Bag1 protein loss sensitizes mouse embryonic fibroblasts to glutathione depletion.

Bcl2-associated athanogene-1 protein (Bag1) acts as a co-chaperone of heat shock protein 70 and heat shock cognate 70 (HSP70/HSC70) and regulates multiple cellular processes, including cell proliferation, apoptosis, environmental stress response, and drug resistance. Since Bag1 knockout mice exhibited fetal lethality, the in vivo function of Bag1 remains unclear. In this study, we established a mouse line expressing Bag1 gene missing exon 5, which corresponds to an encoding region for the interface of HSP70/HSC70. Despite mice carrying homoalleles of the Bag1 mutant (Bag1Δex5) expressing undetectable levels of Bag1, Bag1Δex5 homozygous mice developed without abnormalities. Bag1Δex5 protein was found to be highly unstable in cells and in vitro. We found that the growth of mouse embryonic fibroblasts (MEFs) derived from Bag1Δex5-homo mice was attenuated by doxorubicin and a glutathione (GSH) synthesis inhibitor, buthionine sulfoximine (BSO). In response to BSO, Bag1Δex5-MEFs exhibited a higher dropping rate of GSH relative to the oxidized glutathione level. In addition, Bag1 might mitigate cellular hydrogen peroxide levels. Taken together, our results demonstrate that the loss of Bag1 did not affect mouse development and that Bag1 is involved in intracellular GSH homeostasis, namely redox homeostasis.

Efficacy of Recombinant Methioninase on Late-stage Patient Cancer in the Histoculture Drug Response Assay (HDRA) as a Potential Functional Biomarker of Sensitivity to Methionine-restriction Therapy in the Clinic.

Background/Aim: The present study utilized the three-dimensional histoculture drug response assay (HDRA) to determine the efficacy of recombinant methioninase (rMETase) on tumor tissue resected from patients with late-stage cancer, as a functional biomarker of sensitivity to methionine restriction therapy. Patients and Methods: Resected peritoneal-metastatic cancer, including colorectal cancer, pancreatic cancer, ovarian cancer, and pseudomyxoma were placed on Gelform in RPMI 1640 medium for seven days and treated with rMETase from 2.5 U/ml to 20 U/ml. Cell viability was determined using the MTT assay. A total of 48 patients with late-stage cancer underwent testing for rMETase responsiveness using the HDRA. Results: Colorectal cancer and pseudomyxoma had the highest sensitivity to rMETase. Pancreatic and ovarian cancer also responded to rMETase, but to a lesser degree. Conclusion: Patients with tumors with at least 40% sensitivity to rMETase in the HDRA are being considered as candidates for methionine restriction therapy, which includes the use of rMETase in combination with a low-methionine diet.

Visualization of Modified Bisarylbutadiyne-Tagged Small Molecules in Live-Cell Nuclei by Stimulated Raman Scattering Microscopy.

Visualizing the distribution of small-molecule drugs in living cells is an important strategy for developing specific, effective, and minimally toxic drugs. As an alternative to fluorescence imaging using bulky fluorophores or cell fixation, stimulated Raman scattering (SRS) imaging combined with bisarylbutadiyne (BADY) tagging enables the observation of small molecules closer to their native intracellular state. However, there is evidence that the physicochemical properties of BADY-tagged analogues of small-molecule drugs differ significantly from those of their parent drugs, potentially affecting their intracellular distribution. Herein, we developed a modified BADY to reduce deviations in physicochemical properties (in particular, lipophilicity and membrane permeability) between tagged and parent drugs, while maintaining high Raman activity in live-cell SRS imaging. We highlight the practical application of this approach by revealing the nuclear distribution of a modified BADY-tagged analogue of JQ1, a bromodomain and extra-terminal motif inhibitor with applications in targeted cancer therapy, in living HeLa cells. The modified BADY, methoxypyridazyl pyrimidyl butadiyne (MPDY), revealed intranuclear JQ1, while BADY-tagged JQ1 did not show a clear nuclear signal. We anticipate that the present approach combining MPDY tagging with live-cell SRS imaging provides important insight into the behavior of intracellular drugs and represents a promising avenue for improving drug development.

Extracellular vesicles of Weizmannia coagulans lilac-01 reduced cell death of primary microglia and increased mitochondrial content in dermal fibroblasts in vitro.

We investigated the properties of extracellular vesicles from the probiotic Weizmannia coagulans lilac-01 (Lilac-01EVs). The phospholipids in the Lilac-01EV membrane were phosphatidylglycerol and mitochondria-specific cardiolipin. We found that applying Lilac-01EVs to primary rat microglia in vitro resulted in a reduction in primary microglial cell death (P < .05). Lilac-01EVs, which contain cardiolipin and phosphatidylglycerol, may have the potential to inhibit cell death in primary microglia. The addition of Lilac-01EVs to senescent human dermal fibroblasts suggested that Lilac-01 EVs increase the mitochondrial content without affecting their membrane potential in these cells.

One-carbon metabolizing enzyme ALDH1L1 influences mitochondrial metabolism through 5-aminoimidazole-4-carboxamide ribonucleotide accumulation and serine depletion, contributing to tumor suppression.

Tumor cells generally require large amounts of nucleotides, and thus activate de novo purine synthesis (dnPS). In the dnPS reactions, 10-formyltetrahydorofolate (10-fTHF) supplied by one-carbon metabolism is utilized as a formyl group donor. We focused on aldehyde dehydrogenase 1 family member L1 (ALDH1L1), which metabolizes 10-fTHF to tetrahydrofolate and whose expression is often attenuated in hepatocellular carcinoma (HCC). We generated ALDH1L1-expressing HuH-7 cells to perform metabolome analysis and found that intracellular levels of serine were reduced and glycine was increased. In addition, 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP), a dnPS intermediate, accumulated due to the consumption of 10-fTHF by ALDH1L1, which inhibited ZMP formylation. Importantly, ALDH1L1-expressing cells showed reduced ZMP sensitivity and higher mitochondrial activity. The suppression of mitochondrial serine catabolism by ALDH1L1 expression was speculated to be closely related to this phenotype. Gene set enrichment analysis utilizing The Cancer Genome Atlas data revealed that genes related to oxidative phosphorylation were enriched in HCC patients with high ALDH1L1 expression. Moreover, drug sensitivity data analysis demonstrated that HCC cell lines with low expression of ALDH1L1 were sensitive to ZMP and cordycepin, a structural analog of ZMP and AMP. Our study revealed that ZMP and AMP analogs might be effective in the pharmacotherapy of HCC patients with low expression of ALDH1L1.

Incarceration of a colonoscope in an inguinal hernia: A report of two cases.

We report two cases of the rare complication of a colonoscope incarcerated in an inguinal hernia. The first patient was a 73-year-old man in whom a colonoscope was incarcerated in a left inguinal hernia on attempted withdrawal. The incarcerated colonoscope was successfully reduced manually under fluoroscopic guidance. The hernia was subsequently repaired using an extraperitoneal approach followed by a successful colonoscopy. The second patient was a 74-year-old man in whom the colonoscope became incarcerated in a left inguinal hernia on insertion. Similar to the first case, the colonoscope was manually reduced under fluoroscopy and the entire colonoscopy was then uneventfully performed. An advanced sigmoid cancer was identified and treated with sigmoidectomy. The hernia resolved after this operation. When a colonoscope becomes incarcerated in an inguinal hernia, the manual reduction should be attempted. Subsequent colonoscopy can be safely performed under certain circumstances.

Percutaneous surgical repair for a patient with adult pararectal hernia caused by intractable ascites associated with liver cirrhosis.

Patients with liver cirrhosis are at increased risk of various visceral hernia because of persistent ascites and tissue fragility. Here we report successful treatment in a patient with pararectal hernia due to liver cirrhosis by a less invasive approach via para-anal region. The patient was a 73-year-old woman with a history of chronic hepatitis B that had been untreated for at least 20 years. At the age of 68 years, she was referred to our hospital for treatment of persistent ascites and thrombocytopenia due to advanced liver cirrhosis. Neither diuretics nor cell-free and concentrated ascites reinfusion therapy could decrease the ascites. She needed repeated paracentesis. She was referred to the surgical department due to the painful swelling of the left buttock which was diagnosed as the pararectal hernia. The welling was huge enough with the erosion of the covering skin. Surgery was planned in view of concern about the possible rupture of the hernia. Due to the massive ascites with the advanced liver cirrhosis, we were reluctant to do the laparotomic approach, and simple closure of the hernial orifice via direct approach from the cutaneous side was planned and performed. The patient was fortunately discharged seven days after the operation without any complications. One year later, there has been no recurrence of the hernia. Even in cases with massive ascites, direct simple closure of the hernia by percutaneous approach may be one of the options for the treatment of the pararectal hernia in case of urgent situation.

Antineutrophil Cytoplasmic Antibody-associated Vasculitis after COVID-19 Vaccination with Pfizer-BioNTech.

The extent of rare side effects of mRNA vaccines for coronavirus disease 2019 (COVID-19) remains unclear. Several cases of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) following COVID-19 vaccination have been reported. We herein report a 72-year-old man who presented with a fever after receiving the second dose of the Pfizer-BioNTech COVID-19 vaccine. He was diagnosed with acute kidney injury due to myeloperoxidase-ANCA-associated vasculitis and was treated with intermittent hemodialysis, high-dose prednisolone, and intravenous rituximab. His general symptoms and renal impairment subsequently improved. When systemic symptoms are prolonged or renal abnormalities appear after COVID-19 vaccination, the possibility of AAV should be considered.

Treatment of anomalous arterial supply to the basal segment of left lung with aortic stent graft.

The patient was a 53-year-old man with a history of recurrent sputum. An anomalous systemic arterial supply to the basal segment of the left lung with an aneurysm of the aberrant artery detected on three-dimensional computed tomography angiography. Before left lower pulmonary lobectomy and aberrant artery resection, thoracic endovascular aortic repair was performed to block the blood flow to the aberrant artery aneurysm. Prior blockade of the blood flow to the aneurysm minimized the risk of aneurysm rupture and bleeding during lobectomy, yielding a good postoperative outcome.

Case of a lung collision tumor consisting of squamous cell carcinoma of the lung and diffuse large B-cell lymphoma.

Among the reports of malignant collision tumors, collision tumors consisting of lung cancer and malignant lymphoma are extremely rare. We report case of a lung collision tumor consisting of squamous cell carcinoma of the lung and diffuse large B-cell lymphoma.

Quantification of Bisacurone and Curcuminoids in Turmeric Products by Liquid Chromatography Coupled with Tandem Mass Spectrometry.

Turmeric products have many useful physiological functions and are widely used as health food and food ingredient. Here, we report the use of HPLC-ESI-MS/MS to simultaneously quantify bisacurone and three curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) in turmeric products (high viscosity liquid, granular powder, tablet, and solution). The results showed that the standard values and measured values of curcumin in each product were almost same. Demethoxycurcumin and bisdemethoxycurcumin were contained in each products. Meanwhile, the content of bisacurone differed greatly among the products. In particular, the highest amount of bisacurone was found in the turmeric product A (high viscosity liquid, 9.48 g/100 g product). It would become important to consider the bisacurone content in turmeric products.

Adenoid cystic carcinoma of the sublingual gland developing lung metastasis 20 years after primary treatment: A case report and literature review.

RATIONALE: Adenoid cystic carcinoma (ACC) is a rare malignant tumor that primarily occurs in the salivary glands. Distant metastases can develop despite favorable local control. Moreover, distant metastasis of ACC can occur after a long time interval without local recurrence. We report the first case of ACC of the sublingual gland that developed lung metastasis 20 years after primary treatment. PATIENT CONCERNS: A 52-year-old man was referred to our department with a 1-year history of painful swelling on the right oral floor. DIAGNOSIS: An incisional biopsy was performed, and histopathological examination revealed malignancy. INTERVENTIONS: Surgical excision of the right oral floor and right supra-omohyoid neck dissection with postoperative chemoradiation therapy were performed, and ACC of the sublingual gland was diagnosed. Left pulmonary metastasis was detected 20 years after the primary treatment. Metastasectomy was performed; however, subsequently, skin and bone metastases developed. OUTCOMES: After receiving palliative care, the patient died of multiple organ failure. LESSONS: As late distant metastasis of salivary ACC can develop, patients who undergo primary treatment need a long-term, strict follow-up plan even if locoregional control is favorable.

Removal of chlorophyll and pheophorbide from Chlorella pyrenoidosa by supercritical fluid extraction: potential of protein resource.

This study tried to quantitatively clarify the usefulness of supercritical fluid extraction for removal of chlorophyll and pheophorbide from Chlorella pyrenoidosa. C. pyrenoidosa powder was subjected to supercritical fluid extraction, and chlorophyll a and pheophorbide a in its extracted fractions were measured by HPLC-UV. Chlorophyll a and pheophorbide a in residue after supercritical fluid extraction became below of detection limit.

Cell wall N-glycan of Candida albicans ameliorates early hyper- and late hypo-immunoreactivity in sepsis.

Severe infection often causes a septic cytokine storm followed by immune exhaustion/paralysis. Not surprisingly, many pathogens are equipped with various anti-inflammatory mechanisms. Such mechanisms might be leveraged clinically to control septic cytokine storms. Here we show that N-glycan from pathogenic C. albicans ameliorates mouse sepsis through immunosuppressive cytokine IL-10. In a sepsis model using lipopolysaccharide (LPS), injection of the N-glycan upregulated serum IL-10, and suppressed pro-inflammatory IL-1ß, TNF-α and IFN-γ. The N-glycan also improved the survival of mice challenged by LPS. Analyses of structurally defined N-glycans from several yeast strains revealed that the mannose core is key to the upregulation of IL-10. Knocking out the C-type lectin Dectin-2 abrogated the N-glycan-mediated IL-10 augmentation. Furthermore, C. albicans N-glycan ameliorated immune exhaustion/immune paralysis after acute inflammation. Our results suggest a strategy where the immunosuppressive mechanism of one pathogen can be applied to attenuate a severe inflammation/cytokine storm caused by another pathogen.

Intratumoral gene expression of dihydrofolate reductase and folylpoly-c-glutamate synthetase affects the sensitivity to 5-fluorouracil in non-small cell lung cancer.

BACKGROUND: Various factors related to the sensitivity of non-small cell lung carcinoma (NSCLC) to 5-fluorouracil (5-FU) have been reported, and some of them have been clinically applied. In this single-institutional prospective analysis, the mRNA expression level of five folic acid-associated enzymes was evaluated in surgical specimens of NSCLC. We investigated the correlation between the antitumor effect of 5-FU in NSCLC using an anticancer drug sensitivity test and the gene expression levels of five enzymes. MATERIALS AND METHODS: Forty patients who underwent surgery for NSCLC were enrolled, and the antitumor effect was measured using an in vitro anticancer drug sensitivity test (histoculture drug response assay) using freshly resected specimens. In the same sample, the mRNA expression levels of five enzymes involved in the sensitivity to 5-FU were measured in the tumor using real-time PCR. The expression levels and the result of the sensitivity test were compared. RESULTS: No correlation was found between dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase (OPRT), or DPD/OPRT expression and the antitumor effects of 5-FU. On the other hand, a correlation was found between thymidylate synthase (TS), folylpoly-c-glutamate synthetase (FPGS), and dihydrofolate reductase (DHFR) expression and 5-FU sensitivity. CONCLUSION: Expression of FPGS and DHFR may be useful for predicting the efficacy of 5-FU-based chemotherapy for NSCLC.

Novel LCAT (Lecithin:Cholesterol Acyltransferase) Activator DS-8190a Prevents the Progression of Plaque Accumulation in Atherosclerosis Models.

OBJECTIVE: Enhancement of LCAT (lecithin:cholesterol acyltransferase) activity has possibility to be beneficial for atherosclerosis. To evaluate this concept, we characterized our novel, orally administered, small-molecule LCAT activator DS-8190a, which was created from high-throughput screening and subsequent derivatization. We also focused on its mechanism of LCAT activation and the therapeutic activity with improvement of HDL (high-density lipoprotein) functionality. Approach and Results: DS-8190a activated human and cynomolgus monkey but not mouse LCAT enzymes in vitro. DS-8190a was orally administered to cynomolgus monkeys and dose dependently increased LCAT activity (2.0-fold in 3 mg/kg group on day 7), resulting in HDL cholesterol elevation without drastic changes of non-HDL cholesterol. Atheroprotective effects were then evaluated using Ldl-r KO×hLcat Tg mice fed a Western diet for 8 weeks. DS-8190a treatment achieved significant reduction of atherosclerotic lesion area (48.3% reduction in 10 mg/kg treatment group). Furthermore, we conducted reverse cholesterol transport study using Ldl-r KO×hLcat Tg mice intraperitoneally injected with J774A.1 cells loaded with [3H]-cholesterol and confirmed significant increases of [3H] count in plasma (1.4-fold) and feces (1.4-fold on day 2 and 1.5-fold on day3) in the DS-8190a-treated group. With regard to the molecular mechanism involved, direct binding of DS-8190a to human LCAT protein was confirmed by 2 different approaches: affinity purification by DS-8190a-immobilized beads and thermal shift assay. In addition, the candidate binding site of DS-8190a in human LCAT protein was identified by photoaffinity labeling. CONCLUSIONS: This study demonstrates the potential of DS-8190a as a novel therapeutic for atherosclerosis. In addition, this compound proves that a small-molecule direct LCAT activator can achieve HDL-C elevation in monkey and reduction of atherosclerotic lesion area with enhanced HDL function in rodent.

Conversion hepatectomy for advanced hepatocellular carcinoma after right portal vein transection and lenvatinib therapy.

BACKGROUND: Lenvatinib is a novel tyrosine kinase inhibitor that exhibits an antitumor effect on hepatocellular carcinoma (HCC). An established strategy that involves surgery and usage of lenvatinib for advanced HCC remains elusive. CASE PRESENTATION: A 58-year-old male patient with advanced HCC and untreated hepatitis B was referred to our hospital. The tumor at the right lobe was 10 cm in diameter with right portal vein thrombus. Because of the possible lung metastasis and concern about the remaining hepatic function after extended right hepatectomy, lenvatinib was initiated before surgery. After the confirmation of a sharp decrease of tumor markers during the 3-week lenvatinib therapy, only a right portal vein transection was done leaving the enlargement of the left lobe for improved post-hepatectomy liver function while lenvatinib therapy was continued. The laparotomy revealed that the tumor was invading the right diaphragm. After 7 weeks of lenvatinib administration after right portal vein transection, an extended right hepatectomy with resection of the tumor-invaded diaphragm was successfully done. The lung nodules that were suspected as metastases had disappeared. The patient has been doing well without any sign of recurrence for 1 year. CONCLUSION: The strategy involving the induction of lenvatinib to conversion hepatectomy including the portal vein transection was effective for advanced HCC.

[Internet survey of the actual situation of constipation in Japanese general population REACTION-J:Research for Actual Situation of Constipation in the Japanese].

Although chronic constipation is highly prevalent, its definition remains unclear. Therefore, the prevalence varies depending on reports, and the understanding of actual situations is unclear. Hence, we performed an internet survey on constipation among the Japanese general population to study the background factors and actual situations. Preliminary study on the awareness of constipation was conducted among 10000 people in which 9523 of them was asked if they had constipation at the time of the survey. In this population, 51.5% realized that they had constipation. Multivariate analysis showed the significant association of constipation to age, sex, and past histories or complications of diabetes, hemorrhoids, and cerebrovascular diseases. In a main research composed of 3000 general Japanese population, approximately 30.9% of the subjects reported the use of laxatives to treat constipation, and 43.8% of them were found to use irritant laxatives. Moreover, 67.5% of the subjects purchased laxatives at a pharmacy. The frequency of bowel movement less than 3 times per week was manifested in 36.3% of the subjects, and more than once per week in 21.4%. The percentage of hard (Bristol Stool Form Scale [BSFS] Type 1-2), normal (BSFS Type 3-5), and diarrhea stools (BSFS Type 6-7) was 33.1%, 60.0%, and 6.9%, respectively. The quality of life (QOL) of the subjects with hard and diarrhea stools evaluated by SF-8 was significantly lower than that of those with normal stools. Furthermore, the actual monthly cost for the therapeutic drugs used for treating constipation was less than 1000 yen in 75% of the subjects. Analysis of the IBS-QOL-J indicated that the ≥5000 yen payable group had the lowest satisfaction of defecation among the study groups. At present, many Japanese patients with constipation have not been receiving enough treatment for constipation. Therefore, appropriate medication by physicians as well as instruction to patients is required.

Target identification of hepcidin production inhibitors by a combination of chemical proteomics and radioactive compound binding assay.

Hepcidin is a peptide hormone and has emerged as the central molecule regulating systemic iron homeostasis. Hepcidin inhibition could be a strategy for treating anemia of chronic disease. We previously reported the discovery of DS79182026, a new inhibitor of hepcidin production, from phenotypic screening using the human hepatocyte HepG2 cell line. In this study, we utilized a combination of affinity purification-based chemical proteomics and radioactive compound binding assay, and identified several candidate proteins. Purified recombinant proteins were subjected to radioactive compound binding assays for validation, and ALK2 and ALK3 demonstrated specific binding to the compound. Since ALK2 is known to be related to hepcidin production, we focused on ALK2 and found that its knockdown decreased hepcidin expression; we also found a strong correlation (R = 0.920) between pharmacological activity and compound affinity to ALK2. These results indicate that ALK2 is the primary target protein of our new hepcidin production inhibitors.

Simultaneous Visualization of Vessels and Brain Tumor with Contrast-enhanced Three-dimensional Phase-contrast MR Imaging.

The sequence for concurrently depicting engulfed vessels and a well-enhanced tumor in once-off scanning has never been reported for preoperative magnetic resonance imaging for brain tumor resection. Multimodal fusion techniques have been recently developed, but the risks of misregistration still remain. Here a case is reported where contrast-enhanced three-dimensional phase contrast sequence concurrently depicted an engulfed vessel and metastatic brain tumor in once-off scanning and related technical aspects are discussed.