Development of a potent small-molecule degrader against oncogenic BRAFV600E protein that evades paradoxical MAPK activation.

BRAF mutations are frequently observed in melanoma and hairy-cell leukemia. Currently approved rapidly accelerated fibrosarcoma (RAF) kinase inhibitors targeting oncogenic BRAF V600 mutations have shown remarkable efficacy in the clinic, but their therapeutic benefits are occasionally hampered by acquired resistance due to RAF dimerization-dependent reactivation of the downstream MAPK pathway, which is known as paradoxical activation. There is also a concern that paradoxical activation of the MAPK pathway may trigger secondary cancer progression. In this study, we developed chimeric compounds, proteolysis targeting chimeras (PROTACs), that target BRAFV600E protein for degradation. CRBN(BRAF)-24, the most effective chimera, potently degraded BRAFV600E in a ubiquitin-proteasome system (UPS)-dependent manner and inhibited the proliferation of BRAFV600E -driven cancer cells. In BRAF wild-type cells, CRBN(BRAF)-24 induced neither BRAFWT degradation nor paradoxical activation of the MAPK pathway. Biochemical analysis revealed that CRBN(BRAF)-24 showed more potent and sustained suppression of MAPK signaling than a BRAFV600E inhibitor, PLX-8394, in BRAFV600E -driven cancer cells. Targeted degradation of BRAFV600E by CRBN(BRAF)-24 could be a promising strategy to evade paradoxical activation of the RAF-MAPK pathway.

Relationship between the vertical distribution of fine roots and residual soil nitrogen along a gradient of hardwood mixture in a conifer plantation.

In forest ecosystems, understanding the relationship between the vertical distribution of fine roots and residual soil nitrogen is essential for clarifying the diversity-productivity-water purification relationship. Vertical distributions of fine-root biomass (FRB) and concentrations of nitrate-nitrogen (NO3 -N) in soil water were investigated in a conifer plantation with three thinning intensities (Control, Weak and Intensive), in which hardwood abundance and diversity were low, moderate and high, respectively. Intensive thinning led to the lowest NO3 -N concentration in soil water at all depths (0-100 cm) and highest FRB at shallow depths (0-50 cm). The NO3 -N concentration at a given depth was negatively correlated with total FRB from the surface to the depth at which NO3 -N concentration was measured, especially at shallow depths, indicating that more abundant fine roots led to lower levels of downward NO3 -N leaching. FRB contributed positively to nitrogen content of hardwood leaves. These findings demonstrate that a hardwood mixture in conifer plantations resulted in sufficient uptake of NO3 -N from soil by well developed fine-root systems, and translocation to canopy foliage. This study suggests that productivity and water purification can be achieved through a hardwood mixture in conifer plantations.

Chorea-like symptoms and high blood concentration of ceftriaxone in a patient undergoing hemodialysis: A case report.

Ceftriaxone (CTRX) is a third-generation cephalosporin commonly used to treat infections such as community-acquired pneumonia and urinary tract infections caused by mainly Gram-negative bacteria and some Gram-positive bacteria. Here, we report a case of a patient on hemodialysis who had chorea-like symptoms with high blood concentration of CTRX. A 74-year-old Japanese woman receiving hemodialysis was admitted with obstructive cholangitis and was started on CTRX therapy at a dose of 2 g every 24 hours. On the 6th day after starting administration of CTRX, chorea-like symptoms appeared. We suspected that her symptoms were caused by a high blood concentration of CTRX. We performed a series of blood sampling to determine the concentration of CTRX at different time points before and after discontinuing CTRX administration. CTRX concentrations were higher than those expected in healthy adults, and her chorea-like symptoms had disappeared from the second day of discontinuation of CTRX. The association between CTRX blood concentration and chorea-like symptoms is unclear. However, measuring a series of plasma or serum concentrations from symptom onset to disappearance suggested that chorea-like symptoms appeared when the concentration exceeded approximately 450 µg/mL. Care should be taken when administering CTRX to patients with cholestasis undergoing hemodialysis, as blood CTRX levels may rise unexpectedly and result in complications.

White Matter Lesions as Brain Frailty and Age are Risk Factors for Surgical Clipping of Unruptured Intracranial Aneurysms in the Elderly.

INTRODUCTION: We aimed to identify the risk factors for surgical treatment of unruptured intracranial aneurysms (UIAs) in individuals aged >60 years, particularly focusing on white matter lesions (WMLs). MATERIAL AND METHODS: We investigated a total of 214 patients with UIAs. The patient group comprised 53 males and 151 females with an average age of 68.2 years. UIA size ranged from 2.7 to 26 (mean: 7.3) mm. The primary endpoint of the study was patient prognosis evaluated at the time of discharge using the modified Rankin Scale. We examined the risk factors for poor outcome and WMLs using magnetic resonance imaging. RESULTS: Poor outcome was observed in 23 (10.7%) patients. Significant correlations were observed between poor outcome and UIA size (P < 0.0001), UIAs located posteriorly (P = 0.0204), UIA thrombosis (P = 0.0002), and presence of WMLs (P < 0.0001) in univariate regression analysis. However, no significant correlations were noted between poor outcome and age (P = 0.1438). Multivariate logistic regression analyses showed significant correlations between poor outcome and UIA size (P < 0.0001), presence of WMLs (P = 0.001). Severe WMLs based on the Fazekas classification was correlated to age (P < 0.0001) and atherosclerosis (P = 0.0001). Severe WMLs were associated with ischemia (P < 0.001) and epilepsy (P = 0.0502) as well as length of hospitalization (P < 0.0001). CONCLUSION: Severe WMLs are risk factors for surgical treatment of UIAs in the elderly. Surgical indications must be considered and caution should be taken when managing patients with severe WMLs.

Investigation of Biofilms Formed on Steelmaking Slags in Marine Environments for Water Depuration.

Steelmaking slags are a promising resource as artificial seaweed beds for the reconstitution of marine environments. To grow seaweed well, the formation of biofilms is an essential process in biofouling. This study focused on the formation of initial biofilms on steelmaking slag samples and analyzed the resulting bacterial communities using the next-generation sequencing technique. Three types of steelmaking slag were submerged in an area of Ise Bay in Mie Prefecture, Japan, for 3 and 7 days in the summer and winter seasons to allow the formation of biofilms. The bacterial communities of these biofilms were richer in sulfur-oxidizing bacteria compared to the biofilms formed on polyurethane sponges. It was found that Helicobacteraceae dominantly grew on the biofilms formed on the slag samples. This shows that steelmaking slags have potential to be used as artificial seaweed beds and marine water purifiers.

Outcomes in Elderly Japanese Patients Treated for Aneurysmal Subarachnoid Hemorrhage: A Retrospective Nationwide Study.

OBJECTIVE: Japan has the largest elderly population in the world. As data on the clinical outcomes in elderly patients with aneurysmal subarachnoid hemorrhage (aSAH), including those older than 80 years, are lacking, we analyzed the characteristics of 54,805 aSAH patients and recorded their treatments and clinical outcomes using a Japanese nationwide inpatient database. METHODS: Using the Japanese Diagnostic Procedure Combination database, we identified aSAH patients aged 18 years or older who were hospitalized between July 1, 2010 and March 31, 2016. They were categorized as less than or equal to 60-, 61-70-, 71-80-, 81-90-, and greater than or equal to 91 years of age. The primary outcome was the modified Rankin Scale (mRS) score at discharge. Multivariable logistic regression analysis was performed to examine factors affecting the mRS score at discharge. RESULTS: Of 54,805 patients, 37.5% were aged less than or equal to 60 years; 24.8% were 61-70-, 21.8% were 71-80-, 13.9% were 81-90-, and 2.0% were greater than or equal to 91 years old at the time of the insult. Among 46,107 patients younger than 81 years, 58.9% underwent surgical clipping (SC), 22.9% endovascular coiling (EC), and 18.2% were treated conservatively. There were 8,698 patients aged 81 years or older, 32.4% underwent SC, 23.2% EC, and 44.4% were treated conservatively. A poor mRS score (3-6) at discharge was recorded in 87.2% of patients older than 80 years. Multivariable logistic regression was used to compare their estimated odds ratio (OR) for a poor mRS score at discharge with that of patients aged less than or equal to 60 years. The OR increased by 87% in patients between 61 and 70 years of age (P < .001; OR, 1.87; 95% confidence interval (CI), 1.77-1.98), by 358% in patients aged from 71 to 80 years (P < .001; OR, 4.58; 95%CI, 4.29-4.89), by 1,035% in patients between 81 and 90 years (P < .001; OR, 11.35; 95%CI, 10.32-12.49), and by 1,710% in patients aged more than or equal to 91 years (P < .001; OR, 18.10; 95%CI, 13.96-23.46). CONCLUSIONS: As the treatment outcomes in elderly aSAH patients, especially those 80 years old or older, were poor, the appropriate therapy decisions must be made on a case-by-case basis.

Risk of Out-of-Hospital Cardiac Arrest in Aged Individuals in Relation to Cold Ambient Temperature　- A Report From North Tochigi Experience.

BACKGROUND: The impact of cold ambient temperature on out-of-hospital cardiac arrest (OHCA) in aged individuals caused by cardiovascular events in indoor environments has not been investigated sufficiently.Methods and Results:We conducted a case-crossover study. The relationship between OHCA caused by cardiovascular events and exposure to minimum temperature <0℃ was analyzed. Conditional logistic regression analysis was performed to estimate the odds ratios for the relationship between exposure to minimum temperature <0℃ and the risk of OHCA. Between January 1, 2011, and December 31, 2015, a total of 1,452 cases of OHCA were documented, and patients were screened for enrollment. A total of 458 individuals were enrolled in this analysis, and were divided into 2 groups of 110 (elderly group: 65-74 years old) and 348 (aged group: ≥75 years old). The aged individuals had a significant increased risk of OHCA after exposure to minimum temperature <0℃ (odds ratio [OR]: 1.528, 95% confidence interval [CI] 1.009-2.315, P=0.045). Cold ambient temperature was an especially significant increased risk for OHCA occurrence for males (OR: 1.997, 95% CI 1.036-3.773, P=0.039) and during winter (OR: 2.391, 95% CI 1.312-4.360, P=0.004) in the aged group. CONCLUSIONS: Cold ambient temperature significantly affected aged individuals (≥75 years old) experiencing an OHCA caused by cardiovascular events in indoor environments.

Hydroxyl-Group Identification Using O K-Edge XAFS in Porous Glass Fabricated by Hydrothermal Reaction and Low-Temperature Foaming.

Porous glass was prepared by the hydrothermal reaction of sodium borosilicate glass, and oxygen-ion characterization was used to identify the hydroxyl groups in its surface area. A substantial amount of "water" was introduced into the ionic structure as either OH- groups or H2O molecules through the hydrothermal reaction. When the hydrothermally treated glass was reheated at normal pressures, a porous structure was formed due to the low-temperature foaming resulting from the evaporation of H2O molecules and softening of the glass. Although it was expected that the OH- groups would remain in the porous glass, their distribution required clarification. Oxygen K-edge X-ray absorption fine structure (XAFS) spectroscopy enables the bonding states of oxygen ions in the surface area and interior to be characterized using the electron yield (EY) and fluorescence yield (FY) mode, respectively. The presence of OH- groups was detected in the O K-edge XAFS spectrum of the porous glass prepared by hydrothermal reaction with a corresponding pre-edge peak energy of 533.1 eV. In addition, comparison of the XAFS spectra obtained in the EY and FY modes revealed that the OH- groups were mainly distributed in the surface area (depths of several tens of nanometers).

Pharmacist-led intervention in the multidisciplinary team approach optimizes heart failure medication.

We evaluated the impact of pharmacist-led heart failure (HF) drug recommendations during hospitalization for hospitalized patients with HF. Hospitalized patients with HF were retrospectively reviewed. Patients were hospitalized before (n = 208, non-intervention group) or after (n = 170, intervention group) the launch of the HF multidisciplinary team (HFMDT) approach with pharmacist-led HF medication optimization. There were no significant group differences in patient background characteristics at admission. Patients with HF with reduced ejection fraction who were not on beta blockers or angiotensin-converting enzyme inhibitor/angiotensin receptor blockers (ACE-I/ARB) at admission were significantly more likely to be on beta blockers at the time of discharge in the intervention group (73.3 vs 96.3%, P = 0.027) compared to those in non-intervention group; however, the change in ACE-I/ARB prescriptions was not significant (53.3 vs 63.3%, P = 0.601). The proportion of patients on any drug with recommendations against its use in patients with HF did not change from admission to discharge in the non-intervention group (21.2 vs. 20.2%, P = 0.855), but was significantly reduced in the intervention group (22.9 vs. 12.9%, P = 0.005). There were no group differences in the in-hospital all-cause mortality (non-intervention, 3.4%; intervention, 2.4%; P = 0.761) or length of hospital stay (median: non-intervention, 13 days; intervention, 14 days; P = 0.508). Pharmacist-led HF drug recommendations during hospitalization as part of a HFMDT approach for hospitalized patients with HF can increase beta blocker prescriptions and decrease non-preferred drug prescriptions.

A superficial esophageal cancer in an epiphrenic diverticulum treated by endoscopic submucosal dissection.

BACKGROUND: We report a unique case of a superficial esophageal cancer arising in a single diverticulum, diagnosed with magnifying image-enhanced endoscopy and then successfully treated by endoscopic submucosal dissection (ESD). CASE PRESENTATION: A 66-year-old man with alcohol-related liver injury visited our hospital for endoscopy for investigation of varix. Esophagogastroduodenoscopy showed no varix but a large epiphrenic diverticulum with an area of fainted redness just above the esophagogastric junction. Narrow band imaging revealed a sharply demarcated brownish dotted area, and dilated intra-epithelial papillary capillary loops (IPCL) were subsequently seen after magnification. Chromoendoscopy with 1% Lugol's iodine solution demonstrated a well-demarcated unstained area, approximately 20 mm in diameter. Endoscopic biopsy revealed a squamous cell carcinoma (SCC). CONCLUSION: The tumor was completely resected by ESD without perforation. Histologically, it was an intraepithelial SCC without lympho-vascular invasion of cancer cells. No local recurrence or metastasis was detected at the last follow-up of 42 months.

Retrograde suction decompression of a large internal carotid aneurysm using a balloon guide catheter combined with a blood-returning circuit and STA-MCA bypass: a technical note.

It is difficult to treat large internal carotid aneurysms with simple surgical clipping. Here, we present a retrograde suction decompression (RSD) procedure for large internal carotid aneurysms using a balloon guide catheter combined with a blood-returning circuit and a superficial temporal artery to middle cerebral artery (STA-MCA) bypass.All patients underwent an STA-MCA bypass before the temporary occlusion of the internal carotid artery (ICA). A 6-French sheath was inserted into the common carotid artery (CCA), and a 6-French Patrive balloon catheter was placed into the ICA 5 cm past the bifurcation. Aneurysm exposure was obtained; temporary clips were placed on the proximal M1, A1, and posterior communicating (Pcom) segments; and an extension tube was then connected to the balloon catheter. A three-way stopcock was placed, and aspiration was performed through the device to collapse the aneurysm. The aspirated blood was returned to a venous line with an added heparin to prevent anemia after aspiration. During the decompression, the blood flow to the cortical area was supplied through the STA-MCA bypass. After the aneurysm collapse, the surgeon carefully dissected the perforating artery from the aneurysm dome or neck, and permanent clips were then placed on the aneurysm neck. Our procedure has several advantages, such as STA-MCA bypass without external carotid artery occlusion for preventing ischemic complications of the cortical area, anemia may be avoided because of the return of the aspirated blood, and a hybrid operation room is not required to perform this method.

Synthesis and biological evaluation of novel imidazol-1-ylacetic acid derivatives as non-brain penetrant bombesin receptor subtype-3 (BRS-3) agonists.

Novel compounds based on 1a were synthesized with the focus of obtaining agonists acting upon peripheral BRS-3. To identify potent anti-obesity compounds without adverse effects on the central nervous system (CNS), a carboxylic acid moiety and a labile carboxylic ester with an antedrug functionality were introduced. Through the extensive synthetic exploration and the pharmacokinetic studies of intravenous administration in mice, the ester 2b was selected owing to its most suitable pharmacological profile. In the evaluation of food intake suppression in C57BL/6N mice, 2b showed significant in vivo efficacy and no clear adverse effects on blood pressure change in dogs administered the compound by intravenous infusion.

A transient disruption of fibroblastic transcriptional regulatory network facilitates trans-differentiation.

Transcriptional Regulatory Networks (TRNs) coordinate multiple transcription factors (TFs) in concert to maintain tissue homeostasis and cellular function. The re-establishment of target cell TRNs has been previously implicated in direct trans-differentiation studies where the newly introduced TFs switch on a set of key regulatory factors to induce de novo expression and function. However, the extent to which TRNs in starting cell types, such as dermal fibroblasts, protect cells from undergoing cellular reprogramming remains largely unexplored. In order to identify TFs specific to maintaining the fibroblast state, we performed systematic knockdown of 18 fibroblast-enriched TFs and analyzed differential mRNA expression against the same 18 genes, building a Matrix-RNAi. The resulting expression matrix revealed seven highly interconnected TFs. Interestingly, suppressing four out of seven TFs generated lipid droplets and induced PPARG and CEBPA expression in the presence of adipocyte-inducing medium only, while negative control knockdown cells maintained fibroblastic character in the same induction regime. Global gene expression analyses further revealed that the knockdown-induced adipocytes expressed genes associated with lipid metabolism and significantly suppressed fibroblast genes. Overall, this study reveals the critical role of the TRN in protecting cells against aberrant reprogramming, and demonstrates the vulnerability of donor cell's TRNs, offering a novel strategy to induce transgene-free trans-differentiations.

Exploring factors associated with bleeding events after open heart surgery in patients on dialysis - effects of the presence or absence of warfarin therapy.

BACKGROUND: Perioperative management of patients on dialysis is critical for controlling bleeding and thrombotic risk, in addition to infection control. Postoperative anticoagulation is often difficult to control, and different institutions have different policies. Therefore, in this study, we aimed to investigate factors associated with postoperative bleeding events and whether warfarin (WF) therapy affects the incidence of postoperative bleeding events, total mortality, and stroke. METHODS: Patients who were admitted to the cardiovascular surgery department and underwent valve replacement or plasty were included, and those who underwent mechanical valve introduction were excluded. Thirty-nine patients were included in the study. The primary endpoint was to identify factors associated with the composite endpoint of postoperative bleeding events, and the secondary endpoint was to determine the effect size of WF therapy on postoperative bleeding events, all-cause mortality, and stroke and the strength of association between the crossed endpoints. The strength of the association between the crossed items was examined. RESULTS: Low body weight (p = 0.038) was identified as a factor associated with the primary endpoint of postoperative bleeding events. The secondary endpoint of whether or not patients received WF therapy was largely unrelated to bleeding events, all-cause mortality, and postoperative stroke up to 90 days after surgery. CONCLUSIONS: Preliminary studies suggest that low body weight is a risk factor for postoperative bleeding events in patients on dialysis, although further exploration of other factors will be necessary with the accumulation of similar cases.

Discovery of Novel 11-Membered Templates as Squalene Synthase Inhibitors.

Squalene synthase is one of the most promising pharmaceutical targets to treat hyperlipidemia. Inhibition of the squalene synthase causes a decrease in the hepatic cholesterol concentration. We have already reported the design and synthesis of highly potent benzhydrol-type squalene inhibitors. Although these templates showed unique and potent cyclic active conformations via intramolecular hydrogen bonds, the in vivo cholesterol-lowering efficacy was insufficient. We attempted to improve their potential as an orally active medicine. In our medicinal chemistry effort, cyclized 11-membered ring templates were acquired. The novel series of compounds exhibited potent squalene synthase inhibitory activity, and one of the derivatives, isomer A-(1S, 3R)-14i, showed plasma lipid-lowering efficacy in hamster and marmoset repeated-dose studies. Our findings provide valuable insights into the design and development of novel and unique 11-membered ring-type highly potent squalene synthase inhibitors.

An algorithm-based technique for counting mitochondria in cells using immunohistochemical staining of formalin-fixed and paraffin-embedded sections.

PURPOSE: Visualizing mitochondria in cancer cells from human pathological specimens may improve our understanding of cancer biology. However, using immunohistochemistry to evaluate mitochondria remains difficult because almost all cells contain mitochondria and the number of mitochondria per cell may have important effects on mitochondrial function. Herein, we established an objective system (Mito-score) for evaluating mitochondria using machine-based processing of hue, saturation, and value color spaces. METHODS: The Mito-score was defined as the number of COX4 (mitochondrial inner membrane) immunohistochemistry-positive pixels divided by the number of nuclei per cell. The system was validated using four lung cancer cell lines, normal tissues, and lung cancer tissues (199 cases). RESULTS: The Mito-score correlated with MitoTracker, a fluorescent dye used to selectively label and visualize mitochondria within cells under a microscope (R2 = 0.68) and with the number of mitochondria counted using electron microscopy (R2 = 0.79). Histologically, the Mito-score of small cell carcinoma (57.25) was significantly lower than that of adenocarcinoma (147.5, p < 0.0001), squamous cell carcinoma (120.6, p = 0.0004), and large cell neuroendocrine carcinoma (111.8, p = 0.002). CONCLUSION: The Mito-score method enables the analysis of the mitochondrial status of human formalin-fixed paraffin-embedded specimens and may provide insights into the metabolic status of cancer.

Successful treatment of cerebral venous thrombosis associated with ulcerative colitis.

Thromboembolic complications, such as deep venous thrombosis and pulmonary embolism, are well described in patients with inflammatory bowel disease, but cerebral venous thrombosis (CVT) is a rare but potentially devastating complication. The authors describe the case of a 36-year-old Japanese man presenting with CVT associated with ulcerative colitis (UC) that was successfully treated with a combination of continuous anticoagulant and pulse steroid therapy. Our observations suggest that aggressive therapy for inducing acute UC remission is vitally important for CVT associated with UC.

Mechanical Thrombectomy for Acute Common Carotid Artery Occlusion.

Mechanical thrombectomy (MT) is the standard treatment for acute large occlusion of the cerebral artery. Evidence for the success of this procedure was based on the treatment of patients with internal carotid artery and middle cerebral artery thrombi. There are a few reports on thrombi extending to the common carotid artery (CCA). We document our endovascular procedure and the clinical outcome in seven consecutive patients who underwent MT for CCA thrombi between September 2016 and April 2021. Their mean National Institutes of Health Stroke Scale score was 20.0 (range, 9-30), and the mean diffusion-weighted imaging Alberta Stroke Program Early Computed Tomography Score on magnetic resonance images was 8.7 (range, 7-10). In six patients, MT of the CCA occlusion was successful, and the mean puncture-to-reperfusion time was 84 minutes (range, 39-211 minutes). In five patients, successful reperfusion was obtained. The mean total pass number was 4.1 (range, 2-7). Due to large thrombi, we performed balloon guide catheter (BGC) occlusion in three patients. Sheath occlusion occurred in two, and thrombus migration into the femoral artery around the sheath was observed in two patients. The mean modified Rankin Scale score 3 months post-stroke was 3.6 (range, 2-5). When the removal of a large CCA thrombus is attempted in a single step, catheter and sheath occlusion may occur, and this increases the risk for critical systemic artery occlusion. Therefore, we suggest that MT be combined with the BGC technique and propose the use of a large aspiration catheter to decrease the volume of the thrombus.

Immunoelectron microscopy findings in a patient with C3 glomerulonephritis.

We performed a kidney biopsy in a 36-year-old man to evaluate microscopic hematuria and proteinuria. Light microscopy showed increased mesangial matrix and partial swelling of the glomerular basement membrane (GBM), and immunofluorescence showed positive staining only for C3. Immunoelectron microscopy showed that gold particle-labeled C3 was localized in the electron-dense and moderately electron-dense deposits shown by electron microscopy in the mesangium, the thickened GBM near the paramesangium, and the thickened distal portion of the GBM but was not localized in the non-thickened GBM. Gold-labeled immunoglobulin G, κ, and λ were not seen. C3 glomerulonephritis was more evident in gold-labeled electron microscopy, which further clarified the localization of C3 deposition.

Building promoter aware transcriptional regulatory networks using siRNA perturbation and deepCAGE.

Perturbation and time-course data sets, in combination with computational approaches, can be used to infer transcriptional regulatory networks which ultimately govern the developmental pathways and responses of cells. Here, we individually knocked down the four transcription factors PU.1, IRF8, MYB and SP1 in the human monocyte leukemia THP-1 cell line and profiled the genome-wide transcriptional response of individual transcription starting sites using deep sequencing based Cap Analysis of Gene Expression. From the proximal promoter regions of the responding transcription starting sites, we derived de novo binding-site motifs, characterized their biological function and constructed a network. We found a previously described composite motif for PU.1 and IRF8 that explains the overlapping set of transcriptional responses upon knockdown of either factor.