[Analysis of gut target microbiota and species difference in patients with obstructive sleep apnea based on 16S rRNA sequencing].

OBJECTIVE: To explore the difference in gut microbiota composition between patients with obstructive sleep apnea (OSA) and healthy individuals and the role of gut microbiota in the pathogenesis of OSA. METHODS: Thirty-nine patients with OSA admitted to our hospital between May and December, 2022 and 20 healthy individuals were enrolled in this study. Stool samples were collected from all the participants for analysis of microbiome composition using 16S rRNA high- throughput sequencing analysis. The alpha diversity, beta diversity, and species difference were determined between the two groups and marker species analysis and metabolic pathway function prediction analysis were performed. RESULTS: The species diversity (Shannon and Simpson) indexes, richness (observed species) and evenness (Pielou) of gut microbiota were significantly lower in OSA patients than in the healthy individuals (P < 0.05). The OSA patients had also a significantly lowered community diversity (P < 0.05) with different gut microbial communities from those of the healthy individuals shown by increased relative abundance of potentially pathogenic bacteria such as Pseudomonas and Monocytogenes (P < 0.05). LEfSe analysis showed that the abundance of 23 species of gut microbiota differed significantly between the two groups and the OSA patients had significant increases in the abundance of Pseudomonas, Meganomonas, and Fusobacterium (P < 0.05). The differential marker flora affected host homeostasis. Random Forest and ROC curve analyses confirmed that Pseudomonas could be used as important biomarkers for a differential diagnosis. Metabolic pathway function prediction analysis showed that biosynthesis function had the greatest contribution to maintaining gut microbiota homeostasis, and Pseudomonas affected the occurrence and progression of OSA by participating in aromatic bioamine degradation and ketogluconic acid metabolic pathway. CONCLUSION: OSA patients have obvious gut microbiota disturbances, and Pseudomonas may affect the development of OSA by participating in substance metabolism to serve as the potential target gut bacteria for OSA treatment.

[Interpretation of the third edition Bethesda system for reporting thyroid cytopathology].

Based on the changes of the classification and nomenclature in the fifth edition of the World Health Organization (WHO) classification of thyroid neoplasms, the third edition Bethesda system for reporting thyroid cytopathology (TBSRTC) was revised in June 2023. Two new chapters have been added: one addressing the clinical perspectives and imaging findings in thyroid disease and another summarizing the molecular test for thyroid cytology. A discussion of risk of malignancy (ROM) and clinical management algorithms for pediatric thyroid carcinoma have been added. The third edition provides an average ROM for each category, in addition to the expected range of cancer risk. This paper aims to interpret the main changes in the third edition TBSRTC and to provide guidelines for the clinical management of thyroid nodules.

A pretreatment scheme for plasmid extraction contained sugar, high concentration lysozyme and mild lysozyme removal.

To address the issue of low efficiency in extracting plasmid DNA (pDNA) from Lactobacillus plantarum by breaking the cell wall, we proposed an effective pretreatment scheme. This study investigated the impacts of lysozyme concentrations and glucose, as well as centrifugal forces during lysozyme removal in the pretreatment system. The efficiency of pDNA extraction was assessed using non-staining method, acridine orange staining method (AO staining) and agarose gel electrophoresis (AGE). Furthermore, the glucose high lysozyme method was compared to the commercial kit method and the lysozyme removal method using L. plantarum PC518, 9L15, JS193 and Staphylococcus aureus USA300. The results indicated that the pDNA extraction concentrations from the four tested strains were increased by 8.9, 7.2, 8.5, and 3.6 times, respectively, compared to the commercial kit method. Furthermore, they increased by 1.9, 1.5, 1.8, and 1.4 times, respectively, compared to the lysozyme removal method. The maximum average concentration of pDNA extraction (from L. plantarum PC518) reached 590.8 ± 31.9 ng/ul. In conclusion, the incorporation of sugar, high concentration lysozyme and mild lysozyme removal proved to be effective enhancements in improving the efficiency of pDNA extraction from L. plantarum. Using the pretreatment scheme, the concentration of pDNA extraction was significantly increased, approaching levels comparable to pDNA extraction from Gram-negative bacteria.

[Fibrocartilaginous mesenchymoma: a clinicopathological analysis of four cases].

Objective: To investigate the clinical, radiological, histological and molecular features and the differential diagnosis of fibrocartilaginous mesenchymoma (FM). Methods: Four cases of FM diagnosed in the Department of Pathology, the Sixth People's Hospital Affiliated to Shanghai Jiaotong University School of Medicine from 2020 to 2022 were analyzed. Related literature was also reviewed. Results: Case 1 was a 10-year-old girl with bone destruction in the sacrum and L5 articular processes revealed by CT scan. Case 2 was a 7-year-old girl with an aggressive lesion in her right distal ulna. Case 3 was an 11-year-old boy with a lesion in the metaphysis of his left proximal tibia. Case 4 was an 11-year-old boy with bone destruction in the distal portion of a radius. Microscopically, the four tumors all consisted of numerous spindle cells, hyaline cartilage nodules, and bone trabeculae. The hypocellular to moderately cellular spindle cell component contained elongated cells with slightly hyperchromatic, mildly atypical nuclei arranged in bundles or intersecting fascicles. Benign-appearing cartilaginous nodules of various sizes and shapes were scattered throughout the tumors. There were areas mimicking epiphyseal growth-plate characterized by chondrocytes arranged in parallel columns and areas of enchondral ossification. The stroma was rich in mucus in case 1. Mutation of GNAS and IDH1/IDH2 and amplification of MDM2 gene were not found in any of the three tested cases. Conclusions: FM is very rare and tends to affect young patients. It most frequently occurs in the metaphysis of long tubular bones, followed by the iliac-pubic bones and vertebrae. FM is characterized by a mixed population of spindle cells, hyaline cartilage nodules and trabeculae of bone, without specific immunophenotypes and molecular alternations. As a borderline, locally aggressive neoplasm, surgical removal with a wide margin is generally the treatment of choice for FM.

[Regulatory mechanism of interferon regulatory factor 1 by α-synuclein in mouse Parkinson's disease model].

OBJECTIVE: To investigate the molecular mechanism by which α-synuclein (α-Syn) regulates interferon regulatory factor 1 (IRF-1) expression. METHODS: SH-SY5Y cells overexpressing α-Syn and transgenic mouse model carrying human α-Syn gene with A53T mutation (3 and 6 months old) were examined for IRF-1 mRNA and protein expressions using real-time PCR and Western blotting, respectively. The subcellular localization of IRF-1 was determined with immunofluorescence staining and cytoplasmic/nuclear protein isolation. The optimal concentrations of the proteasome inhibitor MG132 (0.01-2.0 µmol/L) and lysosomal inhibitor chloroquine (5-200 µmol/L) for treatment of SH-SY5Y cells for 24 h were determined by examining the cell viability. SH-SY5Y cells were treated with 0.2 µmol/L MG132 and 30 µmol/L chloroquine for 24 h (the maximum dose that did not cause cell damage), and the changes of IRF-1 protein expressions was analyzed. The effects of α-Syn on MDM2 protein expression and IRF-1 ubiquitylation were analyzed using Western blotting and ubiquitylation assay. RESULTS: α-Syn overexpression did not affect the mRNA level of IRF-1 but significantly increased its protein level (P < 0.01). In α-Synoverexpressing SH-SY5Y cells, IRF-1 translocation was observed from the cytoplasm to the nucleus (P < 0.001). Treatment of the cells with 0.2 µmol/L MG132 significantly aggravated α-Syn-induced increase of IRF-1 protein expression (P < 0.01) while 30 µmol/L chloroquine produced no significant changes in IRF-1 level. α-Syn overexpression caused an obvious decrease of MDM2 protein level and further inhibited the ubiquitylation of IRF-1 (P < 0.01). CONCLUSION: α-Syn blocks MDM2-mediated ubiquitylation of IRF-1 through ubiquitin proteasome pathway, thereby enhancing IRF-1 protein expression.

[Primary study on the mechanism of ductus arteriosus closure in rats: impact of aging pathway].

Objective: To explore the role and mechanism of aging pathway in patent ductus arteriosus closure of rats. Methods: Thirty outbreeding Sprague Dawley rats(20 females, 10-15 weeks old, 270-330 g) underwent random mating and conception. The primary Ductus Arteriosus smooth muscle cells (DASMCs) of pregnant 19 days(E19 group), 21 days(E21 group) and newborn(Day0 group) fetus were extracted and cultured. mRNA expression of cell senescence related markers p16, 21 and 53 genes in each group were detected by real-time fluorescent quantitative PCR(RT-PCR) after 48 hours culture. After hypoxic culture on DASMCs for 3 days, the DASMCs were divided into 3 groups: hypoxic control group(G0 group), 3 hours normal oxygen concentration treatment group(G3 group) and 6 hours normal oxygen concentration treatment group(G6 group). After intervention, mRNA expression of p16, 21 and 53 RT-PCR was detected. The DASMCs of newborn rats(Day0 group) were extracted and divided into 3 groups:low-oxygen culture control group, low-oxygen+siRNA culture group and normal oxygen concentration culture group. The DASMCs migration ability was tested experimentally by Transwell method. Result: The mRNA levels of p16, 21 and 53 in DASMCs were higher in E19 group than in Day0 group(all P<0.01), and the mRNA levels of p16, 21 and 53 in DASMCs were also higher in E21 group than those in Day0 group (all P<0.01). The mRNA levels of p16, 21 and 53 in DASMC were all higher in G0 group than those in G3 group (P<0.05 or 0.01), and the mRNA levels of p16, 21 and 53 in DASMCs were all higher in G0 group than those in G6 group (all P<0.01), and the mRNA levels of p16, 21 and 53 in DASMCs were all higher in G3 group than those in G6 group (all P<0.05). DASMCs migration ability of newborn rats was higher in normal oxygen concentration culture group than that in low-oxygen culture group (P<0.01), and DASMCs migration ability of newborn rats was also higher in low-oxygen+siRNA culture group than that in low-oxygen culture group (P<0.01). Conclusion: The expression of senescence marker of DASMCs decreases with the birth in rats during the process of ductal closure, and the aging pathway may affect ductal closure by inhibiting DASMCs migration in rats.

T helper 22 cells from Han Chinese patients with atopic dermatitis exhibit high expression of inducible T-cell costimulator.

BACKGROUND: Atopic dermatitis (AD) is a heterogeneous disease, characterized by excess T helper (Th) 22 activation in Asian AD. Inducible T-cell costimulator (ICOS) is crucial for T-cell activation and differentiation. However, the role of ICOS in AD and its effect on Th22 cells remain unclear. OBJECTIVES: To gain a better understanding of the role of ICOS and ICOS ligand (ICOSL) in the pathogenesis of Asian AD and its underlying mechanisms. METHODS: We quantified ICOS and ICOSL expression in Han Chinese patients with AD and healthy controls (HC). Then, we assessed the proliferation and the production of the Th22 chemokines CCR4 and CCR10 by ICOSL-stimulated AD peripheral blood mononuclear cells (PBMCs) as well as their effects on keratinocyte filaggrin production. Finally, we explored the link between ICOS-expressing Th22 cells and disease activity and IgE levels in our patients with AD. RESULTS: Our patients with AD showed higher levels of ICOS-expressing Th22 cells as well as ICOSL-expressing CD19+ B cells and CD14+ monocytes compared with HC. ICOSL increased the proliferation and expression of CCR4 and CCR10, and of interleukin (IL)-22 in AD PBMCs. ICOSL treatment also significantly increased the downregulation of filaggrin expression by keratinocytes cocultured with PBMCs from patients with AD. Finally, blood levels of ICOS+ Th22 cells and ICOSL+ B cells in this AD cohort were correlated with disease activity as assessed by the SCORing Atopic Dermatitis index and with total IgE levels. In Han Chinese patients with AD, circulating Th22 cells, serum levels of IL-22 and IL-22+ cells in lesional skin were all markedly increased. CONCLUSIONS: Our findings demonstrate that ICOS/ICOSL expression and effects are linked to Th22 skewing and the pathogenesis of Han Chinese AD, which suggests ICOSL and ICOS as well as Th22 cells and IL-22 as new and promising therapeutic targets. What's already known about this topic? In Asian patients, atopic dermatitis (AD) is characterized by excess T helper (Th) 22 activation. Inducible T-cell costimulator (ICOS) is crucial for T-cell activation and differentiation. What does this study add? This study demonstrates that circulating Th22 cells, serum levels of interleukin (IL)-22 and IL-22+ cells are all markedly increased in lesional skin in Han Chinese patients with AD. In Han Chinese patients with AD, ICOS and ICOS ligand (ICOSL) drive Th22 skewing and increase filaggrin downregulation, and ICOS+ Th22 cells and ICOSL+ B cells are linked to disease activity. What is the translational message? ICOS+ Th22 cells and ICOSL+ B cells are potential clinical biomarkers of disease activity in Han Chinese patients with AD. ICOS- and ICOSL-targeted treatment approaches may benefit Han Chinese patients with AD.

Programmed death-1 ligands 1 and 2 expression in cutaneous squamous cell carcinoma and their relationship with tumour- infiltrating dendritic cells.

The programmed death-1 (PD-1) receptor ligands, PD-L1 and PD-L2, are co-stimulatory molecules that contribute to the negative regulation of T lymphocyte activation. It is still unclear whether there is correlation between PD-L1 or PD-L2 and tumour-infiltrating dendritic cells (TIDCs) in cutaneous squamous cell carcinoma (CSCC). The aim of this study was to analyse PD-L1 and PD-L2 expression and dendritic cells infiltration in tumour tissue of CSCC patients and investigate their clinical significance. Immunohistochemical analysis was used to evaluate the expression of PD-L1, PD-L2, CD1a and CD83 in 61 CSCC tissues. The immunofluoresence double-labelling technique was performed to detect the co-expression of PD-L1 or PD-L2 and CD1a or CD83 in tumour tissues. We found that 25 of 61 cases CSCC (40·98%) exhibited positivity for PD-L1, whereas 37 of 61 cases CSCC (60·66%) exhibited positivity for PD-L2. A higher percentage of CD1a-positive cases were observed on both PD-L1-positive and PD-L2-positive specimens compared with that of CD83-positive cases (92·29% versus 37·60%, 83·20% versus 33·16%). The expression of PD-L1 and PD-L2 on CD1a+ cells was significantly higher than that on CD83+ cells in tumour tissues of CSCC patients. Furthermore, the expression rate of PD-L1 was associated with UICC stage, and the expression rate of PD-L2 was associated with predominant differentiation and tumour size in CSCC. Our results indicated that higher expression of PD-L1 and PD-L2 on CD1a+ cells than that on CD83+ cells in CSCC tumour tissues may contribute to negative regulation in anti-tumour immune responses.

Fabrication of high-efficiency and low-stray-light grating by inductively coupled plasma(ICP) etching-polishing method.

Gratings with stray light of 4.99 × 10-7-5.67 × 10-7 and efficiency of 93%-95% in a wavelength range of 1592 nm-1632 nm on Si-surface-modification SiC, fused silica and BK7 have been fabricated by the method of ICP etching-polishing. The CHF3 and SF6 plasma were used to etch a preliminary grating profile. Ar and O2 plasma with low energy were then used to polish the grating to acquire low surface roughness and groove profiles closer to the ideal profiles. The morphologies of the gratings were characterized by AFM. The efficiencies and stray light were measured quantitatively by self-developed equipment. These results show that the ICP etching-polishing method is a promising candidate for production of good quality gratings into common optical materials.

Capability of GaN based micro-light emitting diodes operated at an injection level of kA/cm².

Different size InGaN/GaN based micro-LEDs (µLEDs) are fabricated. An extremely high injection level above 16 kA/cm2 is achieved for 10 µm-diameter LED. The lateral current density and carrier distributions of the µLEDs are simulated by APSYS software. Streak camera time resolved photoluminescence (TRPL) results show clear evidence that the band-gap renormalization (BGR) effect is weakened by strain relaxation in smaller size µLEDs. BGR affects the relaxation of free carriers on the conduction band bottom in multiple quantum wells (MQWs), and then indirectly affects the recombination rate of carriers. An energy band model based on BGR effect is made to explain the high-injection-level phenomenon for µLEDs.

Identifying Corticothalamic Network Epicenters in Patients with Idiopathic Generalized Epilepsy.

BACKGROUND AND PURPOSE: Corticothalamic networks are considered core pathologic substrates for idiopathic generalized epilepsy; however, the predominant epileptogenic epicenters within these networks are still largely unknown. The current study aims to identify these epicenters by resting-state functional connectivity. MATERIALS AND METHODS: To identify epicenters within the corticothalamic networks in idiopathic generalized epilepsy, we retrospectively studied a large cohort of patients with this condition (n = 97) along with healthy controls (n = 123) by resting-state functional MR imaging. The thalamus was functionally divided into subregions corresponding to distinct cortical lobes for 5 parallel corticothalamic networks. The functional connectivity between each voxel in the cortical lobe and the corresponding thalamic subregion was calculated, and functional connectivity strength was used to evaluate the interconnectivity of voxels in the cortex and thalamus. RESULTS: The projection of 5 cortical lobes to the thalamus is consistent with previous histologic findings in humans. Compared with controls, patients with idiopathic generalized epilepsy showed increased functional connectivity strength in 4 corticothalamic networks: 1) the supplementary motor area, pulvinar, and ventral anterior nucleus in the prefrontal-thalamic network; 2) the premotor cortex and ventrolateral nucleus in motor/premotor-thalamic networks; 3) the visual cortex, posterior default mode regions, and pulvinar in parietal/occipital-thalamic networks; and 4) the middle temporal gyrus in the temporal-thalamic network. CONCLUSIONS: Several key nodes were distinguished in 4 corticothalamic networks. The identification of these epicenters refines the corticothalamic network theory and provides insight into the pathophysiology of idiopathic generalized epilepsy.

Time-shift homotopic connectivity in mesial temporal lobe epilepsy.

BACKGROUND AND PURPOSE: Voxel-mirrored intrinsic functional connectivity allows the depiction of interhemispheric homotopic connections in the human brain, whereas time-shift intrinsic functional connectivity allows the detection of the extent of brain injury by measuring hemodynamic properties. We combined time-shift voxel-mirrored homotopic connectivity analyses to investigate the alterations in homotopic connectivity in mesial temporal lobe epilepsy and assessed the value of applying this approach to epilepsy lateralization and the prediction of surgical outcomes in mesial temporal lobe epilepsy. MATERIALS AND METHODS: Resting-state functional MR imaging data were acquired from patients with unilateral mesial temporal lobe epilepsy (n=62) (31 left- and 31 right-side) and healthy controls (n=33). Dynamic interhemispheric homotopic architecture seeding from each hemisphere was individually calculated by 0, 1, 2, and 3 repetition time time-shift voxel-mirrored homotopic connectivity. Voxel-mirrored homotopic connectivity maps were compared between the patient and control groups by using 1-way ANOVA for each time-shift condition, separately. Group comparisons were further performed on the laterality of voxel-mirrored homotopic connectivity in each time-shift condition. Finally, we correlated the interhemispheric homotopic connection to the surgical outcomes in a portion of the patients (n=20). RESULTS: The patients with mesial temporal lobe epilepsy showed decreased homotopic connectivity in the mesial temporal structures, temporal pole, and striatum. Alterations of the bihemispheric homotopic connectivity were lateralized along with delays in the time-shift in mesial temporal lobe epilepsy. The patients with unsuccessful surgical outcomes presented larger interhemispheric voxel-mirrored homotopic connectivity differences. CONCLUSIONS: This study showed whole patterns of dynamic alterations of interhemispheric homotopic connectivity in mesial temporal lobe epilepsy, extending the knowledge of abnormalities in interhemispheric connectivity in this condition. Time-shift voxel-mirrored homotopic connectivity has the potential for lateralization of unilateral mesial temporal lobe epilepsy and may have the capability of predicting surgical outcomes in this condition.

The decreased self-renewal potential of NPCs during human embryonic brain development with reduced activity of MAPKs.

Study of neural progenitor cells (NPCs) is important for treatment of degerative diseases in central nervous system. One of the key questions in NPCs transplantation therapy is about the understanding of which stage of the NPCs in brain development is ideal. Herein we investigated survival, proliferation and apoptosis of NPCs from 12 w, 16 w and 20 w human embryonic brain, meanwhile, the phosphorylation of mitogen-activated protein kinases (MAPKs) signaling were analyzed. The results showed that the survival, proliferation and cell division of 16 w and 20 w human NPCs significantly decreased comparing with 12 w human NPCs in vitro; and the NPCs apoptosis remarkably increased. Phosphorylation of ERK1/2 of 16 w and 20 w NPCs significantly decreased comparing with 12 w human NPCs, however phosphorylation of p38 MAPK increased. NPCs proliferation increase when ERK1/2 signaling is activated by PMA. The results demonstrated that self-renewal potential of NPCs decreased in culture during human embryonic brain development, the activity of ERK signaling pathway were decreased, and suggest NPCs from 12-week fetuses might be better donor for cell transplantation during the period of 12-20 weeks because of their advantage on survival and proliferation.

The increased expression of metabotropic glutamate receptor 5 in subventricular zone neural progenitor cells and enhanced neurogenesis in a rat model of intracerebral hemorrhage.

The metabotropic glutamate receptor 5 (mGluR5) is closely relative to the proliferation, survival, and differentiation of neural progenitor cells (NPCs). This study primarily examined the mGluR5 expression of NPCs in subventricular zone (SVZ) and the effects of mGluR5 on neurogenesis to intracerebral hemorrhage (ICH) rat. The experiment was designated as the following: (1) The ICH model was established by collagenase infusion into the right striatum of the rats, and the brain tissue was collected to assess the expression of mGluR5 in SVZ NPCs. (2) The rat brains were sampled for immunostaining of doublecortin (DCX) and 5-bromo-2'-deoxyuridine (BrdU) to examine the effects of the (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) on neurogenesis. (3) Behavioral testing was carried out to evaluate the effects of CHPG on neurofunctional recovery. The results of Western blot analysis showed that mGluR5 levels in the ipsilateral SVZ increased as early as at 3 days after ICH, peaked at 14 days. The change of mGluR5 mRNA level in the ipsilateral SVZ was generally similar to the pattern of Western blot analysis. The immunostaining also demonstrated that some nestin-positive cells were co-expressed with mGluR5. The injection of CHPG into ipsilateral ventricle increased DCX levels both in the ipsilateral striatum (STR) and the peri-lesion area of the striatum (PLA). Meanwhile, a significant difference in behavioral score was presented at 28 days after ICH between the CHPG-treated rats and the vehicle-treated or the non-treated rats. Our results demonstrated for the first time that the increased expression of mGluR5 in SVZ NPCs occurred in ICH rat. The CHPG promoted the neurogenesis and improved neurofunctional symptom induced by ICH. These results suggested that the increased expression of mGluR5 on NPCs in SVZ may play an important role in neurogenesis in ICH rat.

Metabotropic glutamate receptor 5 promotes proliferation of human neural stem/progenitor cells with activation of mitogen-activated protein kinases signaling pathway in vitro.

Metabotropic glutamate receptors (mGluRs) regulate neurogenesis in brain, but the mechanisms remain unknown. In this study, we investigated the effect of mGluR5 on the proliferation of human embryonic neural stem/progenitor cells (NPCs), the expression of cyclin D1 and the activation of signaling pathways of mitogen-activated protein kinases (MAPKs). Results showed that mGluR5 agonist (S)-3,5-dihydroxyphenylglycine hydrate (DHPG) increased the proliferation of NPCs by increasing cell activity, diameter of neurospheres and cell division, while mGluR5 siRNA and antagonist 6-methyl-2-(phenylethynyl) pyridine hydrochloride (MPEP) decreased the NPC proliferation. The mRNA and protein expressions of cyclin D1 increased with DHPG treatment and decreased after siRNA or MPEP treatment. It was also found that activation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal protein kinase (JNK) signaling pathways were involved in the proliferation of NPCs. After DHPG treatment, p-ERK1/2 and p-JNK2 levels increased, and meanwhile p-p38 level decreased; but p-ERK1/2 and p-JNK2 levels decreased after siRNA or MPEP treatment, and p-p38 level increased. Our findings demonstrated that mGluR5 promoted the proliferation of human embryonic cortical NPCs and increased cyclin D1 expression with the changes in phosphorylation of MAPKs signaling pathways in vitro, suggesting a novel mechanism for pharmacological study of treatment for ischemic brain injury and neurodegenerative disorders.

Molecular characterization, expression pattern and association analysis of the porcine BTG2 gene.

B-cell translocation gene 2 (BTG2), a member of the B-cell translocation gene family with anti-proliferative properties, have been characterized to be involved in cell growth, differentiation and survival. In this study, we cloned the full length sequences of cDNA and genomic DNA of BTG2 gene from the porcine skeletal muscle. Spatial expression analysis showed that the porcine BTG2 gene is expressed predominantly in muscle. Temporal expression analysis in longissimus dorsi muscle demonstrated that the expression of BTG2 gene has the highest expression at 60 days old in Large White while with a peak expression at 120 days old in Meishan. Temporal analysis also revealed that the expression of BTG2 gene is generally higher in Large White than in Meishan at all the developmental stages tested (65 days of conception and 3, 35, 60, 120, and 180 days of postnatal). A single nucleotide polymorphism (G417C) in the intron of BTG2 gene was then detected by PCR-RFLP in Large White × Meishan F2 resource population and association analysis suggested that this polymorphic site had significant association (P < 0.05) with the buttock fat thickness, fat percentage, lean muscle percentage, ratio of lean to fat and carcass length.

Investigating the binding interaction of azur A with hyaluronic acid via spectrophotometry and its analytical application.

The interaction between azur A (AA) and hyaluronic acid (HA) at AA concentrations from 3.430 x 10(-5) to 8.575 x 10(-5) M and sodium chloride concentrations from 0 to 0.01 M was investigated spectrophotometrically at 620 nm at temperatures from 0 to 50 degrees C. AA was shown to be a useful spectroscopic probe for detecting carboxyl groups in HA macromolecules. The interaction between AA and HA was temperature sensitive and little AA-HA interaction was observed at temperatures higher than 30 degrees C. The interaction of HA with AA was seen to be electrostatic in nature. The maximum binding number decreased with decreasing NaCl concentration, and the absorbance sensitivity decreased with increasing NaCl concentration in aqueous solution. Self-interference from the AA in the AA-HA interaction caused an overestimate of the molar mass of hyaluronic acid. An improved method was proposed to estimate the molar mass of HA, and a molar mass of 1.219 x 10(6) Da was obtained with this improved method for HA.

Simultaneous quantification of seven active metabolites of roxifiban in human plasma by LC/MS/MS in the presence of an interfering displacer at millimolar concentrations.

Roxifiban (DMP 754) is a glycoprotein (GP) IIb/IIIa antagonist. Following oral administration to humans, roxifiban is metabolized to its primary active zwitterionic form, XV459, and several minor, active, hydrolyzed and hydroxylated metabolites, namely, M1a (DPC-AD3508), M1b (DPC-AD6128), M2 (SW156), M3 (DPC-AG2185), M8a (DPC-AF5814), and M8b (DPC-AF5818). Quantification of these metabolites in humans was not workable with a previous analytical method due to ion suppression of at least four of the analytes by a competitive displacer, DMP 728. This compound, which is another GP IIb/IIIa antagonist with very high affinity for the platelet receptor, was added to harvested blood samples in millimolar quantity to liberate XV459 from the GP IIb/IIIa receptor. An automated ion exchange solid phase extraction (IX-SPE) procedure was developed to selectively extract the seven metabolites of roxifiban and its deuterated internal standard while specifically excluding DMP 728. Among the six hydroxylation metabolites, there were two pairs of epimeric diastereomers (M1a/M1b and M8a/M8b) and one pair of geometric isomers (M2/M3), corresponding to three critical chromatographic pairs that needed to be base-line resolved because of the lack of specificity of MS/MS detection for these isomers. A new LC/MS/MS assay was developed to simultaneously quantify the seven metabolites in human plasma. The assay method was validated under GLP conditions over the concentration range of 0.5 to 80 nM for each of the analytes and successfully applied to assaying approximately 500 plasma samples from clinical trials.

Cytotoxic T-lymphocyte antigen 4 gene polymorphisms and susceptibility to acute allograft rejection.

BACKGROUND: Cytotoxic T-lymphocyte antigen 4 (CTLA4) has been shown to play a critical role in the down-regulation of the immune response. We retrospectively examined the association between acute rejection and two polymorphisms in the CTLA4 gene, the dinucleotide (AT)n repeat polymorphism in exon 3 and the single nucleotide polymorphism A/G at position 49 in exon 1, in a cohort of liver and kidney transplant recipients. METHODS AND RESULTS: A total of 207 liver and 167 renal transplant recipients were analyzed. In the case of the (AT)n repeat polymorphism we found an increased incidence of acute rejection in association with allele 3 and 4 in both liver and kidney (P=0.002 and 0.05, respectively). In addition, in liver transplant recipients, allele 7 was associated with acute rejection independent of ethnicity (P<0.05). Allele 1 was less frequently observed in African American as compared with Caucasian liver and kidney transplant recipients, with a frequency of 33.8% and 69%, respectively (P<0.0001). Those patients with allele 1 had a tendency toward a lower rate of rejection at 42% versus 57.8% (P=0.058), suggesting a potential protective effect of allele 1. Analysis of the A/G single nucleotide polymorphism demonstrated no association between either allele and the incidence of acute rejection in the patients studied. CONCLUSION: These initial observations provide the necessary basis to further investigate the risk stratification of transplant recipients based on specific CTLA4 gene polymorphisms.