Toward Interpretable Cell Image Representation and Abnormality Scoring for Cervical Cancer Screening Using Pap Smears.

Screening is critical for prevention and early detection of cervical cancer but it is time-consuming and laborious. Supervised deep convolutional neural networks have been developed to automate pap smear screening and the results are promising. However, the interest in using only normal samples to train deep neural networks has increased owing to the class imbalance problems and high-labeling costs that are both prevalent in healthcare. In this study, we introduce a method to learn explainable deep cervical cell representations for pap smear cytology images based on one-class classification using variational autoencoders. Findings demonstrate that a score can be calculated for cell abnormality without training models with abnormal samples, and we localize abnormality to interpret our results with a novel metric based on absolute difference in cross-entropy in agglomerative clustering. The best model that discriminates squamous cell carcinoma (SCC) from normals gives 0.908±0.003 area under operating characteristic curve (AUC) and one that discriminates high-grade epithelial lesion (HSIL) 0.920±0.002 AUC. Compared to other clustering methods, our method enhances the V-measure and yields higher homogeneity scores, which more effectively isolate different abnormality regions, aiding in the interpretation of our results. Evaluation using an external dataset shows that our model can discriminate abnormality without the need for additional training of deep models.

Design, synthesis, and evaluation of purine and pyrimidine-based KRAS G12D inhibitors: Towards potential anticancer therapy.

Oncogenic RAS mutations, commonly observed in human tumors, affect approximately 30% of cancer cases and pose a significant challenge for effective cancer treatment. Current strategies to inhibit the KRAS G12D mutation have shown limited success, emphasizing the urgent need for new therapeutic approaches. In this study, we designed and synthesized several purine and pyrimidine analogs as inhibitors for the KRAS G12D mutation. Our synthesized compounds demonstrated potent anticancer activity against cell lines with the KRAS G12D mutation, effectively impeding their growth. They also exhibited low toxicity in normal cells, indicating their selective action against cancer cells harboring the KRAS G12D mutation. Notably, the lead compound, PU1-1 induced the programmed cell death of KRAS G12D-mutated cells and reduced the levels of active KRAS and its downstream signaling proteins. Moreover, PU1-1 significantly shrunk the tumor size in a pancreatic xenograft model induced by the KRAS G12D mutation, further validating its potential as a therapeutic agent. These findings highlight the potential of purine-based KRAS G12D inhibitors as candidates for targeted cancer therapy. However, further exploration and optimization of these compounds are essential to meet the unmet clinical needs of patients with KRAS-mutant cancers.

Glucocorticoid-Induced Hyperglycemia: A Neglected Problem.

Glucocorticoids provide a potent therapeutic response and are widely used to treat a variety of diseases, including coronavirus disease 2019 (COVID-19) infection. However, the issue of glucocorticoid-induced hyperglycemia (GIH), which is observed in over one-third of patients treated with glucocorticoids, is often neglected. To improve the clinical course and prognosis of diseases that necessitate glucocorticoid therapy, proper management of GIH is essential. The key pathophysiology of GIH includes systemic insulin resistance, which exacerbates hepatic steatosis and visceral obesity, as well as proteolysis and lipolysis of muscle and adipose tissue, coupled with ß-cell dysfunction. For patients on glucocorticoid therapy, risk stratification should be conducted through a detailed baseline evaluation, and frequent glucose monitoring is recommended to detect the onset of GIH, particularly in high-risk individuals. Patients with confirmed GIH who require treatment should follow an insulin-centered regimen that varies depending on whether they are inpatients or outpatients, as well as the type and dosage of glucocorticoid used. The ideal strategy to maintain normoglycemia while preventing hypoglycemia is to combine basal-bolus insulin and correction doses with a continuous glucose monitoring system. This review focuses on the current understanding and latest evidence concerning GIH, incorporating insights gained from the COVID-19 pandemic.

Improving Tumor-Infiltrating Lymphocytes Score Prediction in Breast Cancer with Self-Supervised Learning.

Tumor microenvironment (TME) plays a pivotal role in immuno-oncology, which investigates the intricate interactions between tumors and the human immune system. Specifically, tumor-infiltrating lymphocytes (TILs) are crucial biomarkers for evaluating the prognosis of breast cancer patients and have the potential to refine immunotherapy precision and accurately identify tumor cells in specific cancer types. In this study, we conducted tissue segmentation and lymphocyte detection tasks to predict TIL scores by employing self-supervised learning (SSL) model-based approaches capable of addressing limited labeling data issues. Our experiments showed a 1.9% improvement in tissue segmentation and a 2% improvement in lymphocyte detection over the ImageNet pre-training model. Using these SSL-based models, we achieved a TIL score of 0.718 with a 4.4% improvement. In particular, when trained with only 10% of the entire dataset, the SwAV pre-trained model exhibited a superior performance over other models. Our work highlights improved tissue segmentation and lymphocyte detection using the SSL model with less labeled data for TIL score prediction.

Heterogeneity of gut microbiome compositions in the third decade of life in Japanese women: insights from a comparative analysis.

The reasons for sex-associated gut microbiota differences have not been determined, and although sex hormones, diet, and other factors are considered to contribute to them, many of these factors are age related. To shed light on this complex interplay, our study aimed to investigate and compare the gut microbial compositions of males and females across a broad range of ages, aiming to identify sex-associated disparities and potential causal factors. Our study encompassed a comprehensive analysis of gut microbiota data obtained from 444 Japanese individuals, ranging from newborns to centenarians, sourced from the DNA Data Bank of Japan. We categorized the subjects into 13 distinct age groups and examined their relative microbial abundances, as well as alpha and beta diversities, in relation to sex and age. No difference was observed between gut microbiota relative abundances or alpha diversities between men and women at any age. However, the study showed that the heterogeneity of gut microbiota among women in their 20s was greater than in men. To confirm the general occurrence of this difference, we conducted additional analyses using seven datasets: three from Japan and four from other countries. Interestingly, this variance was particularly noticeable within Japanese women. We also showed a potential link between the observed heterogeneity and dietary fiber intake. It is hoped this study will provide clues that aid in the identification of factors responsible for sex-associated differences in gut microbiota compositions.

Profiling of Lymphovascular Space Invasion in Cervical Cancer Revealed PI3K/Akt Signaling Pathway Overactivation and Heterogenic Tumor-Immune Microenvironments.

Lymphovascular space invasion (LVSI) is the presence of tumor emboli in the endothelial-lined space at the tumor body's invasive edge. LVSI is one of three Sedlis criteria components-a prognostic tool for early cervical cancer (CC)-essential for indicating poor prognosis, such as lymph node metastasis, distant metastasis, or shorter survival rate. Despite its clinical significance, an in-depth comprehension of the molecular mechanisms or immune dynamics underlying LVSI in CC remains elusive. Therefore, this study investigated tumor-immune microenvironment (TIME) dynamics of the LVSI-positive group in CC. RNA sequencing included formalin-fixed paraffin-embedded (FFPE) slides from 21 CC patients, and differentially expressed genes (DEGs) were analyzed. Functional analysis and immune deconvolution revealed aberrantly enriched PI3K/Akt pathway activation and a heterogenic immune composition with a low abundance of regulatory T cells (Treg) between LVSI-positive and LVSI-absent groups. These findings improve the comprehension of LSVI TIME and immune mechanisms, benefiting targeted LVSI therapy for CC.

Relationship between Human Papillomavirus Status and the Cervicovaginal Microbiome in Cervical Cancer.

Uterine cervical cancer (CC) is a complex, multistep disease primarily linked to persistent infection with high-risk human papillomavirus (HR-HPV). However, it is widely acknowledged that HR-HPV infection alone cannot account for the formation and progression of CC. Emerging evidence suggests that the cervicovaginal microbiome (CVM) also plays a significant role in HPV-related CC. Certain bacteria, such as Fusobacterium spp., Porphyromonas, Prevotella, and Campylobacter, are currently being considered as potential microbiomarkers for HPV-positive CC. However, the composition of the CVM in CC is inconsistent; thus, further studies are needed. This review comprehensively discusses the complex interplay between HPV and the CVM in cervical carcinogenesis. It is postulated that the dynamic interaction between HPV and the CVM creates an imbalanced cervicovaginal microenvironment that triggers dysbiosis, enhances HPV persistence, and promotes cervical carcinogenesis. Moreover, this review aims to provide updated evidence on the potential role of bacteriotherapy, particularly probiotics, in the treatment of CC.

A collaborative study to establish the second national standard for hepatitis B immunoglobulin in Korea.

This study aimed to establish a second national standard for hepatitis B immunoglobulin (HBIG) that can be used for potency assays of hepatitis B and normal immunoglobulin. The candidate material was manufactured using a process approved as Good Manufacturing Practice. The freeze-dried candidate preparation was tested for physicochemical and biological properties, including pH, residual moisture, molecular size distribution, and potency. A collaborative study was performed involving four laboratories, including the National Institute of Food and Drug Safety Evaluation, as an official national control laboratory in Korea and manufacturers. The potency was calibrated against the second international standard for HBIG using two enzyme immunoassays: enzyme-linked immunosorbent assay and electrochemiluminescence immunoassay. Results from 240 assays were obtained from four laboratories, and combined potency estimates were obtained by calculating the geometric means. Intra- and inter-laboratory variability showed acceptable geometric coefficients of variation of 1.3-6.0 and 3.2-3.6%, respectively. The candidate preparation showed satisfactory stability in accelerated thermal degradation and real-time stability tests. Based on these results, the potency value of 105 IU/vial was assigned (95% confidence intervals: 100.0-109.2 IU/vial), and it was deemed suitable to serve as the Korean national standard for HBIG.

Deep Learning Prediction of TERT Promoter Mutation Status in Thyroid Cancer Using Histologic Images.

Background and objectives: Telomerase reverse transcriptase (TERT) promoter mutation, found in a subset of patients with thyroid cancer, is strongly associated with aggressive biologic behavior. Predicting TERT promoter mutation is thus necessary for the prognostic stratification of thyroid cancer patients. Materials and Methods: In this study, we evaluate TERT promoter mutation status in thyroid cancer through the deep learning approach using histologic images. Our analysis included 13 consecutive surgically resected thyroid cancers with TERT promoter mutations (either C228T or C250T) and 12 randomly selected surgically resected thyroid cancers with a wild-type TERT promoter. Our deep learning model was created using a two-step cascade approach. First, tumor areas were identified using convolutional neural networks (CNNs), and then TERT promoter mutations within tumor areas were predicted using the CNN-recurrent neural network (CRNN) model. Results: Using the hue-saturation-value (HSV)-strong color transformation scheme, the overall experiment results show 99.9% sensitivity and 60% specificity (improvements of approximately 25% and 37%, respectively, compared to image normalization as a baseline model) in predicting TERT mutations. Conclusions: Highly sensitive screening for TERT promoter mutations is possible using histologic image analysis based on deep learning. This approach will help improve the classification of thyroid cancer patients according to the biologic behavior of tumors.

Metabarcoding of eDNA for tracking the floral and geographical origins of bee honey.

Authentic honey products have a high commercial value and are often falsified via adulteration. Metabarcoding of environmental DNA (eDNA) from bacterial, floral, and entomological sources has recently been proposed as a useful tool for identifying and authenticating floral and geographical origins of bee honey. In this study, eDNA metabarcoding was applied to reveal the bacterial, plant, and honey bee DNA signatures of 48 commercial honey products from six different geographical origins. Bacterial DNA composition in commercial honey showed different relative abundance of Paenibacillus and Bacillus in geographically different samples, and high abundance of Methylobacterium in chestnut honey implying potential use of bacterial DNA composition for honey authentication. Using the chloroplast trnL (UAA) as a DNA marker, floral origins of commercial honey were investigated. Based on floral DNA signatures, 12 monofloral honey samples were identified among the 45 samples tested. Targeted amplicon sequencing of cytochrome oxidase I (COI) gene from entomological DNA identified three different Apis mellifera sequence variants, specific to geographic origin of honey, suggesting that COI can be implemented as a DNA marker to trace the origin of honey. Therefore, the current study demonstrated the potential of eDNA based metabarcoding as a robust tool for evaluating commercial bee honey by exploring their floral and geographical origins.

Enzymatic Process for the Carrageenolytic Bioconversion of Sulfated Polygalactans into ß-Neocarrabiose and 3,6-Anhydro-d-galactose.

Oligosaccharides and anhydro-sugars derived from carrageenan have great potential as functional foods and drugs showing various bioactivities, including antioxidant, anti-inflammatory, antiviral, antitumor, and cytotoxic activities. Although preparation of sulfated carrageenan oligosaccharides by chemical and enzymatic processes has been widely reported, preparation of nonsulfated ß-neocarrabiose (ß-NC2) and the rare sugar 3,6-anhydro-d-galactose (d-AHG) was not reported in the literature. Based on the carrageenan catabolic pathway in marine heterotrophic bacteria, an enzymatic process was designed and constructed with recombinant κ-carrageenase, GH127/GH129 α-1,3 anhydrogalactosidase, and cell-free extract from marine carrageenolytic bacteria Colwellia echini A3T. The process consisted of three successive steps, namely, (i) depolymerization, (ii) desulfation, and (iii) monomerization, by which carrageenan oligosaccharides, ß-NC2, and d-AHG were obtained from κ-carrageenan. Unlike the chemical process, enzymatic hydrolysis yields oligosaccharides with the desired degree of polymerization facilitates specific removal of sulfated groups, free of toxic byproducts, and avoids chemical modifications. The final optimized enzymatic process produced 0.52 g of ß-NC2 and 0.24 g of d-AHG from 1 g of κ-carrageenan. The carrageenolytic process designed for the enzymatic hydrolysis of κ-carrageenan can be scaled up for the mass production of bioactive carrageeno-oligosaccharides.

Immune Pathway and Gene Database (IMPAGT) Revealed the Immune Dysregulation Dynamics and Overactivation of the PI3K/Akt Pathway in Tumor Buddings of Cervical Cancer.

Tumor budding (TB) is a small cluster of malignant cells at the invasive front of a tumor. Despite being an adverse prognosis marker, little research has been conducted on the tumor immune microenvironment of tumor buddings, especially in cervical cancer. Therefore, RNA sequencing was performed using 21 formalin-fixed, paraffin-embedded slides of cervical tissues, and differentially expressed genes (DEGs) were analyzed. Immune Pathway and Gene Database (IMPAGT) was generated for immune profiling. "Pathway in Cancer" was identified as the most enriched pathway for both up- and downregulated DEGs. Kyoto Encyclopedia of Genes and Genomes Mapper and Gene Ontology further revealed the activation of the PI3K/Akt signaling pathway. An IMPAGT analysis revealed immune dysregulation even at the tumor budding stage, especially in the PI3K/Akt/mTOR axis, with a high efficiency and integrity. These findings emphasized the clinical significance of tumor buddings and the necessity of blocking the overactivation of the PI3K/Akt/mTOR pathway to improve targeted therapy in cervical cancer.

Simple Electric Device to Isolate Nucleic Acids from Whole Blood Optimized for Point of Care Testing of Brain Damage.

BACKGROUND: Detection or monitoring of brain damage is a clinically crucial issue. Nucleic acids in the whole blood can be used as biomarkers for brain injury. Polymerase chain reaction (PCR) which is one of the most commonly used molecular diagnostic assays requires isolated nucleic acids to initiate amplification. Currently used nucleic acid isolation procedures are complicated and require laboratory equipments. OBJECTIVE: In this study, we tried to develop a simple and convenient method to isolate nucleic acids from the whole blood sample using a tiny battery-powered electric device. The quality of the isolated nucleic acids should be suitable for PCR assay without extra preparation. METHODS: A plastic device with separation chamber was designed and printed with a 3D printer. Two platinum electrodes were placed on both sides and a battery was used to supply the electricity. To choose the optimal nucleic acid isolation condition, diverse lysis buffers and separation buffers were evaluated, and the duration and voltage of the electricity were tested. Western blot analysis and PCR assay were used to determine the quality of the separated nucleic acids. RESULTS: 2ul of whole blood was applied to the cathode side of the separation chamber containing 78 ul of normal saline. When the electricity at 5 V was applied for 5 min, nucleic acids were separated from segment 1 to 3 of the separation chamber. The concentration of nucleic acids peaked around 7~8 mm from cathode side. PCR assay using the separation buffer as the template was performed successfully both in conventional and realtime PCR methods. The hemoglobin in the whole blood did not show the inhibitory effect in our separation system and it may be due to structural modification of hemoglobin during electric separation. CONCLUSION: Our simple electric device can separate nucleic acids from the whole blood sample by applying electricity at 5 V for 5 min. The separation buffer solution taken from the device can be used for PCR assay successfully.

Transcriptomic Immune Profiles Can Represent the Tumor Immune Microenvironment Related to the Tumor Budding Histology in Uterine Cervical Cancer.

Tumor budding (TB) histology has become a critical biomarker for several solid cancers. Despite the accumulating evidence for the association of TB histology with poor prognosis, the biological characteristics of TB are little known about in the context related to the tumor immune microenvironment (TIME) in uterine cervical cancer (CC). Therefore, this study aimed to identify the transcriptomic immune profiles related to TB status and further provide robust medical evidence for clinical application. In our study, total RNA was extracted and sequenced from 21 CC tissue specimens. As such, 1494 differentially expressed genes (DEGs) between the high- and low-TB groups were identified by DESeq2. After intersecting the list of DEGs and public immune genes, we selected 106 immune-related DEGs. Then, hub genes were obtained using Least Absolute Shrinkage and Selection Operator regression. Finally, the correlation between the hub genes and immune cell types was analyzed and four candidate genes were identified (one upregulated (FCGR3B) and three downregulated (ROBO2, OPRL1, and NR4A2) genes). These gene expression levels were highly accurate in predicting TB status (area under the curve >80%). Interestingly, FCGR3B is a hub gene of several innate immune pathways; its expression significantly differed in the overall survival analysis (p = 0.0016). In conclusion, FCGR3B, ROBO2, OPRL1, and NR4A2 expression can strongly interfere with TB growth and replace TB to stratify CC patients.

Point of Care Test Technology Suitable for Early Detection and Monitoring of Ischemic Stroke.

BACKGROUND: Stroke is one of the leading causes of death and disability in adulthood worldwide. A simple and convenient diagnostic method is needed for monitoring high-risk patients for stroke. Few POCTs are available for stroke diagnosis. Soluble blood P-selectin is known as a biomarker for platelet aggregation. Increased expression of P-selectin is observed in coronary artery disease, acute myocardial infarction, stroke and peripheral arterial disease. OBJECTIVE: A simple method that can measure the increased expression of P-selectin in stroke patients is intended to be used for diagnosis or early detection and hospital monitoring of ischemic stroke. METHODS: Plasma proteins in blood were separated using a three-layered filter system. Quantum dot and antibody were conjugated to detect biomarkers present in plasma and then measured with a fluorescence spectrophotometer. RESULTS: The detection limit of soluble P-selectin confirmed by immunoassay was 1 ng/ul. In order to increase the sensitivity and simplify the reaction, the detection limit was measured to evaluate the sensitivity of the quantum dot labeled anti P-selectin antibody. As a result, P-selectin of 5 ng/ul or more showed saturation signal intensity, indicating the upper limit of detection, and 10 pg/ul was the lower limit of detection. CONCLUSION: In this study, we proposed a three-layer filter membrane system that can separate biomarker- rich fractions from whole blood, simplifying the analysis process and improving sensitivity by using quantum dot-labeled antibodies to detect biomarkers. We hope that our system complements the advantages of POCT and can be applied to real clinical applications.

Dietary sophorolipid accelerates growth by modulation of gut microbiota population and intestinal environments in broiler chickens.

BACKGROUND: Gut is a crucial organ for the host's defense system due to its filtering action of the intestinal membrane from hazardous foreign substances. One strategy to strengthen the gut epithelial barrier function is to upregulate beneficial microflora populations and their metabolites. Sophorolipid (SPL), which is a glycolipid bio-surfactant, could increase beneficial microflora and decrease pathogenic bacteria in the gastrointestinal tract. Therefore, herein, we conducted an experiment with broiler chickens to investigate the fortifying effects of SPL on the host's gut defense system by modulating the microbiota population. METHODS: A total of 540 1-day-old chicks (Ross 308) were used, and they were immediately allotted into three treatment groups (6 replications with 30 chicks/pen) according to their initial body weight. The dietary treatments consisted of CON (basal diet), BAM (10 mg/kg bambermycin), and SPL (10 mg/kg SPL). During the experiment, birds freely accessed feed and water, and body weight and feed intake were measured at the end of each phase. On d 35, birds (one bird/pen) were sacrificed to collect jejunum and cecum samples. RESULTS: Dietary SPL and BAM supplementation significantly accelerated birds' growth and also significantly improved feed efficiency compared to CON. Intestinal microbial community was significantly separated by dietary SPL supplementation from that of CON, and dietary SPL supplementation significantly increased Lactobacillus spp. and Akkermansia muciniphila. Moreover, birds fed with dietary SPL also showed the highest concentration of cecal butyrate among all treatment groups. Gut morphological analysis showed that dietary SPL significantly increased villus height, ratio of villus height to crypt depth, goblet cell numbers, and the gene expression levels of claudin-1 and mucin 2. Additionally, dietary SPL significantly decreased the mRNA expression level of pro-inflammatory cytokine, interleukin-6, and increased that of anti-inflammatory cytokine, interleukin-10, compared to other treatments. CONCLUSIONS: Dietary SPL increases the beneficial bacterial population and butyrate concentration, which leads to a strengthened gut barrier function. In addition, the intestinal inflammation was also downregulated by dietary SPL supplementation.

Intratumoral budding: A novel prognostic biomarker for tumor recurrence and a potential predictor of nodal metastasis in uterine cervical cancer.

OBJECTIVE: To evaluate the prognostic and predictive significance of lymphovascular invasion (LVI) and lymph node metastasis of intratumoral budding (ITB) and its correlation with clinicopathological parameters in patients with cervical cancer. METHODS: Total 151 patients with cervical cancer who underwent radical hysterectomy with pelvic and/or paraaortic lymphadenectomy were included. We assessed the status of ITB and peritumoral budding (PTB) in all available hematoxylin and eosin-stained specimens. Univariate and multivariate analyses were performed for ITB, PTB, and other clincopathological parameters as predictors of recurrence. RESULTS: ITBhigh (≥3TB/HPF) was significantly associated with large tumor size, deep stromal invasion, LVI, parametrial invasion, and lymph node metastasis. The numbers of ITBs and PTBs were positively correlated (r2 = 0.754, p < 0.0001). ITBhigh was more frequently observed in squamous cell carcinoma compared with adenocarcinoma and adenosquamous cell carcinoma (p = 0.010). ITBhigh was found to be an independent prognostic factor for tumor recurrence by multivariate analysis (hazard ratio, 1.92; 95% confidence interval [CI], 1.37-9.90; p = 0.026). Multiple logistic regression showed association of LVI (odds ratio [OR], 1.85; 95% CI, 1.11-3.06; p = 0.017) and lymph node metastasis (OR, 1.96; 95% CI, 1.26-4.66; p = 0.019). CONCLUSION: ITBhigh is an independent prognostic factor for tumor recurrence. ITB is a surrogate marker for predicting LVI in cervical cancers. The evaluation of ITB may be readily applied in the clinical setting for improved prognosis and to guide the clinical management of patients with cervical cancer.

Clinical usefulness of deep learning-based automated segmentation in intracranial hemorrhage.

BACKGROUND: Doctors with various specializations and experience order brain computed tomography (CT) to rule out intracranial hemorrhage (ICH). Advanced artificial intelligence (AI) can discriminate subtypes of ICH with high accuracy. OBJECTIVE: The purpose of this study was to investigate the clinical usefulness of AI in ICH detection for doctors across a variety of specialties and backgrounds. METHODS: A total of 5702 patients' brain CTs were used to develop a cascaded deep-learning-based automated segmentation algorithm (CDLA). A total of 38 doctors were recruited for testing and categorized into nine groups. Diagnostic time and accuracy were evaluated for doctors with and without assistance from the CDLA. RESULTS: The CDLA in the validation set for differential diagnoses among a negative finding and five subtypes of ICH revealed an AUC of 0.966 (95% CI, 0.955-0.977). Specific doctor groups, such as interns, internal medicine, pediatrics, and emergency junior residents, showed significant improvement with assistance from the CDLA (p= 0.029). However, the CDLA did not show a reduction in the mean diagnostic time. CONCLUSIONS: Even though the CDLA may not reduce diagnostic time for ICH detection, unlike our expectation, it can play a role in improving diagnostic accuracy in specific doctor groups.

Dose-Dependent Effect of Smoking on Risk of Diabetes Remains after Smoking Cessation: A Nationwide Population-Based Cohort Study in Korea.

BACKGROUND: This study aimed to evaluate the dose-dependent effects of smoking on risk of diabetes among those quitting smoking. METHODS: We analyzed clinical data from a total of 5,198,792 individuals age 20 years or older who received health care check-up arranged by the national insurance program of Korea between 2009 and 2016 using the Korean National Health Insurance Service database. Cumulative smoking was estimated by pack-years. Smokers were classified into four categories according to the amount of smoking: light smokers (0.025 to 5 smoking pack-years), medium smokers (5 to 14 smoking pack-years), heavy smokers (14 to 26 smoking pack-years), and extreme smokers (more than 26 smoking pack-years). RESULTS: During the study period, 164,335 individuals (3.2% of the total population) developed diabetes. Compared to sustained smokers, the risk of diabetes was significantly reduced in both quitters (hazard ratio [HR], 0.858; 95% confidence interval [CI], 0.838 to 0.878) and nonsmokers (HR, 0.616; 95% CI, 0.606 to 0.625) after adjustment for multiple risk factors. The risk of diabetes gradually increased with amount of smoking in both quitters and current smokers. The risk of diabetes in heavy (HR, 1.119; 95% CI, 1.057 to 1.185) and extreme smokers (HR, 1.348; 95% CI, 1.275 to 1.425) among quitters was much higher compared to light smokers among current smokers. CONCLUSION: Smoking cessation was effective in reducing the risk of diabetes regardless of weight change. However, there was a potential dose-dependent association between smoking amount and the development of diabetes. Diabetes risk still remained in heavy and extreme smokers even after smoking cessation.

Novel Monomeric Fungal Subtilisin Inhibitor from a Plant-Pathogenic Fungus, Choanephora cucurbitarum: Isolation and Molecular Characterization.

The bacterial protease inhibitor domains known as Streptomyces subtilisin inhibitors (SSI) are rarely found in fungi. Genome analysis of a fungal pathogen, Choanephora cucurbitarum KUS-F28377, revealed 11 SSI-like domains that are horizontally transferred and sequentially diverged during evolution. We investigated the molecular function of fungal SSI-like domains of C. cucurbitarum, designated "choanepins." Among the proteins tested, only choanepin9 showed inhibitory activity against subtilisin as the target protease, accounting for 47% of the inhibitory activity of bacterial SSI. However, the binding affinity (expressed as the dissociation constant [Kd ]) of choanepin9 measured via microscale thermophoresis was 21 nM, whereas that for bacterial SSI is 34 nM. The trend of binding and inhibitory activity suggests that the two inhibitors exhibit different inhibitory mechanisms for subtilisin protease. Interestingly, choanepin9 was identified as a monomer in studies in vitro, whereas bacterial SSI is a homodimer. Based on these observations, we constructed a monomeric bacterial SSI protein with decreased binding affinity to abrogate its inhibitory activity. By altering the reactive sites of choanepin9 deduced from the P1 and P4 sites of bacterial SSI, we reestablished that these residues in choanepins are also crucial for modulating inhibitory activity. These findings suggest that the fungal SSI evolved to target specific cognate proteases by altering the residues involved in inhibitory reactivity (reactive sites) and binding affinity (structural integrity). The function of fungal SSI proteins identified in this study provides not only a clue to fungal pathogenesis via protease inhibition but also a template for the design of novel serine protease inhibitors.IMPORTANCE Until recently, Streptomyces subtilisin inhibitors (SSI) were reported and characterized only in bacteria. We found SSI-like domains in a plant-pathogenic fungus, Choanephora cucurbitarum KUS-F28377, which contains 11 sequentially diverged SSI-like domains. None of these fungal SSI-like domains were functionally characterized before. The active form of fungal SSI-like protein is a monomer, in contrast to the homodimeric bacterial SSI. We constructed a synthetic monomer of bacterial SSI to demonstrate the modulation of its activity based on structural integrity and not reactive sites. Our results suggest the duplication and divergence of SSI-like domains of C. cucurbitarum within the genome to inhibit various cognate proteases during evolution by modulating both binding and reactivity. The molecular functional characterization of fungal SSI-like domains will be useful in understanding their biological role and future biotechnological applications.