Low-frequency somatic copy number alterations in normal human lymphocytes revealed by large-scale single-cell whole-genome profiling.

Genomic-scale somatic copy number alterations in healthy humans are difficult to investigate because of low occurrence rates and the structural variations' stochastic natures. Using a Tn5-transposase-assisted single-cell whole-genome sequencing method, we sequenced over 20,000 single lymphocytes from 16 individuals. Then, with the scale increased to a few thousand single cells per individual, we found that about 7.5% of the cells had large-size copy number alterations. Trisomy 21 was the most prevalent aneuploid event among all autosomal copy number alterations, whereas monosomy X occurred most frequently in over-30-yr-old females. In the monosomy X single cells from individuals with phased genomes and identified X-inactivation ratios in bulk, the inactive X Chromosomes were lost more often than the active ones.

Integrative study of pulmonary microbiome, transcriptome and clinical outcomes in Mycoplasma pneumoniae pneumonia.

BACKGROUND: This study aimed to investigate the interactions among three core elements of respiratory infection-pathogen, lung microbiome, and host response-and their avocation with the severity and outcomes of Mycoplasma pneumoniae pneumonia (MPP) in children. METHODS: We prospectively collected bronchoalveolar lavage fluid from a cohort of 41 children with MPP, including general MPP (GMPP) and complicated MPP (CMPP), followed by microbiome and transcriptomic analyses to characterize the association among pathogen, lung microbiome, and host response and correlate it with the clinical features and outcomes. RESULTS: The lung microbiome of patients with CMPP had an increased relative abundance of Mycoplasma pneumoniae (MP) and reduced alpha diversity, with 76 differentially expressed species. Host gene analysis revealed a key module associated with neutrophil function and several inflammatory response pathways. Patients with a high relative abundance of MP, manifested by a specific lung microbiome and host response type, were more prone to CMPP and had a long imaging recovery time. CONCLUSION: Patients with CMPP have a more disrupted lung microbiome than those with GMPP. MP, lung microbiome, and host response interacts with each other and are closely related to disease severity and outcomes in children with MPP.

Differential effect of interventions in patients with prediabetes stratified by a machine learning-based diabetes progression prediction model.

AIM: To investigate whether stratifying participants with prediabetes according to their diabetes progression risks (PR) could affect their responses to interventions. METHODS: We developed a machine learning-based model to predict the 1-year diabetes PR (ML-PR) with the least predictors. The model was developed and internally validated in participants with prediabetes in the Pinggu Study (a prospective population-based survey in suburban Beijing; n = 622). Patients from the Beijing Prediabetes Reversion Program cohort (a multicentre randomized control trial to evaluate the efficacy of lifestyle and/or pioglitazone on prediabetes reversion; n = 1936) were stratified to low-, medium- and high-risk groups using ML-PR. Different effect of four interventions within subgroups on prediabetes reversal and diabetes progression was assessed. RESULTS: Using least predictors including fasting plasma glucose, 2-h postprandial glucose after 75 g glucose administration, glycated haemoglobin, high-density lipoprotein cholesterol and triglycerides, and the ML algorithm XGBoost, ML-PR successfully predicted the 1-year progression of participants with prediabetes in the Pinggu study [internal area under the curve of the receiver operating characteristic curve 0.80 (0.72-0.89)] and Beijing Prediabetes Reversion Program [external area under the curve of the receiver operating characteristic curve 0.80 (0.74-0.86)]. In the high-risk group pioglitazone plus intensive lifestyle therapy significantly reduced diabetes progression by about 50% at year l and the end of the trial in the high-risk group compared with conventional lifestyle therapy with placebo. In the medium- or low-risk group, intensified lifestyle therapy, pioglitazone or their combination did not show any benefit on diabetes progression and prediabetes reversion. CONCLUSIONS: This study suggests personalized treatment for prediabetes according to their PR is necessary. ML-PR model with simple clinical variables may facilitate personal treatment strategies in participants with prediabetes.

Pulchinenoside B4 ameliorates oral ulcers in rats by modulating gut microbiota and metabolites.

Pulchinenoside B4, a natural saponin monomer from the Pulsatilla plant, plays an important role as an immunomodulator in the treatment of acute inflammation. Oral ulcer (OU) is a common ulcerative injury disease that occurs in the oral mucosa, including mucosal ulceration and abnormalities of lips and tongue. A close correlation exists between gut microbiota and circulating metabolites in patients with OU. However, the correlation between gut microbiota and serum metabolomics is not clear. Therefore, this study aimed to explore the changes in gut microbiota and metabolites in OU. The 16S ribosomal RNA (16S rRNA) gene sequencing was used to detect the changes in the composition of gut microbiota in OU rat model. Moreover, the endogenous small metabolites were explored by collecting the non-targeted serum metabolomics data. A total of 34 OU-related biomarkers were identified, mainly related to fatty acid metabolism and inflammatory pathways. The administration of B4 effectively reduced the occurrence of OU and restored the levels of multiple endogenous biomarkers and key gut microbial species to the normal level. This study demonstrated that the gut microbiota and metabolites were altered in the OU rat model, which were significantly restored to the normal level by B4, thereby showing good application prospects in the treatment of OU. KEY POINTS: • The first investigating the correlation between OU and gut microbiota. • A close correlation between metabolites and gut microbiota in OU disease was successfully identified. • Pulchinenoside B4 ameliorates oral ulcers in rats by modulating gut microbiota and metabolites.

Meta-analysis comparing different ultrasound detection methods to accurately assess wound healing and scar formation after caesarean section.

The accurate assessment of wound healing post-caesarean section, especially in twin pregnancies, remains a pivotal concern in obstetrics, given its implications for maternal health and recovery. Traditional methods, including conventional abdominal ultrasonography (CU), have been challenged by the advent of transvaginal ultrasonography (TU), offering potentially enhanced sensitivity and specificity. This meta-analysis directly compares the efficacy of TU and CU in evaluating wound healing and scar formation, crucial for optimizing postoperative care. Results indicate that TU is associated with significantly better outcomes in wound healing, demonstrated by lower REEDA scores (SMD = -20.56, 95% CI: [-27.34.20, -13.77], p < 0.01), and in scar formation reduction, evidenced by lower Manchester Scar Scale scores (SMD = -25.18, 95% CI: [-29.98, -20.39], p < 0.01). These findings underscore the potential of integrating TU into routine post-caesarean evaluation protocols to enhance care quality and patient recovery.

Impact of diabetes on weight loss outcomes after bariatric surgery: Experience from 5-year follow-up of Michigan Bariatric Surgery Cohort.

OBJECTIVE: For patients with obesity and diabetes, bariatric surgery can lead to the remission of both diseases. However, the possible impact of diabetes on the magnitude of weight loss outcomes after bariatric surgery has not been precisely quantified. RESEARCH DESIGN AND METHODS: Data from Michigan Bariatric Surgery Cohort (MI-BASiC) was extracted to examine the effect of baseline diabetes on weight loss outcomes. Consecutive patients older than 18 years of age undergoing gastric bypass (GB) or sleeve gastrectomy (SG) for obesity at University of Michigan between January 2008 and November 2013 were included. Repeated measures analysis was used to determine if diabetes was a predictor of weight loss outcomes over 5 years postsurgery. RESULTS: Out of the 714 included patients, 380 patients underwent GB [mean BMI 47.3 ± 0.4 kg/m2 , diabetes 149 (39.2%)] and 334 SG [mean BMI 49.9 ± 0.5 kg/m2 , diabetes 108 (32.3%)]. Multivariable repeated measures analysis showed, after adjusting for covariates, that individuals with diabetes had a significantly lower percentage of total (p = .0023) and excess weight loss (p = .0212) compared to individuals without diabetes. CONCLUSIONS: Our data demonstrate that patients with diabetes undergoing bariatric surgery would experience less weight loss than patients without diabetes.

FGF2 isoforms play distinct roles in tubular epithelial-to-mesenchymal transition in diabetic nephropathy.

INTRODUCTION: The role of different isoforms of Fibroblast growth factor-2 (FGF2) in tubular epithelial-to-mesenchymal transition (EMT) in diabetic nephropathy remains unknown. We aimed to evaluate the role of FGF2 isoforms in the pathogenesis of EMT. MATERIALS AND METHODS: Western blot and immunofluorescence were used to assess the expression of FGF2 isoforms in db/db mice and high glucose-stimulated HK2 cells. The effects of specific FGF2 isoforms on EMT were explored via overexpression or knockdown of the corresponding isoform in HK2 cells cultivated in high glucose. RESULTS: Expression of low molecular weight (LMW) FGF2 was up-regulated while high molecular weight (HMW) FGF2 was down-regulated in the kidney of db/db mice and HK2 cells cultured in high glucose that underwent EMT. Overexpression of the LMW FGF2 enhanced EMT changes, while overexpression of the HMW FGF2 attenuated EMT. Knockdown of HMW FGF2 in HK2 cells promoted the EMT process. CONCLUSIONS: The expression and function of LMW and HMW FGF2 differed in the process of EMT in tubular cells. LMW FGF2 contributed to EMT, while HMW FGF2 played a protective role in the EMT process.

Anemoside A3 Inhibits Macrophage M2-Like Polarization to Prevent Triple-Negative Breast Cancer Metastasis.

Triple negative breast cancer (TNBC) exhibits the characteristics of strong metastatic ability and a high recurrence rate, and M2-type macrophages play an important role in this process. Previous research data suggested that Anemoside A3 (A3), a monomeric component of Pulsatilla Chinensis, could prevent and treat TNBC by converting M0 macrophages into M1 immunogen phenotypes. This study showed that A3 significantly restrained the lung metastases of 4 T1-Luc cells with bioluminescence imaging in vivo and Hematoxylin and Eosin (H&E) staining. Meanwhile, the percentage of M2-type macrophages (CD206+ labeled cells) in the lung tissues was evidently decreased through immunohistochemical assay. We further proved that A3 markedly prevented M2-type polarization induced by IL-4 in vitro, as illustrated by the down-regulated expression of the cell surface marker CD206 protein by FACS and Arg-1, and of the Fizz1 and Ym1 genes by RT-PCR in M2-type macrophages. Furthermore, the invasion and migration of 4 T1 cells, which was promoted by the conditioned medium from M2-type macrophages, could be suppressed by A3. Luminex assay demonstrated that A3 treatment resulted in a reduction of the levels of CCL2, VEGF, CCL7, and MMP-9 in conditioned medium. Additionally, the expression of phosphorylated-STAT3 protein was inhibited by A3, which resulted in the macrophage M2-type polarization arrest, while no significant difference in JAK2 phosphorylation was detected. SiRNA transfection experiments suggested that STAT3 might be the target of A3 inhibiting M2-type polarization of macrophages. In conclusion, these results indicate that A3 could attenuate the metastasis of TNBC by inhibiting the M2-type polarization of macrophages, which may be related to the STAT3 pathway.

Pulsatilla Saponins Inhibit Experimental Lung Metastasis of Melanoma via Targeting STAT6-Mediated M2 Macrophages Polarization.

Pulsatilla saponins (PS) extracts from Pulsatilla chinensis (Bge.) Regel, are a commonly used traditional Chinese medicine. In the previous study, we found Pulsatilla saponins displayed anti-tumor activity without side effects such as bone marrow suppression. However, the mechanism of the anti-tumor effect was not illustrated well. Since M2-like tumor-associated macrophages (TAMs) that required activation of the signal transducer and activator of transcription 6 (STAT6) for polarization are the important immune cells in the tumor microenvironment and play a key role in tumor progress and metastasis, this study aimed to confirm whether Pulsatilla saponins could inhibit the development and metastasis of tumors by inhibiting the polarization of M2 macrophages. We investigated the relevance of M2 macrophage polarization and the anti-tumor effects of Pulsatilla saponins in vitro and in vivo. In vitro, Pulsatilla saponins could decrease the mRNA level of M2 marker genes Arg1, Fizz1, Ym1, and CD206, and the down-regulation effect of phosphorylated STAT6 induced by IL-4; moreover, the conditioned medium (CM) from bone marrow-derived macrophages (BMDM) treated with Pulsatilla saponins could inhibit the proliferation and migration of B16-F0 cells. In vivo, Pulsatilla saponins could reduce the number of lung metastasis loci, down-regulate the expression of M2 marker genes, and suppress the expression of phosphorylated STAT6 in tumor tissues. Furthermore, we used AS1517499 (AS), a STAT6 inhibitor, to verify the role of PS on M2 macrophage polarization both in vitro and in vivo. We found that Pulsatilla saponins failed to further inhibit STAT6 activation; the mRNA level of Arg1, Fizz1, Ym1, and CD206; and the proliferation and migration of B16-F0 cells after AS1517499 intervention in vitro. Similar results were obtained in vivo. These results illustrated that Pulsatilla saponins could effectively suppress tumor progress by inhibiting the polarization of M2 macrophages via the STAT6 signaling pathway; this revealed a novel mechanism for its anti-tumor activity.

[Anemoside B4 regulates fatty acid metabolism reprogramming in mice with colitis-associated cancer].

The study aimed to investigate the effect of anemoside B4(B4) on fatty acid metabolism in mice with colitis-associated cancer(CAC). The CAC model was established by azoxymethane(AOM)/dextran sodium sulfate(DSS) in mice. Mice were randomly divided into a normal group, a model group, and low-, medium-, and high-dose anemoside B4 groups. After the experiment, the length of the mouse colon and the size of the tumor were measured, and the pathological alterations in the mouse colon were observed using hematoxylin-eosin(HE) staining. The slices of the colon tumor were obtained for spatial metabolome analysis to analyze the distribution of fatty acid metabolism-related substances in the tumor. The mRNA levels of SREBP-1, FAS, ACCα, SCD-1, PPARα, ACOX, UCP-2, and CPT-1 were determined by real-time quantitative PCR(RT-qPCR). The results revealed that the model group showed decreased body weight(P<0.05) and colon length(P<0.001), increased number of tumors, and increased pathological score(P<0.01). Spatial metabolome analysis revealed that the content of fatty acids and their derivatives, carnitine, and phospholipid in the colon tumor was increased. RT-qPCR results indicated that fatty acid de novo synthesis and ß-oxidation-related genes, such as SREBP-1, FASN, ACCα, SCD-1, ACOX, UCP-2, and CPT-1 mRNA expression levels increased considerably(P<0.05, P<0.001). After anemoside B4 administration, the colon length increased(P<0.01), and the number of tumors decreased in the high-dose anemoside B4 group(P<0.05). Additionally, spatial metabolome analysis showed that anemoside B4 could decrease the content of fatty acids and their derivatives, carnitine, and phospholipids in colon tumors. Meanwhile, anemoside B4 could also down-regulate the expression of FASN, ACCα, SCD-1, PPARα, ACOX, UCP-2, and CPT-1 in the colon(P<0.05, P<0.01, P<0.001). The findings of this study show that anemoside B4 may inhibit CAC via regulating fatty acid metabolism reprogramming.

[UPLC-Q-TOF-MS metabolomic study on improvement of acute myocardial ischemia in rats by Dalbergia cochinchinensis heartwood].

This paper aimed to study the effect of Dalbergia cochinchinensis heartwood on plasma endogenous metabolites in rats with ligation of the left anterior descending coronary artery, and to analyze the mechanism of D. cochinchinensis heartwood in improving acute myocardial ischemic injury. The stability and consistency of the components in the D. cochinchinensis heartwood were verified by the establishment of fingerprint, and 30 male SD rats were randomly divided into a sham group, a model group, and a D. cochinchinensis heartwood(6 g·kg~(-1)) group, with 10 rats in each group. The sham group only opened the chest without ligation, while the other groups established the model of ligation. Ten days after administration, the hearts were taken for hematoxylin-eosin(HE) staining, and the content of heart injury indexes in the plasma creatine kinase isoenzyme(CK-MB) and lactate dehydrogenase(LDH), energy metabolism-related index glucose(Glu) content, and vascular endothelial function index nitric oxide(NO) was determined. The endogenous metabolites were detected by ultra-high-performance liquid chromatography-time-of-flight-mass spectrometry(UPLC-Q-TOF-MS). The results showed that the D. cochinchinensis heartwood reduced the content of CK-MB and LDH in the plasma of rats to relieve myocardial injury, reduced the content of Glu in the plasma, improved myocardial energy metabolism, increased the content of NO, cured the vascular endothelial injury, and promoted vasodilation. D. cochinchinensis heartwood improved the increase of intercellular space, myocardial inflammatory cell infiltration, and myofilament rupture caused by ligation of the left anterior descending coronary artery. The metabolomic study showed that the content of 26 metabolites in the plasma of rats in the model group increased significantly, while the content of 27 metabolites decreased significantly. Twenty metabolites were significantly adjusted after the administration of D. cochinchinensis heartwood. D. cochinchinensis heartwood can significantly adjust the metabolic abnormality in rats with ligation of the left anterior descending coronary artery, and its mechanism may be related to the regulation of cardiac energy metabolism, NO production, and inflammation. The results provide a corresponding basis for further explaining the effect of D. cochinchinensis on the acute myocardial injury.

The efficacy of canagliflozin in diabetes subgroups stratified by data-driven clustering or a supervised machine learning method: a post hoc analysis of canagliflozin clinical trial data.

AIMS/HYPOTHESIS: Data-driven diabetes subgroups have shown distinct clinical characteristics and disease progression, although there is a lack of evidence that this information can guide clinical decisions. We aimed to investigate whether diabetes subgroups, identified by data-driven clustering or supervised machine learning methods, respond differently to canagliflozin. METHODS: We pooled data from five randomised, double-blinded clinical trials of canagliflozin at an individual level. We applied the coordinates from the All New Diabetics in Scania (ANDIS) study to form four subgroups: mild age-related diabetes (MARD); severe insulin-deficient diabetes (SIDD); mild obesity-related diabetes (MOD) and severe insulin-resistant diabetes (SIRD). Machine learning models for HbA1c lowering (ML-A1C) and albuminuria progression (ML-ACR) were developed. The primary efficacy endpoint was reduction in HbA1c at 52 weeks. Concordance of a model was defined as the difference between predicted HbA1c and actual HbA1c decline less than 3.28 mmol/mol (0.3%). RESULTS: The decline in HbA1c resulting from treatment was different among the four diabetes clusters (pinteraction=0.004). In MOD, canagliflozin showed a robust glucose-lowering effect at week 52, compared with other drugs, with least-squares mean of HbA1c decline [95% CI] being 6.6 mmol/mol (4.1, 9.2) (0.61% [0.38, 0.84]) for sitagliptin, 7.1 mmol/mol (4.7, 9.5) (0.65% [0.43, 0.87]) for glimepiride, and 9.8 mmol/mol (9.0, 10.5) (0.90% [0.83, 0.96]) for canagliflozin. This superiority persisted until 104 weeks. The proportion of individuals who achieved HbA1c <53 mmol/mol (<7.0%) was highest in sitagliptin-treated individuals with MARD but was similar among drugs in individuals with MOD. The ML-A1C model and the cluster algorithm showed a similar concordance rate in predicting HbA1c lowering (31.5% vs 31.4%, p=0.996). Individuals were divided into high-risk and low-risk groups using ML-ACR model according to their predicted progression risk for albuminuria. The effect of canagliflozin vs placebo on albuminuria progression differed significantly between the high-risk (HR 0.67 [95% CI 0.57, 0.80]) and low-risk groups (HR 0.91 [0.75, 1.11]) (pinteraction=0.016). CONCLUSIONS/INTERPRETATION: Data-driven clusters of individuals with diabetes showed different responses to canagliflozin in glucose lowering but not renal outcome prevention. Canagliflozin reduced the risk of albumin progression in high-risk individuals identified by supervised machine learning. Further studies with larger sample sizes for external replication and subtype-specific clinical trials are necessary to determine the clinical utility of these stratification strategies in sodium-glucose cotransporter 2 inhibitor treatment. DATA AVAILABILITY: The application for the clinical trial data source is available on the YODA website ( http://yoda.yale.edu/ ).

Clinical features of NOTCH2NLC-related neuronal intranuclear inclusion disease.

BACKGROUND: Abnormal expanded GGC repeats within the NOTCH2HLC gene has been confirmed as the genetic mechanism for most Asian patients with neuronal intranuclear inclusion disease (NIID). This cross-sectional observational study aimed to characterise the clinical features of NOTCH2NLC-related NIID in China. METHODS: Patients with NOTCH2NLC-related NIID underwent an evaluation of clinical symptoms, a neuropsychological assessment, electrophysiological examination, MRI and skin biopsy. RESULTS: In the 247 patients with NOTCH2NLC-related NIID, 149 cases were sporadic, while 98 had a positive family history. The most common manifestations were paroxysmal symptoms (66.8%), autonomic dysfunction (64.0%), movement disorders (50.2%), cognitive impairment (49.4%) and muscle weakness (30.8%). Based on the initial presentation and main symptomology, NIID was divided into four subgroups: dementia dominant (n=94), movement disorder dominant (n=63), paroxysmal symptom dominant (n=61) and muscle weakness dominant (n=29). Clinical (42.7%) and subclinical (49.1%) peripheral neuropathies were common in all types. Typical diffusion-weighted imaging subcortical lace signs were more frequent in patients with dementia (93.9%) and paroxysmal symptoms types (94.9%) than in those with muscle weakness (50.0%) and movement disorders types (86.4%). GGC repeat sizes were negatively correlated with age of onset (r=-0.196, p<0.05), and in the muscle weakness-dominant type (median 155.00), the number of repeats was much higher than in the other three groups (p<0.05). In NIID pedigrees, significant genetic anticipation was observed (p<0.05) without repeat instability (p=0.454) during transmission. CONCLUSIONS: NIID is not rare; however, it is usually misdiagnosed as other diseases. Our results help to extend the known clinical spectrum of NOTCH2NLC-related NIID.

A variation in SORBS1 is associated with type 2 diabetes and high-density lipoprotein cholesterol in Chinese population.

AIM: Sorbin and SH3-domain-containing-1 (SORBS1) play important roles in insulin signalling and cytoskeleton regulation. Variants of the SORBS1 gene have been inconsistently reported to be associated with type 2 diabetes or diabetic kidney disease (DKD). METHODS: Two independent case-control studies based on two randomized sampling cohorts (cohort 1, n = 3345; cohort 2, n = 2282) were used to confirm the association between rs2281939 of SORBS1 and impaired glucose regulation (IGR). An additional hospital-based cohort (cohort 3, n = 2135) and cohort 1 were used to investigate the association between rs2281939 and DKD. The phenotype of rare variants of SORBS1 was explored in 453 patients with early onset type 2 diabetes (diagnosed before 40 years of age, EOD). RESULTS: The G allele was associated with type 2 diabetes (additive model: OR = 1.25, 95% CI [1.03-1.52], p = 0.022) in cohort 1, and IGR in cohort 2 (additive model: OR = 1.22, 95% CI [1.05-1.43], p = 0.01). We found that the G allele was also associated with HDL-c levels in women in both cohort 1 (p = 0.03) and 2 (p = 0.029) in the dominant model. The rare variant carriers also had lower HDL-c and LDL-c levels than non-carriers in patients with EOD. No association between rs2281939 or rare variants and DKD was observed. CONCLUSIONS: The variants in the SORBS1 gene were associated with IGR and HDL-c levels but not with DKD in the Chinese Han population.

Myasthenia gravis coexisting with HINT1-related motor axonal neuropathy without neuromyotonia: a case report.

BACKGROUND: HINT1 mutations cause an autosomal recessive axonal neuropathy with neuromyotonia. This is a first case report of coexistence of myasthenia gravis (MG) and HINT1-related motor axonal neuropathy without neuromyotonia. CASE PRESENTATION: A 32-year-old woman presented with recurrent ptosis for 8 years, diplopia for 2 years and limb weakness for 1 year and a half. Neostigmine test, elevated AChR antibody level and positive repetitive nerve stimulation supported the diagnosis of MG. Electroneurography (ENG) and electromyography (EMG) examinations revealed a motor axonal neuropathy without neuromyotonic or myokymic discharges. Next-generation sequencing and Sanger sequencing were performed to identify the gene responsible for suspected hereditary neuropathy. Genetic testing for a HINT1 mutation was performed and revealed a homozygous mutation at c.278G>T (p. G93V). The patient was treated with pyridostigmine, oral prednisolone and azathioprine. Her ptosis and diplopia have significantly improved at 6-month follow-up. CONCLUSIONS: Concurrence of MG and hereditary motor axonal neuropathy without neuromyotonia is quite rare. Detection of ptosis with or without ophthalmoplegia, distribution of limb weakness, and reflex can help in recognizing the combination of MG and peripheral neuropathy. Early diagnosis is important for initial treatment and prognosis. The novel homozygous variant c.278G>T(p.G93V) contributes to the pathogenic variants spectrum of the HINT1 gene.

Effectiveness and safety of basal insulin therapy in type 2 diabetes mellitus patients with or without metformin observed in a national cohort in China.

BACKGROUND: Though many randomized control trials had examined the effectiveness and safety of taking insulin therapy with or without metformin, there are limited real-world data, especially among Chinese type 2 diabetes patients initiating basal insulin (BI) with uncontrolled hyperglycemia by oral agents. This study was designed to assess the effectiveness and safety of BI therapy combined with or without metformin in a real-world national cohort study. METHODS: Patients with type 2 diabetes mellitus who initiated BI treatment due to uncontrolled hyperglycemia (HbA1c≥7 %) by oral antidiabetic drugs (OADs) were recruited in Chinese real-world settings between 2011 and 2013. A total of 12,358 patients initiated BI without bolus insulin and completed a 6-month follow-up were selected as the study population and divided into BI with metformin or BI without metformin group based on whether metformin was simultaneously prescribed or not at baseline. Propensity score adjustment was used to balance baseline covariates between two groups. A sub-analysis was also conducted among 8,086 patients who kept baseline treatment regimen during the follow-up. Outcomes were HbA1c, hypoglycemia, weight gain and insulin dose in two groups. RESULTS: 53.6 % (6,621 out of 12,358) patients initiated BI therapy concomitant with metformin. After propensity score adjustment, multivariate regression analysis controlled with number of OADs, total insulin dose, physical activity and diet consumption showed that BI with metformin group had a slightly higher control rate of HbA1c <7.0 % (39.9 % vs. 36.4 %, P = 0.0011) at 6-month follow-up, and lower dose increment from baseline to 6-month (0.0064 vs. 0.0068 U/day/kg, P = 0.0035). The sub-analysis with patients remained at same BI therapy further showed that BI with metformin group had higher HbA1c control rate (47.9 % vs. 41.9 %, P = 0.0001), less weight gain (-0.12 vs. 0.15 kg P = 0.0013), and lower dose increment during 6-month follow-up (0.0033 vs. 0.0037 U/day/kg, P = 0.0073) when compared with BI without metformin group. CONCLUSIONS: In alliance with current guidelines, the real-world findings also support the insulin initiation together with metformin. Continuous patients' education and clinicians training are needed to improve the use of metformin when initiating BI treatment.

A Bioinformatics Perspective on the Dysregulation of Ferroptosis and Ferroptosis-related Immune Cell Infiltration in Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent dementia worldwide, but its pathophysiology and molecular events remain unknown. Herein, we first analyzed the differential expression pattern of patients' AD hippocampus through gene expression array data from the GEO database. Notch2nl, TGFB1I1, and LTF were up-regulated in AD patients, while ARPC1A, CHGB, and MPV17 down-regulated. Second, dysregulation of ferroptosis related genes was demonstrated from our data: PCBP2 and FTL significantly up-significant in AD hippocampus, while VDAC2, LPCAT3, GSS, ACSL4, and ACSL6 significantly down-regulated. The protein-protein interactions (PPI) network revealed that FTL was involved in iron metabolism and utilization, while ACSL4 and ACSL6 were involved in a polyunsaturated fatty acids metabolism network. Gene correlation analysis on differential expressed genes (DEGs) indicated that ferroptosis regulates a series of biological processes and pathways related to AD pathogenesis. Third, ferroptosis-related DEGs regulated the immune cell infiltration pattern in the AD hippocampus, characterized by decreased memory B cells, increased memory resting CD4+ T cells, memory activated CD4+ T cells, and resting NK cells. The altered expression of ferroptosis-related DEGs affected the infiltration of specific immune cell types. The model constructed by the seven ferroptosis-related differential genes may accurately predict the outcome of AD occurrence. Finally, qPCR validation on these ferroptosis-related DEGs in APPswe/PSEN1dE9 mice confirmed the dysregulated expression of Pcbp2, FTL, GSS, and ACSL4 in the AD hippocampus and forebrain. In conclusion, our results supported the conception that the AD brain revealed dysregulated ferroptosis and immune cell infiltration.

[Molecular mechanism of Dalbergia cochinchinensis heartwood in regulation of energy metabolism to treat myocardial ischemia based on network pharmacology and experimental validation].

Dalbergia cochinchinensis(DC) is chemically similar to the valuable and scarce Chinese herb Dalbergiae Odoriferae Lignum, and both of them belong to the Dalbergia Leguminosae. DC is used for treating cardiovascular diseases and cancer. However, its potent active ingredient groups and molecular mechanisms in anti-myocardial ischemia are not fully clarified. In this study, the active ingredient groups, targets, and signaling pathways of DC heartwood for the treatment of myocardial ischemia were screened out based on network pharmacology and molecular docking technology, and the effects were verified by the rat model of acute myocardial ischemia induced by isoprenaline(ISO). The molecular mechanism of DC heartwood was elucidated based on the target of multi-ingredient and multi-target pathways. The crossing targets of DC heartwood for the treatment of myocardial ischemia were identified through the screening of active ingredients in DC heartwood and the prediction of targets. The Kyoto Encyclopedia of Genomes(KEGG) pathway enrichment and Gene Ontology(GO) functional annotation were performed. AutoDock was used to bind the active ingredient groups to the pathway targets. Finally, the molecular mechanism of myocardial ischemia treatment by DC heartwood extracts in the treatment of myocardial ischemia was revealed through the rat model of ISO-induced acute myocardial ischemia by performing electrocardiogram(ECG), hemodynamic, cardiac enzymes, hematoxylin-eosin(HE) staining, high-energy phosphate compounds, reverse transcription polymerase chain reaction(RT-PCR), and Western blot pharmacodynamic experiments, based on the multi-ingredient and multi-target action of active ingredient groups and pathway targets. The network pharmacology showed that the 18 ingredients of DC heartwood corresponded to 510 targets, 629 myocardial ischemia-related targets, and 101 cross-targets. GO and KEGG enrichment analyses showed that DC heartwood was involved in the hypoxic response, vasoconstriction, and nitric oxide biosynthesis, and had effects on the molecular functions of hemoglobin binding, protein binding, and adenosine triphosphate(ATP) binding. It regulated the signaling pathways such as hypoxia-inducible factor 1(HIF-1), vascular endothelial growth factor(VEGF), and phosphatidylinositol-3-kinase/protein kinase B(PI3 K/AKT) to act on myocardial ischemia. Experimental studies showed that DC heartwood slowed down the heart rate and ST segment change(ΔST), and increased systolic blood pressure(SBP), diastolic blood pressure(DBP), and mean arterial pressure(MBP) in rats with ISO-induced acute myocardial ischemia. It also reduced plasma lactate dehydrogenase(LDH), creatine kinase isoenzyme MB(CK-MB), and glutamate transaminase(AST) levels, relieved myocardial fiber disorders and inflammatory cell infiltration, and increased ATP and cellular energy(EC) levels. DC heartwood increased the mRNA expressions of calmodulin-dependent protein kinase kinase(CAMKK) in the myocardial tissue, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3(PFKFB3), mammalian target of rapamycin(mTOR), PI3 K, VEGF, endothelial nitric oxide synthase(eNOS), HIF-1α in the myocardial tissue. It decreased the mRNA expression of pyruvate dehydrogenase(PDH), and increased the protein expressions of PFKFB3, VEGFA, and eNOS. Molecular docking showed that liquiritigenin, stigmasterol, isodalbergin, latifolin, 4-methoxydalbergione, dibutyl terephthalate, 2,4-dihydroxy-5-methoxybenzophenone in DC heartwood produced bio-binding activities with epidermal growth factor receptor(EGFR), HIF-1α, CAMKK, PI3 K, mTOR, and PDH, respectively. Therefore, the active ingredient groups of DC heartwood act on the HIF-1 signaling pathway, regulate cardiomyocyte energy metabolism, and increase ATP energy charge in a multi-ingredient and multi-target manner, improving cardiac function and histopathological changes to protect rats with acute myocardial ischemia induced by ISO.

Protective effect of glycyrrhizin on osteoarthritis cartilage degeneration and inflammation response in a rat model.

This study was conducted to investigate the protective effects of glycyrrhizin on a rat model of osteoarthritis and elucidate the underlying mechanism. Rat osteoarthritis was established by using medial meniscectomy (MMx) and an anterior cruciate ligament transaction (ACLT). Glycyrrhizin (2, 4, and 10 mg/kg) was administered by intra-articular knee injection for 12 weeks. Incapacitance test was performed to determine mechanical hyperalgesia. Enzyme-linked immunosorbent assay (ELISA) was performed to measure cartilage degradation and inflammation-related markers. Quantitative reverse transcription PCR (RT-qPCR) and Western blot were performed to determine the mRNA and protein levels of genes, respectively. The results demonstrated that treatment with glycyrrhizin ameliorated mechanical hyperalgesia and bilateral joints oedema in a rat model of osteoarthritis. Treatment with 10 mg/kg glycyrrhizin also suppressed serum enzymes including matrix metalloproteinase (MMP)-1, MMP-3, prostaglandin E2, and C-telopeptide of type II collagen (CTX-II). In addition to inhibition of cartilage matrix catabolic related markers, treatment with glycyrrhizin also decreased the levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, and iNOS in serum and cartilage. The underlying mechanism study demonstrated that treatment with glycyrrhizin inhibited HMGB1 and the TLR4/NF-κB signaling pathway. In summary, treatment with glycyrrhizin ameliorated cartilage degeneration and inflammation in osteoarthritis rats by the regulation of HMGB1 and the TLR4/NF-κB signaling pathway.

Anemoside A3 activates TLR4-dependent M1-phenotype macrophage polarization to represses breast tumor growth and angiogenesis.

The polarization of macrophages has been previously demonstrated to be closely related to immune and inflammatory processes in the tumorigenesis and progression of breast cancer. In the present study, Anemoside A3 (A3), an active compound from Pulsatilla saponins, was screened out and polarized M0 macrophages into the classically activated macrophages (M1-phenotype). We found that A3 is an activator of TLR4/NF-κB/MAPK signaling pathway. A3 increased the expression of CD86+ (a marker of M1 macrophage) in M0 macrophage, and increased the typical M1 macrophage pro-inflammatory cytokines TNF-α, and IL-12 expression in a TLR4-dependent manner. A macrophage-cancer cell co-culture system was established to evaluate whether A3 can could switch tumor-associated macrophages (TAMs) to the M1-phenotype. In the co-culture system, A3 increased the expression of IL-12 in macrophages, whereby suppressing MCF-7 breast cancer cell line proliferation and VEGF-mediated angiogenesis. Moreover, A3 induced M1 macrophage polarization in the 4 T1 murine breast cancer model and effectively inhibited tumor growth and tumor angiogenesis. Collectively, these findings indicated that A3 induced M1 macrophages polarization to repress breast tumorigenesis via targeting the TLR4/NF-κB/MAPK signaling pathway. This study provides a rationale for utilizing traditional Chinese medicine extracts in the immunotherapy of breast cancer.