Design and Experimental Study of a Hybrid Micro-Vibration Isolation System Based on a Strain Sensor for High-Precision Space Payloads.

Micro-vibrations significantly influence the imaging quality and pointing accuracy of high-precision space-borne payloads. To mitigate this issue, vibration isolation technology must be employed to reduce the transmission of micro-vibrations to payloads. In this paper, a novel active-passive hybrid isolation (APHI) system based on a strain sensor is proposed for high-precision space payloads, and corresponding theoretical and experimental studies are implemented. First, a theoretical analysis model of the APHI system is established using a two-degrees-of-freedom system, and an integral control method based on strain sensing is presented. Then, an electromagnetic damper, active piezoelectric actuator, and strain sensor are designed and manufactured. Finally, an APHI experimental system is implemented to validate the effectiveness of electromagnetic damping and strain-sensing active control. Additionally, the control effects of acceleration, displacement, and strain sensors are compared. The results demonstrate that strain sensors can achieve effective active damping control, and the control method based on strain sensors can effectively suppress the payload response while maintaining stability. Both displacement and strain sensors exhibit superior suppression effects compared with the acceleration sensor, with the strain sensor showing greater potential for practical engineering applications than the displacement sensor.

Association between antinuclear antibody and female infertility: A meta-analysis.

Autoimmunity is reported involving in reproductive failures, and antinuclear antibody (ANA) positivity has been regarded as a typical feature of autoimmunity. Published studies on the association of ANA with reproductive failures including infertility are controversial. The aim of this meta-analysis was to analyse whether the presence of ANA positivity increases the risk of infertility in women. We searched the PubMed and Embase databases for relevant literature without any restrictions prior to April 28, 2021. All analyses were performed using the RevMan 5.3 software. Twelve studies with 2734 participants, including 1482 patients with infertility, met the inclusion and exclusion criteria. The total positivity rate of ANA was 23.8% (353/1482) in all infertile patients and 8.5% (107/1252) in the control group. Infertile females had a significantly higher ANA positivity rate than the control group (odds ratio [OR] = 2.90, 95% confidence interval [CI]: 1.72-4.87, I2 = 65%, P < .0001). Several subgroup analyses were performed to reduce the heterogeneity. ANA positivity was associated with female infertility in studies either performed by indirect immunofluorescence (OR = 2.26, 95% CI:1.67-3.06, P < .00001) or by ELISA (OR = 10.76, 95% CI:1.82-63.64, P < .00001). ANA was significantly associated with increased risk of women infertility either after the definite exclusion of individuals with autoimmune diseases (AID) or without exclusion [(OR = 1.99, 95% CI:1.29-3.06, P = .002), (OR = 2.76, 95% CI:1.56-4.88, P = .0005), respectively]. This meta-analysis provides a comprehensive overview of the prevalence of antinuclear antibodies (ANA) in infertile women and suggests that ANA positivity increases the risk of infertility.

Post-Traumatic Stress Disorder Is Associated with Elevated Plasma Cholesterol in Female TT Homozygotes of LDLR rs5925.

To explore the mechanism of inconsistent relationships between plasma lipid profiles and post-traumatic stress disorder (PTSD) reported before, we hypothesized that interplays might exist between PTSD and a variation of rs5925 at low-density lipoprotein receptor (LDLR) gene on plasma lipid profiles. To test our hypothesis, we analyzed the plasma lipid profiles of 709 high school pupils with various genotypes of LDLR rs5925 and with or without PTSD. The results demonstrated that PTSD prevalence in the C allele carriers was higher than that in the TT homozygotes regardless of gender. The C allele carriers had higher levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), ratios of TC to high-density lipoprotein cholesterol (TC/HDL-C) and LDL-C/HDL-C than the TT homozygotes in the male controls, and only higher TC in the female controls, but no differences in the male or female PTSD subjects. PTSD increased TC in the female TT homozygotes but not in the female C allele carriers. PTSD increased TC/HDL-C in the male TT homozygotes but not in the C allele carriers. These results suggest interactions between PTSD and LDLR rs5925 on plasma lipid profiles, which may be among the explanations for previously reported inconsistent relationships between LDLR rs5925 or PTSD and plasma lipid profiles, and facilitate the development of precision medicine interferences in hypercholesterolemia in individuals with different genetic backgrounds and psychiatric status. Psychiatric care or drug supplement may particularly be needed by female hypercholesterolemic subjects with the TT genotype of LDLR rs5925 in Chinese adolescents.

Two modified density gradient centrifugation methods facilitate the isolation of mouse Leydig cells.

Preparation of sufficient mouse Leydig cells (LCs) with high purity is a prerequisite for investigations of the biological/pathological functions of LCs in mouse models. Density gradient centrifugation based on discontinuous Percoll gradients is an effective method (defined as regular method) for LC isolation. In this study, we developed two modified methods for LC isolation and compared their performance with that of the regular method. Modified method 1 integrated the crude LCs into the 50% Percoll solution before centrifugation. Modified method 2 sequentially used 50 and 60% Percoll solutions to isolate LCs. The purity of LCs was approximately 88.4, 91.3, and 79.7% derived from the regular, modified 1, and modified 2 methods, respectively. The yields of LCs in the same respective order were approximately 1.7 × 105, 3.9 × 105, and 11.9 × 105 cells per 108 interstitial cells input. Modified method 1 attained higher purity and yields than those of the regular method. Although the purity of LCs was relatively low for modified method 2, it could be used before further purification by, for example, fluorescence-activated or magnetic-activated cell sorting, owing to its simplicity and high yields. Therefore, our study provided alternative methods to facilitate LC isolation in mice.

Polysaccharides from Discarded Stems of Trollius chinensis Bunge Elicit Promising Potential in Cosmetic Industry: Characterization, Moisture Retention and Antioxidant Activity.

Unconventional polysaccharides as representative active substances from stems of Trollius chinensis Bunge (TC) were studied. Crude polysaccharides from the stems of TC (TCSP) and the petals of TC (TCPP) were extracted, and the moisture retention and antioxidation activities of both TCSP and TCPP in vitro were studied. The weight-average molar masses (Mw) of TCSP (6.07 × 105 Da) were lower than those of TCPP (9.72 × 105 Da). Glucuronic acid and xylose only existed in TCSP, and the molar ratio of galacturonic acid and mannose in TCSP was significantly higher than that in TCPP. No significant differences in moisture retention ability were found between TCSP and TCPP. The reducing capacity and dphenyl picryl hydrazinyl (DPPH) radical scavenging capacity of TCSP were slightly weaker than those of TCPP. The 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging capacity of TCSP can be equivalent to that of TCPP. The moisture retention ability was not different between TCSP and TCPP, which are both highly homologous with traditional humectants. The antioxidation assays in vitro demonstrated that the antioxidant activity of TCSP is stronger compared to that of some plant-derived polysaccharides. The stems of TC can be a promising source of unconventional polysaccharides, which possess moisture retention and antioxidation capacities for the cosmetics industry.

Mutation Analysis of COL1A1 and COL1A2 in Fetuses with Osteogenesis Imperfecta Type II/III.

AIM: To analyze COL1A1/2 mutations in prenatal-onset OI for determine the proportion of mutations in type I collagen genes among prenatal onset OI and to provide additional data for genotype-phenotype analyses. MATERIAL AND METHODS: Ten cases of severe fetal short-limb dwarfism detected by antenatal ultrasonography were referred to our center. Before the termination of pregnancy, cordocentesis was performed for fetal karyotype and COL1A1/2 gene sequencing analysis. Postmortem radiographic examination was performed at all instances for definitive diagnosis. RESULTS: COL1A1 and COL1A2 SNP and mutations were identified in all the cases. Among these, one synonymous SNP and four synonymous SNPs were recognized in COL1A1/2, respectively, seven cases have distinct heterozygous mutations and six new COL1A1/2 gene mutations were identified. CONCLUSION: There has been substantial progress in the identification of the molecular defects responsible for skeletal dysplasias. With the constant increase in the number of identified mutations in COL1A1 and COL1A2, genotype-phenotype correlation is becoming increasingly pertinent.

Clinical phenotype, in silico and biomedical analyses, and intervention for an East Asian population-specific c.370G>A (p.G124S) COQ4 mutation in a Chinese family with CoQ10 deficiency-associated Leigh syndrome.

COQ4 mutations have recently been shown to cause a broad spectrum of mitochondrial disorders in association with CoQ10 deficiency. Herein, we report the clinical phenotype, in silico and biochemical analyses, and intervention for a novel c.370 G > A (p.G124S) COQ4 mutation in a Chinese family. This mutation is exclusively present in the East Asian population (allele frequency of ~0.001). The homozygous mutation caused CoQ10 deficiency-associated Leigh syndrome with an onset at 1-2 months of age, presenting as respiratory distress, lactic acidosis, dystonia, seizures, failure to thrive, and detectable lesions in the midbrain and basal ganglia. No renal impairment was involved. The levels of CoQ10 and mitochondrial respiratory chain complex (C) II + III activity were clearly lower in cultured fibroblasts derived from the patient than in those from unaffected carriers; the decreased CII + III activity could be increased by CoQ10 treatment. Follow-up studies suggested that our patient benefitted from the oral supplementation of CoQ10, which allowed her to maintain a relatively stable health status. Based on the genetic testing, preimplantation and prenatal diagnoses were performed, confirming that the next offspring of this family was unaffected. Our cases expand the phenotypic spectrum of COQ4 mutations and the genotypic spectrum of Leigh syndrome.

Carrier screening for spinal muscular atrophy with a simple test based on melting analysis.

Carrier screening of spinal muscular atrophy (SMA) can provide reproductive options for carriers and prevent the birth defects. Here, we developed a simple screening test based on melting analysis. The test comprises a duplex PCR with two primer pairs and three probes to simultaneous amplify SMN1, SMN2, and CFTR. By analyzing the melting profiles, we were able to determine the SMN1/SMN2 ratio and SMN1 + SMN2 copy number to subsequently determine the copy number of SMN1. Samples with one copy of SMN1 were considered as "high risk for carrier," while samples with ≥2 copies of SMN1 were considered as "low risk for carrier." We evaluated the clinical performance of this test using 215 clinical samples with various genotypes that had been previously confirmed by multiplex ligation-dependent probe amplification (MLPA). The test showed high sensitivity (100%) and specificity (97.1%) as well as high positive (97.3%) and negative (100%) predictive value, and was in perfect agreement with the gold standard test, MLPA (k = 0.97). Moreover, it is rapid, inexpensive, and easy to perform and automate, with high reproducibility and capacity. Therefore, we expect this test will advance carrier screening for SMA.

Citrus Tree Segmentation from UAV Images Based on Monocular Machine Vision in a Natural Orchard Environment.

The segmentation of citrus trees in a natural orchard environment is a key technology for achieving the fully autonomous operation of agricultural unmanned aerial vehicles (UAVs). Therefore, a tree segmentation method based on monocular machine vision technology and a support vector machine (SVM) algorithm are proposed in this paper to segment citrus trees precisely under different brightness and weed coverage conditions. To reduce the sensitivity to environmental brightness, a selective illumination histogram equalization method was developed to compensate for the illumination, thereby improving the brightness contrast for the foreground without changing its hue and saturation. To accurately differentiate fruit trees from different weed coverage backgrounds, a chromatic aberration segmentation algorithm and the Otsu threshold method were combined to extract potential fruit tree regions. Then, 14 color features, five statistical texture features, and local binary pattern features of those regions were calculated to establish an SVM segmentation model. The proposed method was verified on a dataset with different brightness and weed coverage conditions, and the results show that the citrus tree segmentation accuracy reached 85.27% ± 9.43%; thus, the proposed method achieved better performance than two similar methods.

Lychee Fruit Detection Based on Monocular Machine Vision in Orchard Environment.

Due to the change of illumination environment and overlapping conditions caused by the neighboring fruits and other background objects, the simple application of the traditional machine vision method limits the detection accuracy of lychee fruits in natural orchard environments. Therefore, this research presented a detection method based on monocular machine vision to detect lychee fruits growing in overlapped conditions. Specifically, a combination of contrast limited adaptive histogram equalization (CLAHE), red/blue chromatic mapping, Otsu thresholding and morphology operations were adopted to segment the foreground regions of the lychees. A stepwise method was proposed for extracting individual lychee fruit from the lychee foreground region. The first step in this process was based on the relative position relation of the Hough circle and an equivalent area circle (equal to the area of the potential lychee foreground region) and was designed to distinguish lychee fruits growing in isolated or overlapped states. Then, a process based on the three-point definite circle theorem was performed to extract individual lychee fruits from the foreground regions of overlapped lychee fruit clusters. Finally, to enhance the robustness of the detection method, a local binary pattern support vector machine (LBP-SVM) was adopted to filter out the false positive detections generated by background chaff interferences. The performance of the presented method was evaluated using 485 images captured in a natural lychee orchard in Conghua (Area), Guangzhou. The detection results showed that the recall rate was 86.66%, the precision rate was greater than 87% and the F1-score was 87.07%.

Assessment of External Properties for Identifying Banana Fruit Maturity Stages Using Optical Imaging Techniques.

The maturity stage of bananas has a considerable influence on the fruit postharvest quality and the shelf life. In this study, an optical imaging based method was formulated to assess the importance of different external properties on the identification of four successive banana maturity stages. External optical properties, including the peel color and the local textural and local shape information, were extracted from the stalk, middle and tip of the bananas. Specifically, the peel color attributes were calculated from individual channels in the hue-saturation-value (HSV), the International Commission on Illumination (CIE) L*a*b* and the CIE L*ch color spaces; the textural information was encoded using a local binary pattern with uniform patterns (UP-LBP); and the local shape features were described by histogram of oriented gradients (HOG). Three classifiers based on the naïve Bayes (NB), linear discriminant analysis (LDA) and support vector machine (SVM) algorithms were adopted to evaluate the performance of identifying banana fruit maturity stages using the different optical appearance features. The experimental results demonstrate that overall identification accuracies of 99.2%, 100% and 99.2% were achieved using color appearance features with the NB, LDA and SVM classifiers, respectively; overall accuracies of 92.6%, 86.8% and 93.4% were obtained using local textural features for the three classifiers, respectively; and overall accuracies of only 84.3%, 83.5% and 82.6% were obtained using local shape features with the three classifiers, respectively. Compared to the complicated calculation of both the local textural and local shape properties, the simplicity and high accuracy of the peel color property make it more appropriate for identifying banana fruit maturity stages using optical imaging techniques.

Tumor-originated exosomal lncUEGC1 as a circulating biomarker for early-stage gastric cancer.

Conventional tumor markers for non-invasive diagnosis of gastric cancer (GC) exhibit insufficient sensitivity and specificity to facilitate detection of early gastric cancer (EGC). We aimed to identify EGC-specific exosomal lncRNA biomarkers that are highly sensitive and stable for the non-invasive diagnosis of EGC. Hence, in the present study, exosomes from the plasma of five healthy individuals and ten stage I GC patients and from culture media of four human primary stomach epithelial cells and four gastric cancer cells (GCCs) were isolated. Exosomal RNA profiling was performed using RNA sequencing to identify EGC-specific exosomal lncRNAs. A total of 79 and 285 exosomal RNAs were expressed at significantly higher levels in stage I GC patients and GCCs, respectively, than that in normal controls. Through combinational analysis of the RNA sequencing results, we found two EGC-specific exosomal lncRNAs, lncUEGC1 and lncUEGC2, which were further confirmed to be remarkably up-regulated in exosomes derived from EGC patients and GCCs. Furthermore, stability testing demonstrates that almost all the plasma lncUEGC1 was encapsulated within exosomes and thus protected from RNase degradation. The diagnostic accuracy of exosomal lncUEGC1 was evaluated, and lncUEGC1 exhibited AUC values of 0.8760 and 0.8406 in discriminating EGC patients from healthy individuals and those with premalignant chronic atrophic gastritis, respectively, which was higher than the diagnostic accuracy of carcinoembryonic antigen. Consequently, exosomal lncUEGC1 may be promising in the development of highly sensitive, stable, and non-invasive biomarkers for EGC diagnosis.

Evaluation of a Magnetic Cellulose-Based DNA Extraction System to Improve the Performance of HybriBio Human Papillomavirus Genotyping and Screening Tests for Cervical Swab Samples.

BACKGROUND: To ensure the accuracy of clinical human papillomavirus (HPV) testing, the nucleic acid extraction procedure should be thoroughly evaluated for each clinical sample type. Therefore, we evaluated whether the MagCore® Automated Nucleic Acid Extraction system (MagCore system) could improve HybriBio HPV test performance for cervical swab samples. METHODS: We compared the performance of HybriBio HPV genotyping and screening tests using samples prepared with the MagCore system and Cell Lysis Kit, which was provided by the HPV test manufacturer. RESULTS: The MagCore system extracted high quality DNA and outperformed the Cell Lysis Kit in the subsequent analysis. In terms of the HPV genotyping testing, use of the MagCore system-extracted DNA markedly increased the signal intensity compared to that of DNA extracted with the Cell Lysis Kit for low concentrations of HPV DNA. Thus, the analytical sensitivity of testing was increased approximately 10 times by using the MagCore system, and the reproducibility was also increased (97.1% vs. 88.2%). In terms of the HPV screening tests, use of the MagCore system improved the compatibility of the internal control and targets, reducing the risk of false or invalid results. The MagCore system also improved the amplification efficiency and quantification accuracy for HPV16 detection. CONCLUSIONS: We demonstrated that the performance of HybriBio HPV genotyping and screening tests can be improved by using the MagCore system. This study not only provided an alternative, robust DNA extraction method for clinical users but also reemphasized the importance of establishing a reliable DNA extraction procedure for clinical HPV testing.

Whole Exome Sequencing Identifies a c.C2566T Mutation in the Androgen Receptor in a Chinese Family.

BACKGROUND: Whole exome sequencing (WES) is one of the most valuable tools for the detection of Mendelian diseases in clinical laboratory. We performed WES for a family of 46,XY disorders of gender development and compared the applicability of public databases for the subsequent phenotype studies of WES-identified mutations. METHODS: DNA samples from the two patients were analyzed by WES. The mutated protein was studied using the HomoloGene database, Polyphen2, and SIFT. The phenotype of the mutation was studied using ClinVar, the androgen receptor gene mutations database, AR database at Leiden Open Variation Database, and PubMed. RESULTS: A c.C2566T (p.R856C) mutation in the androgen receptor gene was detected for the patients. The in silico studies indicated that the p.R856C mutation is deleterious to the function of the androgen receptor. Unlike those of other databases, the variations listed in the androgen receptor gene mutations database were classified as complete androgen insensitivity-, partial androgen insensitivity-, or mild androgen insensitivity-relevant according to their clinical phenotype. In addition, the publications of the collected mutations in the androgen receptor gene mutations database are complete and easily accessible, which facilitates in depth studies of clinically identified mutations. CONCLUSIONS: We identified a c.C2566T (p.R856C) mutation of the AR gene in cases of familial complete androgen insensitivity by WES, and provided genetic counseling to related family members. This is the first study reporting this mutation in Chinese patients. We also compared the applicability of several public databases for phenotype studies of clinically identified Androgen Receptor mutations and suggest that the androgen receptor gene mutations database best satisfies clinical demands.

Recurrent c.G1636A (p.G546S) mutation of COL2A1 in a Chinese family with skeletal dysplasia and different metaphyseal changes: a case report.

BACKGROUND: Mutations in the COL2A1 gene cause type II collagenopathies characterized by skeletal dysplasia with a wide spectrum of phenotypic severity. Most COL2A1 mutations located in the triple-helical region, and the glycine to bulky amino acid substitutions (e.g., glycine to serine) in the Gly-X-Y repeat were identified frequently. However, the same COL2A1 mutations are associated with different phenotypes and the genotype-phenotype relationship is still poorly understood. Therefore, the studies of more patients about the recurrent mutations in COL2A1 will be needed for further research to provide more comprehensive clinical and genetic data. In this paper, we report a rare recurrent c.G1636A (p.G546S) mutation in COL2A1 associated with different metaphyseal changes in a Chinese family. CASE PRESENTATION: The proband (III-3) was the second child of the family with skeletal dysplasia. She was 2 years and 3 months old with disproportional short stature, short neck, pectus carinatum, genu varum, bilateral pes planus, and obvious waddling gait. Notably, she displayed severe metaphyseal lesions, especially typical "dappling" and "corner fracture" appearance, whereas no particular metaphyseal involvement was detected in the proband's mother (II-3) and elder sister (III-2) in the family. We identified a heterozygous mutation (c.1636G > A) in COL2A1 in the three patients, causing the substitution of glycine to serine in codon 546. Although the same mutation has been reported in two previous studies, the phenotypes of the previous patients were different from those of our patients, and the characteristic "dappling" and "corner fracture" metaphyseal abnormalities were not reported previously. CONCLUSIONS: In this study, we identified a c.G1636A (p.G546S) mutation in the COL2A1 associated with different metaphyseal changes, which was never reported in the literature. Our findings revealed a different causative amino acid substitution (glycine to serine) associated with the "dappling" and "corner fracture" metaphyseal abnormalities, and may provide a useful reference for evaluating the phenotypic spectrum and variability of type II collagenopathies.

Comparison of the accuracy of multiplex digital PCR versus multiplex ligation-dependent probe amplification in quantification of the survival of motor neuron genes copy numbers.

For over two decades, multiplex ligation-dependent probe amplification (MLPA) has served as the gold standard for genetic testing of spinal muscular atrophy. However, there is emerging evidence questioning the reliability of MLPA in determining the copy numbers (CNs) of the survival of motor neuron (SMN) gene in certain cases. Recently, digital polymerase chain reaction (dPCR) has shown potential for better performance in copy number variant detection. This study aimed to compare MLPA and dPCR in quantifying SMN1 and SMN2 CNs, identify reasons for observed discrepancies, and explore the clinical implications of false results. A total of 733 DNA samples, previously subjected to MLPA analysis, were tested using multiplex droplet dPCR assays. Samples exhibiting inconsistent results between the two methods underwent repeated dPCR assays. When inconsistencies persisted, a third method was employed for verification. Digital PCR yielded results consistent with those of MLPA in 94.4% (692/733) of samples. Forty-one cases exhibited quantitative disparities in SMN1 and/or SMN2 CNs between the two methods. Confirmatory tests revealed that 37 inaccurate results were produced by the MLPA analysis, whereas four were attributed to the dPCR method. The dPCR technique exhibits better accuracy than MLPA and is qualified for SMA genetic testing across various clinical scenarios.

A novel TGFbeta/TGILR axis mediates crosstalk between cancer-associated fibroblasts and tumor cells to drive gastric cancer progression.

Transforming growth factor beta (TGFß) signaling plays a critical role in tumorigenesis and metastasis. However, little is known about the biological function of TGFbeta-induced lncRNA in cancer. In this study, we discovered a novel TGFbeta-induced lncRNA, termed TGILR, whose function in cancer remains unknown to date. TGILR expression was directly activated by the canonical TGFbeta/SMAD3 signaling axis, and this activation is highly conserved in cancer. Clinical analysis showed that TGILR overexpression showed a significant correlation with lymph node metastasis and poor survival and was an independent prognostic factor in gastric cancer (GC). Depletion of TGILR caused an obvious inhibitory effect on GC cell proliferation, invasion, and epithelial-mesenchymal transition (EMT) in vitro and in vivo. More importantly, we demonstrated that TGFbeta signaling in GC was overactivated due to cancer-associated fibroblast (CAF) infiltration. Mechanistically, increased level of CAF-secreted TGFbeta activates TGFbeta signaling, leading to TGILR overexpression in GC cells. Meanwhile, TGILR overexpression inhibited the microRNA biogenesis of miR-1306 and miR-33a by interacting with TARBP2 and reducing its protein stability, thereby promoting GC progression via TCF4-mediated EMT signaling. In conclusion, CAF infiltration drives GC metastasis and EMT signaling through activating TGFbeta/TGILR axis. Targeted blocking of CAF-derived TGFbeta should be a promising anticancer strategy in GC.

Development of a low-cost and accurate carrier screening method for spinal muscular atrophy in developing countries.

Heterozygous carriers of the survival of motor neuron 1 (SMN1) gene deletion in parents account for approximately 95% of neonatal spinal muscular atrophy cases. Given the severity of the disease, professional organizations have recommended periconceptional spinal muscular atrophy carrier screening to all couples, regardless of race or ethnicity. However, the prevalence of screening activities in mainland China remains suboptimal, mainly attributed to the limitations of the existing carrier screening methods. Herein, we aimed to develop a low-cost, accessible, and accurate carrier screening method based on duplex droplet digital PCR (ddPCR), to cover a wider population in developing countries, including China. The receiver operating characteristic curve was used to determine the cut-off value of SMN1 copy numbers. Performance validation was conducted for linearity, precision, and accuracy. In total, 482 cases were considered to validate the concordance between the developed ddPCR assay and multiplex ligation-dependent probe amplification. Linear correlations were excellent between the expected concentration of the reference gene and the observed values (R2 > 0.99). Both the intra- and inter-assay precision of our ddPCR assays were less than 6.0%. The multiplex ligation-dependent probe amplification and ddPCR results were consistent in 480 of the 482 cases (99.6%). Two cases with multiplex ligation-dependent probe amplification, suggestive of two copies of SMN1 exon 7, were classified into three copies by ddPCR analysis. The overall correct classification of the samples included in our ddPCR assay was 100%. This study demonstrates that an appropriate cut-off value is an important prerequisite for establishing a semi-quantitative method to determine the SMN1 copy numbers. Compared to conventional methods, our ddPCR assay is low-cost, highly accurate, and has full potential for application in population spinal muscular atrophy carriers screening.

A new high-throughput screening methodology for the discovery of cancer-testis antigen using multi-omics data.

BACKGROUND: Cancer/testis antigens (CTAs), also known as tumor-specific antigens (TSAs) are specifically expressed in cancer cells and exhibit high immunogenicity, making them promising targets for immunotherapy and cancer vaccines. METHODS: A new integrated high-throughput screening methodology for CTAs was proposed in this study through combining DNA methylation and RNA sequencing data. Briefly, the genes with increased transcript level and decreased DNA methylation were identified by multi-omics analysis. RNA sequencing studies in cell lines exposed to DNA methyltransferase (DNMT) inhibitors were performed to validate the inherent causal relationship between DNA hypomethylation and gene expression upregulation. RESULTS: We proposed a new integrated high-throughput screening methodology for identification of CTAs using multi-omics analysis. In addition, we tested the feasibility of this method using gastric cancer (GC) as an example. In GC, we identified over 2000 primary candidate CTAs and ultimately identified 20 CTAs with significant tissue-specificity, including a testis-specific serine protease TESSP1/PRSS41. Integrated analysis confirmed that PRSS41 expression was reactivated in gastrointestinal cancers by promoter DNA hypomethylation at the CpG site (cg08104780). Additionally, DNA hypomethylation of PRSS41 predicted a poor prognosis in GC. CONCLUSION: We propose a new high-throughput screening method for the identification of CTAs in cancer and validate its effectiveness. Our work emphasizes that serine protease PRSS41 is a novel TSA that is reactivated in GC due to promoter DNA hypomethylation.

Simultaneous detection, genotyping, and quantification of human papillomaviruses by multicolor real-time PCR and melting curve analysis.

Long-term infection with high-risk human papillomavirus (HPV) is the leading cause of cervical cancer, while infection with low-risk HPV is the major reason for condylomata acuminata. An accurate, rapid, and convenient assay that is able to simultaneously detect, genotype, and quantify HPV would be of great clinical value yet remains to be achieved. We developed a three-color real-time PCR assay that is able to analyze 30 predominant HPV types in three reactions. The amplification curves indicated the presence of HPV, melting curve analysis identified the HPV genotype, and the quantification cycle value determined the quantity. We applied this assay to 647 cervical swab samples, and the results were compared with those obtained with a commercial genotyping system. The proposed assay had a limit of detection of 5 to 50 copies per reaction and a dynamic range of 5 × 10(1) to 5 × 10(6) copies per reaction. A comparison study showed that the overall sample concordance with the comparison method was 91.6% and the type agreement was greater than 98.7%. The quantification study demonstrated that the loads of HPV type 16 in 30 samples with cervical intraepithelial neoplasia grade III (CIN III) lesions were significantly higher than those in samples with CIN I lesions or CIN II lesions, and the results were concordant with those of the comparison method. The increased information content, high throughput, and low cost would facilitate the use of this real-time PCR-based assay in a variety of clinical settings.