Doxycycline-Mediated Inhibition of Snake Venom Phospholipase and Metalloproteinase.

INTRODUCTION: Warfighters are exposed to life-threatening injuries daily and according to the Joint Trauma System Military Clinical Practice Guideline-Global Snake Envenomation Management snakebites are a concerning threat in all theaters of operation. Snake venom is a complex mixture of toxins including phospholipases A2 (PLA2) and snake venom metalloproteinases (SVMP) that produce myotoxic, hemotoxic, and cytotoxic injuries. Antibody-based antivenom is the standard of care but new approaches including small-molecule inhibitors have gained attention in recent years. Doxycycline is an effective inhibitor of human metalloproteinases and PLA2. The enzymatic activities of 3 phylogenetically distinct snakes: Agkistrodon piscivorus, Naja kaouthia, and Daboia russelii were tested under inhibitory conditions using doxycycline. MATERIALS AND METHODS: Enzymatic activity of PLA2 and SVMP was measured in N. kaouthia, D. russelii, and A. piscivorus venom alone and with doxycycline using EnzChek Phospholipase A2 and Gelatinase Assay Kits. A 1-way ANOVA with Tukey's post-hoc test was used to conduct comparative analysis. The median lethal dose of the venoms, the effective dose of doxycycline, and creatine kinase (CK) inhibition levels were measured in a murine model with adult Bagg Albino (BALB/c) mice using intramuscular injections. Median lethal and effective doses were determined using Spearman-Karber's method and a 1-way ANOVA with Tukey's post-hoc test was used to compare CK inhibition levels. RESULTS: Phospholipases A2 activity was reduced to 1.5% to 44.0% in all 3 venoms in a dose-dependent manner using 0.32, 0.16, and 0.08 mg/mL doxycycline when compared to venom-only controls (P < .0001) (Fig. 1A). Snake venom metalloproteinases activity was reduced to 4% to 62% in all 3 venoms in a dose-dependent manner using 0.32, 0.16, and 0.08 mg/mL doxycycline (P < .0001) (Fig. 1B). The lethal dose (LD50) values of the venoms in the murine model were calculated as follows: A. piscivorus = 20.29 mg/kg (Fig. 2A), N. kaouthia = 0.38 mg/kg (Fig. 2B), and D. russelii = 7.92 mg/kg (Fig. 2C). The effective dose (ED50) of doxycycline in A. piscivorus was calculated to be 20.82 mg/kg and 72.07 mg/kg when treating D. russelii venom. No ED50 could be calculated when treating N. kaouthia venom (Fig. 3). Creatine kinase activity was significantly decreased in all 3 venoms treated with doxycycline (P < .0001) (Fig. 4). CONCLUSION: Doxycycline reduced PLA2- and SVMP-related lethality, particularly in A. piscivorus envenomings and in a limited capacity with D. russelii revealing its promise as a treatment for snakebites. In addition, CK activity, a common indicator of muscle damage was inhibited in mice that received doxycycline-treated venom. The doxycycline concentrations identified in the ED50 studies correspond to 1,456 to 5,061 mg dosages for a 70 kg human. Factors including venom yield and snake species would affect the actual dosage needed. Studies into high-dose doxycycline safety and its effectiveness against several snake species is needed to fully translate its use into humans. Based on this work, doxycycline could be used as a treatment en route to higher echelons of care, providing protection from muscle damage and reducing lethality in different snake species.

Perfluorooctanesulfonic acid exposure leads to downregulation of 3-hydroxy-3-methylglutaryl-CoA synthase 2 expression and upregulation of markers associated with intestinal carcinogenesis in mouse intestinal tissues.

Perfluorooctanesulfonic acid (PFOS) is a widely recognized environment pollutant known for its high bioaccumulation potential and a long elimination half-life. Several studies have shown that PFOS can alter multiple biological pathways and negatively affect human health. Considering the direct exposure to the gastrointestinal (GI) tract to environmental pollutants, PFOS can potentially disrupt intestinal homeostasis. However, there is limited knowledge about the effect of PFOS exposure on normal intestinal tissues, and its contribution to GI-associated diseases remains to be determined. In this study, we examined the effect of PFOS exposure on the gene expression profile of intestinal tissues of C57BL/6 mice using RNAseq analysis. We found that PFOS exposure in drinking water significantly downregulates mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting ketogenic enzyme, in intestinal tissues of mice. We found that diets containing the soluble fibers inulin and pectin, which are known to be protective against PFOS exposure, were ineffective in reversing the downregulation of HMGCS2 expression in vivo. Analysis of intestinal tissues also demonstrated that PFOS exposure leads to upregulation of proteins implicated in colorectal carcinogenesis, including ß-catenin, c-MYC, mTOR and FASN. Consistent with the in vivo results, PFOS exposure leads to downregulation of HMGCS2 in mouse and human normal intestinal organoids in vitro. Furthermore, we show that shRNA-mediated knockdown of HMGCS2 in a human normal intestinal cell line resulted in increased cell proliferation and upregulation of key proliferation-associated proteins such as cyclin D, survivin, ERK1/2 and AKT, along with an increase in lipid accumulation. In summary, our results suggest that PFOS exposure may contribute to pathological changes in normal intestinal cells via downregulation of HMGCS2 expression and upregulation of pro-carcinogenic signaling pathways that may increase the risk of colorectal cancer development.

Efficient Generation of Skin Organoids from Pluripotent Cells via Defined Extracellular Matrix Cues and Morphogen Gradients in a Spindle-Shaped Microfluidic Device.

Pluripotent stem cell-derived skin organoids (PSOs) emerge as a developmental skin model that is self-organized into multiple components, such as hair follicles. Despite their impressive complexity, PSOs are currently generated in the absence of 3D extracellular matrix (ECM) signals and have several major limitations, including an inverted anatomy (e.g., epidermis inside/dermis outside). In this work, a method is established to generate PSOs effectively in a chemically-defined 3D ECM environment. After examining various dermal ECM molecules, it is found that PSOs generated in collagen -type I (COLI) supplemented with laminin 511 (LAM511) exhibit increased growth compared to conventional free-floating conditions, but fail to induce complete skin differentiation due in part to necrosis. This problem is addressed by generating the PSOs in a 3D bioprinted spindle-shaped hydrogel device, which constrains organoid growth longitudinally. This culture system significantly reduces organoid necrosis and leads to a twofold increase in keratinocyte differentiation and an eightfold increase in hair follicle formation. Finally, the system is adapted as a microfluidic device to create asymmetrical gradients of differentiation factors and improves the spatial organization of dermal and epidermal cells. This study highlights the pivotal role of ECM and morphogen gradients in promoting and spatially-controlling skin differentiation in the PSO framework.

Ring Enhancement in Non-Neoplastic Breast Tissue on MRI During Neoadjuvant Chemotherapy for Breast Cancer: Incidence and Clinical Implications.

RATIONALE AND OBJECTIVES: This study aimed to describe new lesions called ring enhancement in non-neoplastic breast tissue on breast magnetic resonance imaging (MRI) after neoadjuvant chemotherapy (NAC) in breast cancer patients, and to investigate the factors influencing their occurrence. MATERIALS AND METHODS: We retrospectively reviewed 811 consecutive patients (mean age; 50.0 [range, 24-81] years) with breast cancer who had undergone NAC between January 2020 and December 2021, identifying cases with new ring enhancement on post-NAC MRI. We analyzed the MRI findings and identified factors that were potentially associated with ring enhancement through statistical analyses using the chi-square test, univariate and multivariate logistic regression analysis. RESULTS: Forty-seven (5.8%) patients developed new ring enhancement on post-NAC MRI. The variables associated with ring enhancement were premenopausal status (p = 0.0007), younger age (p = 0.0011), high mammographic density (p = 0.0076), and high background parenchymal enhancement (BPE) on baseline MRI (p = 0.0001). Among these, high BPE was independently associated with the occurrence of ring enhancement (p = 0.0294, OR = 2.08; CI: 1.08-4.03). In a subset of high BPE patients, an association between HER2-positive cancers and ring enhancement was observed (odds ratio = 5.51 vs. 2.54). New lesion development exhibited no association with any specific NAC drug (p = 0.1676-0.7583 per drug). CONCLUSION: Ring enhancement often occurs on post-NAC MRI and mostly disappears on subsequent MRI scans. High BPE on MRI was associated with this finding and HER2-positive cancers potentiated it. Knowledge of this finding can prevent unnecessary biopsies.

POU2AF3-rearranged sarcomas: A novel tumor defined by fusions of EWSR1 or FUS to a gene formerly designated COLCA2.

Gene fusions involving EWSR1 or FUS as the 5' partner have been reported in a diverse array of sarcomas. Here, we characterize the histopathology and genomics of six tumors harboring a gene fusion between EWSR1 or FUS and POU2AF3, an understudied, putative colorectal cancer predisposition gene. Striking morphologic features reminiscent of synovial sarcoma were observed including a biphasic appearance with variable fusiform to epithelioid cytomorphology and staghorn-type vasculature. RNA sequencing demonstrated variable breakpoints in EWSR1/FUS along with similar breakpoints in POU2AF3 that encompassed a 3' portion of this gene. For cases in which additional information was available, the behavior of these neoplasms was aggressive with local spread and/or distant metastases. Although further studies are needed to confirm the functional significance of our findings, POU2AF3 fusions to EWSR1 or FUS may define a novel type of POU2AF3-rearranged sarcomas with aggressive, malignant behavior.

Loss of Peroxiredoxin IV Protects Mice from Azoxymethane/Dextran Sulfate Sodium-Induced Colorectal Cancer Development.

Peroxiredoxin IV (Prx4), a typical two-cysteine-containing member of the peroxidase family, functions as an antioxidant to maintain cellular redox homeostasis through the reduction of reactive oxygen species (ROS) via cycles of oxidation-reduction reactions. Under oxidative stress, all Prxs including Prx4 are inactivated as their catalytic cysteines undergo hyperoxidation, and hyperoxidized two-cysteine Prxs can be exclusively repaired and revitalized through the reduction cycle catalyzed by sulfiredoxin (Srx). Previously, we showed that Prx4 is a preferred substrate of Srx, and knockout of Srx in mice leads to resistance to azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colon carcinogenesis. To further understand the significance of the Srx/Prx4 axis in colorectal cancer development, Prx4-/- mice were established and subjected to standard AOM/DSS protocol. Compared with wildtype littermates, mice with Prx4-/- genotype had significantly fewer and smaller tumors. Histopathological analysis revealed that loss of Prx4 leads to increased cell death through lipid peroxidation and lower infiltration of inflammatory cells in the knockout tumors compared to wildtype. Treatment with DSS alone also showed decreased infiltration of macrophages and lymphocytes in the colon of knockout mice, suggesting a role for Prx4 in inflammatory response. In addition, loss of Prx4 caused alterations in plasma cytokines and chemokines after DSS and AOM/DSS treatments. These findings suggest that loss of Prx4 protects mice from AOM/DSS-induced colon tumorigenesis. Thus, targeting Prx4 may provide novel strategies for colon cancer prevention and treatment.

The effect of myofascial release in patients with breast cancer-related lymphedema: a cross-over randomized controlled trial.

AIM: This study aimed to compare the effects of myofascial release (MFR) on upper extremity volume in patients with breast cancer-related lymphedema (BCRL). DESIGN: A randomized, single-blinded, cross-over, controlled trial. SETTING: An outpatient rehabilitation clinical setting. POPULATION: Thirty patients with BCRL. METHODS: Within a crossover design with randomized treatment sequences, fifteen subjects received MFR for 4 weeks, followed by 4 weeks of washout period, and then received placebo MFR and the other fifteen subjects received interventions in the reverse order. Each session had a 60 min process including either MFR or placebo MFR for 30 min, followed by complete decongestive therapy for 30 min twice a week. Upper limb volume as the primary outcome and subjective pain, shoulder range of motion (ROM), chest mobility, shoulder function, and quality of life as secondary outcomes were assessed before and at the end of each intervention period. RESULTS: There were significant differences in upper limb volume after both MFR and placebo MFR (P<0.05) while no significant difference between MFR and placebo MFR treatments was found (P>0.05). MFR-based treatment also achieved a greater improvement than placebo MFR-based treatment in subjective pain and shoulder ROM (P<0.05), except for internal rotation, and shoulder function. CONCLUSIONS: MFR-based treatment showed clinical improvement in shoulder function, induced by decreased edema volume and pain, and improved ROM and chest mobility. However, a further study with parallel randomized controlled trials to confirm what was achieved in the present study. CLINICAL REHABILITATION IMPACT: MFR-based treatment is considered an important part of BCRL rehabilitation. Moreover, MFR-based treatment may be safe for patients with BCRL.

A Rare and Late Complication of Subureteric Teflon Injection With Non-Animal Stabilized Hyaluronic Acid/Dextranomer Gel.

Primary non-syndromic vesicoureteral reflux (VUR) is the commonest paediatric anomaly of the urinary tract. Complications of high-grade VUR include recurrent urinary tract infections, pyelonephritis, reflux nephropathy, and irreversible renal failure. The primary aim of its management centres on minimizing the number of urinary tract infections and renal scarring via surgical correction or continuous antibiotic prophylaxis. A rare complication of surgical treatment by subureteric Teflon injection with non-animal stabilized hyaluronic acid/dextranomer gel (NASHA/Dx) is ureteric obstruction. We report the case of a 38-year-old female who was diagnosed with ureteric obstruction secondary to subureteric injection with Deflux injection 30 years after endoscopic correction of VUR. She was successfully treated with ureteric reimplantation. Although considered efficient and safe, subureteric injection of bulking agent Deflux can be associated with delayed ureteric obstruction. This case highlights the need for long-term follow-up to allow timely detection and management of delayed ureteric obstruction. The possibility of late complication must also be addressed when obtaining pre-operative informed consent.

Associations between breastfeeding intention, breastfeeding practices and post-natal depression during the COVID-19 pandemic: A multi-country cross-sectional study.

Associations between breastfeeding intention, duration and post-natal depression (PND) have been shown in pre-COVID-19 studies. However, studies during COVID-19 have not examined the associations between breastfeeding intention, breastfeeding practices, and PND in an international sample of post-natal women, taking into consideration COVID-19 related factors. This is the first study to address this gap as both PND and breastfeeding may be affected by COVID-19, and have important long-term effects on women's and infant's health. A cross-sectional internet-based survey was conducted with 3253 post-natal women from five countries: Brazil, South Korea, Taiwan, Thailand, and the United Kingdom from July to November 2021. The results showed that women who intended to breastfeed during pregnancy had lower odds of having PND than women who did not intend to. Women who had no breastfeeding intention but actually breastfed had greater odds (AOR 1.75) of having PND than women who intended to breastfeed and actually breastfed. While there was no statistical significance in expressed breast milk feeding in multivariable logistic regression models, women who had shorter duration of breastfeeding directly on breast than they planned had greater odds (AOR 1.58) of having PND than those who breastfed longer than they planned even after adjusting for covariates including COVID-19-related variables. These findings suggested the importance of working with women on their breastfeeding intention. Tailored support is required to ensure women's breastfeeding needs are met and at the same time care for maternal mental health during and beyond the pandemic.

Effects of a Two-Step Silver Diamine Fluoride Varnish on Shear Bond Strength of Restorations, Dentin and Enamel Hardness, and Biofilm Formation.

INTRODUCTION: Dental caries are a limiting factor in maintaining dental and medical readiness in the military. Untreated dental caries can lead to dire health consequences. Consistent and comprehensive access to dental care is often limited due to the intensive operational demands on our nation's warfighters. The standard of care for dental caries is a surgical model where diseased tooth tissue is surgically removed and restored with appropriate restorative materials. While effective, it is not practical in the military operational environment, especially under time constraints. Dental restoratives offer military personnel a simple and preventive treatment of dental caries and are suitable as self-applied first aids. The purpose of this study was to measure the shear bond strengths of two dental restorative materials to human teeth paired with two different fluoride treatments and the hardness and biofilm formation on teeth after applying the fluoride varnishes. MATERIALS AND METHODS: Specimens were made of human molar teeth treated with each of the following four materials: glass ionomer cement GC Fuji II LC Capsules, Filtek Z250, Riva Star steps 1 and 2, or Mark3 NaF varnish. Step 1 of Riva Star consists of silver diamine fluoride and step 2 contains potassium iodide. On human molar slabs, 10 circular specimens of 5 cm in diameter were prepared with restoratives according to manufacturer procedures. Etch-Rite and a proprietary aluminum chloride-based cavity conditioner were used as etchants on tooth surfaces for the Filtek Z250 and glass ionomer cement, respectively. After at least 24 hours underwater, each assembly was removed, and the shear bond strength of the adhesive was measured according to International Organization for Standardization (ISO) 29022.The hardness was measured according to ISO 14233. Hardness measurements were performed before varnish application, then after storage in an incubator at 37 °C for 4 hours in a demineralization solution (pH = 4.5), and after 1 day in a mineralization solution (pH = 7). A crystal violet staining assay was used to measure biofilm formation of Streptococcus mutans bacteria on human molar teeth after the application of fluoride varnish. RESULTS: We report a 16% increase in shear bond strength of the Filtek Z250/Riva Star coupled treatment compared to the Filtek Z250/Mark3 NaF coupled treatment. We also demonstrate a significant 84% decrease in bond strength with a GC Fuji II LC/Mark3 NaF treatment compared to control (P = .0002), while Riva Star remains statistically unchanged. Enamel and dentinal hardness are significantly improved when Riva Star is applied compared to NaF varnish. A 25%-35% (P < .0001) decrease in oral biofilm formation was observed on samples where a Riva Star or NaF varnish was applied. CONCLUSIONS: Mechanical and antimicrobial testing indicated Riva Star, compared favorably with and in some cases, performed better in the laboratory than a Mark3 NaF varnish. Hardness measurements indicated Riva Star is more effective in dentin tubule occlusion compared to NaF varnish. Our findings help provide practical suggestions to dental treatment, particularly to the unique dental environments seen in the military. Riva Star may be used as an adjunctive treatment prior to placing a final restoration. This study supports the use of Riva Star in conjunction with GC Fuji II LC or Filtek Z250 restorative materials, making it a promising treatment in military dental applications.

Upregulation of CD36, a Fatty Acid Translocase, Promotes Colorectal Cancer Metastasis by Increasing MMP28 and Decreasing E-Cadherin Expression.

Altered fatty acid metabolism continues to be an attractive target for therapeutic intervention in cancer. We previously found that colorectal cancer (CRC) cells with a higher metastatic potential express a higher level of fatty acid translocase (CD36). However, the role of CD36 in CRC metastasis has not been studied. Here, we demonstrate that high expression of CD36 promotes invasion of CRC cells. Consistently, CD36 promoted lung metastasis in the tail vein model and GI metastasis in the cecum injection model. RNA-Seq analysis of CRC cells with altered expression of CD36 revealed an association between high expression of CD36 and upregulation of MMP28, a novel member of the metallopeptidase family of proteins. Using shRNA-mediated knockdown and overexpression of CD36, we confirmed that CD36 regulates MMP28 expression in CRC cells. siRNA-mediated knockdown of MMP28 decreases invasion of CRC cells, suggesting that MMP28 regulates the metastatic properties of cells downstream of CD36. Importantly, high expression of MMP28 leads to a significant decrease in active E-cadherin and an increase in the products of E-cadherin cleavage, CTF1 and CTF2. In summary, upregulation of CD36 expression promotes the metastatic properties of CRC via upregulation of MMP28 and an increase in E-cadherin cleavage, suggesting that targeting the CD36-MMP28 axis may be an effective therapeutic strategy for CRC metastasis.

Female reproductive factors and risk of joint replacement arthroplasty of the knee and hip due to osteoarthritis in postmenopausal women: a nationwide cohort study of 1.13 million women.

OBJECTIVES: Previous studies of the relationships between female reproductive factors and osteoarthritis (OA) have shown conflicting results. In this study, we aimed to explore the relationships between reproductive factors and joint replacement arthroplasty of the knee (TKRA) and hip (THRA) in a large nationwide population-based cohort of postmenopausal Korean women. METHODS: We included 1,134,680 subjects who participated in national health examinations in 2009 in the study. The study outcomes were incident THRA or TKRA due to severe hip or knee OA. The relationships between reproductive factors and THRA or TKRA were evaluated using a multivariable-adjusted proportional hazards model. RESULTS: During a mean follow-up duration of 8.2 years, 1,610 incident THRA cases and 60,670 incident TKRA cases were observed. Later age at menarche, longer breastfeeding, HRT and OC use were associated with increased risk of TKRA for severe knee OA, while later age at menopause and longer reproductive span were associated with decreased risk. With regard to THRA for severe hip OA, later menarche, longer breastfeeding, HRT more than 5 years, and OC use more than 1 year were associated with higher risk. The associations between reproductive factors and severe OA were more pronounced in underweight and younger subjects. CONCLUSION: We found that shorter estrogen exposure was associated with higher risk of TKRA due to severe knee OA, and such associations were more pronounced in underweight and younger subjects. The association between shorter estrogen exposure and THRA was not robust.

Active Learning of Contrast Sensitivity to Assess Visual Function in Macula-off Retinal Detachment.

PURPOSE: To characterize the contrast sensitivity function (CSF) in patients with successful repair of macula-off rhegmatogenous retinal detachment (RD) using an adaptive computerized contrast testing device. METHODS: CSF was prospectively measured in macula-off RD patients following successful repair and age-matched controls at W. K. Kellogg Eye Center and Massachusetts Eye and Ear, employing the active learning device Manifold Contrast Vision Meter (Adaptive Sensory Technology, San Diego, CA). Outcome measures included average area under the CSF curve (AULCSF), CS thresholds at 1-18 cycles per degree (cpd) and best correctd visual acuity (BCVA) in RD eyes fellow eyes and controls. A sub-analysis was performed in eyes with BCVA of 20/30 or better. RESULTS: Twenty-three macula-off RD eyes status post repair, fellow healthy eyes and 45 age-matched control eyes underwent CSF testing. The mean BCVA of the 23 RD eyes was 0.250 logMAR, significantly reduced compared to fellow eyes 0.032 (p<0.001) and controls 0.026 (p< 0.00001). There was a statistically significant reduction in AULCSF in RD eyes compared to the fellow eyes (p<0.0001) and to age-matched controls (Z-score -0.90, p<0.0001) and CSF reduction across all spatial frequencies. In the 15 RD eyes with BCVA of 20/30 or better, the mean CSF was significantly reduced compared to fellow eyes (p=0.0158) and controls (p=0.0453). CONCLUSIONS: CSF in macula-off RD eyes following repair was significantly reduced compared to fellow eyes and age-matched controls. CSF seems to be a promising visual function endpoint with potential applications in the clinical practice and future clinical trials.

Potent Synergistic Effect on C-Myc-Driven Colorectal Cancers Using a Novel Indole-Substituted Quinoline with a Plk1 Inhibitor.

Developing effective treatments for colorectal cancers through combinations of small-molecule approaches and immunotherapies present intriguing possibilities for managing these otherwise intractable cancers. During a broad-based, screening effort against multiple colorectal cancer cell lines, we identified indole-substituted quinolines (ISQ), such as N7,N7 -dimethyl-3-(1-methyl-1H-indol-3-yl)quinoline-2,7-diamine (ISQ-1), as potent in vitro inhibitors of several cancer cell lines. We found that ISQ-1 inhibited Wnt signaling, a main driver in the pathway governing colorectal cancer development, and ISQ-1 also activated adenosine monophosphate kinase (AMPK), a cellular energy-homeostasis master regulator. We explored the effect of ISQs on cell metabolism. Seahorse assays measuring oxygen consumption rate (OCR) indicated that ISQ-1 inhibited complex I (i.e., NADH ubiquinone oxidoreductase) in the mitochondrial, electron transport chain (ETC). In addition, ISQ-1 treatment showed remarkable synergistic depletion of oncogenic c-Myc protein level in vitro and induced strong tumor remission in vivo when administered together with BI2536, a polo-like kinase-1 (Plk1) inhibitor. These studies point toward the potential value of dual drug therapies targeting the ETC and Plk-1 for the treatment of c-Myc-driven cancers.

Changes in natural killer cell activity after surgery and predictors of its recovery-failure.

BACKGROUND AND OBJECTIVES: We evaluated the changes in natural killer cell activity (NKA) during the entire treatment period of patients with resectable biliopancreatic cancers and investigated the predictors of the failure of recovery of NKA after surgery. METHODS: A total of 202 patients who underwent curative resection for biliopancreatic cancer were enrolled in the study. NKA levels were measured six times during the treatment period. We investigated whether there was any difference in postoperative NKA recovery according to the period-by-time NKA value. RESULTS: NKA decreased after surgery (mean, 40 pg/ml) compared to the NKA value at admission (200.2 pg/ml), then began to increase from 3 weeks after surgery (139.7 pg/ml) and rose to normal NKA levels at 5 weeks (217.1 pg/ml). The pattern of NKA changes was distinct according to the NKA values at admission. In multivariate analysis, NKA values of less than 250 pg/ml at admission (odds ratio = 5.898, p = 0.044) were a predictor of NKA recovery failure 5 weeks after surgery. CONCLUSIONS: NKA rapidly decreased after curative surgery for biliopancreatic cancer and recovered to normal levels about 5 weeks later. Clinicians should be aware and cautious that patients with low NKA at admission may fail to recover NKA postoperatively.

Ketogenesis alleviates TNFα-induced apoptosis and inflammatory responses in intestinal cells.

The disturbance of strictly regulated self-regeneration in mammalian intestinal epithelium is associated with various intestinal disorders, particularly inflammatory bowel diseases (IBDs). TNFα, which plays a critical role in the pathogenesis of IBDs, has been reported to inhibit production of ketone bodies such as ß-hydroxybutyrate (ßHB). However, the role of ketogenesis in the TNFα-mediated pathological process is not entirely known. Here, we showed the regulation and role of HMGCS2, the rate-limiting enzyme of ketogenesis, in TNFα-induced apoptotic and inflammatory responses in intestinal epithelial cells. Treatment with TNFα dose-dependently decreased protein and mRNA expression of HMGCS2 and its product, ßHB production in human colon cancer cell lines HT29 and Caco2 cells and mouse small intestinal organoids. Moreover, the repressed level of HMGCS2 protein was found in intestinal epithelium of IBD patients with Crohn's disease and ulcerative colitis as compared with normal tissues. Furthermore, knockdown of HMGCS2 enhanced and in contrast, HMGCS2 overexpression attenuated, the TNFα-induced apoptosis and expression of pro-inflammatory chemokines (CXCL1-3) in HT29, Caco2 cells and DLD1 cells, respectively. Treatment with ßHB or rosiglitazone, an agonist of PPARγ, which increases ketogenesis, attenuated TNFα-induced apoptosis in the intestinal epithelial cells. Finally, HMGCS2 knockdown enhanced TNFα-induced reactive oxygen species (ROS) generation. In addition, hydrogen peroxide, the major ROS contributing to intestine injury, decreased HMGCS2 expression and ßHB production in the intestinal cells and mouse organoids. Our findings demonstrate that increased ketogenesis attenuates TNFα-induced apoptosis and inflammation in intestinal cells, suggesting a protective role for ketogenesis in TNFα-induced intestinal pathologies.