[Identification of rare variants in exons of NLRC4 gene in patients with type 1 diabetes and their impact on gene function].

Objective: To identify rare variants in exon and exon-intron boundary of containing NLR family CARD domain protein 4 (NLRC4) in type 1 diabetes (T1DM) patients, and to explore their effects on gene function. Methods: A total of 508 T1DM patients and 527 healthy controls in the Department of Metabolic Endocrinology, Second Xiangya Hospital of Central South University from August 2017 to September 2020 were selected. The case group included 264 males and 244 females, and the age [M (Q1, Q3)] was [27 (11, 43)] years. The control group included 290 males and 237 females, and their ageï¼»M（Q1，Q3）］was [47 (36, 60)] years old. Identification of rare variants in exons of NLRC4 gene in T1DM patients and healthy controls was performed and verified by next-generation sequencing and sanger sequencing. The NLRC4 gene wild-type and mutant plasmids were constructed and transfected into 293T cells. Western blot (WB) was used to detect the expression of NLRC4 protein and cleavage products of pro-cysteinyl aspartate specific proteinase(procaspase-1). Cycloheximide (CHX) was added to 293T cells transfected with wild-type or mutant NLRC4 plasmid to detect the degradation of NLRC4 protein. The localization of NLRC4 protein was detected by immunofluorescence, and the concentration of IL-1ß in the cell supernatant was detected by enzyme-linked immunosorbent assay (ELISA). Results: The sequencing results showed that 4 patients and 2 healthy controls had a heterozygous variant c.208C>T in exon 3 of the NLRC4 gene. Two patient had a heterozygous variant c.1564T>C in exon 4, and 1 patients had c.1219G>C in exon 4. These three variants might be pathogenic variants in T1DM. In 293T cells transfected with NLRC4 wild-type and c.208C>T、c.1564T>Cc.1219G>C mutant plasmids, the expression level, degradation rate, localization of NLRC4 protein and the content of cleavage products of procaspase-1 did not change significantly. However, the concentration of IL-1ß secreted by 293T cells transfected with c.1219G>C and c.208C>T plasmid [M(Q1, Q3)] was 15.25 (12.98, 17.52) and 15.44 (13.81, 17.07) ng/L, respectively, which was lower than 18.70 (16.59, 20.81) ng/L of 293T cells transfected wild-type plasmid (P=0.020, 0.010). Conclusions: NLRC4 gene rare variants c.208C>T, c.1564T>C and c.1219G>C may not change the protein expression, degradation and localization, but c.208C>T and c.1219G>C may inhibit the secretion of IL-1ß. This result suggests that NLRC4 rare variants may have an impact on gene function.

[Adult femur CT modeling and 3D automatic measurement of anatomical parameters].

Objective: To reconstruct a 3D model from adult femur CT scan data and automate a measurement of femoral anatomical parameters to study the characteristics of Chinese femur anatomical parameters. Methods: Using Mimics17.0, models from the CT data of 148 adult patients were established. The completed model was imported into Geomagic Studio and the anatomical landmarks of the femur were extracted to establish 3D coordinate system and unified coordinate system. Programmed with Matlab, using the nearest point iterative ICP algorithm and the 3D automatic extraction algorithm of anatomical landmarks to provide precision positioning, the femoral anatomical parameters were automatically measured and analyzed. The data were compared by using independent sample t test. Results: In this group, the diameter of the male femoral condyle was (84.1±3.6) mm, and it was (74.8±3.3) mm in the female; the anteroposterior diameter of the male femoral condyle was (66.5±3.7) mm, and it was (61.2±3.5) mm in the female; the diameter of the male ball head was (48.8±2.1) mm and it was (43.4±2.2) mm in the female; the differences between the two genders were all statistically significant (t=16.21, 8.84, 15.20, all P<0.05). The male femoral moment radius was (112.5±24.5) mm, and it was (124.7±19.2) mm in the female (t=3.30, P=0.002). The neck angle in male participants was 124.9°±4.0°, and it was 126.1°±5.5° in the female (t=1.40, P>0.05). As the height growed, most of the anatomical parameters increased accordingly. Conclusions: The 3D automatic measurement of femoral anatomical parameters is more reproducible and accurate than manual measurement. It is necessary to establish and enrich the femoral anatomical database to design and develop internal fixation products that meet the needs of Chinese people.

[Development of a method to detect serum unmethylated insulin DNA].

Objective: To develop a specific and sensitive method to detect serum unmethylated insulin (INS) DNA for monitoring pancreatic beta cell death and evaluate its clinical practice value. Methods: This study validated and modified the Akirav method and the Husseiny method. Six type 1 diabetes mellitus (T1DM) patients, who were diagnosed with T1DM in the Second Xiangya Hospital, Central South University between February 2014 and February 2015, and another 8 sex- and age-matched healthy controls, were enrolled in the study. Patients' serum samples were examined by the modified Akirav method. Results: Methylation-independent PCR of the Akirav method cannot amplify the specific product. The efficiency of primers for quantitative methylation-specific PCR used in Husseiny method was (76.98±2.88)%, which was lower than 80% (recommend efficiency), resulting in poor reliability. This study applied primers for the Husseiny method into the procedures of the Akirav method.The results showed that the level of unmethylated INS DNA in T1DM group is significantly higher than that in the healthy controls[demethylation index: 0.039(0.028-0.083) vs 0.016(0.003-0.029), P=0.005]. Conclusions: The modified Akirav method can effectively detect the level of unmethylated INS DNA. The level of unmethylated INS DNA in T1DM patients is higher than that in the healthy controls.

[Identification of HLA class â ¡ susceptible alleles and genotypes in latent autoimmune diabetes in adults].

Objective: To identify human leukocyte antigen (HLA)-DRB1, DQA1 and DQB1 susceptible alleles and genotypes in latent autoimmune diabetes in adults (LADA) patients of Chinese Han nationality. Methods: All subjects including 652 LADA patients and 1 181 healthy controls from 1999 to 2015 in Han nationality region of Hunan province were genotyped with high resolution at HLA-DRB1, DQA1 and DQB1 locus by PCR-sequence based typing (PCR-SBT). Frequencies of genotypes between patients and controls were compared by chi square test. Results: The DQA1 susceptible allele was DQA1*03 (OR=1.23, P(c)=0.028); the DQB1 susceptible alleles were DQB1*0201 (OR=2.24, P(c)<0.001), DQB1*0303 (OR=1.30, P(c)=0.030), DQB1*0304 (OR=10.23, P(c)=0.004) and DQB1*0401(OR=1.94, P(c)<0.001); and the DRB1 susceptible alleles were DRB1*0301 (OR=2.10, P(c)<0.001), DRB1*0405(OR=1.89, P(c)<0.001) and DRB1*0901(OR=1.36, P(c)=0.008), respectively in Chinese Han nationality LADA patients. The HLA-Ⅱ susceptible genotypes were DQA1*03/05 (OR=1.81, P(c)=0.007), DQB1*0201/0201(OR=5.74, P(c)<0.001), DQB1*0201/0303 (OR=2.58, P(c)=0.010), DRB1*0301/0901(OR=3.43, P(c)=0.028) and DRB1*0901/0901 (OR=1.82, P(c)=0.021), respectively in LADA patients. DQB1*0201 and DRB1*0301 were shared susceptible alleles for Chinese Han and Caucasian LADA patients, while DQA1*03, DQB1*0303, DQB1*0304, DQB1*0401, DRB1*0405 and DRB1*0901 were specific susceptible alleles for Chinese Han LADA patients. Interestingly, the DQB1*0303 allele was susceptible in Chinese while protective in Caucasian (OR: 1.30 vs 0.29). Conclusion: Susceptible alleles are DQA1*03, DQB1*0201, DQB1*0303, DQB1*0304, DQB1*0401, DRB1*0301, DRB1*0405 and DRB1*0901, and susceptible genotypes are DQA1*03/05, DQB1*0201/0201, DQB1*0201/0303, DRB1*0301/0901 and DRB1*0901/0901 in Chinese LADA patients.

Association of 3q13.32 variants with hip trochanter and intertrochanter bone mineral density identified by a genome-wide association study.

We performed a GWAS of trochanter and intertrochanter bone mineral density (BMD) in the Framingham Heart Study and replicated in three independent studies. Our results identified one novel locus around the associated variations at chromosomal region 3q13.32 and replicated two loci at chromosomal regions 3p21 and 8q24. Our findings provide useful insights that enhance our understanding of bone development, osteoporosis, and fracture pathogenesis. INTRODUCTION: Hip trochanter (TRO) and intertrochanter (INT) subregions have important clinical relevance to subtrochanteric and intertrochanteric fractures but have rarely been studied by genome-wide association studies (GWASs). METHODS: Aiming to identify genomic loci associated with BMD variation at TRO and INT regions, we performed a GWAS utilizing the Framingham Heart Study (FHS, N = 6,912) as discovery sample and utilized the Women's Health Initiative (WHI) African-American subsample (N = 845), WHI Hispanic subsample (N = 446), and Omaha osteoporosis study (N = 971), for replication. RESULTS: Combining the evidence from both the discovery and the replication samples, we identified one novel locus around the associated variations at chromosomal region 3q13.32 (rs1949542, discovery p = 6.16 × 10-8, replication p = 2.86 × 10-4 for INT-BMD; discovery p = 1.35 × 10-7, replication p = 4.16 × 10-4 for TRO-BMD, closest gene RP11-384F7.1). We also replicated two loci at chromosomal regions 3p21 (rs148725943, discovery p = 6.61 × 10-7, replication p = 5.22 × 10-4 for TRO-BMD, closest gene CTNNB1) and 8q24 (rs7839059, discovery p = 2.28 × 10-7, replication p = 1.55 × 10-3 for TRO-BMD, closest gene TNFRSF11B) that were reported previously. We demonstrated that the effects at both 3q13.32 and 3p21 were specific to the TRO, but not to the femoral neck and spine. In contrast, the effect at 8q24 was common to all the sites. CONCLUSION: Our findings provide useful insights that enhance our understanding of bone development, osteoporosis, and fracture pathogenesis.

Arthroscopic reconstruction of the anterior cruciate ligament with the LARS artificial ligament: thirty-six to fifty-two months follow-up study.

OBJECTIVES: This study aims to analyze the operating methods and fundamental clinical effects of the reconstruction of the anterior cruciate ligament (ACL) with the Ligament Advancement Reinforcement System (LARS) artificial ligaments using an arthroscopy. PATIENTS AND METHODS: Fifty-three patients with ACL rupture who were treated using LARS artificial ligaments were enrolled in this retrospective study. The mean age at the time of reconstruction was 31.2 y (range, 22-51y). Average time from injury to surgery was 18 d (range, 5-51 d). Average follow-up period was 45 mo (range 36-52 months). Follow-up examinations included the Lysholm Knee Score and the International Knee Documentation Committee (IKDC) score. RESULTS: The average Lysholm Knee Score was 53.1±5.8 preoperatively (range, 47-76) versus 93.2±3.4 three years after operation (range, 80-100). Fifty-one of 53 patents (96.2%) showed good or excellent results at final assessment. The final IKDC score 3 years after operation were normal in 28 patients (52.8%), nearly normal in 23 patients (43.4%), and abnormal in 2 patients (3.8%). No postoperative complications, such as infection, ligament rupture, or ligament cinch occurred. CONCLUSIONS: The results suggest that LARS artificial ligament appears to be an effective graft for ACL reconstruction leading to good knee function and stability. Long-term follow-up should be performed to confirm the durable stability of the knee and the tolerance of the knee to the LARS artificial ligament.

Effect of air abrasion and dye on the surface element ratio and resin bond of zirconia ceramic.

The purpose of this study was to evaluate the effect of the surface element composition and roughness of dyed or non-dyed zirconia ceramic by air abrasion on the bond durability to resin luting cement. The sintered zirconia ceramic specimens were divided into four groups as follows: polished, dyed/polished, abraded, dyed/abraded. The surface roughness of each group was measured by a 3D laser scanning microscope. Energy-dispersive spectroscopy was applied for identifying and quantifying the elemental composition of the zirconia ceramic surfaces Each group had 20 shear test specimens of zirconia to Panavia F and was divided into two subgroups (n = 10) after 0 or 10 000 thermal cycles to receive the shear test. Compared with polishing, air abrasion significantly reduced the oxygen atom and weight ratio, increased the zirconium atom and weight ratio, increased R(a) and R(y) of zirconia ceramic surface, and also increased the bond strength of dyed and non-dyed zirconia ceramic to Panavia F after 0 and 10 000 thermal cycles. The dying procedure could affect the abraded and non-abraded zirconia ceramic surface oxygen and zirconia element ratios and surface characteristics. However, the dying procedure does not affect the resin bond strength and durability of zirconia ceramic.Corrections were made to this article on 3 November 2011. The footnote for the corresponding authors was incorrectly labelled.

High dielectric loss in graphite-coated Ti nanocapsules.

Graphite-coated Ti nanocapsules, with Ti nanoparticles as core and onion-like graphite layers as shell, have been prepared by a modified arc-discharge method in ethanol atmosphere, and characterized by means of X-ray diffraction, transmission electron microscopy and Raman spectroscopy. The dielectric properties of the graphite-coated Ti nanocapsules have been investigated in the 2-18 GHz range. An equivalent circuit model was used to interpret the non-linear dielectric resonance behavior of the graphite-coated Ti nanocapsules. The high dielectric loss is mainly attributed to conductance loss and dipole-relaxation loss in the graphite-coated Ti nanocapsules. The graphite-coated Ti nanocapsules exhibit promising properties for application as a new type of shield or absorbent of electromagnetic waves.

Influence of a single quantum dot state on the characteristics of a microdisk laser.

We report a quantum dot microcavity laser with a cw sub-microW lasing threshold, where a significant reduction of the lasing threshold is observed when a single quantum dot (QD) state is aligned with a cavity mode. The quality factor exceeds 15,000 before the system lases. When no QD states are resonant, below threshold the cavity mode initially degrades with increasing pump power, after which saturation occurs and then the cavity mode recovers. We associate the initial cavity mode spoiling with QD state broadening that occurs with increasing pump power.