BAPCP: A comprehensive and user-friendly web tool for identifying biomarkers from protein microarray technologies.

BACKGROUND AND OBJECTIVE: Proteome microarrays are one of the popular high-throughput screening methods for large-scale investigation of protein interactions in cells. These interactions can be measured on protein chips when coupled with fluorescence-labeled probes, helping indicate potential biomarkers or discover drugs. Several computational tools were developed to help analyze the protein chip results. However, existing tools fail to provide a user-friendly interface for biologists and present only one or two data analysis methods suitable for limited experimental designs, restricting the use cases. METHODS: In order to facilitate the biomarker examination using protein chips, we implemented a user-friendly and comprehensive web tool called BAPCP (Biomarker Analysis tool for Protein Chip Platforms) in this research to deal with diverse chip data distributions. RESULTS: BAPCP is well integrated with standard chip result files and includes 7 data normalization methods and 7 custom-designed quality control/differential analysis filters for biomarker extraction among experiment groups. Moreover, it can handle cost-efficient chip designs that repeat several blocks/samples within one single slide. Using experiments of the human coronavirus (HCoV) protein microarray and the E. coli proteome chip that helps study the immune response of Kawasaki disease as examples, we demonstrated that BAPCP can accelerate the time-consuming week-long manual biomarker identification process to merely 3 min. CONCLUSIONS: The developed BAPCP tool provides substantial analysis support for protein interaction studies and conforms to the necessity of expanding computer usage and exchanging information in bioscience and medicine. The web service of BAPCP is available at https://cosbi.ee.ncku.edu.tw/BAPCP/.

Prebiotic Formation of Peptides Through Bubbling and Arc Plasma.

The abiotic synthesis of peptides, widely regarded as one of the key chemical reactions on the prebiotic Earth, is thermodynamically constrained in solution. Herein, a simulation of the lightning phenomenon on the sea surface using bubble bursting and arc plasma under ambient conditions enables dipeptide formation of six amino acids with conversion ratios ranging from 2.6 % to 25.5 %. Additionally, we observed the formation of biologically active tripeptides and investigated the stereoselectivity of the dipeptide formation reaction. By utilizing a mixture of 20 amino acids in the reaction, 102 possible dipeptides were generated. These results establish experimental constructions to mimic achievable prebiotic conditions and provide a credible pathway for endogenous biopolymer synthesis on prebiotic Earth.

Ortho C-H Bond Activations in an Atmospheric Microwave Plasma Ion Source.

C-H bond ortho-substitution reaction has always been a significant and challenging topic in organic chemistry. We proposed a synthesis method based on microwave plasma torches. High-resolution mass spectrometry was used to monitor rapid reaction products. 2-Alkylbenzimidazole can be formed through the reaction of phenylnitrenium ion and nitriles on a millisecond scale. This reaction can achieve the one-step formation of benzimidazoles from benzene ring single-substituted compounds without the addition of external oxidants or catalysts. A similar C-H bond activation reaction can be accomplished with ketones. Meanwhile, the microwave plasma reactor was modified, and the resulting 2-methylbenzimidazole was successfully collected, indicating the device has good application potential in organic reactions such as C-H bond activation reaction.

Influencing Factors on the Efficacy of Aerobic Exercise Therapy in Depression: A Single-Centre Cross-Sectional Study.

BACKGROUND: Aerobic exercise therapy can improve brain function and promote positive emotions in patients with depression. This study aims to improve the efficacy of aerobic exercise therapy in depression by investigating the influencing factors therein. METHODS: This study conducted a single-centre cross-sectional analysis of clinical data from 211 depression patients who received aerobic exercise therapy at Heze Mudan People's Hospital from May 2021 to May 2023. After excluding 10 patients who did not meet the inclusion criteria, the final cohort included 201 patients. The clinical data of patients with effective treatment and patients with ineffective treatment were collected, such as socio-demographic characteristics, disease status, depression degree, treatment compliance, and social support. Binary Logistic regression was used to analyze the factors affecting the clinical efficacy of aerobic exercise therapy in patients. RESULTS: Among the 201 patients, the effective and ineffective rates of aerobic exercise therapy were 80.10% (161/201) and 19.90% (40/201), respectively. When analysed as groups, no significant differences were observed between the effective group (EG) and ineffective group (IG) regarding gender, body mass index, course of depression, age of first onset, mental symptoms, years of education, personal income, or marital status (p > 0.05). However, the EG exhibited a reduced probability of family history of mental illness, smoking history, and severity of depression (p < 0.05), along with higher treatment compliance and Social Support Rating Scale (SSRS) scores compared to the IG (p < 0.05). Binary logistic regression analysis revealed that smoking history, family history of mental illness, treatment compliance, depression, and SSRS score were the primary influencing factors on the efficacy of aerobic exercise therapy (p < 0.05). CONCLUSION: Smoking history, family history of mental illness, treatment compliance, severity of depression, and social support are the primary influencing factors on the efficacy of aerobic exercise therapy in patients with depression. These factors should be emphasized during follow-up treatment to optimize the clinical treatment effect.

Identification and Characterization of Chemical Constituents from Ammopiptanthus nanus Stem and Their Metabolites in Rats by UHPLC-Q-TOF-MS/MS.

Ammopiptanthus nanus as a Kirgiz medicine is widely used for the treatment of frostbite and chronic rheumatoid arthritis. However, due to a lack of systematic research on the chemical components of A. nanus and their metabolites, the bioactive components in it remain unclear. Herein, a reliable strategy based on UHPLC-Q-TOF-MS/MS was established to comprehensively analyze the chemical components and their metabolites in vivo. In total, 59 compounds were identified from A. nanus stem extract, among which 14 isoflavones, 10 isoprenylated isoflavones, 4 polyhydroxy flavonoids, 9 alkaloids and 1 polyol were characterized for the first time. After oral administration of A. nanus stem extract, 30 prototype constituents and 28 metabolites (12 phase I and 16 phase II metabolites) were speculated on and identified in rat serum, urine and feces. Furthermore, the metabolic pathways of the chemical components were systematically analyzed and proposed. In conclusion, the chemical components from A. nanus stem and their metabolites in vivo were first studied, which may provide useful chemical information for further study on the effective material basis and pharmacological mechanism of A. nanus.

Deep Learning-based Diagnosis and Localization of Pneumothorax on Portable Supine Chest X-ray in Intensive and Emergency Medicine: A Retrospective Study.

PURPOSE: To develop two deep learning-based systems for diagnosing and localizing pneumothorax on portable supine chest X-rays (SCXRs). METHODS: For this retrospective study, images meeting the following inclusion criteria were included: (1) patient age ≥ 20 years; (2) portable SCXR; (3) imaging obtained in the emergency department or intensive care unit. Included images were temporally split into training (1571 images, between January 2015 and December 2019) and testing (1071 images, between January 2020 to December 2020) datasets. All images were annotated using pixel-level labels. Object detection and image segmentation were adopted to develop separate systems. For the detection-based system, EfficientNet-B2, DneseNet-121, and Inception-v3 were the architecture for the classification model; Deformable DETR, TOOD, and VFNet were the architecture for the localization model. Both classification and localization models of the segmentation-based system shared the UNet architecture. RESULTS: In diagnosing pneumothorax, performance was excellent for both detection-based (Area under receiver operating characteristics curve [AUC]: 0.940, 95% confidence interval [CI]: 0.907-0.967) and segmentation-based (AUC: 0.979, 95% CI: 0.963-0.991) systems. For images with both predicted and ground-truth pneumothorax, lesion localization was highly accurate (detection-based Dice coefficient: 0.758, 95% CI: 0.707-0.806; segmentation-based Dice coefficient: 0.681, 95% CI: 0.642-0.721). The performance of the two deep learning-based systems declined as pneumothorax size diminished. Nonetheless, both systems were similar or better than human readers in diagnosis or localization performance across all sizes of pneumothorax. CONCLUSIONS: Both deep learning-based systems excelled when tested in a temporally different dataset with differing patient or image characteristics, showing favourable potential for external generalizability.

Progression of the PI3K/Akt signaling pathway in chronic obstructive pulmonary disease.

Chronic Obstructive Pulmonary Disease (COPD) is a chronic respiratory disease characterized by a slow progression and caused by the inhalation of harmful particulate matter. Cigarette smoke and air pollutants are the primary contributing factors. Currently, the pathogenesis of COPD remains incompletely understood. The PI3K/Akt signaling pathway has recently emerged as a critical regulator of inflammation and oxidative stress response in COPD, playing a pivotal role in the disease's progression and treatment. This paper reviews the association between the PI3K/Akt pathway and COPD, examines effective PI3K/Akt inhibitors and novel anti-COPD agents, aiming to identify new therapeutic targets for clinical intervention in this disease.

Radical-Induced Dissociation for Oligonucleotide Sequencing by TiO2/ZnAl-Layered Double Oxide-Assisted Laser Desorption/Ionization Mass Spectrometry.

De novo sequencing of oligonucleotides remains challenging, especially for oligonucleotides with post-transcriptional or synthetic modifications. Mass spectrometry (MS) sequencing can reliably detect and locate all of the modification sites in oligonucleotides via m/z variance. However, current MS-based sequencing methods exhibit complex spectra and low ion abundance and usually require coupled instrumentation. Herein, we demonstrate a method of oligonucleotide sequencing using TiO2/ZnAl-layered double oxide (LDO)-assisted laser desorption/ionization (LDI)-MS based on radical-induced dissociation (RID). ·CH2OH radicals can be produced on the surface of a TiO2/ZnAl-LDO matrix via ultraviolet light, inducing an attack on the active site of the oligonucleotide phosphate skeleton to create typical "a-, a-B-, c·-, d-, w-, and y"-type fragments. Compared with the spectra obtained via collision-based methods, such as collision-induced dissociation and higher-energy collisional dissociation, the LDI-MS spectra based on RID exhibit single-charged signals, fewer types of fragments, and a lower proportion of unknown noise peaks. We demonstrate full sequence coverage for a 6-mer 2'-O-methyl-modified oligonucleotide and a 21-mer small interfering RNA and show that RID can sequence oligonucleotides with modifications. Importantly, the mechanism responsible for the RID of the oligonucleotide phosphate skeleton was investigated through offline experiments, demonstrating consistent results with density functional theory calculations.

Patchouli alcohol against renal fibrosis of spontaneously hypertensive rats via Ras/Raf-1/ERK1/2 signalling pathway.

OBJECTIVES: The present study was designed to obverse the protection of patchouli alcohol (PA) ameliorates hypertensive nephropathy in spontaneously hypertensive rats (SHR) and reveals potential mechanism. METHODS: Briefly, the adult spontaneously hypertensive rats (SHR) or Wistar-Kyoto (WKY) rats (half male and half female) were intragastric gavaged or not with PA (80, 40 and 20 mg/kg) for 8 weeks. Body weight, blood pressure (BP), renal weight, renal function and renal morphology were measured. Further, western blotting and immunohistochemical analysis were used to study the underlying mechanism. KEY FINDINGS: Compared with the WKY group, plasmatic levels of renin, angiotensin II (Ang-II), transforming growth factor beta 1(TGF-ß1), plasminogen activator inhibitor-1(PAI-1), creatinine (Cr), blood urea nitrogen (BUN), renal index, mRNA levels of ERK1/2 and α-SMA were significantly increased in SHR. Histology results showed that renal tubular injury and tubulointerstitial fibrosis occurred in SHR. After administration, SBP of captopril group decreased at each week after administration, especially at 3, 5, 6 7 and 8 weeks (P < 0.05 or P < 0.01). There is no significant effect was assessed in the olive oil group. Decreased plasma Cr, Renin, Ang-II, TGF-ß1, PAI-1, SCFAs and Renin, TGF-ß1, PAI-1 in renal tissues were observed significantly in captopril (P <0.05 or P < 0.01). Plasma BUN, Ang-II, TGF-ß1 and PAI-1 in renal tissues decreased in the olive oil group significantly (P <0.05 or P < 0.01). PA (80, 40 and 20 mg/kg) lowered BP and plasmatic levels of Renin, Ang-II, TGF-ß1 and PAI-1. Treatment with PA (40, 20 mg/kg) decreased levels of Cr, BUN and suppressed of activation of pro-fibrosis cytokines including TGF-ß1 in kidney. There is no ameliorative change in the olive oil group and the captopril group (P > 0.05) while PA treatment alleviated renal tubular injury and produced dramatic collagen fibre area reductions in mesangial membrane, basement membrane, and renal interstitium obviously (P < 0.05 or P < 0.01). Treatment of SHR with PA-inhibited MFB activation and downregulated mRNA of α-SMA. Treatment with PA suppressed excessive production of the extracellular matrix (ECM) via decreasing Col I, III and FN, downregulating mRNA of tissue inhibitor of TIMP-1 along with upregulating mRNA of MMP-9. The expression of Col III and MMP-9 mRNA-reduced in the captopril group (P < 0.05). In addition, the expression of ERK1/2 and pERK1/2 also reduced in the captopril group significantly (P < 0.05 or P < 0.01). Treatment with PA (20 mg/kg) downregulated proteins expression of Raf-1, ERK1/2 and pERK1/2 and mRNA expression of Ras, Raf-1 and ERK1/2. CONCLUSIONS: Overall, PA restored normal BP, alleviated renal dysfunction and renal fibrosis, possibly by suppressing Ang II and TGF-ß1-mediated Ras/Raf-1/ERK1/2 signalling pathway.

Serum TSH Levels are Associated with Hyperactivity Behaviors in Children with Attention Deficit/Hyperactivity Disorder.

Objective: Previous studies indicated that maternal thyroid dysfunction increase the offspring's risk for attention deficit/hyperactivity disorder (ADHD). However, the relationship between thyroid function and symptoms in children with ADHD remains unclear. Methods: A total of 49 children with ADHD were enrolled. The Conners 3 scale was used to estimate the symptoms associated with ADHD. Correlation between thyroid hormones and the scores of the Conners 3 scale was evaluated by Pearson correlation analysis. Then, ADHD children were divided into two groups according to the hyperactivity index (HI) of the Conners 3 scale: ADHD children with hyperactivity behaviors (HB) (HI > 1.5) and ADHD children without HB (HI < 1.5). The demographic characteristics, thyroid hormones, and routine laboratory parameters between the two groups were collected. To distinguish HI-related factors, a univariate analysis and a binary logistic regression predictive model were used. The discriminative ability of thyroid stimulating hormone (TSH) in predicting ADHD children with HB from ADHD children without HB was investigated using the receiver operating characteristic (ROC) curve method. Results: The levels of TSH were positively correlated to the scores of the Conners 3 scale (r = 0.338, P = 0.033) and HI (r = 0.371, P = 0.019). Moreover, the levels of TSH, serum ferritin, and lactic acid were significantly increased in ADHD children with HB compared to ADHD children without HB (all P < 0.05). Furthermore, the results of binary logistic regression found that TSH (OR 2.243 (CL 1.052-4.783)) and lactic acid (OR 1.018 (CI 1.003-1.032)) were independently associated with HI. Additionally, ROC analysis indicated the potential diagnostic value of TSH in discriminating ADHD children with HB from ADHD children without HB with an AUC of 0.684. Conclusion: These results suggested that the serum TSH levels may be related to the HB in children with ADHD.

Treatment of Irritable Bowel Syndrome by Chinese Medicine: A Review.

Irritable bowel syndrome (IBS) is a common refractory disease. Chinese medicine (CM) has remarkable efficacy and advantages on the treatment of IBS. This review summarized the articles focusing on the treatment of IBS with CM to sum up the latest treatment methods for IBS and the underlying mechanisms. Literature analysis showed that prescriptions, acupuncture, and moxibustion are the primary methods of CM treatment for IBS. The potential mechanism centers on the regulation of the enteric nervous system, the alleviation of visceral hypersensitivity, the stability of intestinal flora, and the regulation of the immune system.

Innovative Hybrid-Alignment Annotation Method for Bioinformatics Identification and Functional Verification of a Novel Nitric Oxide Synthase in Trichomonas vaginalis.

Both the annotation and identification of genes in pathogenic parasites are still challenging. Although, as a survival factor, nitric oxide (NO) has been proven to be synthesized in Trichomonas vaginalis (TV), nitric oxide synthase (NOS) has not yet been annotated in the TV genome. We developed a witness-to-suspect strategy to identify incorrectly annotated genes in TV via the Smith-Waterman and Needleman-Wunsch algorithms through in-depth and repeated alignment of whole coding sequences of TV against thousands of sequences of known proteins from other organisms. A novel NOS of TV (TV NOS), which was annotated as hydrogenase in the NCBI database, was successfully identified; this TV NOS had a high witness-to-suspect ratio and contained all the NOS cofactor-binding motifs (NADPH, tetrahydrobiopterin (BH4), heme and flavin adenine dinucleotide (FAD) motifs). To confirm this identification, we performed in silico modeling of the protein structure and cofactor docking, cloned the gene, expressed and purified the protein, performed mass spectrometry analysis, and ultimately performed an assay to measure enzymatic activity. Our data showed that although the predicted structure of the TV NOS protein was not similar to the structure of NOSs of other species, all cofactor-binding motifs could interact with their ligands with high affinities. We clearly showed that the purified protein had high enzymatic activity for generating NO in vitro. This study provides an innovative approach to identify incorrectly annotated genes in TV and highlights a novel NOS that might serve as a virulence factor of TV.

Pharmacological Activities of Safflower Yellow and Its Clinical Applications.

Background: Safflower is an annual herb used in traditional Chinese herbal medicine. It consists of the dried flowers of the Compositae plant safflower. It is found in the central inland areas of Asia and is widely cultivated throughout the country. Its resistance to cold weather and droughts and its tolerance and adaptability to salts and alkalis are strong. Safflower has the effect of activating blood circulation, dispersing blood stasis, and relieving pain. A natural pigment named safflower yellow (SY) can be extracted from safflower petals. Chemically, SY is a water-soluble flavonoid and the main active ingredient of safflower. The main chemical constituents, pharmacological properties, and clinical applications of SY are reviewed in this paper, thereby providing a reference for the use of safflower in preventing and treating human diseases. Methods: The literature published in recent years was reviewed, and the main chemical components of SY were identified based on chemical formula and structure. The pharmacological properties of hydroxysafflor yellow A (HSYA), SYA, SYB, and anhydrosafflor yellow B (AHSYB) were reviewed. Results: The main chemical constituents of SY included HSYA, SYA, SYB, and AHSYB. These ingredients have a wide range of pharmacological activities. SY has protective effects on the heart, kidneys, liver, nerves, lungs, and brain. Moreover, its effects include, but are not limited to, improving cardiovascular and cerebrovascular diseases, abirritation, regulating lipids, and treating cancer and diabetic complications. HSYA is widely recognised as an effective ingredient to treat cardiovascular and cerebrovascular diseases. Conclusion: SY has a wide range of pharmacological activities, among which improving cardiovascular and cerebrovascular diseases are the most significant.

Lewis-Acid-Catalyzed Selective Friedel-Crafts Reaction or Annulation between Anilines and Glyoxylates.

A Lewis-acid-catalyzed selective reaction between anilines and glyoxylates was developed for synthesis of diarylmethanes or oxoimidazolidines. Under the catalysis of AgOTf, a series of anilines-based diarylmethanes, including primary, secondary, and tertiary anilines, were obtained in moderate to good yields. Moreover, stereoselective oxoimidazolidines were performed with the catalysis of Cu(OAc)2·H2O.

KDmarkers: A biomarker database for investigating epigenetic methylation and gene expression levels in Kawasaki disease.

Kawasaki disease (KD) is a form of acute systemic vasculitis that primarily affects children and has become the most common cause of acquired heart disease. While the etiopathogenesis of KD remains unknown, the diagnostic criteria of KD have been well established. Nevertheless, the diagnosis of KD is currently based on subjective clinical symptoms, and no molecular biomarker is yet available. We have previously performed and combined methylation array (Illumina HumanMethylation450 BeadChip) and transcriptome array (Affymetrix GeneChip Human Transcriptome Array 2.0) to identify genes that are differentially methylated/expressed in KD patients compared with control subjects. We have found that decreased methylation levels combined with elevated gene expression can indicate genes (e.g., toll-like receptors and CD177) involved in the disease mechanisms of KD. In this study, we constructed a database called KDmarkers to allow researchers to access these valuable potential KD biomarkers identified via methylation array and transcriptome array. KDmarkers provides three search modes. First, users can search genes differentially methylated and/or differentially expressed in KD patients compared with control subjects. Second, users can check the KD patient groups in which a given gene is differentially methylated and/or differentially expressed. Third, users can explore the DNA methylation levels and gene expression levels in all samples (KD patients and controls) for a particular gene of interest. We further demonstrated that the results in KDmarkers are strongly associated with KD immune responses. All analysis results can be downloaded for downstream experimental designs. KDmarkers is available online at https://cosbi.ee.ncku.edu.tw/KDmarkers/.

Protective effect of the combination of essential oil from patchouli and tangerine peel against gastric ulcer in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Essential oil (EO) is the main extract of patchouli and tangerine peel with antiinflammatory, antiulcer, and other functions. However, the efficacy and mechanism of the combination of EO from patchouli and tangerine peel against gastric ulcer (GU) are unclear. AIM OF THE STUDY: This study aims to reveal the protective effect of the combination of EO from patchouli and tangerine peel against GU in rats, as well as explore the optimal ratio and possible mechanism of EO in GU treatment. MATERIALS AND METHODS: The GU model is executed via water immersion and restraint stress. The repair effect of EO in different proportions on gastric mucosa injury and the effects on serum gastrin (GAS), pepsinogen C (PGC), prostaglandin E2 (PGE2), and 5-hydroxytryptamine in GU rats were observed. The optimal ratio obtained was used in the second part to set different dose groups for further experiment. The effects of the different EO doses on gastric mucosal ulcer formation and gastric acid secretion were evaluated. The morphology of chief and parietal cells were observed via transmission electron microscopy. The contents of GAS, PGC, substance P (SP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), cholecystokinin (CCK), PGE2, and motilin (MTL) in serum in different groups were detected via enzyme-linked immunosorbent assay. Expressions of epidermal growth factor (EGF) and trefoil factor 2 (TFF2) protein in gastric tissues were detected via immunohistochemistry, and expressions of c-Jun N-terminal kinase (JNK), P53, Bcl-2-associated X protein (Bax), and Caspase-3 protein in gastric tissues were detected via western blotting. RESULTS: The EO from patchouli and tangerine peel at 1:2 ratio of compatibility significantly improved gastric mucosal injury, decreased serum GAS and PGC contents, and increased the PGE2 level in serum (p < 0.05). The mixture of EO from patchouli and tangerine peel (Mix-EO) can reduce the formation of gastric mucosal ulcers, reduce gastric mucosal injury, improve the expansion of the endoplasmic reticulum of the chief cells, repair mitochondrial damage, and inhibit the secretion of gastric acid by parietal cells. Mix-EO at 300 mg/kg can reduce the expression of serum GAS, PGC, SP, CCK, and cAMP/cGMP (p < 0.05 or 0.01); increase the expression of EGF and TFF2 protein in gastric tissues (p < 0.01); and inhibit the expression of JNK, p53, Bax, and Caspase-3 proteins (p < 0.01). CONCLUSION: The combination of EO from patchouli and tangerine peel can repair the gastric mucosal damage in GU rats and prevent the occurrence of ulcers by inhibiting the secretion of gastric acid, enhancing the defensive ability of gastric mucosa, and suppressing the apoptosis of gastric epithelial cells. Moreover, the optimal compatible ratio of patchouli and tangerine peel is 1:2.

Incorporating Post-Cessation Weight-Control Coaching into Smoking Cessation Therapy to Reduce Type 2 Diabetes Risk.

Post-cessation weight gain (PCWG) facilitates short-term type 2 diabetes (T2D) risk in prediabetic smokers in the absence of complementary measures. In this shared decision-making-based non-randomized controlled trial, prediabetic smokers joined the Fight Tobacco and Stay Fit (FIT2) program or received usual care. The 16-week FIT2 program combined smoking cessation therapy with individualized coaching in diet and physical activity strategies for PCWG restriction (NCT01926041 at ClinicalTrials.gov). During a mean follow-up period of 1316 days, 217 participants (36.8%) developed T2D, and 68 (11.5%) regressed to normoglycemia. In the intention-to-treat analysis (n = 589), the FIT2 program was associated with a reduced T2D risk (HR, 0.58; 95% CI, 0.40-0.84) and a higher probability of regression to normoglycemia (HR, 1.91; 95% CI, 1.04-3.53) compared with usual care. The post-program quitters were at lower T2D risk (HR, 0.63; 95% CI, 0.44-0.92) and were more likely to regress to normoglycemia (HR, 1.83; 95% CI, 1.01-3.30) compared with the controls in the time-varying analysis (n = 532). We demonstrated that the FIT2 program was negatively associated with long-term T2D risk and positively associated with the probability of regression to normoglycemia compared with usual care. To prevent T2D development, we recommend simultaneously promoting smoking abstinence and lifestyle coaching for PCWG restriction.