3k) showed significant cytotoxic potential against HeLa cells with an inhibition of cell viability that ranged between 28.54 and 44.67% (P < 0.001). Albeit statistically different, the anti-proliferative effect of 3c was in close match with that of the reference drug doxorubicin. Likewise, the test compounds showed profound pro-apoptotic potential with an apoptotic index that ranged between 52.86 and 75.61%. Besides, the docking studies revealed a higher efficiency for compounds (3a and 3h) owing to their better affinity and inhibition constant (Ki = 4.397 and 3.713 nmol) respectively. The antioxidant potential of synthesized benzoxazinones (3a-k) was in close agreement with the experimental anticancer results with a percent inhibition from 34.45 to 85.93% as compared to standard (90.56%).

Exploring the Potential of Overexpressed OsCIPK2 Rice as a Nitrogen Utilization Efficient Crop and Analysis of Its Associated Rhizo-Compartmental Microbial Communities.

Nitrogen (N) is one of the indispensable factors in rice growth and development. China holds a premier position in the production of rice and at the same time also faces higher N fertilizer costs along with serious damage to the environment. A better solution is much needed to address these issues, without disrupting the production of rice as an important cereal, while minimizing all the deleterious effects on the environment. Two isogenic lines Kitaake (WT) and its genetically modified line CIPK2 (RC), overexpressing the gene for Calcineurin B-like interacting protein kinase 2 (OsCIPK2) with better nitrogen use efficiency (NUE), were compared for their growth and development under low versus normal levels of N. NUE is a complex trait mainly related to a plant's efficiency in extraction, assimilation, and recycling of N from soil. The microbial population was analyzed using high-throughput Illumina Miseq 16S rRNA sequencing and found that RC with CIPK2, specifically expressed in rice root, not only performed better without nitrogen fertilizer (LN) but also increased the diversity of bacterial communities in rice rhizosphere compartments (rhizosphere, rhizoplane, and endosphere). The relative abundance of beneficial bacteria phyla increased, which are known to promote the circulation and transformation of N in rhizosphere soil. To further explore the potential of RC regarding better performance under LN, the ion fluxes in root apical were detected by non-invasive micro-test technique (NMT). We found that RC can absorb more Ca2+ and NO3- under LN as compared to WT. Finally, compared to WT, RC plants exhibited better growth of root and shoot, and increased yield and N uptake under LN, whereas there was no significant difference in the growth of two rice lines under normal nitrogen (NN) treatment. We are able to get preliminary results, dealing with the OsCIPK2 overexpressed rice line, by studying the rice molecular, physiological, and chemical parameters related to NUE. The results laid the foundation for further research on N absorption and utilization in rice from the soil and the interaction with microbial communities.

Protein Phosphatase (PP2C9) Induces Protein Expression Differentially to Mediate Nitrogen Utilization Efficiency in Rice under Nitrogen-Deficient Condition.

Nitrogen (N) is an essential element usually limiting in plant growth and a basic factor for increasing the input cost in agriculture. To ensure the food security and environmental sustainability it is urgently required to manage the N fertilizer. The identification or development of genotypes with high nitrogen utilization efficiency (NUE) which can grow efficiently and sustain yield in low N conditions is a possible solution. In this study, two isogenic rice genotypes i.e., wild-type rice kitaake and its transgenic line PP2C9TL overexpressed protein phosphatase gene (PP2C9) were used for comparative proteomics analysis at control and low level of N to identify specific proteins and encoding genes related to high NUE. 2D gel electrophoresis was used to perform the differential proteome analysis. In the leaf proteome, 30 protein spots were differentially expressed between the two isogenic lines under low N level which were involved in the process of energy, photosynthesis, N metabolism, signaling, and defense mechanisms. In addition, we have found that protein phosphatase enhances nitrate reductase activation by downregulation of SnRK1 and 14-3-3 proteins. Furthermore, we showed that PP2C9TL exhibits higher NUE than WT due to higher activity of nitrate reductase. This study provides new insights on the rice proteome which would be useful in the development of new strategies to increase NUE in cereal crops.

Cancer and non-cancer risk assessment of trihalomethanes in urban drinking water supplies of Pakistan.

This study aims at monitoring and risk assessment of trihalomethanes (THMs) such as chloroform, bromodichloromethane, dibromochloromethane and bromoform, in the drinking water supplies of Rawalpindi and Islamabad. THMs were monitored at twenty locations in these twin cities using solid phase micro extraction-gas chromatography (SPME-GC). Total concentration of THMs was ranged between 21 and 373µg/L, whereas both cities had an average total THMs concentration of 142 and 260µg/L, respectively. Chloroform was found as one the major contributor to the THMs concentration (>85%). The occurrence of THMs followed the given order: chloroform, bromodichloromethane>dibromochloromethane>bromoform. Lifetime cancer risk assessment of THMs was carried out using prediction models via different exposure routes (ingestion, inhalation and dermal). An average lifetime cancer risk was found to be 0.74×10(-4) and 1.24×10(-4) for Rawalpindi and Islamabad, respectively. The number of expected cancer cases per year could reach two cases for each city. Hazard index values were found below unity for both the cities implying that there would be no considerable non-cancer risk. Oral ingestion was found to be one of the main routes of exposure for both types of risk which was followed by inhalation and dermal routes.

Performance Evaluation of UF Membranes Derived from Recycled RO Membrane, a Step towards Circular Economy in Desalination.

Reverse osmosis (RO) spiral wound membrane generation reached 93.5% in 2020, resulting in 14,000 tons of used RO membranes being discarded annually into landfills, which is unprecedented. The current study aims to chemically convert the end-of-life RO membrane, followed by its performance evaluation and microbial removal efficiency on three different sources of water, i.e., tap water (TW), integrated constructed wetland permeate (ICW-P), and membrane bio-rector permeate (MBR-P), respectively. This was accomplished by selecting 6 years of spent Filmtech (LC-LE-4040) thin film composite type brackish water reverse osmosis (BWRO) membrane, followed by alkaline and acidic cleaning for 2 h. Finally, the conversion was carried out by 6% sodium hypochlorite (NaOCl) with 300,000 ppm/h exposure by active system (AS) using the clean in place CIP pump at 2 bars for 10 h duration. The membrane demonstrated 67% water recovery and 1% saltwater rejection, which means RO membrane now converted into recycled RO (R-RO) or (UF) by removal of the polyamide (PA) layer. Water recovery was 67% for TW, 68% for ICW-P, and 74% for MBR-P, respectively, with the consistent saltwater rejection rate of 1% being observed, while R-RO exhibited an effective COD removal of 65.79%, 62.96%, and 67.72% in TW, ICW-P, and MBR-P, respectively. The highest turbidity removal of 96% in the ICW-P was also recorded for R-RO. For morphological properties, SEM analysis of the R-RO membrane revealed a likewise appearance as a UF membrane, while pore size is also comparable with the UF membrane. The most probable number (MPN) also showed complete removal of total coliforms after passing through the R-RO membrane. These features made the R-RO membrane an excellent choice for drinking water treatment and wastewater treatment polishing steps. This solution can help developing nations to be efficient in resource recovery and contribute to the circular economy.

Transient osteoporosis of the hip.

Transient Osteoporosis of Hip (TOH) is an uncommon disorder of idiopathic nature, particularly in the Asian population. It has been described to mostly occur in middle aged men and women in their third trimester of pregnancy. A distinctive hallmark of this condition is that it is self limiting and resolves in a few months. The patient presents to the physician with pain on movement and impaired mobility of the affected joint, developing without any history of trauma. MRI is the main diagnostic tool. We report herein a case of a forty five year old male, who developed transient osteoporosis of the hip, and was managed conservatively.

Current understanding of treatment and management protocol for adult diabetic in-patients at a tertiary care hospital.

OBJECTIVE: To assess the current understanding of treatment and management protocols for adult diabetic inpatients at a tertiary care hospital. METHODS: This cross-sectional study, conducted at the Civil Hospital Karachi from July to September 2009, involved 450 participants, who were interviewed through a well-structured questionnaire regarding the patient's demography, clinical features, past medical history, type of diabetes mellitus, duration, associated complications, and also involved patient notes for laboratory tests and management. SPSSv15.0 was used for descriptive analysis. RESULTS: The study population of 450 diabetics had 144 (32%) males and 306 (68%) females. Of the total, 435 (96.7%) patients had type 2 diabetes. There were 231 (51%) patients using insulin, 168 (37.3%) oral hypoglycaemic drugs, and 51 (11.3%) using both. Among patients using insulin, regular insulin usage stood at 30% followed by a combination of regular insulin and NPH (26.7%) and NPH alone at 6%. The most popular drug used was metformin (27.3%) and the least used drug was glitazones (4%). In the study population, 73.3% patients controlled their diabetes with diet, and 24.7% with regular exercise. CONCLUSION: Majority of the study population had type 2 diabetes with a female preponderance. Insulin was prescribed for half the patients. Metformin was the most frequently used oral hypoglycaemic drug.

The role of organic acids on microbial deterioration in the Radix pseudostellariae rhizosphere under continuous monoculture regimes.

A three-year field monoculture trial of Radix pseudostellariae and complementary laboratory studies were conducted to further elucidate the underlying mechanism responsible for significant decreases in the biomass yield and quality of R. pseudostellariae under continuous monoculture regimes. HPLC analysis indicated that continuous monoculture soil was rich in organic acids, which had cumulative effects over time. Further analysis suggested that the application of a mixture of organic acids significantly promoted growth of pathogenic fungi, and increased the expression of chemotaxis-related gene (cheA) and biofilm formation of the specific pathogenic Kosakonia sacchari. However, opposite reactions were observed in the case of Bacillus megaterium and Bacillus pumilus. Concurrently, the present results revealed that the mixed organic acids stimulated the production of toxins, as well as H2O2 in the pathogenic fungi. Furthermore, the presence of organic acids reflecting environmental conditions under monocropping had negative effects on the expression of the biocontrol-related genes, which resulted in attenuated antagonistic activities of plant growth-promoting rhizobacteria (PGPR) to suppress mycelial growth of the pathogenic fungi. These results help to unveil the mechanisms associated with how accumulated organic acids differentially mediate deterioration of soil microbial composition and structure in monocropping system.