1,4,9,9-tetramethyloctahydro-4,7-(epoxymethano)azulen-5(1H)-one, a natural product as a potential inhibitor of COVID-19: Extraction, crystal structure, and virtual screening approach.

In the present work, we describe the extraction of a natural product namely 1,4,9,9-tetramethyloctahydro-4,7-(epoxymethano)azulen-5(1H)-one, and its structure was confirmed by single crystal X-ray diffraction analysis. The conformations of the 5-, 6-, and 7-membered rings in the title compound, C15H24O2, have been probed by a Cremer-Pople puckering analysis. C-H···O hydrogen bonds generate chains in the crystal that stretch along the c-axis direction. The Hirshfeld surface analysis method was used to stabilize the crystal packing of the natural compound. Accompanied by experimental studies, quantum chemical calculations were also performed to compare the structural elucidation and the results of these geometrical parameters exhibited excellent agreement. The compound was also docked with several drug targets of the SARS-CoV-2 virus and found to show the best binding with the main protease enzyme, having a binding energy of -12.31 kcal/mol and interacting with His41 and Cys145 residues. The dynamic stability deciphered the complex to be stable with an average RMSD of 3.8 Å. The compound dynamics with the enzyme showed the compound conformation to be highly stable. The intermolecular binding free energy determined the compound-main protease enzyme to show high interaction energy of < 40 kcal/mol. Together, these studies demonstrate the compound to be a lead structure against SARS-CoV-2.

Pattern of infectious diseases among Khalwa (Quran boarding school) students in Khartoum State, Sudan.

Khalawi are one of the most ancient non-governmental educational institutions that are commonly found all over Sudan playing a major role in the Sudanese society. The majority of Khalwa students are children. This study was part of a mixed methods project, conducted by Khartoum Medical Students' Association, aiming to investigate the health status of Khalwa students. It is a cross-sectional study carried out in Mustafa Alfadni Khalwa, Sharg Al-Neel suburb in Khartoum State. The participants were clinically assessed by a doctor and data were collected by using a data entry tool made specially to screen for major infectious diseases among students. The results showed a mean age of 13.7 years. Most of the students (47.7%) were originally from Darfur, and 27.5% of them had only Khalwa education. Respiratory clinic results revealed that 10% were complaining of cough, and 3.4% were diagnosed with pneumonia. Additionally, 44.6% of the students had tinea capitis and 21% had scabies. On examination, 2.3% and 1.1% had mild splenomegaly and hepatomegaly, respectively. Infectious conjunctivitis and trachoma were reported among 11.7% and 10.9%, respectively. Blood films for malaria were positive in 51.3%. Macroscopic and microscopic haematuria was evident among 13.4% and 10.8% of the participants, respectively. Stool examination results showed that 1.4% of the students had worms, 1.5% had ova and 18.8% had red blood cells in stools. Infectious diseases are very common among Khalwa students and their living environment is facilitating the transmission of these diseases. Agent control and sanitation improvement are crucial to decrease such infectious diseases.

Studying the Influence of Copper on the Growth Behavior, Antioxidative Status, and Histology of the Intestine and Liver of Striped Catfish (Pangasianodon hypophthalmus).

Microelements are well recognized as an essential approach in the field of aquaculture nutrition. Thus, this study aimed to evaluate copper (Cu) inclusion (0, 0.5, 1, and 2 mg/kg) on Striped catfish performances. Fish fed the Cu-incorporated diets for 60 days, then their growth behavior, antioxidative capacity, and intestinal and liver histological features were evaluated. The results showed a marked enhancement in Striped catfish's growth behavior fed 1-2 mg/kg of Cu, as shown by the final weight, weight gain, and specific growth rate. The feed and protein efficiency ratios were significantly affected by Cu in a dose-dependent manner. The highest level of Cu was accumulated in the whole body, muscle, liver, and gills of fish fed 2 mg/kg of Cu. The carcass composition of Striped catfish showed higher protein content in groups received 0.5, 1, and 2 mg/kg Cu in a linear and quadratic manner (p=0.001). The ash content was quadratically increased in Striped catfish fed 2 mg/kg Cu (p=0.001). However, no marked effects were observed on the moisture and lipid contents and the somatic indices (p>0.05). The incorporation of Cu showed meaningfully increased superoxide dismutase, catalase, and glutathione peroxidase but decreased malondialdehyde level in Striped catfish. The villous height exhibited visible growth and branching with increased doses of Cu without a significant increase in the goblet cells. No abnormal features were observed in the liver and hepatocytes of fish treated with Cu. It can be concluded that Cu is required at 1-2 mg/kg for better performances of Striped catfish.

Physiological Roles of Red Carrot Methanolic Extract and Vitamin E to Abrogate Cadmium-Induced Oxidative Challenge and Apoptosis in Rat Testes: Involvement of the Bax/Bcl-2 Ratio.

The precise analysis of the contents of the red carrot is still ambiguous and its role in the maintenance of male fertility needs to be further reconnoitered. Hence, this study targets the physiological impacts of either red carrot methanolic extract (RCME) or vitamin E (Vit. E), co-administrated with cadmium chloride (CdCl2) on rat testes, specifically those concerned with apoptosis and oxidative challenge. Four groups of adult male rats (n = 12) are used; control, CdCl2, CdCl2 + Vit. E and CdCl2 + RCME. LC-MS analysis of RCME reveals the presence of 20 different phytochemical compounds. Our data clarify the deleterious effects of CdCl2 on testicular weights, semen quality, serum hormonal profile, oxidative markers and Bax/Bcl-2 ratio. Histopathological changes in testicular, prostatic and semen vesicle glandular tissues are also observed. Interestingly, our data clearly demonstrate that co-administration of either RCME or Vit. E with CdCl2 significantly succeeded in the modulation (p < 0.05) of all of these negative effects. The most striking is that they were potent enough to modulate the Bax/Bcl-2 ratio as well as having the ability to correct the impaired semen picture, oxidant status and hormonal profile. Thus, RCME and Vit. E could be used as effective prophylactic treatments to protect the male reproductive physiology against CdCl2 insult.

Hemodynamic Safety of Continuous Infusion Labetalol Versus Esmolol Combination Therapies for Type B Aortic Dissections.

BACKGROUND: Medical management for type B aortic dissections (TBADs) require aggressive blood pressure and heart rate control to minimize further dissection extension and to restore perfusion to vital organs. Current guidelines recommend ß-blockers (BB) as first-line therapy, however do not differentiate an ideal agent for use. OBJECTIVE: This study evaluated the hemodynamic safety of continuous infusion labetalol compared to esmolol combination (EC) therapies for TBADs. METHODS: This single-center, retrospective analysis identified patients with a TBAD who received high dose continuous intravenous labetalol (HD-CIVL) or EC therapies. Patients who received HD-CIVL or EC therapies for a minimum of 2 hours, during which a minimum of 4 blood-pressure readings were recorded, were included. The primary end point was the incidence of hemodynamic instability with the use of HD-CIVL versus EC therapies. RESULTS: A total of 20 patients receiving HD-CIVL and 22 patients receiving EC therapy were included in the analysis. Ten (50%) of patients receiving HD-CIVL and 7 (32%) of patients receiving EC therapies met the clinical definition of hemodynamic instability (P = .23). Patients experiencing hemodynamic instability were all due to hypotension, with one also being due to bradycardia. Over half the patients in both groups had discontinued therapy ( P = .06) and were administered bolus fluids (P = .27). Only one patient receiving HD-CIVL required vasopressor administration while none in the EC group (P = .48). CONCLUSION: Our study suggests that HD-CIVL is associated with a nonstatistical significant higher incidence of hemodynamic instability compared to an EC regimen in TBADs. Further studies are warranted in this patient population.

Pharmacokinetic Assessment of Pre- and Post-Oxygenator Vancomycin Concentrations in Extracorporeal Membrane Oxygenation: A Prospective Observational Study.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) is a form of cardiopulmonary life support frequently utilized in catastrophic lung and or cardiac failure. Patients on ECMO often receive vancomycin therapy for treatment or prophylaxis against Gram-positive organisms. It is unclear if ECMO affects vancomycin pharmacokinetics, thus we modeled the pharmacokinetic behavior of vancomycin according to ECMO-specific variables. METHODS: Adult patients receiving vancomycin and Veno-Arterial-ECMO between 12/1/2016 and 10/1/2017 were prospectively enrolled. Extracorporeal membrane oxygenation settings and four sets of pre- and post-oxygenator vancomycin concentrations were collected for each patient. Compartmental models were built and assessed ECMO flow rates on vancomycin clearance and potential circuit sequestration. Bayesian posterior concentrations of the pre- and post-oxygenator concentrations were obtained for each patient, and summary pharmacokinetic parameters were calculated. Simulations were performed from the final model for efficacy and toxicity predictions. RESULTS: Eight patients contributed 64 serum concentrations. Patients were a median (interquartile range) age of 58.5 years (50.8-62.3) with a calculated creatinine clearance of 39 mL/min (29.5-62.5) and ECMO flow rates of 3980 mL/min (interquartile range = 3493.75-4132.5). A three-compartment model best fit the data (Bayesian: plasma pre-oxygenation R2 = 0.99, post-oxygenation R2 = 0.99). Vancomycin clearance was not impacted by ECMO flow rate (p = 0.7). Simulations demonstrated that vancomycin 1 g twice daily was rarely sufficient for minimum inhibitory concentrations > 0.5 mg/L. Doses ≥ 1.5 g twice daily often exceeded toxicity thresholds for exposure. CONCLUSIONS: Extracorporeal membrane oxygenation flow rates did not influence vancomycin clearance between flow rates of 3500 and 5000 mL/min and vancomycin was not sequestered in ECMO. Common vancomycin regimens resulted in suboptimal efficacy and/or excessive toxicity. Individual therapeutic drug monitoring is recommended for patients on ECMO.

New Rare Ent-Clerodane Diterpene Peroxides from Egyptian Mountain Tea (Qourtom) and Its Chemosystem as Herbal Remedies and Phytonutrients Agents.

Genus Stachys, the largest genera of the family Lamiaceae, and its species are frequently used as herbal teas due to their essential oils. Tubers of some Stachys species are also consumed as important nutrients for humans and animals due to their carbohydrate contents. Three new neo-clerodane diterpene peroxides, named stachaegyptin F-H (1, 2, and 4), together with two known compounds, stachysperoxide (3) and stachaegyptin A (5), were isolated from Stachys aegyptiaca aerial parts. Their structures were determined using a combination of spectroscopic techniques, including HR-FAB-MS and extensive 1D and 2D NMR (1H, 13C NMR, DEPT, 1H-1H COSY, HMQC, HMBC and NOESY) analyses. Additionally, a biosynthetic pathway for the isolated compounds (1-5) was discussed. The chemotaxonomic significance of the isolated diterpenoids of S. aegyptiaca in comparison to the previous reported ones from other Stachys species was also studied.

Synthesis of Nanocomposites Layered Double Hydroxide via Ocimum basilicum and Its Acaricidal Efficacy Against Multi-Resistance Rhipicephalus annulatus Tick.

A layered double hydroxide (LDH) incorporated with Ocimum basilicum essential oil (OB) was prepared and its acaricide efficacy against Rhipicephalus annulatus tick was investigated. The OB essential oil was extracted from plants farmed in the study area. Zn-Al LDH/Gallate was prepared by co-precipitation method then OB oil was added to obtain OB/LDH nano-composite. The obtained product was characterized by X-Ray Diffraction (XRD), Fourier-transform infrared (FT-IR) and scanning electron microscopy (SEM). The acaricidal activity of different concentrations of the obtained OB/LDH nanocomposite (6.25, 12.5, 25, 50, 100, 200 and 300 l/mL) was evaluated via the adult immersion test (AIT), egg hatchability test (EHT), larval packet test (LPT) and repellency test. OB oil and OB/LDH nano-composite showed 100% adult mortality and prevent egg deposition at a dose of 300 l/mL. Also, eggs hatching was fully inhibited at 300 l/mL while at the concentration of 200 l/mL the inhibition rate was 95%. In LPT, OB/LDH showed a high toxicological effect which leading to 100% and 43% larval mortality at doses of 200 µ l/mL and 100 µl/mL, respectively. Meanwhile OB alone showed 100% and 45% larval mortality at doses of 300 µl/mL and 200 µl/mL, respectively. The LC50, LC90 and LC99 values of OB/LDH and OB were found to be 107.46 versus 209.908 µl/mL, 171.22 versus 282.63 µl/mL and 196.15 versus 298.26 µl/mL, respectively. The repellence activity was lasted for 3 h after application against larvae. OB/LDH was found to be more repellent than OB as evidenced by the RC50 values after 2 h and 3 h (48.82 vs. 79.99 µl/mL and 89.47 vs. 185.32µ l/mL, respectively). Overall, our results proved that LDH enhanced the larvaicidal and repellent efficacy of OB against R. annulatus tick larvae.

In-Hospital Outcomes with Transfemoral Versus Transapical Access for Transcatheter Aortic Valve Replacement in Patients with Peripheral Arterial Disease.

BACKGROUND: There is a paucity of data regarding outcomes with transfemoral (TF) versus transapical (TA) access for transcatheter aortic valve replacement (TAVR) in patients with peripheral artery disease (PAD). METHODS: We queried the national inpatient sample database (NIS) (2012-2013) to identify patients with PAD who underwent TAVR. We conducted a propensity matching analysis using 25 clinical variables to compare TF-TAVR versus TA-TAVR. The main outcome was in-hospital mortality. RESULTS: The analysis included 22,349 patients who underwent TAVR, among those 6692 (29.9%) had PAD. In the matched cohort, in-hospital mortality was similar between TF-TAVR and TA-TAVR groups (4.8% vs. 5.1%, OR 0.95; 95%CI 0.74-1.21). TF-TAVR was associated with lower rates of cardiogenic shock (OR 0.64; 95%CI 0.50-0.82), use of mechanical circulatory support (OR 0.56; 95%CI 0.42-0.75), acute kidney injury (OR 0.76; 95%CI 0.67-0.86), hemodialysis (OR 0.51; 95%CI 0.36-0.71), major bleeding (OR 0.72; 95%CI 0.64-0.80), blood transfusion (OR 0.65; 95%CI 0.58-0.73), discharge to a skilled nursing facility (OR 0.61; 95%CI 0.54-0.68) as well as shorter length of hospital stay (8.13 ±â€¯6.76 vs. 10.11 ±â€¯7.80 days) compared with TA-TAVR. However, TF-TAVR was associated with higher rate of vascular complications (11.7% vs. 3.7%, OR 3.40; 95%CI 2.63-4.38), complete heart block (OR 1.52; 95%CI 1.23-1.87), and pacemaker insertion (OR = 1.58; 95%CI: 1.28-1.94). There was no difference between both groups in the rate of cerebrovascular accidents (OR 1.26; 95%CI 0.93-1.72). CONCLUSION: In this observational analysis from a large national database, there was no difference in in-hospital mortality between TF-TAVR and TA-TAVR among patients with PAD. Further studies are encouraged to identify the optimal access for TAVR in patients with PAD.

The Anti-Proliferative Activity of Anisosciadone: A New Guaiane Sesquiterpene from Anisosciadium lanatum.

BACKGROUND: The increase in cancer rate and the development of resistant tumors require a continuous search for new anticancer agents. AIMS: This study aimed to analyze and identify the chemical constituents of Anisosciadium lanatum, and to investigate the antiproliferative activity of the identified constituents against various human cell lines (HepG2, MCF7, HT29, A549, and PC3) along with the possible molecular mechanisms involved. METHODS: The structure of the isolated compounds was determined by spectroscopic techniques including HRFABMS, GC-MS, IR, and 400 MHz 1D and 2D NMR analyses (1H, 13C NMR, DEPT, 1H-1H COSY, HMQC, HMBC and NOESY). The antiproliferative activity and IC50 value of the isolated compounds were measured and compared to doxorubicin. RESULTS: A new guaiane sesquiterpene containing a rare epoxide structural element, 10ß,11ß-epoxy-1α,4ß,5ß,7αΗ- guaiane-9-one, anisosciadone (1), and stigmasterol (2) have been isolated from the plant. Anisosciadone (1) showed a significant antiproliferative activity against liver, colon, and lung cells only, while stigmasterol (2) had a significant activity against liver, colon, and breast cells. Both 1 and 2 caused no cytotoxicity to normal fibroblasts. Anisosciadone elevated the expression and activity of Caspase 3 as well as p53 expression without affecting Caspase 9 in HepG2 cells. It also caused ~ 50% downregulation in cdk1 expression. CONCLUSION: Taken together, anisosciadone was specific in action against cancer cells and induced apoptosis in liver cells. It also has a unique feature by elevating the expression and activity of Caspase 3 without affecting the initiator Caspase 9. Therefore, anisosciadone deserves more investigation as a targeted therapy for cancer.

The Use of Kcentra® in the Reversal of Coagulopathy of Chronic Liver Disease.

We aim to describe our experience with the four-factor prothrombin complex concentrates (4F-PCC) Kcentra® at differing doses in patients with liver cirrhosis requiring emergent hemostasis in the setting of major or life-threatening bleeding. An automated query of patients who received Kcentra between January 2014 and March 2016 was performed. Patients who had clinically significant bleeding and received Kcentra for treatment of coagulopathy of chronic liver disease (CCLD) were included in the study. Baseline patient demographics, administration indication, pertinent laboratory values, and other reversal therapies were collected. Four patients met inclusion for analysis. One patient presented with hemopericardium, cardiac tamponade, and shock, and 3 patients presented with intracranial hemorrhage. Each patient experienced an improvement in international normalized ratio (INR) and at least a period of clinical hemostasis after Kcentra administration without complications referable to Kcentra. Kcentra may be a safe, rapid, and effective treatment option for hemorrhagic emergencies associated with CCLD. Further research is needed to determine the ideal monitoring and dosing regimen for use in CCLD.

Perioperative Calcium, Magnesium, and Phosphorus Levels in Live Donors For Liver Transplant.

OBJECTIVES: The increased number of liver transplants in Egypt has increased the focus on perioperative complications in live donors. An important but not yet well-investigated complication is electrolyte disturbances, which are common in such patients, need intervention, and affect the outcome. We retrospectively analyzed data of perioperative calcium, magnesium, and phosphorus levels in live liver donors at our center. MATERIALS AND METHODS: We collected perioperative laboratory results from 44 living donors for liver transplant who were at our center from February 2009 to August 2013. We analyzed results of perioperative calcium, magnesium, and phosphorus levels before transplant, on the day of transplant (defined as day 0), and at 1 and 2 days after the surgical procedure. RESULTS: Mean serum calcium level was 2.31 mmol/L before transplant, 1.97 mmol/L on day 0, and 1.99 mmol/L on day 1, and 2.05 mmol/L on day 2 after transplant. Serum calcium level was significantly reduced at day 0 and on postoperative days 1 and 2 (P < .0001). Mean magnesium level was 0.8 mmol/L before transplant, 0.58 mmol/L on day 0, and 0.83 mmol/L on day 1, and 0.79 mmol/L on day 2 after the surgical procedure. The day 0 level was significantly reduced versus before transplant (P < .0001). Mean phosphorus level was 1.23 mmol/L before transplant, 1.11 mmol/L on day 0, and 0.97 mmol/L on day 1, and 0.76 mmol/L on day 2 after transplant, with significant declines on day 0 and on postoperative days 1 and 2 (P < .0001). CONCLUSIONS: Living liver donors showed significantly decreased levels of calcium and phosphorus on day 0 and on postoperative days 1 and 2, whereas magnesium level was significantly decreased on day 0 only.

The Conyza triloba extracts with high chlorophyll content and free radical scavenging activity had anticancer activity in cell lines.

The discovery of anticancer agents paradigm has been shifted to natural resources to overcome the toxicity of many synthetic agents at early clinical stages. In the present study, the antimutagenic, anticancer, phytochemistry, and free radical scavenging activities of five extracts of Conyza triloba were investigated. Extracts II (water : methanol), III (methylene chloride), and IV (methylene chloride : methanol) had the highest chlorophyll content and the highest superoxide scavenging, and metal chelating activities comparable to that of trolox. They also showed DPPH(•) scavenging activities better than that of α -tocopherol. Virtually all extracts exerted a strong (>40% reduction) antimutagenic activity against sodium azide and benzopyrene. Extracts II, III, and IV showed a remarkable growth inhibition profile with GI50 of 0.07-0.87 µg for Hepa1c1c7 and H4IIE1, A549, HT29, and PC3 cell lines and totally abated the growth of all cell lines, except for the breast cells, at 0.3-7.0 µg. The present study found a strong correlation between the chlorophyll content of Conyza extracts and their DDPH scavenging, metal chelating, and in vitro cytotoxic and cytostatic activities most probably through triggering apoptosis. This study could offer a platform for future studies and help selecting the vital features that identify the extract with potential anticancer activities.

Biological activity of a phloroglucinol glucoside derivative from Conyza aegyptiaca.

The phloroglucinol glucoside derivative [2,4-dihydroxy-6-(beta-D-glucopyranosyloxy)phenyl]-butan-1-one (1), roseoside (2), and kaempferol-3-O-beta-D-glucopyranoside (3) were isolated from the aerial parts of Conyza aegyptiaca (L.). To the best of our knowledge, this is the first isolation of compounds 1-3 from C. aegyptiaca. Their structures were determined by spectroscopic techniques including, IR, HR-EIMS, and extensive 500 MHz 1D- and 2D-NMR analyses (1H, 13C NMR, DEPT, 1H-1H COSY, HMQC and HMBC experiments). The antioxidant activity of 1, using the DPPH assay, was investigated; in addition, 1 was investigated against different types of cell lines, including Hep-G2, HCT-116, and RAW 264.7 for its cytotoxic effects. Also, this is the first report on the activity of 1.

Ketoisophorone transformation by Marchantia polymorpha and Nicotiana tabacum cultured cells.

Stereospecific olefin (C=C) and carbonyl (C=O) reduction of the readily available prochiral compound ketoisophorone (2,2,6-trimethyl-2-cyclohexene-1,4-dione) (1) by Marchantia polymorpha and Nicotiana tabacum cell suspension cultures produce the chiral products (6R)-levodione (2), (4R,5S)-4-hydroxy-3,3,5-trimethylcyclohexanone (3), and (4R,6R)-actinol (4) as well as the minor components (4R)-hydroxyisophorone (5) and (4S)-phorenol (6).

Interspecific rice hybrid of Oryza sativa x Oryza nivara reveals a significant increase in seed protein content.

Wild species offer a potential reservoir of genetic variation for crop improvement. Besides the valuable genes for disease resistance that the wild species have provided for rice improvement, recent studies have shown that these wild species could also provide favorable alleles for the improvement of yield and yield-related traits. The present study reports yet another potential of wild relatives of rice, which involves the improvement of seed protein content. A significant increase in seed protein content was observed in an interspecific hybrid between Oryza sativa ssp. indica and the wild species Oryza nivara. The hybrid showed a protein content of 12.4%, which was 28 and 18.2% higher than those of the parents O. nivara and IR 64, respectively. The increase in protein content was dependent on the genetic background of the rice variety used in the hybridization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of seed storage proteins demonstrated that a significant increase in prolamins and glutelins was mainly responsible for the elevated protein content of the hybrid. Amino acid analysis of seed proteins revealed that the hybrid had net gains of 19.5% in lysine and 19.4% in threonine over the O. nivara parent on a seed dry weight basis. Molecular analysis indicated that the increase in protein content of the hybrid was not a result of chromosomal rearrangements or transposable element activation, at least in the chromosomal regions containing seed storage protein genes. A preliminary genetic analysis of the F 2 segregating population showed that the inheritance of the increased protein content was polygenic in nature. The development of this interspecific hybrid offers a great potential for selecting new rice cultivars that combine the high yield and superior cooking quality of IR 64 with improved seed protein content.

A new germacranolide-type sesquiterpene lactone from Tanacetum santolinoides.

A new germacranolide-type sesquiterpene lactone, 1alpha-hydroxy-3-oxo-7alpha,11betaH-germacra-4Z,9Z-dien-12,6alpha-olide (1), was isolated from the CH2Cl2--MeOH extract of the aerial parts of Tanacetum santolinoides. Its structure was determined by spectroscopic techniques including, IR, high-resolution-EIMS, and extensive 400 MHz one- and two-dimensional NMR-analysis (1H, 13C-NMR, DEPT, 1H--1H COSY, HMQC, HMBC, and NOE experiments).

Identification of glycinin and beta-conglycinin subunits that contribute to the increased protein content of high-protein soybean lines.

Seed protein concentration of commercial soybean cultivars calculated on a dry weight basis ranges from approximately 37 to 42% depending on genotype and location. A concerted research effort is ongoing to further increase protein concentration. Several soybean plant introductions (PI) are known to contain greater than 50% protein. These PIs are exploited by breeders to incorporate the high-protein trait into commercial North American cultivars. Currently, limited information is available on the biochemical and genetic mechanisms that regulate high-proteins. In this study, we have carried out proteomic and molecular analysis of seed proteins of LG00-13260 and its parental high-protein lines PI 427138 and BARC-6. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that the high-protein lines accumulated increased amounts of beta-conglycinin and glycinins, when compared with Williams 82. High-resolution two-dimensional electrophoresis utilizing pH 4-7 and pH 6-11 ampholytes enabled improved resolution of soybean seed proteins. A total of 38 protein spots, representing the different subunits of beta-conglycinin and glycinin, were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. High-protein was correlated with an increase in the accumulation of most of the subunits representing beta-conglycinin and glycinin. Comparisons of the amino acid profiles of high-protein soybean lines revealed that the concentration of sulfur amino acids, a reflection of protein quality, was not influenced by the protein concentration. Southern blot analysis showed the presence of genotypic variation at the DNA level between PI 427138 and BARC-6 for the genes encoding group1 glycinin, beta-conglycinin, Bowman-Birk inhibitor (BBI), and the Kunitz trypsin inhibitor (KTI). LG00-13260 inherited the allelic variants of the parental line PI 427138 for glycinin, beta-conglycinin, and KTI, while BBI was inherited from the parental line BARC-6. The results of our study indicate that high-seed protein concentration is attributed to greater accumulation of specific components of beta-conglycinin and glycinin subunits presumably mediated by preferential expression of these genes during seed development.

Assessment of indigenous Nepalese soybean as a potential germplasm resource for improvement of protein in North American cultivars.

Soybeans contain approximately 40% protein and 20% oil and represents an important source of protein in animal rations and human diets. Attempts are being made to increase further the overall protein content of soybeans by utilization of exotic germplasms. In this study, soybean cultivars from Nepal have been characterized and their potential as a germplasm resource for improvement of the protein content and quality of North American cultivars assessed. Soybean cultivars 'Sathia', 'Seti', 'Kavre', and 'Soida Chiny', indigenous to various regions of Nepal, contained 42-45% protein, which is significantly higher in comparison to that of the North American cultivar 'Williams 82' (39%). Fractionation of seed protein by high-resolution two-dimensional gel electrophoresis revealed differences in the protein profiles of these cultivars. Various isoelectric forms of glycinin and beta-conglycinin were identified by comparing the matrix-assisted laser desorption ionization time-of-flight mass fingerprinting data against the National Center for Biotechnology Information nonredundant database. Nepalese cultivar Sathia was distinct, lacking some isoelectric forms of acidic and basic glycinin subunits while expressing other unique forms. The contribution of these unique protein spots present in either Sathia or Williams 82 to the total protein content was quantified using scanning laser densitometry. Distinct restriction fragment length polymorphisms (RFLP) for group 1 glycinin genes were observed among the tested Nepalese genotypes, indicating sequence variation among the cultivars. Conversely, evaluation of RFLP for the genes encoding group 2 glycinins, beta-conglycinin, and Bowman-Birk proteinase inhibitors indicated a high degree of conservation in these genes. Determination of amino acid composition, a reflection of protein quality, indicated that the arginine content of the Nepalese soybeans ranged from 7.7 to 8.1%, which was 5-10% higher than the 7.4% expressed in Williams 82. Additionally, Karve and Seti contained significantly more cysteine than Williams 82. Nepalese high-protein soybeans having a desirable amino acid composition hold potential to increase the protein quality and diversity of North American cultivars.