Analytical validation of the DropXpert S6 system for diagnosis of chronic myelocytic leukemia.

Digital PCR is a powerful method for absolute nucleic acid quantification and is widely used in the absolute quantification of viral copy numbers, tumor marker detection, and prenatal diagnosis. However, for most of the existing droplet-based dPCR systems, the droplet generation, PCR reaction, and droplet detection are performed separately using different instruments. Making digital PCR both easy to use and practical by integrating the qPCR workflow into a superior all-in-one walkaway solution is one of the core ideas. A new innovative and integrated digital droplet PCR platform was developed that utilizes cutting-edge microfluidics to integrate dPCR workflows onto a single consumable chip. This makes previously complex workflows fast and simple; the whole process of droplet generation, PCR amplification, and droplet detection is completed on one chip, which meets the clinical requirement of "sample in, result out". It provides high multiplexing capabilities and strong sensitivity while all measurements were within the 95% confidence interval. This study is the first validation of the DropXpert S6 system and focuses primarily on verifying its reliability, repeatability, and consistency. In addition, the accuracy, detection limit, linearity, and precision of the system were evaluated after sample collection. Among them, the accuracy assessment by calculating the absolute bias of each target gene yielded a range from -0.1 to 0.08, all within ±0.5 logarithmic orders of magnitude; the LOB for the assay was set at 0, and the LoD value calculated using probit curves is MR4.7 (0.002%); the linearity evaluation showed that the R2 value of the BCR-ABL was 0.9996, and the R2 value of the ABL metrics calculated using the ERM standard was 0.9999; and the precision evaluation showed that all samples had a CV of less than 4% for intra-day, inter-day, and inter-instrument variation. The CV of inter-batch variation was less than 7%. The total CV was less than 5%. The results of the study demonstrate that dd-PCR can be applied to molecular detection and the clinical evaluation of CML patients and provide more precise personal treatment guidance, and its reproducibility predicts the future development of a wide range of clinical applications.

Protective role of hydrogen sulfide against diabetic cardiomyopathy by inhibiting pyroptosis and myocardial fibrosis.

Diabetic cardiomyopathy (DCM) contributes significantly to the heightened mortality rate observed among diabetic patients, with myocardial fibrosis (MF) being a pivotal element in the disease's progression. Hydrogen sulfide (H2S) has been shown to mitigate MF, but the specific underlying mechanisms have yet to be thoroughly understood. A connection has been established between the evolution of DCM and the incidence of cardiomyocyte pyroptosis. Our research offers insights into H2S protective impact and its probable mode of action against DCM, analyzed through the lens of MF. In this study, a diabetic rat model was developed using intraperitoneal injections of streptozotocin (STZ), and hyperglycemia-stimulated cardiomyocytes were employed to replicate the cellular environment of DCM. There was a marked decline in the expression of cystathionine γ-lyase (CSE), a catalyst for H2S synthesis, in both the STZ-induced diabetic rats and hyperglycemia-stimulated cardiomyocytes. Experimental results in vivo indicated that H2S ameliorates MF and enhances cardiac functionality in diabetic rats by mitigating cardiomyocyte pyroptosis. In vitro assessments highlighted the induction of cardiomyocyte pyroptosis and the subsequent decline in cell viability under hyperglycemic conditions. However, the administration of sodium hydrosulfide (NaHS) curtailed cardiomyocyte pyroptosis and augmented cell viability. In contrast, propargylglycine (PAG), a CSE inhibitor, reversed the effects rendered by NaHS administration. Additional exploration indicated that the mitigating effect of H2S on cardiomyocyte pyroptosis is modulated through the ROS/NLRP3 pathway. In essence, our findings corroborate the potential of H2S in alleviating MF in diabetic subjects. This therapeutic effect is likely attributable to the regulation of cardiomyocyte pyroptosis via the ROS/NLRP3 pathway. This discovery furnishes a prospective therapeutic target for the amelioration and management of MF associated with diabetes.

Causes and management strategies for elevated intraocular pressure after implantable collamer lens implantation.

With the widespread application of Implantable Collamer Lens (ICL) implantation surgery in the field of myopia correction, a comprehensive understanding of its potential complications, especially those related to intraocular pressure (IOP), becomes crucial. This article systematically reviews various complications that may lead to IOP elevation after ICL surgery. Firstly, common complications after ICL surgery, including residual viscoelastic, steroid response, and excessive vault of the ICL, are detailed, emphasizing their potential impact on intraocular pressure. Regarding residual viscoelastic, we delve into its direct relationship with postoperative elevated IOP and possible preventive measures. For steroid response, we stress the importance of timely adjustment of steroid therapy and monitoring intraocular pressure. Additionally, excessive vault of the ICL is considered a significant potential issue, and we elaborate on its mechanism and possible management methods. In further discussion, we focus on relatively rare complications such as Toxic Anterior Segment Syndrome (TASS), Urrets-Zavalia Syndrome (UZS), Pigment Dispersion Syndrome (PDS), and malignant glaucoma. For these relatively rare complications, this review thoroughly explores their potential mechanisms, emphasizes the importance of prevention, and provides guidance for early diagnosis and treatment. This is a comprehensible review that aims to offer eye care professionals a comprehensive understanding and effective management guidance for complications of elevated IOP after ICL surgery, ultimately providing optimal care for patients' visual health.

Characterization of a DsbA family protein reveals its crucial role in oxidative stress tolerance of Listeria monocytogenes.

IMPORTANCE: The adaption and tolerance to various environmental stresses are the fundamental factors for the widespread existence of Listeria monocytogenes. Anti-oxidative stress is the critical mechanism for the survival and pathogenesis of L. monocytogenes. The thioredoxin (Trx) and glutaredoxin (Grx) systems are known to contribute to the anti-oxidative stress of L. monocytogenes, but whether the Dsb system has similar roles remains unknown. This study demonstrated that the DsbA family protein Lmo1059 of L. monocytogenes participates in bacterial oxidative stress tolerance, with Cys36 as the key amino acid of its catalytic activity and anti-oxidative stress ability. It is worth noting that Lmo1059 was involved in the invading and cell-to-cell spread of L. monocytogenes. This study lays a foundation for further understanding the specific mechanisms of oxidative cysteine repair and antioxidant stress regulation of L. monocytogenes, which contributes to an in-depth understanding of the environmental adaptation mechanisms for foodborne bacterial pathogens.

Increasing global precipitation whiplash due to anthropogenic greenhouse gas emissions.

Precipitation whiplash, including abrupt shifts between wet and dry extremes, can cause large adverse impacts on human and natural systems. Here we quantify observed and projected changes in characteristics of sub-seasonal precipitation whiplash and investigate the role of individual anthropogenic influences on these changes. Results show that the occurrence frequency of global precipitation whiplash is projected to be 2.56 ± 0.16 times higher than in 1979-2019 by the end of the 21st Century, with increasingly rapid and intense transitions between two extremes. The most dramatic increases of whiplash show in the polar and monsoon regions. Changes in precipitation whiplash show a much higher percentage change than precipitation totals. In historical simulations, anthropogenic greenhouse gas (GHG) and aerosol emissions have increased and decreased precipitation whiplash occurrences, respectively. By 2079, anthropogenic GHGs are projected to increase 55 ± 4% of the occurrences risk of precipitation whiplash, which is driven by shifts in circulation patterns conducive to precipitation extremes.

NONO regulates multiple cytokine production in sepsis via the ERK1/2 signaling pathway.

The massive release of pro-inflammatory cytokines is a crucial step in triggering the inflammatory cascade in sepsis. Exploring the key molecules regulating the expression and release of multiple cytokines has important value for revealing the mechanism of the cytokine storm in sepsis. This study aimed to investigate the role of multifunctional nuclear protein non-POU domain containing octamer-binding protein (NONO) in the sepsis cytokine storm and to elucidate the underlying mechanism. We found that NONO expression in tissues and cells of sepsis mice was significantly upregulated. Downregulation of NONO expression inhibited the mRNA expression of multiple cytokines, including IL-6, IL-1ß, MCP-1, MIP-1α, and MIP-1ß in inflammatory cells from mice and human leukemic monocyte-THP1 cells challenged with lipopolysaccharide (LPS), and significantly decreased the level of these cytokines and TNF-α in the supernatant of THP1 cells challenged by LPS. Nono knockout also reduced the levels of TNF-α, IL-6, MIP-1α, and MIP-1ß in serum, alleviated hepatocyte edema, and improved the survival rate of sepsis mice. Reduced NONO expression decreased the phospho-ERK1/2 level in inflammatory cells from sepsis mice or THP1 cells challenged by LPS. Phospho-ERK1/2 inhibitor decreased the mRNA expression and concentration of cytokines in the culture supernatant of LPS-induced THP1 cells, similar to the effect of NONO knockdown. After LPS challenge, the levels of phospho-ERK1/2 and NONO were increased, with obvious colocalization in the nucleus and vesicular-like organelles in macrophages. NONO knockdown decreased nuclear translocation of phospho-ERK1/2 in LPS-challenged THP1 cells. These results suggest that NONO is a potentially critical molecule involved in multiple cytokine production in sepsis. Upregulated NONO in sepsis may promote the expression and release of multiple cytokines to participate in a sepsis cytokine storm by promoting ERK1/2 phosphorylation.

Rapid screening of antioxidant from natural products by AAPH-Incubating HPLC-DAD-HR MS/MS method: A case study of Gardenia jasminoides fruit.

A new AAPH-Incubating HPLC-DAD-HR MS/MS method was developed for the rapid and high-throughput screening of antioxidants directly in natural products and applied to Gardenia jasminoides fruit. This method was assumed that the peak areas of compounds with potential antioxidant activity in HPLC chromatograms would be significantly reduced or disappeared after incubating with the AAPH which can release ROO at physiological conditions (37 °C, pH 7.4). Additionally, the activity of antioxidants can be evaluated by comparing the peak reduction rates and the screened components can be further identified by HRMS/MS. Then, 17 potential natural antioxidants from the crude extract of GJF was screened. Among them, three major components including crocin I, crocin II and crocetin showed excellent ROO scavenging activity, which were further validated by the ORAC assay. In conclusion, our study provided a simple and effective strategy to rapidly screen antioxidants in natural products.

FOXD1-dependent RalA-ANXA2-Src complex promotes CTC formation in breast cancer.

BACKGROUND: Early metastasis is a key factor contributing to poor breast cancer (BC) prognosis. Circulating tumor cells (CTCs) are regarded as the precursor cells of metastasis, which are ultimately responsible for the main cause of death in BC. However, to date molecular mechanisms underlying CTC formation in BC have been insufficiently defined. METHODS: RNA-seq was carried out in primary tissues from early-stage BC patients (with CTCs≥5 and CTCs = 0, respectively) and the validation study was conducted in untreated 80 BC patients. Multiple in vitro and in vivo models were used in functional studies. Luciferase reporter, ChIP-seq, CUT&Tag-seq, and GST-pulldown, etc. were utilized in mechanistic studies. CTCs were counted by the CanPatrol™ CTC classification system or LiquidBiospy™ microfluidic chips. ERK1/2 inhibitor SCH772984 was applied to in vivo treatment. RESULTS: Highly expressed FOXD1 of primary BC tissues was observed to be significantly associated with increased CTCs in BC patients, particularly in early BC patients. Overexpressing FOXD1 enhanced the migration capability of BC cells, CTC formation and BC metastasis, via facilitating epithelial-mesenchymal transition of tumor cells. Mechanistically, FOXD1 was discovered to induce RalA expression by directly bound to RalA promotor. Then, RalA formed a complex with ANXA2 and Src, promoting the interaction between ANXA2 and Src, thus increasing the phosphorylation (Tyr23) of ANXA2. Inhibiting RalA-GTP form attenuated the interaction between ANXA2 and Src. This cascade culminated in the activation of ERK1/2 signal that enhanced metastatic ability of BC cells. In addition, in vivo treatment with SCH772984, a specific inhibitor of ERK1/2, was used to dramatically inhibit the CTC formation and BC metastasis. CONCLUSION: Here, we report a FOXD1-dependent RalA-ANXA2-Src complex that promotes CTC formation via activating ERK1/2 signal in BC. FOXD1 may serve as a prognostic factor in evaluation of BC metastasis risks. This signaling cascade is druggable and effective for overcoming CTC formation from the early stages of BC.

Trilobatin alleviates non-alcoholic fatty liver disease in high-fat diet plus streptozotocin-induced diabetic mice by suppressing NLRP3 inflammasome activation.

Diabetes mellitus (DM) is a factor with great risk in the course of non-alcoholic fatty liver disease (NAFLD) due to its high glucotoxicity and lipotoxicity. Trilobatin, a glycosylated dihydrochalcone derived from the leaves of the Chinese sweet tea Lithocarpus polystachyus Rehd, is reported to possess various pharmacological activities. Nevertheless, it is still unclear regarding if trilobatin can alleviate liver injury in diabetic mice with NAFLD and its mechanism. Our aim was to investigative the protective effects of trilobatin against DM with NAFLD and its mechanism of action. A DM mice model was established by high-fat diet (HFD) feeding with streptozocin (STZ) injections, and treated with trilobatin for 10 weeks. The biochemical results showed that trilobatin restored glucose metabolic disorder and liver function in diabetic mice. The histopathological evaluation revealed that trilobatin improved liver injury by alleviating lipid accumulation and liver fibrosis. Mechanistically, trilobatin decreased expression of NLRP3, p65 NF-κB, cleaved-Caspase-1 and N-GSDMD, as well as the release of IL-18 and IL-1ß, leading to a alleviation of inflammation and pyroptosis. Taken together, we determined for the first time found that trilobatin could prevent liver injury in diabetic mice with NAFLD by suppressing NLRP3 inflammasome activation to reduce inflammation and pyroptosis.

Eimeria acervulina Microneme Protein 3 Inhibits Apoptosis of the Chicken Duodenal Epithelial Cell by Targeting the Casitas B-Lineage Lymphoma Protein.

Eimeria acervulina (E. acervulina) causes coccidiosis in poultry which persists as economic pain worldwide. Most damage to the intestinal mucosa results from apoptosis of the infected intestinal epithelial cells. The Microneme protein 3 (MIC3) protein is a key virulence factor in some parasites involved in host cell apoptosis inhibition. Here, we studied whether and how MIC3 affects the apoptosis in E. acervulina infected chicken duodenal epithelial cells. Through flow cytometry (FCM), we found that the presence of merozoites and the overexpression of MIC3 significantly decreased apoptosis and the activity of caspase-3 in chicken duodenal epithelial cells at 4, 6, and 8 h post merozoite infection (P < 0.01). Silencing the Casitas B-lineage lymphoma (CBL) protein, a host receptor for MIC3 with shRNA was shown to promote apoptosis in the chicken duodenal epithelial cells. The early apoptotic rate of host cells in the lentiviral-MIC3 group was significantly lower than that in the lentiviral-MIC3 + shRNA CBL group at 4 h after MIC3 expression (P < 0.01), and it was moderately decreased in the lentiviral-MIC3 + shRNA CBL group compared with that in the shRNA CBL group. Our data indicated that MIC3 inhibited early apoptosis of E. acervulina infected chicken duodenal epithelial cells by targeting host receptor-CBL protein. These findings unveiled one of the mechanisms of how intracellular parasites affect the apoptosis of infected host cells, which provided a deeper understanding of their pathogenesis.

Factors influencing the implementation of a pilot smoking cessation intervention among migrant workers in Chinese factories: a qualitative study.

BACKGROUND: Tobacco control intervention with Chinese internal migrants, especially those working in factories has rarely been investigated. This study aims to identify aids and barriers to implementing a comprehensive pilot intervention aimed at reducing smoking among migrant workers working in factories in China. METHOD: Twenty in-depth interviews were conducted 3 months into the intervention, with managers, migrant workers and team leaders in two factories, where the pilot intervention was implemented, in Zhongshan city in Guangdong, a southern Chinese province. Data analysis was based on the thematic approach. RESULTS: This study identifies the societal, individual and programmatic factors that could influence the implementation of a pilot smoking cessation intervention among migrant workers in the two Chinese factories. At the societal level, social customs and relationships where smoking is seen as essential in social communications was the most important barrier to the implementation of smoking cessation intervention. At the individual level, migrant-related features such as low education, high mobility and poor integration with local residents, together with poor health beliefs and attitudes added to the challenges of implementing smoking cessation intervention. At the programmatic level, the role of small-team leaders was generally positive, although limited due to their busy work patterns and poor powers of enforcement. CONCLUSION: Achieving successful smoking cessation intervention in factories could be challenging with many migrants, as multi-level factors including social context, intervention delivery, individual and migrants' characteristics play an important role in shaping the implementation of the intervention. Our study suggests the importance of tailoring interventions for the migrant factory workers. TRIAL REGISTRATION: ChiCTR-OPC-17011637 at Chinese Clinical Trial Registry. Retrospectively registered on 12th June 2017.

A preliminary exploration of plain-film radiography in scapular dyskinesis evaluation.

BACKGROUND: Evaluation of scapular dyskinesis is of clinical interest because it is believed to be associated with pathologies of the shoulder. This study investigated the feasibility of plain-film radiography in evaluating scapular dyskinesis. METHODS: Subjects with unilateral disorders of the shoulder (n = 186) who underwent plain-film radiography of bilateral scapulae were divided into 4 categories of scapular dyskinesis patterns according to the Kibler classification and analyzed. Coracoid upward shift distance (CUSD), length of the scapular spine line (LSS), and scapular upward rotation angle (SURA) were measured on the radiographs. Intrarater and inter-rater reliability were tested, and the characteristics of these parameters in each type were analyzed. The differences (d) between bilateral scapulae (d-CUSD, d-LSS, and d-SURA) among the 4 categories were compared. RESULTS: Intrarater and inter-rater reliability were excellent for all parameters. Significant differences between the scapulae were observed in CUSD in type I and in LSS in type II categories. No significant difference in any of the parameters was found in type III. Compared with the other categories, d-CUSD in type I and d-LSS in type II were significantly larger. The cutoff values of d-CUSD and d-LSS were 1.1 mm and 1.2 mm, respectively. No significant difference in d-SURA was found among the 4 categories. CONCLUSIONS: The measurement of CUSD, LSS, and SURA on plain-film radiography had excellent reliability. d-CUSD and d-LSS were characteristic parameters of type I and type II, respectively; however, type III had no distinguishing characteristics among the parameters.

Implementation of a comprehensive intervention for patients at high risk of cardiovascular disease in rural China: A pragmatic cluster randomized controlled trial.

OBJECTIVE: This study aims to assess whether a standard intervention package of cardiovascular disease (CVD) care was being delivered effectively, and if it was associated with improved lifestyle and biomedical indicators. METHODS: In rural China, we implemented a pragmatic cluster randomized controlled trial for 12 months, randomized at the township hospital level, and compared with usual care. Intervention case management guideline, training and performance monitoring meeting and patient support activities were designed to fit within the job description of family doctors in the township hospitals and comprised: 1) prescription of a standardised package of medicines targeted at those with hypertension or diabetes; 2) advice about specific lifestyle interventions; and 3) advice about medication adherence. Participants were 50-74 years old, had hypertension and CVD risk scores >20% or diabetes, but were excluded if a history of severe CVD events. We also randomly selected 100 participants from six selected clusters per arm as a panel to collect intermediate biomedical indicators over time. RESULTS: A total of 28,130 participants, in 33 intervention and 34 control township hospitals, were recruited. Compared with the control arm, participants in the intervention arm had substantially improved prescribing rates of anti-hypertensives, statins and aspirin (P<0.001), and had higher medication taking rates of aspirin and statins (P<0.001). Mean systolic and diastolic blood pressures were similar across both arms (0.15 mmHg, P = 0.79, and 0.52 mmHg, P = 0.05, respectively). In the panel, (950) rates of smoking (OR = 0.23, P = 0.02) and salt intake (OR = 2.85, P = 0.03) were significantly reduced in the intervention versus control arms, but there were no statistically significant improvement over the 12 month follow-up period in biomedical indicators (P>0.05). CONCLUSION: Implementation of the package by family doctors was feasible and improved prescribing and some lifestyle changes. Additional measures such as reducing medication costs and patient education are required. TRIAL REGISTRATION: Current Controlled Trials ISRCTN58988083.

[Application of three dimensional model in treatment of superolateral dislocation of mandibular condyle].

OBJECTIVE: To assess the application of three-dimensional jaw model in treatment of superolateral dislocation of condyle. METHODS: Nine patients with superolateral dislocation of condyle and symphyseal fractures were admitted in hospital from June 2008 to June 2013. Spiral CT scan on maxilla and mandible was performed，and three-dimensional jaw model was constructed with rapid prototyping technology. According to conditions of condylar superolateral dislocation model，the operation scheme was designed. The symphyseal fracture line was made and fracture was reduced on three-dimensional jaw model. Individualized prefabricated reconstructive titanium plate was made on symphyseal fracture model. Symphyseal fracture was replaced with individualized prefabricated mandibular reconstruction titanium plate according to occlusal relationship. At the same time，reduction of condylar superolateral dislocation was accomplished. RESULTS: Operations were successfully completed in 9 cases under guidance of three dimensional jaw model surgery. Operative incision was primarily healed，and occlusal relationship was restored. Postoperative CT scan showed that the locations of condyle in 9 cases were completely restored. Patients were followed-up for 6-28 months with a median of 18 months. The maximum of mouth opening was 3.1- 4.2 cm with a medians of 3.3 cm，and the occlusal relationship of all patients was normal after surgery. CONCLUSION: Three-dimensional jaw model can show complete status of condylar superolateral dislocation and symphyseal fracture. The model can accurately shape symphyseal reconstructive titanium plate and restore condylar superolateral dislocation during reductive surgery.

Material assignment in finite element modeling: heterogeneous properties of the mandibular bone.

PURPOSE: The properties of the biomaterial are normally characterized by heterogeneity on all scales influencing the function and biomechanics. Elastic modulus (EM), which is one of the most important mechanical properties of material, is necessary for finite element modeling and needed to be determined by some methods. The aim of this study is to demonstrate the feasibility of assessment of EM from GrayValue (GV) of computed tomographic image and assignment of material properties in heterogeneous finite element modeling for studying the performance of the mandibular bones. METHODS: Three mandibles obtained from fresh human cadavers were used in this study. All mandibular bones were scanned using computed tomography, and the original data were stored in optical disks. The finite element modeling of the 3 mandibles was meshed using Materialise Mimics 10.01 and Abaqus 6.10 software. Using the empirical expression on relationship between GV and EM, the empirical EM of the meshed mandibular elements were calculated. To verify the empirical EM, actual EM of the three was determined by nanoindentation test using Oliver and Pharr method. The dependence of EM on test regions and loading directions were also discussed. RESULTS: The empirical EM of the mandible element is in the range of 3.7 to 23.4 Gpa, and EM of the cortical element is in the range of 8.6 to 13.6 Gpa. In comparison, the actual EM of cortical bone tested by nanoindentation method is in the range of 10.0 to 22.0 Gpa. The tested EM is varied with the test regions and loading directions. The difference in the values of EM determined by the empirical analysis and by the nanoindentation test is approximately 5.0 Gpa. CONCLUSIONS: A mandibular finite element model with heterogeneous material properties is built. By analyzing the EM value of this model, it is concluded that the actual EM in anterior-posterior direction in mandibular ramus and EM in superior-inferior direction in mandibular body match the empirical EM better than the other directions.