Biocatalytic Generation of o-Quinone Imines in the Synthesis of 1,4-Benzoxazines and Its Comparative Green Chemistry Metrics.

1,4-Benzoxazines are important motifs in many pharmaceuticals and can be formed by a reaction sequence involving the oxidation of o-aminophenols to their corresponding quinone imine followed by an in situ inverse electron demand Diels-Alder (IEDDA) cycloaddition with a suitable dienophile. Reported herein is the development of a reaction sequence that employs horseradish peroxidase to catalyze the oxidation of the aminophenols prior to the IEDDA as a more sustainable alternative to the use of conventional stoichiometric oxidants. The synthesis of 10 example benzoxazines is demonstrated in this "one-pot, two-step" procedure with yields between 42% and 92%. The green chemistry metrics, including the E-factor and generalized reaction mass efficiency, for this biocatalytic reaction were compared against the conventional chemical approach. It was found that the reported biocatalytic route was approximately twice as green by these measures.

Whole bone subcutaneous transplantation as a strategy to study precisely the bone marrow niche.

Hematopoietic stem cells are maintained in a specialized microenvironment, known as the 'niche', within the bone marrow. Understanding the contribution of cellular and molecular components within the bone marrow niche for the maintenance of hematopoietic stem cells is crucial for the success of therapeutic applications. So far, the roles of crucial mechanisms within the bone marrow niche have been explored in transgenic animals in which genetic modifications are ubiquitously introduced in the whole body. The lack of precise tools to explore genetic alterations exclusively within the bone marrow prevents our determination of whether the observed outcomes result from confounding effects from other organs. Here, we developed a new method - 'whole bone subcutaneous transplantation'- to study the bone marrow niche in transgenic animals precisely. Using immunolabeling of CD45.1 (donor) vs. CD45.2 (recipient) hematopoeitic stem cells, we demonstrated that hematopoeitic stem cells from the host animals colonize the subcutaneously transplanted femurs after transplantation, while the hematopoietic stem cells from the donor disappear. Strikinlgy, the bone marrow niche of these subcutaneously transplanted femurs remain from the donor mice, enabling us to study specifically cells of the bone marrow niche using this model. We also showed that genetic ablation of peri-arteriolar cells specifically in donor femurs reduced the numbers of hematopoietic stem cells in these bones. This supports the use of this strategy as a model, in combination with genetic tools, to evaluate how bone marrow niche specific modifications may impact non-modified hematopoietic stem cells. Thus, this approach can be utilized for genetic manipulation in vivo of specific cell types only within the bone marrow. The combination of whole bone subcutaneous transplantation with rodent transgenic models will facilitate a more precise, complex and comprehensive understanding of existing problems in the study of the hematopoietic stem cell bone marrow niche.

Learning Curve Associated with ClearPoint Neuronavigation System: A Case Series.

BACKGROUND: The ClearPoint neuronavigation system affords real-time magnetic resonance imaging (MRI) guidance during stereotactic procedures. While such information confers potential clinical benefits, additional operative time may be needed. METHODS: We conducted a retrospective analysis of procedural time associated with ClearPoint Stereotaxis, with hypothesis that this procedural time is comparable with that associated with frame-based biopsy. RESULTS: Of the 52 patients evaluated, the total procedural time for ClearPoint stereotactic biopsy averaged 150.0 (±40.4) minutes, of which 111.5 (±16.5) minutes were dedicated to real-time MRI acquisition and trajectory adjustment. This procedural time is within the range of those reported for frame-based needle biopsies. Approximately 5 minutes of the procedural time is related to the mounting of the MRI-compatible stereotactic frame. Based on the procedural time, we estimate that four cases are required in the learning curve to achieve this efficiency. Efficient algorithms for distortion corrections and isocenter localization are keys to ClearPoint stereotaxis. Routine quality assurance/control after each MRI software update and institutional information technology maintenance also contribute to efficiency. Real-time MRI is essential for definitive diagnosis in select cases. CONCLUSIONS: ClearPoint stereotactic needle biopsy can be achieved in time frames comparable to frame-based stereotaxis. However, procedural efficiency requires 4 "learning curve" cases as well as vigilance in terms of MR distortion correction and information technology maintenance.

Genetic associated complications of type 2 diabetes mellitus.

According to the International Diabetes Federation, the number of adults (age of 20-79) being diagnosed with diabetes mellitus (DM) have increased from 285 million in year 2009 to 463 million in year 2019 which comprises of 95% type 2 DM patient (T2DM). Research have claimed that genetic predisposition could be one of the factors causing T2DM complications. In addition, T2DM complications cause an incremental risk to mortality. Therefore, this article aims to discuss some complications of T2DM in and their genetic association. The complications that are discussed in this article are diabetic nephropathy, diabetes induced cardiovascular disease, diabetic neuropathy, diabetic foot ulcer (DFU) and Alzheimer's disease (AD). According to the information obtained, genes associated with diabetic nephropathy (DN) are gene GABRR1 and ELMO1 that cause injury to glomerular. Replication of genes FRMD3, CARS and MYO16/IRS2 shown to have link with DN. The increase of gene THBS2, NGAL, PIP, TRAF6 polymorphism, ICAM-1 encoded for rs5498 polymorphism and C667T increase susceptibility towards DN in T2DM patient. Genes associated with cardiovascular diseases are adiponectin gene (ACRP30) and apolipoprotein E (APOE) polymorphism gene with ξ2 allele. Haptoglobin (Hp) 1-1 genotype and mitochondria superoxide dismutase 2 (SOD2) plays a role in cardiovascular events. As for genes related to diabetic neuropathy, janus kinase (JAK), mutation of SCN9A and TRPA1 gene and destruction of miRNA contribute to pathogenesis of diabetic neuropathy among T2DM patients. Expression of cytokine IL-6, IL-10, miR-146a are found to cause diabetic neuropathy. Besides, A1a16Va1 gene polymorphism, an oxidative stress influence was found as one of the gene factors. Diabetic retinopathy (DR) is believed to have association with monocyte chemoattractant protein-1 (MCP-1) and insulin-like growth factor 1 (IGF1). Over-expression of gene ENPP1, IL-6 pro-inflammatory cytokine, ARHGAP22's protein rs3844492 polymorphism and TLR4 heterozygous genotype are contributing to significant pathophysiological process causing DR, while research found increases level of UCP1 gene protects retina cells from oxidative stress. DFU is manifested by slowing in re-epithelialization of keratinocyte, persistence wound inflammation and healing impairment. Re-epithelialization disturbance was caused by E2F3 gene, reduction of Tacl gene encoded substance P causing persistence inflammation while expression of MMp-9 polymorphism contributes to healing impairment. A decrease in HIF-1a gene expression leads to increased risk of pathogenesis, while downregulation of TLR2 increases severity of wound in DFU patients. SNPs alleles has been shown to have significant association between the genetic dispositions of T2DM and AD. The progression of AD can be due to the change in DNA methylation of CLOCK gene, followed with worsening of AD by APOE4 gene due to dyslipidemia condition in T2DM patients. Insulin resistance is also a factor that contributes to pathogenesis of AD.

Nectin-4 Interactions Govern Measles Virus Virulence in a New Model of Pathogenesis, the Squirrel Monkey (Saimiri sciureus).

In addition to humans, only certain nonhuman primates are naturally susceptible to measles virus (MeV) infection. Disease severity is species dependent, ranging from mild to moderate for macaques to severe and even lethal for certain New World monkey species. To investigate if squirrel monkeys (Saimiri sciureus), which are reported to develop a course of disease similar to humans, may be better suited than macaques for the identification of virulence determinants or the evaluation of therapeutics, we infected them with a green fluorescent protein-expressing MeV. Compared to cynomolgus macaques (Macaca fascicularis) infected with the same virus, the squirrel monkeys developed more-severe immunosuppression, higher viral load, and a broader range of clinical signs typical for measles. In contrast, infection with an MeV unable to interact with the epithelial receptor nectin-4, while causing immunosuppression, resulted in only a mild and transient rash and a short-lived elevation of the body temperature. Similar titers of the wild-type and nectin-4-blind MeV were detected in peripheral blood mononuclear cells and lymph node homogenates, but only the wild-type virus was found in tracheal lavage fluids and urine. Thus, our study demonstrates the importance of MeV interactions with nectin-4 for clinical disease in the new and better-performing S. sciureus model of measles pathogenesis.IMPORTANCE The characterization of mechanisms underlying measles virus clinical disease has been hampered by the lack of an animal model that reproduces the course of disease seen in human patients. Here, we report that infection of squirrel monkeys (Saimiri sciureus) fulfills these requirements. Comparative infection with wild-type and epithelial cell receptor-blind viruses demonstrated the importance of epithelial cell infection for clinical disease, highlighting the spread to epithelia as an attractive target for therapeutic strategies.

Waist circumference percentiles for Singaporean children and adolescents aged 6-17 years.

AIM: Population-specific waist circumference (WC) percentiles are crucial for screening children at higher obesity-related metabolic risk. This study aimed to develop age- and gender-specific WC percentile curves for Singaporean children and adolescents. METHODS: 3029 participants (boys, 1506; girls, 1523) from different population strata of Singapore were recruited. Stature, weight and WC were measured and BMI calculated. Smoothed WC percentile curves and cut-offs for the 3rd, 5th, 10th, 25th, 50th, 75th, 85th, 90th, 95th, 97th were constructed using the Cole's LMS method. RESULTS: WC and BMI increased with age in both sexes and boys had higher WC than girls at every age. Comparison of 50th and 90th percentiles with other populations showed distinct difference in WC curve patterns and values of Singaporean children. CONCLUSIONS: We present the first working WC percentile curves and age- and gender-specific cut-offs of Singaporean children and adolescents. These cut-offs and curves can serve as valuable reference for screening and identify children at a higher metabolic risk, for international comparisons and to better understand secular trends in paediatric obesity.

Nectin-4-dependent measles virus spread to the cynomolgus monkey tracheal epithelium: role of infected immune cells infiltrating the lamina propria.

After the contagion measles virus (MV) crosses the respiratory epithelium within myeloid cells that express the primary receptor signaling lymphocytic activation molecule (SLAM), it replicates briskly in SLAM-expressing cells in lymphatic organs. Later, the infection spreads to epithelia expressing nectin-4, an adherens junction protein expressed preferentially in the trachea, but how it gets there is not understood. To characterize the mechanisms of spread, we infected groups of 5 or 6 cynomolgus monkeys (Macaca fascicularis) with either a wild-type MV or its "N4-blind" derivative, which is unable to enter nectin-4-expressing cells because of the targeted mutation of two hemagglutinin residues. As expected, both viruses caused similar levels of immunosuppression, as monitored by reductions in white blood cell counts and lymphocyte proliferation activity. However, monkeys infected with the N4-blind MV cleared infection more rapidly. Wild-type virus-infected monkeys secreted virus, while marginal virus titers were detected in tracheal lavage fluid cells of N4-blind MV-infected hosts. Analyses of tracheal rings obtained at necropsy (day 12) documented widespread infection of individual cells or small cell clusters in the subepithelial lamina propria of monkeys infected with either virus. However, only wild-type MV spread to the epithelium, forming numerous infectious centers comprised of many contiguous columnar cells. Infected CD11c(+) myeloid (macrophage or dendritic) cells were frequently observed in the lamina propria below epithelial infectious centers. Thus, MV may use myeloid cells as vehicles not only immediately after contagion but also to infect epithelia of tissues expressing nectin-4, including the trachea.

Canine distemper virus epithelial cell infection is required for clinical disease but not for immunosuppression.

To characterize the importance of infection of epithelial cells for morbillivirus pathogenesis, we took advantage of the severe disease caused by canine distemper virus (CDV) in ferrets. To obtain a CDV that was unable to enter epithelial cells but retained the ability to enter immune cells, we transferred to its attachment (H) protein two mutations shown to interfere with the interaction of measles virus H with its epithelial receptor, human nectin-4. As expected for an epithelial receptor (EpR)-blind CDV, this virus infected dog and ferret epithelial cells inefficiently and did not cause cell fusion or syncytium formation. On the other hand, the EpR-blind CDV replicated in cells expressing canine signaling lymphocyte activation molecule (SLAM), the morbillivirus immune cell receptor, with similar kinetics to those of wild-type CDV. While ferrets infected with wild-type CDV died within 12 days after infection, after developing severe rash and fever, animals infected with the EpR-blind virus showed no clinical signs of disease. Nevertheless, both viruses spread rapidly and efficiently in immune cells, causing similar levels of leukopenia and inhibition of lymphocyte proliferation activity, two indicators of morbillivirus immunosuppression. Infection was documented for airway epithelia of ferrets infected with wild-type CDV but not for those of animals infected with the EpR-blind virus, and only animals infected with wild-type CDV shed virus. Thus, epithelial cell infection is necessary for clinical disease and efficient virus shedding but not for immunosuppression.

Adherens junction protein nectin-4 is the epithelial receptor for measles virus.

Measles virus is an aerosol-transmitted virus that affects more than 10 million children each year and accounts for approximately 120,000 deaths. Although it was long believed to replicate in the respiratory epithelium before disseminating, it was recently shown to infect initially macrophages and dendritic cells of the airways using signalling lymphocytic activation molecule family member 1 (SLAMF1; also called CD150) as a receptor. These cells then cross the respiratory epithelium and transport the infection to lymphatic organs where measles virus replicates vigorously. How and where the virus crosses back into the airways has remained unknown. On the basis of functional analyses of surface proteins preferentially expressed on virus-permissive human epithelial cell lines, here we identify nectin-4 (ref. 8; also called poliovirus-receptor-like-4 (PVRL4)) as a candidate host exit receptor. This adherens junction protein of the immunoglobulin superfamily interacts with the viral attachment protein with high affinity through its membrane-distal domain. Nectin-4 sustains measles virus entry and non-cytopathic lateral spread in well-differentiated primary human airway epithelial sheets infected basolaterally. It is downregulated in infected epithelial cells, including those of macaque tracheae. Although other viruses use receptors to enter hosts or transit through their epithelial barriers, we suggest that measles virus targets nectin-4 to emerge in the airways. Nectin-4 is a cellular marker of several types of cancer, which has implications for ongoing measles-virus-based clinical trials of oncolysis.

[Construction of expression vector with antisense Rubisco activase gene and its genetic transformation in rice].

In this research, Rubisco activase gene (rca) was amplified using specific primers and inserted into pGEM T-easy vector, and then cut with EcoRI after confirming by sequencing. The fragment was subcloned into pBluescript KS+, digested with the enzyme BamHI and inserted into the binary expression vector pCAMBIA1301, and the resulting construction with antisense rca was named pCAMR02. The pCAMR02 vector was introduced into Agrobacterium tumefaciens strain EHA105 by electroporation and transformed to embryos of rice (Oryza. Sativa L.ssp.japonica) cultivar Zhonghua11 via Agrobacterium tumefaciens system. Plantlets were regenerated in vitro by resistance selection on medium containing various concentrations of hygromycin. Both GUS histochemical assays and PCR amplification demonstrated that antisense rca was integrated into T0 genomes and inherited to T1. The measurement of phenotypes of transgenic rice plants with antisense rca showed that most of them could hardly survive at ambient CO2 concentration, even could not grow. The antisense plants that survived under natural condition were dwarf and grew slower than the wild-type controls, and their contents of RCA and Rubisco changed significantly. These plants generated in this experiment will be used to study the relationship between RCA and Rubisco and their regulation.