Preoperative prognostic predictors and treatment strategies for surgical procedure focused on the sequential organ failure assessment score in nonocclusive mesenteric ischemia: a multicenter retrospective cohort study.

BACKGROUND: Several large-scale studies have assessed endovascular and surgical treatment methods for nonocclusive mesenteric ischemia (NOMI); however, the prognostic factors for NOMI remain unclear. Therefore, this study aimed to evaluate risk factors for in-hospital mortality among patients with NOMI who underwent laparotomy and to examine therapeutic strategies that may improve the prognosis. MATERIALS AND METHODS: In this multicenter retrospective study, the authors reviewed the electronic medical records retrieved from the inpatient database of patients with NOMI at eight district general hospitals between January 2011 and January 2021. A total of 88 patients who underwent laparotomies were divided into survivor and nonsurvivor groups, and statistical analysis was performed to determine clinical and physiological factors. RESULTS: Exploratory laparotomy based on second-look surgery was the first treatment choice. The overall mortality rate was 48.8%, with a male-to-female ratio of 1.1:1. The median Sequential Organ Failure Assessment (SOFA) score was 8 [interquartile range: 3.75-14.2], and the median SOFA scores were 5 [3-7] in the survivor group and 13 [9-17.5] in the nonsurvivor group. Univariate analysis revealed a significant difference in BMI ( P <0.001), hypoglycemia ( P =0.0012), previous cardiovascular surgery ( P =0.0019), catecholamine use ( P <0.001), SOFA score ( P <0.001), platelet count ( P =0.0023), and lactate level ( P <0.001). Logistic regression analysis using the factors with significant differences revealed that SOFA score ≥10 (odds ratio 23.3; 95% CI: 1.94-280.00; P =0.013) was an independent prognostic factor. In addition, catecholamine use was suggested as a factor with a SOFA score greater than or equal to 10. CONCLUSION: This study confirmed that a SOFA score of greater than or equal to 10 may be associated with increased mortality. While closely monitoring low blood pressure and renal dysfunction, survival rates may be improved if surgical intervention is performed before the SOFA score reaches greater than or equal to 10.

Development of a Monoclonal Antibody Targeting HTLV-1 Envelope gp46 Glycoprotein and Its Application to Near-Infrared Photoimmuno-Antimicrobial Strategy.

Human T-cell leukemia virus type 1 (HTLV-1), a retrovirus, causes adult T-cell leukemia-lymphoma, HTLV-1 associated myelopathy/tropical spastic paraparesis, and HTLV-1 uveitis. Currently, no antiretroviral therapies or vaccines are available for HTLV-1 infection. This study aimed to develop an antibody against the HTLV-1 envelope protein (Env) and apply it to a near-infrared photoimmuno-antimicrobial strategy (NIR-PIAS) to eliminate HTLV-1 infected cells. We established mouse monoclonal antibodies (mAbs) against HTLV-1 Env by immunization with a complex of liposome and the recombinant protein. Detailed epitope mapping revealed that one of the mAbs bound to the proline-rich region of gp46 and exhibited no obvious neutralizing activity to inhibit viral infection. Instead, the mAb was rarely internalized intracellularly and remained on the cell surface of HTLV-1-infected cells. The antibody conjugated to the photosensitive dye IRDye700Dx recognized HTLV-1 infected cells and killed them following NIR irradiation. These results suggest that the novel mAb and NIR-PIAS could be developed as a new targeted therapeutic tool against HTLV-1 infected cells.

Detection of hepatocarcinogens by combination of liver micronucleus assay and histopathological examination in 2-week or 4-week repeated dose studies.

BACKGROUND: Currently, revisions to the ICH S1 guidance on rodent carcinogenicity testing are being proposed. Application of this approach would reduce the use of animals in accordance with the 3Rs principles (reduce/refine/replace). The method would also shift resources to focus on more scientific mechanism-based carcinogenicity assessments and promote safe and ethical development of new small molecule pharmaceuticals. In the revised draft, findings such as cellular hypertrophy, diffuse and/or focal cellular hyperplasia, persistent tissue injury and/or chronic inflammation, preneoplastic changes, and tumors are listed as histopathology findings of particular interest for identifying carcinogenic potential. In order to predict hepatocarcinogenicity of test chemicals based on the results from 2- or 4-week repeated dose studies, we retrospectively reanalyzed the results of a previous collaborative study on the liver micronucleus assay. We focused on liver micronucleus induction in combination with histopathological changes including hypertrophy, proliferation of oval cells or bile duct epithelial cells, tissue injuries, regenerative changes, and inflammatory changes as the early responses of hepatocarcinogenesis. For these early responses, A total of 20 carcinogens, including 14 genotoxic hepatocarcinogens (Group A) and 6 non-liver-targeted genotoxic carcinogens (Group B) were evaluated. RESULTS: In the Group A chemicals, 5 chemicals (NPYR, MDA, NDPA, 2,6-DNT, and NMOR) showed all of the 6 early responses in hepatocarcinogenesis. Five chemicals (DMN, 2,4-DNT, QUN, 2-AAF, and TAA) showed 4 responses, and 4 chemicals (DAB, 2-NP, MCT, and Sudan I) showed 3 responses. All chemicals exhibited at least 3 early responses. Contrarily, in the Group B chemicals (6 chemicals), 3 of the 6 early responses were observed in 1 chemical (MNNG). No more than two responses were observed in 3 chemicals (MMC, MMS, and KA), and no responses were observed in 2 chemicals (CP and KBrO3). CONCLUSION: Evaluation of liver micronucleus induction in combination with histopathological examination is useful for detecting hepatocarcinogens. This assay takes much less time than routine long-term carcinogenicity studies.

All-Trans Retinoic Acid Exhibits Antiviral Effect against SARS-CoV-2 by Inhibiting 3CLpro Activity.

The pandemic of COVID-19 caused by SARS-CoV-2 continues to spread despite the global efforts taken to control it. The 3C-like protease (3CLpro), the major protease of SARS-CoV-2, is one of the most interesting targets for antiviral drug development because it is highly conserved among SARS-CoVs and plays an important role in viral replication. Herein, we developed high throughput screening for SARS-CoV-2 3CLpro inhibitor based on AlphaScreen. We screened 91 natural product compounds and found that all-trans retinoic acid (ATRA), an FDA-approved drug, inhibited 3CLpro activity. The 3CLpro inhibitory effect of ATRA was confirmed in vitro by both immunoblotting and AlphaScreen with a 50% inhibition concentration (IC50) of 24.7 ± 1.65 µM. ATRA inhibited the replication of SARS-CoV-2 in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 2.69 ± 0.09 µM in the former and 0.82 ± 0.01 µM in the latter. Further, we showed the anti-SARS-CoV-2 effect of ATRA on the currently circulating variants of concern (VOC); alpha, beta, gamma, and delta. These results suggest that ATRA may be considered as a potential therapeutic agent against SARS-CoV-2.

Development of Acquired Factor V Inhibitor After Surgical Procedure Without the Use of Fibrin Tissue Adhesives: A Case Report.

Development of acquired factor V (FV) inhibitor is a rare coagulation disorder. Production of heteroantibodies against bovine FV, a contaminant in fibrin tissue adhesives, is a common cause of this condition in the field of surgery. The development of recombinant thrombin eliminated contamination of bovine FV, and infrequent use of bovine thrombin has decreased the risk of FV inhibitor development. Here, we report the case of a 43-year-old man who had marked prolongation of prothrombin time and activated partial thromboplastin time after surgery. Mixing coagulation studies with normal plasma and patient's plasma suggested the presence of an inhibitor. Clotting factor assays revealed that FV activity decreased to <1% with positive FV inhibitor titer (9.2 Bethesda units). The diagnosis of the FV inhibitor was confirmed. Overt bleeding was not observed during the course of hospitalization. His coagulation abnormalities rapidly normalized without any medical intervention. A careful review of his medical records revealed that no tissue adhesives were used in the patient, and the FV inhibitor would likely be autoantibodies. Antibiotic use during the perioperative period or the surgical procedure itself may trigger the occurrence of FV inhibitors. This case highlights that FV inhibitor may develop after the surgical procedure even without a history of the use of fibrin tissue adhesives. Surgeons and hematologists should be aware that this rare but potentially life-threatening condition may occur after the surgical procedure.

Two different regimes in alcohol-induced coil-helix transition: effects of 2,2,2-trifluoroethanol on proteins being either independent of or enhanced by solvent structural fluctuations.

Inhomogeneous distribution of constituent molecules in a mixed solvent has been known to give remarkable effects on the solute, e.g., conformational changes of biomolecules in an alcohol-water mixture. We investigated the general effects of 2,2,2-trifluoroethanol (TFE) on proteins/peptides in a mixture of water and TFE using melittin as a model protein. Fluctuations and Kirkwood-Buff integrals (KBIs) in the TFE-H2O mixture, quantitative descriptions of inhomogeneity, were determined by small-angle X-ray scattering investigation and compared with those in the aqueous solutions of other alcohols. The concentration fluctuation for the mixtures ranks as methanol < ethanol ≪ TFE < tert-butanol < 1-propanol, indicating that the inhomogeneity of molecular distribution in the TFE-H2O mixture is unexpectedly comparable to those in the series of mono-ols. On the basis of the concentration dependence of KBIs between the TFE molecules, it was found that a strong attraction between the TFE molecules is not necessarily important to induce helix conformation, which is inconsistent with the previously proposed mechanism. To address this issue, by combining the KBIs and the helix contents reported by the experimental spectroscopic studies, we quantitatively evaluated the change in the preferential binding parameter of TFE to melittin attributed to the coil-helix transition. As a result, we found two different regimes on TFE-induced helix formation. In the dilute concentration region of TFE below ∼2 M, where the TFE molecules are not aggregated among themselves, the excess preferential binding of TFE to the helix occurs due to the direct interaction between them, namely independent of the solvent fluctuation. In the higher concentration region above ∼2 M, in addition to the former effect, the excess preferential binding is significantly enhanced by the solvent fluctuation. This scheme should be held as general cosolvent effects of TFE on proteins/peptides.

[A Case of Appendiceal Intussusception Induced by Early Appendiceal Carcinoma].

Appendiceal intussusception is an uncommon pathologic condition. We report herein a case of appendiceal intussusception induced by appendiceal carcinoma. A 76-year-old woman was admitted to hospital because of epigastric pain. CT scan showed multiple concentric ring sign in ascending colon and enhanced tumor in transverse colon. Colonoscopy showed invagination of polypoid lesion which was pushed back to cecum endoscopically. Laparoscopy-assisted ileocecal resection with regional lymph node dissection was performed for cecal cancer. During surgery, the appendix was found to be inverted completely into the cecum. The tumor was 70×35 mm in size in the cecal cavity, and the appendix had completely invaginated into the cecum at its base. Histopathologic examination revealed early appendiceal carcinoma. The patient is healthy without recurrence.

EURL ECVAM Genotoxicity and Carcinogenicity Database of Substances Eliciting Negative Results in the Ames Test: Construction of the Database.

The bacterial reverse mutation test (Ames test) is the most commonly used genotoxicity test; it is a primary component of the chemical safety assessment data required by regulatory agencies worldwide. Within the current accepted in vitro genotoxicity test battery, it is considered capable of revealing DNA reactivity, and identifying substances that can produce gene mutations via different mechanisms. The previously published consolidated EURL ECVAM Genotoxicity and Carcinogenicity Database, which includes substances that elicited a positive response in the Ames test, constitutes a collection of data that serves as a reference for a number of regulatory activities in the area of genotoxicity testing. Consequently, we considered it important to expand the database to include substances that fail to elicit a positive response in the Ames test, i.e., Ames negative substances. Here, we describe a curated collection of 211 Ames negative substances, with a summary of complementary data available for other genotoxicity endpoints in vitro and in vivo, plus available carcinogenicity data. A descriptive analysis of the data is presented. This includes a representation of the chemical space formed by the Ames-negative database with respect to other substances (e.g. REACH registered substances, approved drugs, pesticides, etc.) and a description of the organic functional groups found in the database. We also provide some suggestions on further analyses that could be made.

Double-parabolic-reflectors acoustic waveguides for high-power medical ultrasound.

High intensity focused ultrasound therapeutics are widely used to noninvasively treat various types of primary tumors and metastasis. However, ultrasound penetration depth is shallowed with increasing frequency which limits the therapeutic accuracy for deep tissues. Although acoustic waveguides are commonly inserted into tissue for localized therapy, powerful ultrasound delivery is difficult. Here, we invent double-parabolic-reflectors acoustic waveguides, where high-power ultrasound emission and large mechanical vibration enhance the therapeutic efficiency. High-energy-density ultrasound with around 20 times amplification by two parabolic reflectors propagates through the thin waveguide between 1 to 2 MHz, and wideband large mechanical vibration at the waveguide tip from 1 kHz to 2.5 MHz accelerates the therapeutics. This fundamental work serves as a milestone for future biomedical applications, from therapeutics to diagnostics. Since the high-power ability at high frequencies, our waveguide will also open up new research fields in medical, bio, physics and so on.

In vivo genotoxicity testing strategies: Report from the 7th International workshop on genotoxicity testing (IWGT).

The working group reached complete or majority agreement on many issues. Results from TGR and in vivo comet assays for 91 chemicals showed they have similar ability to detect in vivo genotoxicity per se with bacterial mutagens and Ames-positive carcinogens. TGR and comet assay results were not significantly different when compared with IARC Group 1, 2 A, and unclassified carcinogens. There were significantly more comet assay positive responses for Group 2B chemicals, and for IARC classified and unclassified carcinogens combined, which may be expected since mutation is a sub-set of genotoxicity. A liver comet assay combined with the bone marrow/blood micronucleus (MNviv) test would detect in vivo genotoxins that do not exhibit tissue-specific or site-of-contact effects, and is appropriate for routine in vivo genotoxicity testing. Generally for orally administered substances, a comet assay at only one site-of-contact GI tract tissue (stomach or duodenum/jejunum) is required. In MNviv tests, evidence of target tissue exposure can be obtained in a number of different ways, as recommended by ICH S2(R1) and EFSA (Hardy et al., 2017). Except for special cases the i.p. route is inappropriate for in vivo testing; for risk evaluations more weight should be given to data from a physiologically relevant administration route. The liver MN test is sufficiently validated for the development of an OECD guideline. However, the impact of dosing animals >6 weeks of age needs to be evaluated. The GI tract MN test shows promise but needs more validation for an OECD guideline. The Pig-a assay detects systemically available mutagens and is a valuable follow-up to in vitro positive results. A new freeze-thaw protocol provides more flexibility. Mutant reticulocyte and erythrocyte frequencies should both be determined. Preliminary data are available for the Pig-a assay in male rat germ cells which require validation including germ cell DNA mutation origin.

Evaluation of the novel liver micronucleus assay using formalin-fixed tissues.

BACKGROUND: The repeated-dose liver micronucleus (RDLMN) assay is an effective and important in vivo test for detecting genotoxic compounds, particularly for those that require metabolic activation to show genotoxicity. In a collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS) - Mammalian Mutagenicity Study Group (MMS), micronucleus induction of 22 chemicals with the RDLMN assay employing the collagenase digestion method was examined and reported on. Recently, we have developed a method which enables retrospective evaluation of micronucleus induction in formalin-fixed liver tissues (the formalin-fixed method) obtained in general toxicity studies completed in the past. Using this method, we were able to easily evaluate clastogenic potential of chemicals from the formalin-fixed tissues obtained in the general toxicity studies.In this study, to evaluate the usefulness of the formalin-fixed method, we have conducted a liver micronucleus assay using the formalin-fixed liver samples obtained from the above collaborative study (18 of 22 test chemicals) and carried out a comparison with the results obtained by the collagenase digestion method. RESULTS: Comparison of the collagenase digestion and formalin-fixed methods was conducted using the results of the micronucleus assays with a total of 18 test chemicals which included 12 genotoxic hepatocarcinogens (Group A), 4 genotoxic carcinogens but not liver targeted (Group B), and 2 nongenotoxic hepatocarcinogens (Group C). The formalin-fixed method obtained the similar results as the collagenase digestion method in 10 out of the 12 chemicals of Group A, and all chemicals of Group B and Group C. Although the results were statistically contradictive due to different levels of concurrent negative control, the 2 other chemicals of Group A showed comparable responses between the two methods. CONCLUSION: The present study shows that the formalin-fixed method is capable of detecting liver carcinogens with sensitivity equal to or higher than that of the collagenase digestion method. We recommend use of the formalin-fixed method because of its capability of enabling retrospective evaluation of micronucleus induction in the formalin-fixed liver tissues obtained in general toxicity studies completed in the past.

Interaction potential surface between Raman scattering enhancing nanoparticles conjugated with a functional copolymer.

A novel Raman scattering enhancement was discovered using colloid nanoparticles conjugated with an amine-based copolymer. The interaction potential surface between Raman scattering enhancing nanoparticles was clarified by combining a small-angle scattering method and a model-potential-free liquid-state theory as an in situ observation in the solution state. The potential surface indicates that the most stable position is located around 0.9 nm from the particle surface, suggesting the existence of a nanogap structure between the nanocomposites. The change in Raman scattering enhancement was also acquired during the dispersion process of the aggregated nanocomposites through a glutathione-triggered nanosensing reaction.

[Successful One-Stage Operation for Transverse Colon Cancer with Gastrocolic Fistula and Synchronous Rectal Cancer].

A 76-year-old man visited our hospital with complaints of appetite loss and diarrhea. Abdominal computed tomography (CT)showed a large transverse colon tumor at the splenic flexure and a gastrocolic fistula. Upper gastrointestinal series and gastroscopy demonstrated a type 2 tumor in the transverse colon and a gastrocolic fistula as the scope was inserted into the transverse colon. Colonoscopy showed a type 2 tumor of the rectum. Based on the diagnosis of advanced transverse colon cancer with gastrocolic fistula and synchronous rectal cancer, a one-stage curative operation was performed. Pathologically, both cancers were well-differentiated adenocarcinomas, but the transvers colon cancer was partially mucinous. Lymph node metastasis was absent. Gastrocolic fistula complicating colon cancer is rare, to our knowledge, with only 29 cases reported in Japan. A curative operation was performed in 73%of these cases, including ours, and lymph node metastasis was observed in only 22%. This suggests that colon cancer with a gastrocolic fistula might undergo less lymph node metastasis despite increased invasion depth, and that a curative operation for the colon cancer and gastrocolic fistula should be considered.

In silico prediction of chromosome damage: comparison of three (Q)SAR models.

Two major endpoints for genotoxicity tests are gene mutation and chromosome damage (CD), which includes clastogenicity and aneugenicity detected by chromosomal aberration (CA) test or micronucleus (MN) test. Many in silico prediction systems for bacterial mutagenicity (i.e. Ames test results) have been developed and marketed. They show good performance for prediction of Ames mutagenicity. On the other hand, it seems that in silico prediction of CD does not progress as much as Ames prediction. Reasons for this include different mechanisms and detection methods, many false positives and conflicting test results. However, some (quantitative) structure-activity relationship ((Q)SAR) models (e.g. Derek Nexus [Derek], ADMEWorks [AWorks] and CASE Ultra [MCase]) can predict CA test results. Therefore, performances of the three (Q)SAR models were compared using the expanded Carcinogenicity Genotoxicity eXperience (CGX) dataset for understanding current situations and future development. The constructed dataset contained 440 chemicals (325 carcinogens and 115 non-carcinogens). Sensitivity, specificity, accuracy or applicability of each model were 56.0, 86.9, 68.6 or 89.1% in Derek, 67.7, 61.5, 65.2 or 99.3% in AWorks, and 91.0, 64.9, 80.5 or 97.7% in MCase, respectively. The performances (sensitivity and accuracy) of MCase were higher than those of Derek or AWorks. Analysis of predictivity of (Q)SAR models of certain chemical classes revealed no remarkable differences among the models. The tendency of positive prediction by (Q)SAR models was observed in alkylating agents, aromatic amines or amides, aromatic nitro compounds, epoxides, halides and N-nitro or N-nitroso compounds. In an additional investigation, high sensitivity but low specificity was noted in in vivo MN prediction by MCase. Refinement of test data to be used for in silico system (e.g. consideration of cytotoxicity or re-evaluation of conflicting test results) will be needed to improve performance of CD prediction.

A decision tree-based integrated testing strategy for tailor-made carcinogenicity evaluation of test substances using genotoxicity test results and chemical spaces.

Genotoxicity evaluation has been widely used to estimate the carcinogenicity of test substances during safety evaluation. However, the latest strategies using genotoxicity tests give more weight to sensitivity; therefore, their accuracy has been very low. For precise carcinogenicity evaluation, we attempted to establish an integrated testing strategy for the tailor-made carcinogenicity evaluation of test materials, considering the relationships among genotoxicity test results (Ames, in vitro mammalian genotoxicity and in vivo micronucleus), carcinogenicity test results and chemical properties (molecular weight, logKow and 179 organic functional groups). By analyzing the toxicological information and chemical properties of 230 chemicals, including 184 carcinogens in the Carcinogenicity Genotoxicity eXperience database, a decision tree for carcinogenicity evaluation was optimised statistically. A decision forest model was generated using a machine-learning method-random forest-which comprises thousands of decision trees. As a result, balanced accuracies in cross-validation of the optimised decision tree and decision forest model, considering chemical space (71.5% and 75.5%, respectively), were higher than balanced accuracy of an example regulatory decision tree (54.1%). Moreover, the statistical optimisation of tree-based models revealed significant organic functional groups that would cause false prediction in standard genotoxicity tests and non-genotoxic carcinogenicity (e.g., organic amide and thioamide, saturated heterocyclic fragment and aryl halide). In vitro genotoxicity tests were the most important parameters in all models, even when in silico parameters were integrated. Although external validation is required, the findings of the integrated testing strategies established herein will contribute to precise carcinogenicity evaluation and to determine new mechanistic hypotheses of carcinogenicity.

Weight of contribution of in vitro chromosomal aberration assay for evaluation of pesticides: Experience of risk assessment at the Food Safety Commission of Japan.

Due to the course of registration of pesticides in Japan, the Food Safety Commission (FSC) has the responsibility to make a risk assessment of residual pesticides and related chemicals through foods. Among the set of safety evaluations for pesticides, genotoxicity assay data are mandatory. The standard test battery for this evaluation consists of a bacterial gene mutation assay, in vitro mammalian chromosomal aberrations and/or other chromosome damage assay, and in vivo rodent micronucleus assay. These assay outcomes are used for mechanistic consideration of carcinogenicity, if any. As a rule, if a certain substance is carcinogenic and the mechanism of it includes genotoxicity, the FSC might decide it is not possible to establish the acceptable daily intake of that pesticide. Therefore, the information about genotoxicity is critical for potentially carcinogenic chemicals, whether the applied substance will be adopted and permitted for use or not as pesticides. It is important to assess fairly, carefully, and transparently, but feasible, rapid, and efficient assessment also should be taken into account. Therefore, needless to say, the assay(s) should have the sensitivity to detect potent mutagens. It is also important to be aware that the required data set should be consisted of reliable assays without certain assay(s) that give(s) false positive information or offer less of a contribution for the safety assessment.

Genotoxicity assessment of 4-methylimidazole: regulatory perspectives.

4-Methylimidazole (4-MI) is formed as a result of the Maillard reaction process, and therefore is found in many foods and beverages. It is also found in soft drinks (i.e., cola) as a by-product in the production of some caramel colors. NTP bioassays revealed clear evidence of lung carcinogenicity of 4-MI in male and female mice, but not in rats and then IARC classified 4-MI as group 2B carcinogen. Genotoxicity studies with 4-MI were negative in the Ames tests and in the erythrocyte micronucleus tests with mice or rats. US California EPA (CEPA) evaluated the testing has not been adequately comprehensive to rule out a genotoxic mode of action; as target tissue of the carcinogenicity of 4-MI was lung, the lung should be used as a source tissue for in vitro metabolic activation system. Thus, CEPA defined the No Significant Risk Level (NSRL) for 10-5 lifetime risk level of cancer by 4-MI as 29 µg/day based on the non-threshold approach. As higher levels of 4-MI than the NSRL were identified in some kinds of cola, health concerns of 4-MI were drawn the attention. On the other hand, other regulatory bodies (e.g., European Food Safety Authority, EFSA) showed no concerns of 4-MI from the use of caramel colors in food. EFSA evaluated 4-MI is not genotoxic, so, non-observed adverse effect level of 4-MI was considered to be 80 mg/kg/day. In this paper, genotoxic assessments of 4-MI in different regulatory bodies are presented and the risk evaluation of 4-MI is discussed based on new genotoxicity data.