Linkage analysis and residual heterozygotes derived near isogenic lines reveals a novel protein quantitative trait loci from a Glycine soja accession.

Modern soybean [Glycine max (L.) Merr] cultivars have low overall genetic variation due to repeated bottleneck events that arose during domestication and from selection strategies typical of many soybean breeding programs. In both public and private soybean breeding programs, the introgression of wild soybean (Glycine soja Siebold and Zucc.) alleles is a viable option to increase genetic diversity and identify new sources for traits of value. The objectives of our study were to examine the genetic architecture responsible for seed protein and oil using a recombinant inbred line (RIL) population derived from hybridizing a G. max line ('Osage') with a G. soja accession (PI 593983). Linkage mapping identified a total of seven significant quantitative trait loci on chromosomes 14 and 20 for seed protein and on chromosome 8 for seed oil with LOD scores ranging from 5.3 to 31.7 for seed protein content and from 9.8 to 25.9 for seed oil content. We analyzed 3,015 single F4:9 soybean plants to develop two residual heterozygotes derived near isogenic lines (RHD-NIL) populations by targeting nine SNP markers from genotype-by-sequencing, which corresponded to two novel quantitative trait loci (QTL) derived from G. soja: one for a novel seed oil QTL on chromosome 8 and another for a novel protein QTL on chromosome 14. Single marker analysis and linkage analysis using 50 RHD-NILs validated the chromosome 14 protein QTL, and whole genome sequencing of RHD-NILs allowed us to reduce the QTL interval from ∼16.5 to ∼4.6 Mbp. We identified two genomic regions based on recombination events which had significant increases of 0.65 and 0.72% in seed protein content without a significant decrease in seed oil content. A new Kompetitive allele-specific polymerase chain reaction (KASP) assay, which will be useful for introgression of this trait into modern elite G. max cultivars, was developed in one region. Within the significantly associated genomic regions, a total of eight genes are considered as candidate genes, based on the presence of gene annotations associated with the protein or amino acid metabolism/movement. Our results provide better insights into utilizing wild soybean as a source of genetic diversity for soybean cultivar improvement utilizing native traits.

Transforming medical education to strengthen the health professional training in Viet Nam: A case study.

The competency-based undergraduate curriculum reform at the University of Medicine and Pharmacy at Ho Chi Minh City, Faculty of Medicine (UMP-FM) is detailed and reviewed in reference to the instructional and institutional reforms, and enabling actions recommended by the Lancet 2010 Commission for Health Professional Education. Key objectives are to: revise the overall 6-year curriculum to be more integrated and competency-based; reinforce students' knowledge application, problem-solving, clinical competence, self-directed learning and soft skills; develop a comprehensive and performance-based student assessment programme; and establish a comprehensive quality monitoring programme to facilitate changes and improvements. New features include early introduction to the practice of medicine, family- and community-based medicine, professionalism, interprofessional education, electives experiences, and a scholarly project. Institutional reform introduces a faculty development programme, joint planning mechanism, a "culture of critical inquiry", and a transparent faculty reward system. Lessons learnt from the curriculum reform at UMP-FM could be helpful to medical schools from low- and middle-income countries considering transitioning from a traditional to a competency-based curriculum. Funding: This work receives no external funding.

Safety of Continuous Erector Spinae Catheters in Chest Trauma: A Retrospective Cohort Study.

BACKGROUND: The erector spinae block is an efficacious analgesic option for the management of rib fracture--related pain. Despite there being minimal published data specifically addressing the safety profile of this block, many societies have made statements regarding its safety and its use as an alternative to traditional regional anesthesia techniques in patients at risk of complications. The primary aim of this study was to characterize the safety profile of erector spinae plane block catheters by determining the incidence of early complications. The secondary aim of this study was to characterize the incidence of late adverse events, as well as the erector spinae plane block catheter failure rate. METHODS: We analyzed electronic medical record data of patients who had an erector spinae plane block catheter inserted for the management of rib fractures between November 2017 and September 2020. To assess early adverse events, data collection included hypotension, hypoxemia, local anesthetic systemic toxicity, and pneumothorax thought to be associated with erector spinae plane block catheter insertion. Late complications included catheter site infection and catheter site hematoma. RESULTS: A total of 224 patients received 244 continuous erector spinae catheters during the study period. After insertion of the erector spinae, there were no immediate complications such as hypotension, hypoxia, local anesthetic toxicity, or pneumothorax. Of all blocks inserted, 7.7% were removed due to catheter failure (8.4 per 100 catheters; 95% confidence interval [CI], 5.1-13.9 per 100 catheters). This resulted in a failure rate of 1.9 per 1000 catheter days (95% CI, 1.1-6.7 catheter days). Late complications included 2 erythematous catheter sites and 2 small hematomas not requiring intervention. The incidence of a minor late complication was 16.7 per 1000 catheters (95% CI, 6.1-45.5 per 1000 catheters). CONCLUSIONS: This study supports the statements made by regional anesthesia societies regarding the safety of the erector spinae plane block. Based on the results presented in this population of trauma patients, the erector spinae plane block catheter is a low-risk analgesic technique that may be performed in the presence of abnormal coagulation status or systemic infection.

Health Care Worker Preferences and Perspectives on Doses per Container for 2 Lyophilized Vaccines in Senegal, Vietnam, and Zambia.

INTRODUCTION: Limited information exists on health care workers' (HCWs) perceptions about use of multidose vaccine vials and their preferences about doses per container (DPC). We present findings from qualitative studies conducted in Senegal, Vietnam, and Zambia to explore HCWs' behavior regarding opening vials and their perceptions and preferences for the number of doses in vials of BCG and measles-containing vaccine (MCV). Zambia and Senegal currently offer MCV in 10-dose vials and BCG in 20-dose vials; 10-dose vials are used for both vaccines in Vietnam. Unused doses in vials of these reconstituted vaccines must be discarded within 6 hours. METHODS: Key informant interviews (KIIs) were conducted with frontline HCWs in Senegal, Vietnam, and Zambia. In Senegal and Vietnam, the KIIs were conducted as part of broader formative research; in Zambia, KIIs were conducted in control districts using 10-dose MCV vials only and in intervention districts that switched from 10- to 5-dose vials during the study. During analysis, themes common to all 3 countries were synthesized. Critical themes relevant to country contexts were also examined. RESULTS: HCWs in all 3 countries preferred containers with fewer doses for BCG and MCV to reduce wastage and increase the likelihood of vaccinating every eligible child. HCWs in Senegal and HCWs using 10-dose vials in Zambia reported sending unvaccinated children away because not enough children were present to warrant opening a new vial. In Vietnam, where sessions are typically held monthly, and in Zambia when the 5-dose vials were used, almost all HCWs reported opening a vial of MCV for even 1 child. DISCUSSION: HCWs prefer vials with fewer DPC. Their concerns about balancing coverage and wastage influence their decisions to vaccinate every eligible child; and their perspectives are crucial to ensuring that all target populations are reached with vaccines in a timely manner.

Induction opioids for caesarean section under general anaesthesia: a systematic review and meta-analysis of randomised controlled trials.

INTRODUCTION: The adverse effects of induction opioids on the neonate are poorly characterised. The study aim was to investigate whether induction opioids can be used in caesarean section without adversely affecting the neonate. METHODS: Six databases were systematically searched from inception until January 2019. Included studies compared induction opioids and placebo in caesarean section. Results were presented as odds ratios (95% confidence intervals) for dichotomous outcomes and weighted mean difference for continuous outcomes. An I2 statistic of >50% was significant for heterogeneity. The primary outcome was Apgar score (1 and 5 min). Secondary outcomes included neonatal adverse events, cord blood gas analyses, maternal haemodynamic parameters (systolic blood pressure (SBP), mean arterial pressure (MAP), heart rate (HR) and catecholamine concentrations. RESULTS: Seventeen studies (n=987) were included in the meta-analysis. Remifentanil 0.5-1 µg/kg or 2-3 µg/kg/h, alfentanil 7.5-10 µg/kg and fentanyl 0.5-1 µg/kg were compared to placebo. There was no significant difference in Apgar scores at 1 min (P=0.25, 0.58 and 0.89 respectively) for all three opioids or at 5 min for remifentanil and alfentanil (P=0.08 and 0.21 respectively). Fentanyl significantly reduced 5 min Apgar scores (P=0.002). There was no difference in neonatal airway interventions with remifentanil or alfentanil (P <0.05). All three induction opioids caused a significant reduction in maximum SBP (P <0.0001), MAP (P <0.00001) and HR (P <0.00001). CONCLUSION: Induction opioids are effective sympatholytic agents. Remifentanil and alfentanil appear to be safe, with no significant effect on Apgar scores or neonatal airway intervention, but a well-powered trial is required to confirm these findings.

Effect of feeding cassava and/or Stylosanthes foliage on the performance of crossbred growing cattle.

The effect of feeding different levels of cassava foliage (Manihot esculenta, Crantz) and/or Stylosanthes guianensis foliage on the growth and digestibility was studied using twenty eight 6-month-old crossbred growing cattle (50% local Yellow cattle and 50% Sindhi) (both Bos indicus) weighing on average 114 kg at start. All animals were fed a basal diet consisting of urea treated rice straw (URTRS) fed ad libitum, 0.87 kg concentrate and 0.22 kg molasses on dry matter (DM) basis. The treatments were four supplements: soybean meal, cassava foliage, stylosanthes foliage or a mix of stylosanthes foliage and cassava foliage all giving the same nitrogen intake. The consumption of tannins and hydrogen cyanide (HCN) were significantly higher in groups fed a mixture of foliages compared with only cassava foliage, respectively. There were 30% and 19%, respectively, higher live weight gain in the group fed a mixture of foliages as compared to the groups fed only cassava or stylosanthes. The factors of low organic matter and high level of HCN in the diet when feeding only cassava foliage might explain the negative effects on intake, neutral detergent fibre digestibility and nitrogen retention, and resulted in lower growth rates.

Structural characterization of the active form of PerR: insights into the metal-induced activation of PerR and Fur proteins for DNA binding.

In Bacillus subtilis, the transcription factor PerR is an iron dependant sensor of H(2)O(2). The sensing mechanism relies on a selective metal catalysed oxidation of two histidine residues of the regulatory site. Here we present the first crystal structure of the active PerR protein in complex with a Mn(2+) ion. In addition, X-ray absorption spectroscopy experiments were performed to characterize the corresponding iron form of the protein. Both studies reveal a penta-coordinate arrangement of the regulatory site that involves three histidines and two aspartates. One of the histidine ligand belongs to the N-terminal domain. Binding of this residue to the regulatory metal allows the protein to adopt a caliper-like conformation suited to DNA binding. Since this histidine is conserved in all PerR and a vast majority of Fur proteins, it is likely that the allosteric switch induced by the regulatory metal is general for this family of metalloregulators.

Dengue epidemic in southern Vietnam, 1998.

A widespread epidemic of dengue hemorrhagic fever (DHF) occurred in southern Vietnam in 1998, with 438.98 cases/100,000 population and 342 deaths. The number of DHF cases and deaths per 100,000 population increased 152.4% and 151.8%, respectively, over a 1997 epidemic. Dengue viruses were isolated from 143 patient blood samples; DEN-3 virus was identified as the predominant serotype, although a resurgence of DEN-4 was noted.

Alterations in plasminogen activation correlate with epithelial cell dysplasia grading in colorectal adenomas.

Proteases are important for neoplastic invasion but a specific role for the plasminogen activator system in the progression of colorectal epithelial dysplasia to adenomatous lesions remains unclear. Consecutive tissue cryosections of 51 adenomas, 49 distant mucosa samples and five mucosa samples from control subjects were histopathologically analysed for dysplasia grade and tissue type, urokinase plasminogen activator levels and plasminogen activator inhibitor type 1 (PAI-1) using immunosorbent methods. Plasminogen activation and urokinase-mediated proteolytic activity levels were assessed using in situ zymography. Plasminogen activation and tissue-type activator levels were lower in adenomas than in mucosae (P < 0.001). PAI-1 concentration and urokinase levels were higher in adenomas than in mucosae (P < 0.001 and P < 0.001 respectively). In adenomas, urokinase concentration increased in parallel with PAI-1, but only the urokinase levels correlated with the dysplasia grade (P < 0.01). Thus, the alterations in plasminogen activation correlated with epithelial cell dysplasia grading. In the mucosa to adenoma transition, a marked decrease in tissue-type plasminogen activator occurred. In adenomas, this decrease was accompanied by a concomitant increase in urokinase and PAI-1. The urokinase level only continued to rise in parallel with the dysplasia grade. Resulting protease-antiprotease imbalance in high-grade dysplasia may represent the phenotypic change associated with malignant transformation and invasive behaviour.

Role of integrins and evidence for two distinct mechanisms mediating human colorectal carcinoma cell interaction with peritoneal mesothelial cells and extracellular matrix.

Peritoneal carcinomatosis involves a series of events including tumor cell interactions with mesothelial cells and the extracellular matrix (ECM). We have studied the adhesive and invasive properties of four human colorectal carcinoma cell lines (Co115, HT29, SW480, SW620) confronted in vitro with a human mesothelial cell monolayer or with the ECM proteins collagen IV, laminin-1, fibronectin, tenascin-C and vitronectin. Quantitation was achieved following staining of tumor cells with the calcein-AM fluorescent dye. We found that all four cell lines rapidly adhered to a mesothelial cell monolayer. This adhesion event was not inhibitable by anti-integrin and anti-CD44 antibodies. Following initial attachment, the SW480 and SW620 cells invaded the mesothelial cell monolayer more aggressively than HT29 and Co115 cells. All cell lines adhered to ECM proteins with each one exhibiting an individual adhesion pattern. Adhesion to matrix was completely integrin-dependent. When tested in an invasion assay, HT29 and Co115 cells crossed Matrigel-coated filters while SW480 and SW620 cells did not. This invasion was inhibited by anti-beta 1 integrin antibodies. Taken together, our results demonstrate that the initial colorectal tumor cell-mesothelial cell interaction occurs through an integrin-independent mechanism while adhesion to matrix proteins and invasion through Matrigel are integrin-dependent events. Furthermore, the different invasive capacity of SW480 and SW620 versus HT29 and Co115 cells upon interaction with a mesothelial cell monolayer or Matrigel suggests that these two invasion events may be mediated by distinct mechanisms.

Plasmin-induced proteolysis of tenascin-C: modulation by T lymphocyte-derived urokinase-type plasminogen activator and effect on T lymphocyte adhesion, activation, and cell clustering.

Proteolysis and remodeling of the extracellular matrix occur physiologically in processes such as tissue morphogenesis and repair and may participate in the regulation of complex cell functions, including proliferation and differentiation. While matrix degradation appears to be relevant to T lymphocyte migration through tissues, little is known about whether degraded matrix affects T lymphocyte function. We have studied the interaction between T lymphocytes and tenascin-C (TN-C), a matrix protein we have previously reported to inhibit T lymphocyte activation, in the context of plasmin-induced degradation. Here we report that plasmin efficiently cleaves TN-C. Peripheral blood T lymphocytes stimulated with phorbol ester, anti-CD28, or anti-CD3 Ab, induce, within 24 to 48 h, a strong plasminogen-dependent proteolysis of TN-C. We demonstrate that stimulated T lymphocytes activate plasminogen by secreting the urokinase-type plasminogen activator (u-PA). Plasminogen activation by T lymphocyte-derived u-PA occurs efficiently in fluid phase in the absence of cells. We investigate the consequences of plasmin-induced proteolysis on three of the effects of TN-C in relation to lymphocyte functions. Plasmin proteolysis converts TN-C from a nonadhesive into an adhesive substrate for T lymphocytes and abolishes its aggregating activity on PBMC. In contrast, the inhibitory effect of TN-C on T lymphocyte activation remains unaffected. These observations demonstrate that stimulated T lymphocytes induce plasminogen-dependent proteolysis of TN-C by secreting u-PA and suggest that proteolysis of TN-C may represent a mechanism by which to regulate some of its effects on T lymphocyte functions.

In situ stromal expression of the urokinase/plasmin system correlates with epithelial dysplasia in colorectal adenomas.

An increase of urokinase-type plasminogen activator (uPA) and a decrease of tissue-type PA (tPA) have been associated with the transition from normal to adenomatous colorectal mucosa. Serial sections from 25 adenomas were used to identify PA-related caseinolytic activities by in situ zymography, blocking selectively uPA or tPA. The distribution of uPA, tPA, and type 1 PA inhibitor mRNAs was investigated by nonradioactive in situ hybridization, and the receptor for uPA was detected by immunostaining. Low- and high-grade epithelial cell dysplasia was mapped histologically. Results show that 23 of 25 adenomas expressed uPA-related lytic activity located predominantly in the periphery whereas tPA-related activity was mainly in central areas of adenomas. In 15 of 25 adenomas, uPA mRNA was expressed in stromal cells clustered in foci that coincided with areas of uPA lytic activity. The probability of finding uPA mRNA-reactive cells was significantly higher in areas with high-grade epithelial dysplasia. uPA receptor was mainly stromal and expressed at the periphery. Type 1 PA inhibitor mRNA cellular expression was diffuse in the stroma, in endothelial cells, and in a subpopulation of alpha-smooth muscle cell actin-reactive cells. These results show that a stromal up-regulation of the uPA/plasmin system is associated with foci of severe dysplasia in a subset of colorectal adenomas.

Modulation of the plasminogen activation system by inflammatory cytokines in human colon carcinoma cells.

Inflammation may promote malignant invasion by enhancing cancer cell-associated proteolysis. Here we present the effect of inflammatory cytokines on the plasminogen activation system of eight human colon carcinoma cell lines. Tumour necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) increased in several, but not all, cell lines the production of urokinase-type plasminogen activator (uPA), tissue-type PA (tPA) and plasminogen activator inhibitor type 1 (PAI-1) as analysed by zymography, enzyme immunoassays and Northern analysis. Interleukin 6 (IL-6) had no effect. uPA receptor (uPAR) mRNA levels were also upregulated. However, each individual cell line responded differently following exposure to TNF-alpha or IL-1 beta. For example, there was a dose-dependent up-regulation of uPA and PAI-1 in SW 620 cells, whereas increased uPA production in SW 1116 cells was not accompanied by an increase in PAI-1. The TNF-alpha stimulatory effect was blocked by anti-TNF-alpha Fab fragments. All cell lines expressed both types of TNF receptor mRNAs, whereas no transcript for TNF-alpha, IL-1 beta, IL-6, IL-6 receptor or the IL-1 receptors was found. Our results demonstrate that TNF-alpha and IL-1 beta stimulate the plasminogen activation system in tumour cell but the responses differed even in cells derived from the same tissue origin.

Human Co115 colon carcinoma cells potentiate the degradation of laminin mediated by tissue-type plasminogen activator.

The plasminogen activation (PA) system of human Co115 colon carcinoma cells was investigated. Analysis at the levels of protein and mRNA of cultured cells and of histozymography of tumor xenografts in nude mice showed that Co115 cells produce only tissue type PA (t-PA) and no urokinase (u-PA). Also, mRNA for the u-PA receptor and for PA inhibitor type 2 (PAI-2), but not for PAI-1, were detected. We developed a quantitative degradation assay using glutaraldehyde-immobilized 125I-laminin to investigate the capacity of Co115 cells to degrade laminin. Laminin degradation by Co115 cells was completely inhibited by 100 micrograms/ml of polyclonal anti-t-PA IgG, by the plasmin inhibitors aprotinin (100 micrograms/ml) or epsilon-aminocaproic acid (EACA; at 0.3 M), but not by antibodies against u-PA or u-PAR nor by nonimmune IgG. Cycloheximide-treated Co115 cells were unable to degrade laminin but increased laminin degradation induced by conditioned medium of Co115 cells or recombinant t-PA. No potentiation was observed when Co115 cells and laminin were kept separated by Transwell inserts. Our results suggest that Co115 human colon carcinoma cells degrade laminin by potentiating t-PA-mediated plasminogen activation at the cell surface which requires close contact between tumor cells and laminin substrate.

Laminin degradation by human colon carcinoma cells: a role for urinary and tissue plasminogen activators.

The human colon carcinoma cell lines Co112 and Co115 are both invasive in nude mice following intraperitoneal implantation. Co115 cells only exhibit metastasis capacity under this condition. Characterization of the plasminogen activation system demonstrates that Co112 cells express the urinary-type plasminogen activator (uPA) and Co115 cells the tissue-type (tPA), exclusively. Immunocytochemical analyses revealed that the in vitro plasminogen-dependent lysis of exogenous basement membrane laminin induced by Co112 cells displayed a gradient-like pattern, whereas, in the case of Co115 cells, it was sharply confined to the pericellular area. Double-labeling experiments showed that uPA on Co112 and tPA on Co115 cells are cell-surface-associated constituents. The cellular distribution of laminin expressed by tumor cells themselves appears to be distributed homogeneously in the cytoplasm of both cell types. We suggest that the extracellular matrix degradation induced by tumor cell surface-associated plasmin implies two different mechanisms which are specifically related to uPA or to tPA, both contributing to matrix degradation and malignant invasion.

Plasminogen activators and plasminogen activator inhibitors in liver deficiencies caused by chronic alcoholism or infectious hepatitis.

Plasma concentrations of tissue-type plasminogen activator (t-PA), urokinase (u-PA), plasminogen activator inhibitor 1 (PAI-1) and PAI-2 were studied in 53 patients with liver deficiency caused by chronic alcoholism (n = 40), viral hepatitis (n = 10) or malignant disease of the liver (n = 3) and compared to that of a control group (n = 20) of healthy subjects. u-PA and PAI-1 levels were significantly increased in all patients with chronic alcoholism, whereas high t-PA was only observed in combination with disturbed liver function tests or with liver cirrhosis (two and six-fold above control values, respectively). A good correlation was observed between t-PA and gamma glutamyl transferase (r = 0.615; p less than 0.001). In patients with infectious hepatitis or with malignant disease of the liver t-PA was normal whereas u-PA and PAI-1 were increased. PAI-2 levels were close to or below the detection limit (15 ng/ml) in the control group and in most patients. However, in two patients with alcohol induced cirrhosis PAI-2 levels were approximately 45 ng/ml and in one patient with hepatocarcinoma even 66 ng/ml. Thus, in liver disease, marked elevations of t-PA, u-PA and PAI-1 levels may occur, with increased PAI-1 as an early marker of liver defects and t-PA a marker of severe liver defects.