A Variant in the IRF6 Promoter Associated with the Risk for Orofacial Clefting.

The single-nucleotide polymorphism (SNP) rs2235371 (IRF6 V274I) is associated with nonsyndromic cleft lip with or without cleft palate (NSCL/P) in Han Chinese and other populations but appears to be without a functional effect. To find the common etiologic variant or variants within the haplotype tagged by rs2235371, we carried out targeted sequencing of an interval containing IRF6 in 159 Han Chinese with NSCL/P. This study revealed that the SNP rs12403599, within the IRF6 promoter, is associated with all phenotypes of NSCL/P, especially nonsyndromic cleft lip (NSCLO) and a subphenotype of it, microform cleft lip (MCL). This association was replicated in 2 additional much larger cohorts of cases and controls from the Han Chinese. Conditional logistic analysis indicated that association of rs2235371 with NSCL/P was lost if rs12403599 was excluded. rs12403599 contributes the most risk to MCL: its G allele is responsible for 38.47% of the genetic contribution to MCL, and the odds ratios of G/C and G/G genotypes were 2.91 and 6.58, respectively, for MCL. To test if rs12403599 is functional, we carried out reporter assays in a fetal oral epithelium cells (GMSM-K). Unexpectedly, the risk allele G yielded higher promoter activity in GMSM-K. Consistent with the reporter studies, expression of IRF6 in lip tissues from NSCLO and MCL patients with the G/G phenotype was higher than in those from patients with the C/C phenotype. These results indicate that rs12403599 is tagging the risk haplotype for NSCL/P better than rs2235371 in Han Chinese and supports investigation of the mechanisms by which the allele of rs12403599 affects IRF6 expression and tests of this association in different populations.

Single cell transcriptomics in human osteoarthritis synovium and in silico deconvoluted bulk RNA sequencing.

OBJECTIVES: To reveal the heterogeneity of different cell types of osteoarthritis (OA) synovial tissues at a single-cell resolution, and determine by novel methodology whether bulk-RNA-seq data could be deconvoluted to create in silico scRNA-seq data for synovial tissue analyses. METHODS: OA scRNA-seq data (102,077 synoviocytes) were provided by 17 patients undergoing total knee arthroplasty; 9 tissues with matched scRNA-seq and bulk RNA-seq data were used to evaluate six in silico gene deconvolution tools. Predicted and observed cell types and proportions were compared to identify the best deconvolution tool for synovium. RESULTS: We identified seven distinct cell types in OA synovial tissues. Gene deconvolution identified three (of six) platforms as suitable for extrapolating cellular gene expression from bulk RNA-seq data. Using paired scRNA-seq and bulk RNA-seq data, an "arthritis" specific signature matrix was created and validated to have a significantly better predictive performance for synoviocytes than a default signature matrix. Use of the machine learning tool, Cell-type Identification By Estimating Relative Subsets of RNA Transcripts x (CIBERSORTx), to analyze rheumatoid arthritis (RA) and OA bulk RNA-seq data yielded proportions of T cells and fibroblasts that were similar to the gold standard observations from RA and OA scRNA-seq data, respectively. CONCLUSION: This novel study revealed heterogeneity of synovial cell types in OA and the feasibility of gene deconvolution for synovial tissue.

[Serum procalcitonin in patients with pulmonary infection and central nervous system injury].

Objective: To evaluate the influence of serum procalcitonin in the diagnosis and treatment of pulmonary infection in patients with central nervous system injury. Methods: From October 2014 to February 2017, a retrospective study was performed. A total of 1 852 patients were screened in Department of Intensive Care Unite, First Affiliated Hospital of Sun Yat-sen University.Among them, 173 patients were identified with different kinds of infection. Finally, a total of 42 patients with pulmonary infection were enrolled. The clinical data of patients with pulmonary infection and central nervous system (CNS) injury was collected. A univariate and multivariate regression analysis was performed to study the correlation of serum procalcitonin (PCT) with clinical symptoms and signs of the pulmonary infection, body temperature(T), white blood cell count (WBC), percentage of neutrophils (NEU) and the severity of the pulmonary infection (CPIS). The relationship of serum PCT with type of CNS injury, GCS, and exogenous glucocorticoid was further studied. Results: During the period of pulmonary infection, the peak PCT was 0.83 (0.29, 2.79) µg/L and the CPIS was 5.50 (5.00, 7.00). In 9 of 42 patients, the peak PCT was less than 0.25 µg/L. In 7 of 42 patients, the peak PCT was ranged from 0.25 to 0.5 µg/L. In 12 of 42 patients, PCT was ranged from 0.5 to 2 µg/L. Only 10 patients had a PCT 2-10 µg/L and 4 patients had a PCT more than 10 µg/L. There is no correlation between serum PCT and body temperature, white blood cell, percentage of neutrophils and CPIS. There was no significant differences in patients with PCT<0.5 or ≥0.5 µg/L regarding the body temperature, white blood cell, percentage of neutrophils and CPIS. However, serum PCT in patients with pulmonary infection had independent correlation with the post CNS injury day (ß=0.17, 95% CI (0.02, 0.32), P<0.05). The serum PCT was 1.26 (0.47, 2.7) µg/L and 29.41% patients with a PCT less than 0.5 µg/L within 3 days post CNS injury. Serum PCT level was 0.23 (0.16, 0.39) µg/L, and 77.78% patients with a PCT less than 0.5 µg/L at day 4 to day 7 post-injury. The PCT level was 0.52 (0.33, 1.12) µg/L, and 44.44% patients with a PCT less than 0.5 µg/L at day 8 to day 14. The PCT was 3.26 (2.07, 12.40) µg/L, and no patient with a PCT less than 0.5 µg/L after day 15 post-injury. There were no significant relationship found between serum PCT level and type of the disease and surgery, GCS, and use of exogenous glucocorticoid. Conclusions: Serum PCT had no significant increase and was not able to be used in guiding the antibiotics use in patients with CNS injury and pulmonary infection.

Biological evaluation of boronated unnatural amino acids as new boron carriers.

There is a pressing need for new and more efficient boron delivery agents to tumor cells for use in boron neutron capture therapy (BNCT). A class of boronated unnatural cyclic amino acids has demonstrated a remarkable selectivity toward tumors in animal and cell culture models, far superior to currently used agents in clinical BNCT. One of these amino acids, 1-amino-3-boronocyclopentanecarboxylic acid (ABCPC), has shown a tumor to blood ratio of 8 and a tumor to normal brain ratio of nearly 21 in a melanoma bearing mouse model. This work represents further biological characterization of this compound for tumor targeting in an EMT6 murine mammary carcinoma mouse model and a T98G human glioblastoma cell line. Female BALB/c mice bearing EMT6 tumors were injected with the fructose complex form of racemic mixtures of cis and trans isomers of ABCPC in identical concentrations. Boron concentrations were measured in the tumor, blood, brain, skin, and liver tissues at 1, 3, and 5 h post-injection. These observations revealed a remarkable difference in racemic mixtures of cis and trans isomers in tumor targeting by boron. This implies that further separation of the L and D forms of this compound may enhance tumor targeting to an even higher degree than that provided by the racemic mixtures. Since the uptake measurements were made in homogenized tumor and normal tissues, little is known about the subcellular location of the boron arising from the various isomeric forms of the amino acid. To study subcellular delivery of boron from ABCPC in T98G human glioblastoma cells, we employed secondary ion mass spectrometry (SIMS) based technique of ion microscopy, which is capable of quantitatively imaging isotopic (elemental) gradients in cells and tissues at 500 nm spatial resolution. The T98G cells were exposed to the nutrient medium containing 100 ppm boron equivalent of a mixture of both L and D isomers of ABCPC in the form of a fructose complex for 1 h. Following this treatment, the cells were fast frozen, freeze-fractured, and freeze-dried for SIMS analysis. Within an hour of exposure, ABCPC provided partitioning of intracellular to extracellular boron of 3/1. SIMS imaging revealed that boron from ABCPC was distributed throughout the cell, including the nucleus. This level of boron delivery within an hour of exposure is superior to p-boronophenylalanine (BPA) and sodium borocaptate (BSH), which have been previously studied by SIMS in the same cell line. These encouraging observations provide compelling support for further isomeric separations of ABCPC into the D and L forms for enhanced tumor targeting and continued testing of these compounds as new boron carriers in BNCT.

The syntheses and in vivo biodistribution of novel boronated unnatural amino acids.

A series of boronated, unnatural amino acids were prepared and their biodistribution determined in melanoma bearing mice. The unnatural amino acids were prepared utilizing recently developed borylation. The majority of the syntheses utilize metal catalyzed additions of diboron agents to unsaturated carbonyl compounds. Biodistribution studies in mice bearing melanoma tumors indicated that all the boronated amino acids were taken up by the melanoma tumors. The data for the cyclic five-membered ring analogue, 1-amino-3-boronocyclopentanecarboxylic acid, was most striking, exhibiting a nearly 22:1 ratio of boron concentration for tumor to brain at the 2 h time point, dropping to 7.3 after 6 h. The tumor to blood and tumor to skin ratios were also quite high. It is important to note that all of the amino acids were synthesized as racemic and diastereomeric mixtures. Thus there is a high probability that a single enantiomer of 1-amino-3-boronocyclopentanecarboxylic acid might exhibit far higher selectivity.