Shortcut barcoding and early pooling for scalable multiplex single-cell reduced-representation CpG methylation sequencing at single nucleotide resolution.

DNA methylation is essential for a wide variety of biological processes, yet the development of a highly efficient and robust technology remains a challenge for routine single-cell analysis. We developed a multiplex scalable single-cell reduced representation bisulfite sequencing (msRRBS) technology. It allows cell-specific barcoded DNA fragments of individual cells to be pooled before bisulfite conversion, free of enzymatic modification or physical capture of the DNA ends, and achieves read mapping rates of 62.5 ± 3.9%, covering 60.0 ± 1.4% of CpG islands and 71.6 ± 1.6% of promoters in K562 cells. Its reproducibility is shown in duplicates of bulk cells with close to perfect correlation (R = 0.97-0.99). At a low 1 Mb of clean reads, msRRBS provides highly consistent coverage of CpG islands and promoters, outperforming the conventional methods with orders of magnitude reduction in cost. Here, we use this method to characterize the distinct methylation patterns and cellular heterogeneity of six cell lines, plus leukemia and hepatocellular carcinoma models. Taking 4 h of hands-on time, msRRBS offers a unique, highly efficient approach for dissecting methylation heterogeneity in a variety of multicellular systems.

[Anatomical observation and CT three-dimensional reconstruction of Five-shu acupoints of Shaoyin Heart Meridian in rabbit's forelimb].

OBJECTIVE: To investigate the location and anatomical structure of "Shaochong"(HT9), "Shaofu"(HT8), "Shenmen"(HT7), "Lingdao"(HT4) and "Shaohai"(HT3) in the rabbit's forelimb. METHODS: Sixteen rabbits (half male and half female) were used in the present study. By referring to the national standards on the location of acupoints in the human body and the literature about the location of acupoints in the rabbit, and by using the method of comparative anatomy, the location and needling operation of the Five-shu acupoints of Shaoyin Heart Meridian on the rabbit's forelimb were defined, and these acupoints were needled and CT three-dimensional reconstruction were conducted. Then, the rabbits were killed, and intravascular perfusion was performed, followed by inserting acupuncture needles into these five acupoints for observing the anatomical relationship between the inserted acupuncture needle and the structure of surrounding tissues. RESULTS: HT9 is located at the medial side of the little finger of forelimb, about 1 mm beside the nail root, and is adjacent to the superficial flexor tendon of the finger, the dorsal branches of the proper palmar digital artery and vein, and the endings of dorsal branch of palmar digital proper nerve of the ulnar nerve on the fifth finger side. HT8 is located at the palm side of the forelimb, horizontally parallel to the proximal end of the 5th metacarpophalangeal joint and between the 4th and 5th metacarpal bones, and is adjacent to the lumbricalis, the 4th and 5th interossei, and common palmar digital artery and vein and the palmar digital proper nerve of the ulnar nerve. HT7 is located at the medial margin of the extensor carpal tendon on the ulnar side, between the distal end of the ulna and the ulnar carpal bone, and is adjacent to the tendons of flexor carpi ulnaris and extensor carpi ulnaris, ulnar artery, ulnar vein and ulnar nerve. HT4 is located at the medial border of the ulnar flexor tendon, about 1.5 cun superior to HT7, and is adjacent to extensor carpi ulnaris, flexor carpi ulnaris, flexor digitorum superficialis, flexor digitorum profundus, ulnar artery, vein and ulnar nerve. HT3 is located at the depression, medial to the condyle of humerus when the elbow is bent at 90°, its neighbor structure is composed of pronator teres, biceps brachii, brachial artery and vein, radial collateral artery, radial collateral vein, medial antebrachial cutaneous nerve and median nerve. CONCLUSION: In the rabbit, there is a close relationship between HT9, HT8, HT7, HT4 and HT3 regions and brachial vascular and its branches, cephalic vein and its branches, medial antebrachial cutaneous nerve, median nerve and ulnar nerve, which is the morphological basis of the Five-shu acupoints of Shaoyin Heart Meridian for treating some related clinical disorders.

Air pollution in heavy industrial cities along the northern slope of the Tianshan Mountains, Xinjiang: characteristics, meteorological influence, and sources.

The spatiotemporal characteristics, relationship with meteorological factors, and source distribution of air pollutants (January 2017-December 2021) were analyzed to better understand the air pollutants on the northern slope of the Tianshan Mountains (NSTM) in Xinjiang, a heavily polluted urban agglomeration of heavy industries. The results showed that the annual mean concentrations of SO2, NO2, CO, O3, PM2.5, and PM10 were 8.61-13.76 µg m-3, 26.53-36.06 µg m-3, 0.79-1.31 mg m-3, 82.24-87.62 µg m-3, 37.98-51.10 µg m-3, and 84.15-97.47 µg m-3. The concentrations of air pollutants (except O3) showed a decreasing trend. The highest concentrations were in winter, and in Wujiaqu, Shihezi, Changji, Urumqi, and Turpan, the concentrations of particulate matter exceeded the NAAQS Grade II during winter. The west wind and the spread of local pollutants both substantially impacted the high concentrations. According to the analysis of the backward trajectory in winter, the air masses were mainly from eastern Kazakhstan and local emission sources, and PM10 in the airflow had a more significant impact on Turpan; the rest of the cities were more affected by PM2.5. Potential sources included Urumqi-Changj-Shihezi, Turpan, the northern Bayingol Mongolian Autonomous Prefecture, and eastern Kazakhstan. Consequently, the emphasis on improving air quality should be on reducing local emissions, strengthening regional cooperation, and researching transboundary transport of air pollutants.

Comparison of Primary B/NKT Non-Hodgkin Lymphomas in Nasopharynx, Nasal Cavity, and Paranasal Sinuses.

OBJECTIVE: We aimed to compare clinical and survival differences between B-cell (B-NHL) and NKT-cell non-Hodgkin lymphomas (NKT-NHL) located in the nasal cavity (NC), nasopharynx, and paranasal sinuses, which are always categorized as one sinonasal type. STUDY DESIGN: Patients diagnosed with primary B-NHL and NKT-NHL in the nasal cavity, nasopharynx, and paranasal sinuses from Surveillance, Epidemiology, and End Results (SEER) database were included (1975-2017). SETTING: Population-based cohort study. METHODS: We conducted univariate and multivariate Cox regressions and Kaplan-Meier analysis to examine survival outcomes of B/NKT-NHL in the nasal cavity, nasopharynx, and paranasal sinuses, respectively. RESULTS: Overall, most B-NHL cases originated from the nasopharynx, while the majority of NKT-NHL cases occurred in the nasal cavity. Notably, the cancer-special survival (CSS) outcomes improved significantly in all sinonasal B-NHL cases over time, whereas no such improvement trend was observed in each sinonasal NKT-NHL type. Additionally, increasing age was linked with an elevated risk of death in B-NHL, particularly in the nasal cavity (Hazard ratio [HR]: 3.37), rather than in NKT-NHL. Compared with B-NHL, the adverse effect of a higher stage on CSS was more evident in NKT-NHL, particularly in its nasopharynx site (HR: 5.12). Furthermore, radiotherapy was beneficial for survival in patients with sinonasal B-NHL and NKT-NHL, except in the nasopharynx NKT-NHL. However, chemotherapy has only been beneficial for CSS in patients with paranasal sinuses B-NHL (HR: 0.42) since 2010, rather than in other types of B/NKT-NHL. CONCLUSION: Although B-NHL and NKT-NHL in the nasal cavity, nasopharynx and paranasal sinuses have similar anatomical locations, their clinicodemographics and prognoses are largely different and should be treated and studied as distinct diseases.

Finite element analysis of the biomechanical effect of clear aligners in extraction space closure under different anchorage controls.

INTRODUCTION: Clear aligners (CAs) have attracted increasing attention from patients and orthodontists because of their excellent esthetics and comfort. However, treating tooth extraction patients with CAs is difficult because their biomechanical effects are more complicated than those of traditional appliances. This study aimed to analyze the biomechanical effect of CAs in extraction space closure under different anchorage controls, including moderate, direct strong, and indirect strong anchorage. It could provide several new cognitions for anchorage control with CAs through finite element analysis, further directing clinical practice. METHODS: A 3-dimensional maxillary model was generated by combining cone-beam computed tomography and intraoral scan data. Three-dimensional modeling software was used to construct a standard first premolar extraction model, temporary anchorage devices, and CAs. Subsequently, finite element analysis was performed to simulate space closure under different anchorage controls. RESULTS: Direct strong anchorage was beneficial for reducing the clockwise occlusal plane rotation, whereas indirect anchorage was conducive for anterior teeth inclination control. In the direct strong anchorage group, an increase in the retraction force would require more specific anterior teeth overcorrection to resist the tipping movement, mainly including lingual root control of the central incisor, followed by distal root control of the canine, lingual root control of the lateral incisor, distal root control of the lateral incisor, and distal root control of the central incisor. However, the retraction force could not eliminate the mesial movement of the posterior teeth, possibly causing a reciprocating motion during treatment. In indirect strong groups, when the button was close to the center of the crown, the second premolar presented less mesial and buccal tipping but more intrusion. CONCLUSIONS: The 3 anchorage groups showed significantly different biomechanical effects in both the anterior and posterior teeth. Specific overcorrection or compensation forces should be considered when using different anchorage types. The moderate and indirect strong anchorages have a more stable and single-force system and could be reliable models in investigating the precise control of future tooth extraction patients.

Diversity of clinical phenotypes in a cohort of Han Chinese patients with PAX6 variants.

The PAX6 gene plays an important role in ocular development. Mutations of the PAX6 gene may result in a series of ocular abnormalities, including congenital aniridia, anterior segment dysgenesis (ASD), progressive corneal opacification, glaucoma, and hypoplasia of the fovea and optic nerve, leading to reduced visual acuity and even blindness. This study aimed to describe the diversity of clinical features caused by PAX6 pathogenic variants in 45 Han Chinese patients from 23 unrelated families. All patients underwent detailed clinical assessment. Genetic testing was performed to identify pathogenic variations in the PAX6 gene by next-generation sequencing, minigene splicing assay, RT-qPCR, and long-range PCR. Twenty pathogenic variations were detected in the PAX6 gene from 12 pedigrees and 11 sporadic patients, of which 12 were previously reported and 8 were novel. The clinical phenotypes obtained as a result of the PAX6 gene mutations were complicated and vary among patients, even among those who carried the same variants. Genetic testing is helpful for differential diagnosis. Our genetic findings will expand the spectrum of pathogenic variations in the PAX6 gene. PAX6 pathogenic variants not only cause defects in ocular tissues, such as the iris and retina, but also lead to maldevelopment of the whole eye, resulting in microphthalmia.

The Relationship Between Sleep Traits and Tinnitus in UK Biobank: A Population-Based Cohort Study.

OBJECTIVES: Understanding the association between sleep traits and tinnitus could help prevent and provide appropriate interventions against tinnitus. Therefore, this study aimed to assess the relationship between different sleep patterns and tinnitus. DESIGN: A cross-sectional analysis using baseline data (2006-2010, n = 168,064) by logistic regressions was conducted to evaluate the association between sleep traits (including the overall health sleep score and five sleep behaviors) and the occurrence (yes/no), frequency (constant/transient), and severity (upsetting/not upsetting) of tinnitus. Further, a prospective analysis of participants without tinnitus at baseline (n = 9581) was performed, who had been followed-up for 7 years (2012-2019), to assess the association between new-onset tinnitus and sleep characteristics. Moreover, a subgroup analysis was also carried out to estimate the differences in sex by dividing the participants into male and female groups. A sensitivity analysis was also conducted by excluding ear-related diseases to avoid their confounding effects on tinnitus (n = 102,159). RESULTS: In the cross-sectional analysis, participants with "current tinnitus" (OR: 1.13, 95% CI: 1.04-1.22, p = 0.004) had a higher risk of having a poor overall healthy sleep score and unhealthy sleep behaviors such as short sleep durations (OR: 1.09, 95% CI: 1.04-1.14, p < 0.001), late chronotypes (OR: 1.09, 95% CI: 1.05-1.13, p < 0.001), and sleeplessness (OR: 1.16, 95% CI: 1.11-1.22, p < 0.001) than those participants who "did not have current tinnitus." However, this trend was not obvious between "constant tinnitus" and "transient tinnitus." When considering the severity of tinnitus, the risk of "upsetting tinnitus" was obviously higher if participants had lower overall healthy sleep scores (OR: 1.31, 95% CI: 1.13-1.53, p < 0.001). Additionally, short sleep duration (OR: 1.22, 95% CI: 1.12-1.33, p < 0.001), late chronotypes (OR: 1.13, 95% CI: 1.04-1.22, p = 0.003), and sleeplessness (OR: 1.43, 95% CI: 1.29-1.59, p < 0.001) showed positive correlations with "upsetting tinnitus." In the prospective analysis, sleeplessness presented a consistently significant association with "upsetting tinnitus" (RR: 2.28, p = 0.001). Consistent results were observed in the sex subgroup analysis, where a much more pronounced trend was identified in females compared with the males. The results of the sensitivity analysis were consistent with those of the cross-sectional and prospective analyses. CONCLUSIONS: Different types of sleep disturbance may be associated with the occurrence and severity of tinnitus; therefore, precise interventions for different types of sleep disturbance, particularly sleeplessness, may help in the prevention and treatment of tinnitus.

The prognostic value of machine learning techniques versus cox regression model for head and neck cancer.

BACKGROUND: Accurate prognostic prediction for head and neck cancer (HNC) is important for the improvement of clinical management. We aimed to compare the prognostic value of various machine learning techniques (MLTs) and statistical Cox regression model for different types of HNC. METHODS: Clinical data of HNC patients were extracted from the Surveillance, Epidemiology, and End Results (SEER) database from 1974 to 2016. The prediction performance of five ML models, including random forest (RF), gradient boosting decision tree (GBDT), support vector machine (SVM), neural network (NN) and deep learning (DL), were compared with the statistical Cox regression model by estimating the concordance index (C-index), integrated Brier score (IBS), time-dependent receiver operating characteristic (ROC) curve and the area under the curve (AUC). RESULTS: Our results showed that the RF model outperformed all other models in prognostic prediction for all tumor sites of HNC, particularly for major salivary gland cancer (MSGC, C-index: 88.730 ± 0.8700, IBS: 7.680 ± 0.4800), oral cavity cancer (OCC, C-index: 84.250 ± 0.6700, IBS: 11.480 ± 0.3300) and oropharyngeal cancer (OPC, C-index: 82.510 ± 0.5400, IBS: 10.120 ± 0.1400). Meanwhile, we analyzed the importance of each clinical variable in the RF model, in which age and tumor size presented the strongest positive prognostic effects. Additionally, similar results can be observed in the internal (6th edition of the AJCC TNM staging system cohort) and external validations (the TCGA HNC cohort). CONCLUSIONS: The RF model is a promising prognostic prediction tool for HNC patients, regardless of the anatomic subsites.

Impact of Allergic Rhinitis and Asthma on COVID-19 Infection, Hospitalization, and Mortality.

BACKGROUND: It remains unclear if patients with allergic rhinitis (AR) and/or asthma are susceptible to corona virus disease 2019 (COVID-19) infection, severity, and mortality. OBJECTIVE: To investigate the role of AR and/or asthma in COVID-19 infection, severity, and mortality, and assess whether long-term AR and/or asthma medications affected the outcomes of COVID-19. METHODS: Demographic and clinical data of 70,557 adult participants completed SARS-CoV-2 testing between March 16 and December 31, 2020, in the UK Biobank were analyzed. The rates of COVID-19 infection, hospitalization, and mortality in relation to pre-existing AR and/or asthma were assessed based on adjusted generalized linear models. We further analyzed the impact of long-term AR and/or asthma medications on the risk of COVID-19 hospitalization and mortality. RESULTS: Patients with AR of all ages had lower positive rates of SARS-CoV-2 tests (relative risk [RR]: 0.75, 95% confidence interval [CI]: 0.69-0.81, P < .001), with lower susceptibility in males (RR: 0.74, 95% CI: 0.65-0.85, P < .001) than females (RR: 0.8, 95% CI: 0.72-0.9, P < .001). However, similar effects of asthma against COVID-19 hospitalization were only major in participants aged <65 (RR: 0.93, 95% CI: 0.86-1, P = .044) instead of elderlies. In contrast, patients with asthma tested positively had higher risk of hospitalization (RR: 1.42, 95% CI: 1.32-1.54, P < .001). Neither AR nor asthma had an impact on COVID-19 mortality. None of conventional medications for AR or asthma, for example, antihistamines, corticosteroids, or ß2 adrenoceptor agonists, showed association with COVID-19 infection or severity. CONCLUSION: AR (all ages) and asthma (aged <65) act as protective factors against COVID-19 infection, whereas asthma increases risk for COVID-19 hospitalization. None of the long-term medications had a significant association with infection, severity, and mortality of COVID-19 among patients with AR and/or asthma.

Glutamine Regulates Cell Growth and Casein Synthesis through the CYTHs/ARFGAP1-Arf1-mTORC1 Pathway in Bovine Mammary Epithelial Cells.

In the dairy industry, glutamine (Gln) is often used as a feed additive to increase milk yield and quality; however, the molecular regulation underneath needs further clarification. Here, with bovine mammary epithelial cells (BMECs), the effects and mechanisms of Gln on cell growth and casein synthesis were assessed. When Gln was added or depleted from BMECs, both cell growth and ß-casein (CSN2) expression were increased or decreased, respectively. Overexpressing or inhibiting the mechanistic target of rapamycin (mTOR) revealed that Gln regulated cell growth and CSN2 synthesis through the mTORC1 pathway. A similar intervention of ADP-ribosylation factor 1 (Arf1) uncovered that Gln activated the mTORC1 pathway through Arf1. We next observed that both guanine nucleotide exchange factors, Cytohesin-1/2/3 (CYTH1/2/3, CYTHs) and ADP-ribosylation factor GTPase activating protein 1 (ARFGAP1), interacted with Arf1. Inhibiting CYTHs or ARFGAP1 showed that Gln supplement or depletion activated or inactivated Arf1 through CYTHs or ARFGAP1, respectively. Collectively, this study demonstrated that Gln positively regulated cell growth and casein synthesis in BMECs, which works through the CYTHs/ARFGAP1-Arf1-mTORC1 pathway. These results greatly enhanced current understanding regarding the regulation of the mTOR pathway and provided new insights for the processes of cell growth and casein synthesis by amino acids, particularly Gln.

Review of the distribution and detection methods of heavy metals in the environment.

Heavy metals can be enriched in living organisms and seriously endanger human health and the ecological environment, which has evolved into a significant global environmental problem. Based on summarizing the spatial distribution of heavy metals in the environment, this review introduces heavy metal detection technologies such as inductively coupled plasma mass spectrometry/atomic emission spectrometry, atomic absorption spectrometry, atomic fluorescence spectrometry, and laser-induced breakdown spectrometry. It summarizes their respective advantages, characteristics, and applicability. Besides, atmospheric pressure discharge plasma as a potential heavy metal detection technology is also introduced and discussed in this review. The current research mainly focuses on improving the analytical performance and optimizing the practical application. Furthermore, this review not only summarizes the advantages of atmospheric pressure discharge plasma in the field of element analysis but also summarizes the principal scientific and technical problems to be solved urgently.

Mesencephalic astrocyte-derived neurotrophic factor ameliorates steatosis in HepG2 cells by regulating hepatic lipid metabolism.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a global metabolism-associated liver disease. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a newly discovered secreted protein that is involved in metabolic homeostasis. However, much remains to be discovered about its function in hepatic lipid metabolism; thus, we assessed whether MANF could regulate hepatic metabolism. AIM: To establish in vivo and in vitro NAFLD models to explore the role of MANF in hepatic lipid metabolism. METHODS: HepG2 cells treated with free fatty acids (FFAs) and ob/ob mice were used as NAFLD models. Liver tissues collected from wild type and ob/ob mice were used to detect MANF expression. Cells were treated with FFAs for different durations. Moreover, we used lentiviral constructs to establish overexpression and knockdown cell models in order to interfere with MANF expression levels and observe whether MANF influences hepatic steatosis. Western blot analysis and quantitative real-time PCR were used to detect protein and gene expression, and oil red O staining was used to visualize intracellular lipid droplets. RESULTS: Hepatic MANF protein and mRNA expression in wild type mice were 10-fold and 2-fold higher, respectively, than those in ob/ob mice. The MANF protein was temporarily increased by 1.3-fold after stimulation with FFAs for 24 h and gradually decreased to 0.66-fold that of the control at the 72 h time point in HepG2 cells. MANF deficiency upregulated the expression of genes involved in fatty acid synthesis, cholesterol synthesis, and fatty acid uptake and aggravated HepG2 cell steatosis, while MANF overexpression inhibited fatty acid synthesis and uptake and cholesterol synthesis, and rescued HepG2 cells from FFAs-induced steatosis. Furthermore, a significant decrease in triglyceride levels was observed in the MANF overexpression group compared with the control group (0.4288 ± 0.0081 mmol/g vs 0.3746 ± 0.0121 mmol/g, P < 0.05) upon FFAs treatment. There was also a 17% decrease in intracellular total cholesterol levels between the MANF overexpression group and the control group (0.1301 ± 0.0059 mmol/g vs 0.1088 ± 0.0009 mmol/g, P < 0.05) upon FFAs treatment. Moreover, MANF suppressed lipid deposition in HepG2 cells. CONCLUSION: Our findings indicate that MANF improves the phenotype of liver cell steatosis and may be a potential therapeutic target in hepatic steatosis processes.

Increased plasma osteopontin levels are associated with nonalcoholic fatty liver disease in patients with type 2 diabetes mellitus.

Nonalcoholic fatty liver disease (NAFLD) commonly occurs in patients with type 2 diabetes mellitus (T2DM). Osteopontin (OPN) is a multifunctional protein with pleiotropic physiological functions. This study aimed to investigate the interrelation between circulating OPN and NAFLD in T2DM patients. Overall, 249 subjects were classified into 4 groups: 53 patients with NAFLD and T2DM; 57 with newly diagnosed T2DM; 59 with NAFLD; and 80 healthy age- and sex-matched controls. Serum OPN was measured by ELISA. The OPN distribution in the pooled data was divided into quartiles; significant trends across increasing quartiles were estimated by the Cochran-Armitage trend test. Compared with the controls, circulating OPN concentrations were significantly elevated in NAFLD patients and T2DM patients with or without NAFLD. Serum OPN levels were higher in the overweight/obese group than that in the lean group. Circulating OPN levels were positively correlated with CRP, age, BMI, SBP, DBP, HbA1c, UA, TGs, WBCs, neutrophils, FBG, and HOMA-IR and negatively correlated with ADP, albumin and HDL. Age, albumin, HbA1c, HDL and hsCRP were independently related to circulating OPN. The relative risks for NAFLD, T2DM and T2DM with NAFLD increased significantly along with increasing OPN quartiles based on the Cochran-Armitage trend test. OPN is an optimal predictor in the diagnosis of T2DM with NAFLD and T2DM and may contribute to the aggravation of the metabolic state.

Clinicopathological features and prognostic analysis of 49 cases with crescentic glomerulonephritis.

Rapidly progressive glomerulonephritis (RPGN), characterized by rapid kidney dysfunction caused by aggressive glomerulonephritis, is usually associated with crescentic glomerulonephritis (CrGN). In the present study, the data from patients with CrGN were retrospectively analyzed at a tertiary medical center in China with the aim of investigating the clinicopathological features and the association of the type of CrGN with the prognosis. The renal biopsies of 49 patients diagnosed with CrGN were obtained between December 2011 and July 2016. Of the 49 patients, 11 patients (22.45%) had type I CrGN, 19 (38.78%) had type II CrGN and 19 (38.78%) had type III CrGN. The majority of CrGN patients exhibited multiple-system involvement and 28 patients (57.14%) had kidney enlargement. Proportions of patients with acute kidney injury (AKI), acute kidney diseases without AKI, and chronic kidney disease were 28.57, 46.94 and 24.49%, respectively. Among the 3 types of CrGN, patients with type I CrGN tended to have a higher proportion of AKI with more cellular crescent formation, and higher serum creatinine and retinol binding protein. Circulating anti-GBM antibodies were present in all type I CrGN patients and anti-neutrophilic cytoplasmic autoantibodies were detected in 84.21% of patients with type III CrGN. Type III CrGN patients had a superior kidney survival, whereas type I CrGN patients had the worst kidney prognosis (P<0.001). There was no significant difference in overall patient survival among the 3 types of CrGN. CrGN remains the primary cause of critical illness in RPGN patients. There was much heterogeneity between the different subtypes of CrGN. Patients with type I tended to have an acute onset and had the poorest kidney survival.