Genetic analysis of the variation for mineral accumulation in the leaves and seeds of natural germplasm of Brassica rapa L. (AA) and the its derived forms extracted from an allotetraploid B.juncea L.(AABB).

Brassica rapa L. (2n = 20; AA) is a vegetable and oilseed crop that is grown all over the world. Its leaves, shoots, and seeds store significant amounts of minerals. We used inductively coupled plasma-optical emission spectroscopy (ICP-OES) to determine the concentrations of eleven minerals in the leaves and seeds of 195 advanced generation inbred lines, of which 92 represented natural (NR) B. rapa and the remaining 103 were derived (DR) from a set of mother genotypes originally extracted from an allotetraploid B. juncea (2n = 36; AABB). The inbred lines differed for the composition of leaf and seed minerals. Leaf concentrations of N, K, Zn, and Se were higher in the DR subpanel as compared to NR subpanel, along with high seed accumulations of K and Se. DArT genotyping and genome wide association mapping led to the identification of SNPs associated with leaf and seed mineral compositions. Chromosomes A03, A05, and A10 harboured the most associated loci. Annotations of the regions adjacent to respective GWAS peaks allowed prediction of genes known for acquisition, transport, and accumulation of minerals and heavy metal detoxification. Transcriptome analysis revealed differential expression patterns of the predicted candidates, with most genes either down-regulated in derived genotypes relative to natural forms or their expression being comparable between the two. General downregulation may be a consequence of extracting B. rapa from allotetraploid B. juncea through genome resection. Some of the identified SNPs may be used as DNA markers for breeding programmes designed to modify the leaf and seed mineral compositions.

STUDY OF RELATIONSHIP OF PSYCHOSOCIAL FACTORS WITH SMOKING IN NORTHERN POPULATION.

OBJECTIVE: The aim: Of our study was to identify the relationship between the main risk factors for heart disease and social factors among northern population. PATIENTS AND METHODS: Materials and methods: We polled 3092 native and non-native habitants of the Sakha Republic (Yakutia). The poll consists of several sections. It includes questions related to social, demographic and life record data, heredity, physical activity and unhealthy habits. RESULTS: Results: Among the people diagnosed with hypertension (HT), there are more smokers than among the people without this medical condition. On the contrary, in the group of people diagnosed with HT at the examination for taking blood pressure, there were fewer smokers than in the group, in which HT was not registered. Patients with CHD, Myocardial infarction (MI) in past medical history, cerebrovascular accident (CVA) and type 2 diabetes (T2D), are smokers to the same degree as the other group. This indicates that this category of the examined are exposed to the risk of cardiovascular aggravations. We determined a high spread of psychosocial risk factors for the examined respondents - the relationship between smoking, overweight, obesity, abdominal obesity and HT, and the level of education, marital status and labor specificity. The increase in the level of education is associated with fewer amounts of smokers among both non-native and native habitants. Overweight is more frequently observed for the people who are not single, have low level of education and are engaged in manual labor. In particular, it is applicable to native habitants. CONCLUSION: Conclusions: Abdominal obesity did not have any relationship with psychosocial factors for native habitants. However, in relation to non-native habitants, overweight, obesity, abdominal obesity and HT are associated with marital status (married) and with manual labor.

Association genetics of the parameters related to nitrogen use efficiency in Brassica juncea L.

KEY MESSAGE: Genome wide association studies allowed prediction of 17 candidate genes for association with nitrogen use efficiency. Novel information obtained may provide better understanding of genomic controls underlying germplasm variations for this trait in Indian mustard. Nitrogen use efficiency (NUE) of Indian mustard (Brassica juncea (L.) Czern & Coss.) is low and most breeding efforts to combine NUE with crop performance have not succeeded. Underlying genetics also remain unexplored. We tested 92 SNP-genotyped inbred lines for yield component traits, N uptake efficiency (NUPEFF), nitrogen utilization efficiency (NUTEFF), nitrogen harvest index (NHI) and NUE for two years at two nitrogen doses (No without added N and N100 added @100 kg/ha). Genotypes IC-2489-88, M-633, MCP-632, HUJM 1080, GR-325 and DJ-65 recorded high NUE at low N. These also showed improved crop performance under high N. One determinate mustard genotype DJ-113 DT-3 revealed maximum NUTEFF. Genome wide association studies (GWAS) facilitated recognition of 17 quantitative trait loci (QTLs). Environment specificity was high. B-genome chromosomes (B02, B03, B05, B07 and B08) harbored many useful loci. We also used regional association mapping (RAM) to supplement results from GWAS. Annotation of the genomic regions around peak SNPs helped to predict several gene candidates for root architecture, N uptake, assimilation and remobilization. CAT9 (At1g05940) was consistently envisaged for both NUE and NUPEFF. Major N transporter genes, NRT1.8 and NRT3.1 were predicted for explaining variation for NUTEFF and NUPEFF, respectively. Most significant amino acid transporter gene, AAP1 appeared associated with NUE under limited N conditions. All these candidates were predicted in the regions of high linkage disequilibrium. Sequence information of the predicted candidate genes will permit development of molecular markers to aid breeding for high NUE.

Association Mapping of Seed Quality Traits Under Varying Conditions of Nitrogen Application in Brassica juncea L. Czern & Coss.

Indian mustard (Brassica juncea) is a major source of vegetable oil in the Indian subcontinent. The seed cake left after the oil extraction is used as livestock feed. We examined the genetic architecture of oil, protein, and glucosinolates by conducting a genome-wide association study (GWAS), using an association panel comprising 92 diverse genotypes. We conducted trait phenotyping over 2 years at two levels of nitrogen (N) application. Genotyping by sequencing was used to identify 66,835 loci, covering 18 chromosomes. Genetic diversity and phenotypic variations were high for the studied traits. Trait performances were stable when averaged over years and N levels. However, individual performances differed. General and mixed linear models were used to estimate the association between the SNP markers and the seed quality traits. Population structure, principal components (PCs) analysis, and discriminant analysis of principal components (DAPCs) were included as covariates to overcome the bias due to the population stratification. We identified 16, 23, and 27 loci associated with oil, protein, and glucosinolates, respectively. We also established LD patterns and haplotype structures for the candidate genes. The average block sizes were larger on A-genome chromosomes as compared to the B- genome chromosomes. Genetic associations differed over N levels. However, meta-analysis of GWAS datasets not only improved the power to recognize associations but also helped to identify common SNPs for oil and protein contents. Annotation of the genomic region around the identified SNPs led to the prediction of 21 orthologs of the functional candidate genes related to the biosynthesis of oil, protein, and glucosinolates. Notable among these are: LACS5 (A09), FAD6 (B05), ASN1 (A06), GTR2 (A06), CYP81G1 (B06), and MYB44 (B06). The identified loci will be very useful for marker-aided breeding for seed quality modifications in B. juncea.

Long-term functional outcomes and knee alignment of computer-assisted navigated total knee arthroplasty.

INTRODUCTION: This retrospective study examined the relationship between the mechanical axis throughout a functional arc of motion and functional outcome scores in patients undergoing computer-assisted navigation-based total knee arthroplasty (CAN-TKA) at 6-year follow-up. MATERIALS AND METHODS: The Stryker eNact Precision Knee Navigation System was utilized to obtain pre- and postoperative alignment measurements throughout the functional arc of motion. Patients were contacted via telephone and asked to complete the Short Form-12 and Western Ontario and McMaster Universities, which have been demonstrated to be reliable, valid, and sensitive assessment tools in this patient population. Statistical analysis was performed to determine the correlation between arc alignment and patient-reported functional outcome measures. RESULTS: A total of 47 patients at a mean of 76.1 (±6.3)-month follow-up and mean age of 65.9 (±7.9) years were surveyed. No correlation was found between the postoperative alignment or degree of intraoperative correction and the functional outcome scores. In a planned subgroup analysis of patients with a mean functional arc alignment greater than 3° from neutral, mean intraoperative degree of correction correlated with decreasing physical function (Spearman's ρ = 0.772, p = 0.04) and mean postoperative arc alignment positively correlated with increasing stiffness (ρ = 0.798, p = 0.03). CONCLUSION: This study suggests that patients undergoing CAN-TKA with mean functional arc range of motion greater than 3° may be at increased risk for suboptimal patient-reported functional outcomes. This study also illustrates the ability of CAN-TKA to measure the varus or valgus alignment of the knee throughout the entire range of motion.

Health state of adolescents in the Republic Sakha (Yakutia).

INTRODUCTION: According to the state policy in the field of health care, one of the priority directions is the improving of medical care for children's population [1]. It should be noted, that the state of health of the population and the development of society as a whole is largely determined by the level of adolescents health who have a significant impact on the health of the nation as a whole, and form the cultural, intellectual, industrial and reproductive potential [2, 3]. THE AIM: This article analyzes the health status indicators of adolescent population of the Republic Sakha (Yakutia), including the dynamics of the disease, including socially conditioned diseases, disability and mortality among the teenage population. MATERIAL AND METHODS: To study of health status of adolescents, social condition of life and there dynamic we use statistic materials of Yakut Republican Medical informative-analytic center, carry out medical prophylactic examination and survey research of 748 adolescents in 7 districts of republic, using questionnaires Global epidemic surveillance system health status of schoolchildren with support from WHO / UNAIDS / UNICEF / UNESCOCO. RESULTS: Established negative trend of deceasing of adolescent population, the increase in performance of both primary and general morbidity. Out of the total incidence of first place ranking among teenagers occupied by respiratory diseases, diseases of the eye and diseases of the digestive system. The most intensive increased prevalence of neurological diseases, cardiovascular system diseases, injuries and poisoning, and others external causes. Over the years remain unstable indicators of socio-caused diseases (alcoholism, drug addiction, tuberculosis, sexually transmitted infections). Despite the positive dynamics of some socially constructed and socially significant diseases, the relevance of this problem persists. Analysis of the dynamics of disability among children and adolescents showed a reduction in performance. In the structure of disability dominate diseases of the nervous system and sensory organs, congenital malformations and mental illness. Among the main causes of deaths in adolescence are external causes, there is no downward trend. There were significant gender differences, the increase in mortality associated with substance use among children died prevail children Yakut nationality. CONCLUSION: These results let us to plan and conduct the prophylactic work to decrease the pathology level among adolescents, and also to detect the need in medical staff and other resources.

Mixed pulmonary infection in an immunocompromised patient: a rare case report.

Patients who are immunocompromised are predisposed to a variety of common and uncommon pulmonary infections. We report a case of mixed pulmonary infection by drug resistant tuberculosis with a nocardiosis in a 49-year-old man who was a known case of chronic obstructive pulmonary disease, on prolonged corticosteroid use with diabetes mellitus. Chronic use of corticosteroids is a predisposing factor for opportunistic infections, such as nocardiosis or tuberculosis. Since such a mixed infection is rare, maybe a combined approach to therapy early in the course of disease would be effective in such cases.

Impact of a modified carbonated beverage on human dental plaque and salivary pH: an in vivo study.

BACKGROUND: To assess the plaque and salivary pH changes at different time intervals in vivo after consumption of a carbonated beverage modified with sodium fluoride and calcium phosphate. MATERIALS AND METHODS: Twenty-four subjects aged 18-25 years were recruited and randomly assigned to three groups (group A, original drink Sprite; group B, Sprite with sodium fluoride; group C, Sprite with calcium phosphate). Collection of pooled plaque and unstimulated saliva was done before and after the drinks were consumed by the subjects at 5-, 10-, 20- and 30-minute intervals. RESULTS: The pH rise was higher with group C for plaque and group B for saliva. CONCLUSIONS: Modification of the test carbonated beverage with calcium phosphate and fluoride may exert some protective potential, especially in high caries risk candidates.

Can IVUS-virtual histology improve outcomes of percutaneous carotid treatment?

Several previous studies focusing on comparison between outcomes of carotid artery stenting (CAS) and carotid endoarterectomy (CEA) have put forward conflicting results about the non-inferiority of CAS compared to CEA. Likely outcomes after CAS have been greatly limited by incomplete knowledge of atherosclerotic carotid pathology and probably inappropriate patient selection criteria. In the current practice, only the degree of lumen obstruction is indication to an invasive treatment (CEA or CAS) in symptomatic or asymptomatic patients, but it has been recently demonstrated that histology of carotid plaques also plays a major role. Indeed, plaque morphology and composition seem to influence more importantly outcomes of CAS than those of CEA. Angiography is a poor diagnostic tool to detect the severity and composition of atherosclerotic lesions. Virtual histology (VH) is a new technology incorporated in the latest intravascular ultrasound (IVUS) equipment that allows a validated histological characterization of plaques by performing a spectral, objective and highly-reproducible analysis of the radiofrequency and amplitude data of the ultrasound waves that cross different tissues. This manuscript reports authors' experience with the use of IVUS-VH during CAS. This new technology, by characterizing morphology, extension and histology of carotid plaque, seems to provide important information for confirming percentage of carotid stenosis and judging its embolic potential, tailoring the procedure and guiding the choice of stent and finally for checking stent apposition and complete covering of vulnerable plaques. According to authors' opinion IVUS-VH has the potential to optimize patients' and lesions' selection criteria for CAS in order to improve its outcomes.

Cerebral venous thrombosis in ulcerative colitis.

Cerebral venous thrombosis is a rare complication of ulcerative colitis. We report a case of 29 year old male who developed superior sagittal, left lateral and sigmoid sinus thrombosis secondary to ulcerative colitis. He was successfully treated with low molecular weight heparin and steroids.

Juvenile myoclonic epilepsy--an experience from north western India.

OBJECTIVES: The clinical data on cases of Juvenile Myoclonic Epilepsy (JME) were analysed. Response to initial small dosages (lower than usual) of sodium valproate and further lower maintenance dosages in patients who were seizure free for 2 years on drug were assessed. MATERIAL AND METHODS: Seventy-six patients who were diagnosed to have Juvenile Myoclonic Epilepsy on definite criteria were studied. All patients were put on sodium valproate in dosages (lower than usual) for initial control and further lower maintenance dosage and response evaluated. RESULTS: The clinical profile was found to be similar as in other parts of India. There was a female preponderance and average delay of 4.9 years in final diagnosis. Forty-eight (63.1%) patients showed good control on 15 mg/kg/day dosages of sodium valproate. After a seizure free interval of 2 years, 58% of patients could be maintained on small dosages ranging from 3-5 mg/kg/day to 6-8 mg/kg/day. CONCLUSION: The majority of JME patients responded well not only to sodium valproate in dosages lower than usually prescribed but required very small dosages for maintenance after a seizure free period of 2 years.

Physician assisted suicide--the perspective of a neurologist.

Modern medicine has resulted in a better control and treatment of many diseases. This has provided a better life for patients with treatable disorders, but at the same times has prolonged the suffering of the patients with diseases that are not treatable. This is of particular importance for the patients with neurological diseases, as some of these conditions have a relentlessly progressive course and cause significant distress to the patient. Recent times have seen an expansion of the scope of the various "rights of the patient" and according to some, this includes the "right to die". As a result of this concept of regarding the autonomy of the patient, the phenomenon of physician assisted suicide has emerged and is a topic of intense debate amongst the physicians, social workers and politicians all over the world. The present article puts forth the current concepts and brings out the exact definitions of the terms like physician-assisted suicide, voluntary active euthanasia and voluntary passive euthanasia. It presents the statements made by the various societies and organisations regarding the PAS. It also brings forth the point of view of the authors about this aspect of medicine. The authors share the view of the various scientific organisations, and opine that though the idea of physician assisted suicide may be morally justifiable in certain circumstances, it should not be made legal.

Only a small fraction of purified hepatitis C RNA-dependent RNA polymerase is catalytically competent: implications for viral replication and in vitro assays.

The enzymatic activity of a C-terminally truncated form of the RNA-dependent RNA polymerase, termed NS5B(Delta21), of the hepatitis C virus (strain BK) has been investigated using both homopolymeric and heteropolymeric RNA templates. Incorporation of nucleotides into a heteropolymeric RNA template as catalyzed by NS5B(Delta21) is characterized by biphasic reaction time courses. At high concentrations of nucleoside triphosphate in reactions allowing a preincubation of NS5B(Delta21) and RNA template, an initial rapid phase of the reaction is followed by a slower linear phase. The amplitude of the first phase of the reaction varies directly with the concentration of the enzyme in the reaction. It is shown here that full-length copies of the template are produced during the first phase of the reaction. Our results reveal that NS5B(Delta21) is processive but only a small fraction, less than 1%, of the purified enzyme present participates productively in the reaction. Most importantly, the turnover number for the hepatitis C NS5B(Delta21) is comparable to those observed for other polymerases such as the HIV-1 reverse transcriptase. The combined results reconcile in part the apparent discrepancy of the low, observed specific activity of the purified enzyme and the rapid generation of HCV in vivo.

X-ray structure of simian immunodeficiency virus integrase containing the core and C-terminal domain (residues 50-293)--an initial glance of the viral DNA binding platform.

The crystal structure of simian immunodeficiency virus (SIV) integrase that contains in a single polypeptide the core and the C-terminal deoxyoligonucleotide binding domain has been determined at 3 A resolution with an R-value of 0.203 in the space group P2(1)2(1)2(1). Four integrase core domains and one C-terminal domain are found to be well defined in the asymmetric unit. The segment extending from residues 114 to 121 assumes the same position as seen in the integrase core domain of avian sarcoma virus as well as human immunodeficiency virus type-1 (HIV-1) crystallized in the absence of sodium cacodylate. The flexible loop in the active site, composed of residues 141-151, remains incompletely defined, but the location of the essential Glu152 residue is unambiguous. The residues from 210-218 that link the core and C-terminal domains can be traced as an extension from the core with a short gap at residues 214-215. The C(alpha) folding of the C-terminal domain is similar to the solution structure of this domain from HIV-1 integrase. However, the dimeric form seen in the NMR structure cannot exist as related by the non-crystallographic symmetry in the SIV integrase crystal. The two flexible loops of the C-terminal domain, residues 228-236 and residues 244-249, are much better fixed in the crystal structure than in the NMR structure with the former in the immediate vicinity of the flexible loop of the core domain. The interface between the two domains encompasses a solvent-exclusion area of 1500 A(2). Residues from both domains purportedly involved in DNA binding are narrowly distributed on the same face of the molecule. They include Asp64, Asp116, Glu152 and Lys159 from the core and Arg231, Leu234, Arg262, Arg263 and Lys264 from the C-terminal domain. A model for DNA binding is proposed to bridge the two domains by tethering the 228-236 loop of the C-terminal domain and the flexible loop of the core.

Purification, solution properties and crystallization of SIV integrase containing a continuous core and C-terminal domain.

The C-terminal two-thirds segment of integrase derived from the simian immunodeficiency virus has been cloned, expressed in Escherichia coli, and purified to greater than 95% homogeneity. The protein encompasses amino-acid residues 50-293 and contains a F185H substitution to enhance solubility. In dilute solutions at concentrations below 1 mg ml(-1), the enzyme is predominantly dimeric. At the higher concentrations (>10 mg ml(-1)) required to enable crystallization, the enzyme self-associates to form species with molecular weights greater than 200 kDa. Despite the apparent high aggregation in solution, the enzyme crystallizes from a 8%(v/v) polyethylene glycol (molecular weight 6000) solution in a form suitable for X-ray diffraction studies. The resulting single crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 79.76, b = 99.98, c = 150.2 A, alpha = beta = gamma = 90 degrees and Z = 4. Under X-ray irradiation generated with a rotating-anode generator, the crystals diffract to 2.8 A resolution and allow collection of a native 3 A resolution diffraction data set.

An uniquely purified HCV NS3 protease and NS4A(21-34) peptide form a highly active serine protease complex in peptide hydrolysis.

The N-terminal domain of the hepatitis C virus (HCV) polyprotein containing the NS3 protease (residues 1027 to 1206) was expressed in Escherichia coli as a soluble protein under the control of the T7 promoter. The enzyme has been purified to homogeneity with cation exchange (SP-Sepharose HR) and heparin affinity chromatography in the absence of any detergent. The purified enzyme preparation was soluble and remained stable in solution for several weeks at 4 degrees C. The proteolytic activity of the purified enzyme was examined, also in the absence of detergents, using a peptide mimicking the NS4A/4B cleavage site of the HCV polyprotein. Hydrolysis of this substrate at the expected Cys-Ala scissile bond was catalyzed by the recombinant protease with a pseudo second-order rate constant (k(cat)/K(M)) of 205 and 196,000 M(-1) s(-1), respectively, in the absence and presence of a central hydrophobic region (sequence represented by residues 21 to 34) of the NS4A protein. The rate constant in the presence of NS4A peptide cofactor was two orders of magnitude greater than reported previously for the NS3 protease domain. A significantly higher activity of the NS3 protease-NS4A cofactor complex was also observed with a substrate mimicking the NS4B/5A site (k(cat)/K(M) of 5180 +/- 670 M(-1) s(-1)). Finally, the optimal formation of a complex between the NS3 protease domain and the cofactor NS4A was critical for the high proteolytic activity observed.

Complex of NS3 protease and NS4A peptide of BK strain hepatitis C virus: a 2.2 A resolution structure in a hexagonal crystal form.

The crystal structure of the NS3 protease of the hepatitis C virus (BK strain) has been determined in the space group P6(3)22 to a resolution of 2.2 A. This protease is bound with a 14-mer peptide representing the central region of the NS4A protein. There are two molecules of the NS3(1-180)-NS4A(21'-34') complex per asymmetric unit. Each displays a familiar chymotrypsin-like fold that includes two beta-barrel domains and four short alpha-helices. The catalytic triad (Ser-139, His-57, and Asp-81) is located in the crevice between the beta-barrel domains. The NS4A peptide forms an almost completely enclosed peptide surface association with the protease. In contrast to the reported H strain complex of NS3 protease-NS4A peptide in a trigonal crystal form (Kim JL et al., 1996, Cell 87:343-355), the N-terminus of the NS3 protease is well-ordered in both molecules in the asymmetric unit of our hexagonal crystal form. The folding of the N-terminal region of the NS3 protease is due to the formation of a three-helix bundle as a result of crystal packing. When compared with the unbound structure (Love RA et al., 1996, Cell 87:331-342), the binding of the NS4A peptide leads to the ordering of the N-terminal 28 residues of the NS3 protease into a beta-strand and an alpha-helix and also causes local rearrangements important for a catalytically favorable conformation at the active site. Our analysis provides experimental support for the proposal that binding of an NS4A-mimicking peptide, which increases catalytic rates, is necessary but not sufficient for formation of a well-ordered, compact and, hence, highly active protease molecule.