Abemaciclib and Vacuolin-1 decrease aggregate-prone TDP-43 accumulation by accelerating autophagic flux.

(Macro)autophagy is a cellular degradation system for unnecessary materials, such as aggregate-prone TDP-43, a central molecule in neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Abemaciclib (Abe) and vacuolin-1 (Vac) treatments are known to induce vacuoles characterized by an autophagosome and a lysosome component, suggesting that they facilitate autophagosome-lysosome fusion. However, it remains unknown whether Abe and Vac suppress the accumulation of aggregate-prone TDP-43 by accelerating autophagic flux. In the present study, the Abe and Vac treatment dose-dependently reduced the GFP/RFP ratio in SH-SY5Y neuroblastoma cells stably expressing the autophagic flux marker GFP-LC3-RFP-LC3ΔG. Abe and Vac also increased the omegasome marker GFP-ATG13 signal and the autophagosome marker mCherry-LC3 localized to the lysosome marker LAMP1-GFP. The Abe and Vac treatment decreased the intracellular level of the lysosome marker LAMP1-GFP in SH-SY5Y cells stably expressing LAMP1-GFP, but did not increase the levels of LAMP1-GFP, the autophagosome marker LC3-II, or the multivesicular body marker TSG101 in the extracellular vesicle-enriched fraction. Moreover, Abe and Vac treatment autophagy-dependently inhibited GFP-tagged aggregate-prone TDP-43 accumulation. The results of a PI(3)P reporter assay using the fluorescent protein tagged-2 × FYVE and LAMP1-GFP indicated that Abe and Vac increased the intensity of the PI(3)P signal on lysosomes. A treatment with the VPS34 inhibitor wortmannin (WM) suppressed Abe-/Vac-facilitated autophagic flux and the degradation of GFP-tagged aggregate-prone TDP-43. Collectively, these results suggest that Abe and Vac degrade aggregate-prone TDP-43 by accelerating autophagosome formation and autophagosome-lysosome fusion through the formation of PI(3)P.

In vivo heat production dynamics during a contraction-relaxation cycle in rat single skeletal muscle fibers.

Skeletal muscle generates heat via contraction-dependent (shivering) and independent (nonshivering) mechanisms. While this thermogenic capacity of skeletal muscle undoubtedly contributes to the body temperature homeostasis of animals and impacts various cellular functions, the intracellular temperature and its dynamics in skeletal muscle in vivo remain elusive. We aimed to determine the intracellular temperature and its changes within skeletal muscle in vivo during contraction and following relaxation. In addition, we tested the hypothesis that sarcoplasmic reticulum Ca2+ ATPase (SERCA) generates heat and increases the myocyte temperature during a transitory Ca2+-induced contraction-relaxation cycle. The intact spinotrapezius muscle of anesthetized adult male Wistar rats (n = 18) was exteriorized and loaded with the fluorescent probe Cellular Thermoprobe for Fluorescence Ratio (49.3 µM) by microinjection over 1 s. The fluorescence ratio (i.e., 580 nm/515 nm) was measured in vivo during 1) temperature increases induced by means of an external heater, and 2) Ca2+ injection (3.9 nL, 2.0 mM). The fluorescence ratio increased as a linear function of muscle surface temperature from 25 °C to 40 °C (r2 = 0.97, P < 0.01). Ca2+ injection (3.9 nL, 2.0 mM) significantly increased myocyte intracellular temperature: An effect that was suppressed by SERCA inhibition with cyclopiazonic acid (CPA, Ca2+: 38.3 ± 1.4 °C vs Ca2++CPA: 28.3 ± 2.8 °C, P < 0.01 at 1 min following injection). While muscle shortening occurred immediately after the Ca2+ injection, the increased muscle temperature was maintained during the relaxation phase. In this investigation, we demonstrated a novel model for measuring the intracellular temperature of skeletal muscle in vivo and further that heat generation occurs concomitant principally with SERCA functioning and muscle relaxation.

In vivo cytosolic H2O2 changes and Ca2+ homeostasis in mouse skeletal muscle.

Hydrogen peroxide (H2O2) and calcium ions (Ca2+) are functional regulators of skeletal muscle contraction and metabolism. Although H2O2 is one of the activators of the type-1 ryanodine receptor (RyR1) in the Ca2+ release channel, the interdependence between H2O2 and Ca2+ dynamics remains unclear. This study tested the following hypotheses using an in vivo model of mouse tibialis anterior (TA) skeletal muscle. 1) Under resting conditions, elevated cytosolic H2O2 concentration ([H2O2]cyto) leads to a concentration-dependent increase in cytosolic Ca2+ concentration ([Ca2+]cyto) through its effect on RyR1; and 2) in hypoxia (cardiac arrest) and muscle contractions (electrical stimulation), increased [H2O2]cyto induces Ca2+ accumulation. Cytosolic H2O2 (HyPer7) and Ca2+ (Fura-2) dynamics were resolved by TA bioimaging in young C57BL/6J male mice under four conditions: 1) elevated exogenous H2O2; 2) cardiac arrest; 3) twitch (1 Hz, 60 s) contractions; and 4) tetanic (30 s) contractions. Exogenous H2O2 (0.1-100 mM) induced a concentration-dependent increase in [H2O2]cyto (+55% at 0.1 mM; +280% at 100 mM) and an increase in [Ca2+]cyto (+3% at 1.0 mM; +8% at 10 mM). This increase in [Ca2+]cyto was inhibited by pharmacological inhibition of RyR1 by dantrolene. Cardiac arrest-induced hypoxia increased [H2O2]cyto (+33%) and [Ca2+]cyto (+20%) 50 min postcardiac arrest. Compared with the exogenous 1.0 mM H2O2 condition, [H2O2]cyto after tetanic muscle contractions rose less than one-tenth as much, whereas [Ca2+]cyto was 4.7-fold higher. In conclusion, substantial increases in [H2O2]cyto levels evoke only modest Ca2+ accumulation via their effect on the sarcoplasmic reticulum RyR1. On the other hand, contrary to hypoxia secondary to cardiac arrest, increases in [H2O2]cyto from muscle contractions are small, indicating that H2O2 generation is unlikely to be a primary factor driving the significant Ca2+ accumulation after, especially tetanic, muscle contractions.NEW & NOTEWORTHY We developed an in vivo mouse myocyte H2O2 imaging model during exogenous H2O2 loading, ischemic hypoxia induced by cardiac arrest, and muscle contractions. In this study, the interrelationship between cytosolic H2O2 levels and Ca2+ homeostasis during muscle contraction and hypoxic conditions was revealed. These results contribute to the elucidation of the mechanisms of muscle fatigue and exercise adaptation.

PHI-1, an Endogenous Inhibitor Protein for Protein Phosphatase-1 and a Pan-Cancer Marker, Regulates Raf-1 Proteostasis.

Raf-1, a multifunctional kinase, regulates various cellular processes, including cell proliferation, apoptosis, and migration, by phosphorylating MAPK/ERK kinase and interacting with specific kinases. Cellular Raf-1 activity is intricately regulated through pathways involving the binding of regulatory proteins, direct phosphorylation, and the ubiquitin-proteasome axis. In this study, we demonstrate that PHI-1, an endogenous inhibitor of protein phosphatase-1 (PP1), plays a pivotal role in modulating Raf-1 proteostasis within cells. Knocking down endogenous PHI-1 in HEK293 cells using siRNA resulted in increased cell proliferation and reduced apoptosis. This heightened cell proliferation was accompanied by a 15-fold increase in ERK1/2 phosphorylation. Importantly, the observed ERK1/2 hyperphosphorylation was attributable to an upregulation of Raf-1 expression, rather than an increase in Ras levels, Raf-1 Ser338 phosphorylation, or B-Raf levels. The elevated Raf-1 expression, stemming from PHI-1 knockdown, enhanced EGF-induced ERK1/2 phosphorylation through MEK. Moreover, PHI-1 knockdown significantly contributed to Raf-1 protein stability without affecting Raf-1 mRNA levels. Conversely, ectopic PHI-1 expression suppressed Raf-1 protein levels in a manner that correlated with PHI-1's inhibitory potency. Inhibiting PP1 to mimic PHI-1's function using tautomycin led to a reduction in Raf-1 expression. In summary, our findings highlight that the PHI-1-PP1 signaling axis selectively governs Raf-1 proteostasis and cell survival signals.

Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43.

The cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43 kDa (TDP-43) has been linked to the progression of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 secreted into the extracellular space has been suggested to contribute to the cell-to-cell spread of the cytoplasmic accumulation of TDP-43 throughout the brain; however, the underlying mechanisms remain unknown. We herein demonstrated that the secretion of TDP-43 was stimulated by the inhibition of the autophagy-lysosomal pathway driven by progranulin (PGRN), a causal protein of frontotemporal lobar degeneration. Among modulators of autophagy, only vacuolar-ATPase inhibitors, such as bafilomycin A1 (Baf), increased the levels of the full-length and cleaved forms of TDP-43 and the autophagosome marker LC3-II (microtubule-associated proteins 1A/1B light chain 3B) in extracellular vesicle fractions prepared from the culture media of HeLa, SH-SY5Y, or NSC-34 cells, whereas vacuolin-1, MG132, chloroquine, rapamycin, and serum starvation did not. The C-terminal fragment of TDP-43 was required for Baf-induced TDP-43 secretion. The Baf treatment induced the translocation of the aggregate-prone GFP-tagged C-terminal fragment of TDP-43 and mCherry-tagged LC3 to the plasma membrane. The Baf-induced secretion of TDP-43 was attenuated in autophagy-deficient ATG16L1 knockout HeLa cells. The knockdown of PGRN induced the secretion of cleaved TDP-43 in an autophagy-dependent manner in HeLa cells. The KO of PGRN in mouse embryonic fibroblasts increased the secretion of the cleaved forms of TDP-43 and LC3-II. The treatment inducing TDP-43 secretion increased the nuclear translocation of GFP-tagged transcription factor EB, a master regulator of the autophagy-lysosomal pathway in SH-SY5Y cells. These results suggest that the secretion of TDP-43 is promoted by dysregulation of the PGRN-driven autophagy-lysosomal pathway.

Simple new clinical score to predict hepatocellular carcinoma after sustained viral response with direct-acting antivirals.

The time point of the most precise predictor of hepatocellular carcinoma (HCC) development after viral eradication with direct-acting antiviral (DAA) therapy is unclear. In this study we developed a scoring system that can accurately predict the occurrence of HCC using data from the optimal time point. A total of 1683 chronic hepatitis C patients without HCC who achieved sustained virological response (SVR) with DAA therapy were split into a training set (999 patients) and a validation set (684 patients). The most accurate predictive scoring system to estimate HCC incidence was developed using each of the factors at baseline, end of treatment, and SVR at 12 weeks (SVR12). Multivariate analysis identified diabetes, the fibrosis-4 (FIB-4) index, and the α-fetoprotein level as independent factors at SVR12 that contributed to HCC development. A prediction model was constructed with these factors that ranged from 0 to 6 points. No HCC was observed in the low-risk group. Five-year cumulative incidence rates of HCC were 1.9% in the intermediate-risk group and 15.3% in the high-risk group. The prediction model at SVR12 most accurately predicted HCC development compared with other time points. This simple scoring system combining factors at SVR12 can accurately evaluate HCC risk after DAA treatment.

Discussion on Electron Temperature of Gas-Discharge Plasma with Non-Maxwellian Electron Energy Distribution Function Based on Entropy and Statistical Physics.

Electron temperature is reconsidered for weakly-ionized oxygen and nitrogen plasmas with its discharge pressure of a few hundred Pa, with its electron density of the order of 1017m-3 and in a state of non-equilibrium, based on thermodynamics and statistical physics. The relationship between entropy and electron mean energy is focused on based on the electron energy distribution function (EEDF) calculated with the integro-differential Boltzmann equation for a given reduced electric field E/N. When the Boltzmann equation is solved, chemical kinetic equations are also simultaneously solved to determine essential excited species for the oxygen plasma, while vibrationally excited populations are solved for the nitrogen plasma, since the EEDF should be self-consistently found with the densities of collision counterparts of electrons. Next, the electron mean energy U and entropy S are calculated with the self-consistent EEDF obtained, where the entropy is calculated with the Gibbs's formula. Then, the "statistical" electron temperature Test is calculated as Test=[∂S/∂U]-1. The difference between Test and the electron kinetic temperature Tekin is discussed, which is defined as [2/(3k)] times of the mean electron energy U=⟨ϵ⟩, as well as the temperature given as a slope of the EEDF for each value of E/N from the viewpoint of statistical physics as well as of elementary processes in the oxygen or nitrogen plasma.

Subclassification of pT3 upper tract urothelial carcinoma: a multicenter retrospective study.

PURPOSE: The prognosis of patients with pT3 upper tract urothelial carcinoma (UTUC) varies. The current study aimed to further classify patients with pT3 UTUC into different survival outcome groups based on tumor location and site of invasion. METHODS: This retrospective study included 323 patients with pT3 UTUC who underwent nephroureterectomy at 11 hospitals in Japan. Histological and clinical data were obtained via a chart review. Univariate and multivariate Cox proportional hazards analyses showed the effect of different variables on recurrence-free survival (RFS), cancer-specific survival (CSS), and overall survival (OS). RESULTS: The median age of the patients was 72 years. Patients with pT3 UTUCs were divided into two groups: those with renal parenchymal invasion only (pT3a, n = 95) and those with peripelvic or periureteral fat invasion (pT3b, n = 228). pT3b UTUC was significantly associated with hydronephrosis, low preoperative estimated glomerular filtration rate (eGFR), histological nodal metastasis, nuclear grade 3, lymphovascular invasion (LVI), carcinoma in situ, and positive surgical margin. Based on the univariate analyses, patients with pT3b UTUC had a significantly lower 5-year RFS (42.4% vs. 70.1%, p < 0.0001), 5-year CSS (54.3% vs. 80.0%, p = 0.0002), and 5-year OS (47.8% vs. 76.8%, p < 0.0001) than those with pT3a UTUC. According to the multivariate analyses, nodal metastasis, LVI, adjuvant chemotherapy, preoperative eGFR, nuclear grade (RFS only), surgical margin (RFS only), and Charlson comorbidity index (OS only), but not pT3b stage, were associated with survival. CONCLUSION: Compared with pT3a UTUC, pT3b UTUC was significantly associated with worse histological features, consequently resulting in unsatisfactory survival outcomes.

Thrombocytopenia, anasarca, and renal insufficiency as severe and rare complications of Hodgkin lymphoma: a case report.

BACKGROUND: Patients with Hodgkin lymphoma exhibit various clinical presentations. Needle biopsy of the lymph nodes is a minimally invasive procedure and a useful diagnostic method for malignant lymphomas. However, at times it is difficult to differentiate malignant lymphomas from reactive lymph node changes using a small amount of biopsy material. CASE PRESENTATION: A 77-year-old Japanese man was referred to the emergency department of our hospital owing to high fever and disturbance of consciousness. We diagnosed sepsis due to an acute biliary tract infection because he presented with Charcot's triad-fever, jaundice, and right-sided abdominal pain. However, he did not respond well to antimicrobial therapy and his high fever persisted. Considering the swelling of the right cervical, mediastinal, and intraperitoneal lymph nodes and splenomegaly detected on computed tomography, a differential diagnosis of malignant lymphoma was needed. Hence, we performed a needle biopsy of the right cervical lymph node; however, the amount of sample obtained was insufficient in establishing a definitive diagnosis of malignant lymphoma. Furthermore, during hospitalization, the patient developed thrombocytopenia, anasarca, and renal insufficiency. These symptoms seemed to be the typical signs of the thrombocytopenia, anasarca, fever, reticulin fibrosis or renal insufficiency, and organomegaly syndrome. Next, an external incisional mass biopsy of the right cervical lymph node was performed, which helped identify Hodgkin and Reed-Sternberg cells. Collectively, we established a definitive diagnosis of Hodgkin lymphoma with lymphoma-associated hemophagocytic syndrome. CONCLUSIONS: This case highlights the importance of performing an external incisional mass biopsy of the lymph nodes for the early diagnosis and treatment, if malignant lymphoma is strongly suspected.

Patients with Chronic Liver Disease under Surveillance for Hepatocellular Carcinoma Have a Favorable Long-Term Outcome for Pancreatic Cancer Due to Early Diagnosis and High Resection Rate.

Patients with viral hepatitis-related chronic liver disease (CLD) under surveillance for hepatocellular carcinoma (HCC) are often diagnosed with pancreatic cancer (PC) at an early stage. However, the long-term outcomes of these patients are unclear. We aimed to clarify the long-term outcomes of patients with PC with viral hepatitis-related CLD using a chart review. Data collection included the Union for International Cancer Control (UICC) stage at PC diagnosis, hepatitis B virus and hepatitis C virus status, and long-term outcomes. The distribution of the entire cohort (N = 552) was as follows: early stage (UICC 0-IB; n = 52, 9.5%) and non-early stages (UICC IIA-IV; n = 500, 90.5%). At diagnosis, the HCC surveillance group (n = 18) had more patients in the early stages than the non-surveillance group (n = 534) (50% vs. 8.0%), leading to a higher indication rate for surgical resection (72.2% vs. 29.8%) and a longer median survival time (19.0 months vs. 9.9 months). We confirmed that patients with viral hepatitis-related CLD under HCC surveillance were diagnosed with PC at an early stage. Because of the higher indication rate for surgical resection in these patients, they had favorable long-term outcomes for PC.

[A Case of Perforation at the Stump of Descending Colon after Hartmann's Operation Performed after Transverse Colostomy].

A 77-year-old man with complaining of anemia and abdominal pain was admitted to our hospital. An abdominal computed tomography showed the sigmoid colon tumor with bowel obstruction. Laparoscopic transverse colostomy was performed to release intestinal obstruction. After first operation, he was diagnosed the sigmoid colon cancer: cT4b(bladder), cN0, cM0, and cStage Ⅱc. Radical laparoscopic operation(Hartmann's operation)was performed. On the 4th postoperative day, fecal juice was discharged from the abdominal drain placed in the Douglas fossa, so emergency laparotomy was performed. The intraoperative findings showed perforation in the blind end of the descending colon. The descending colon was resected from a site approximately 5 cm anal side of the transverse colostomy to the blind end. It was thought that perforation occurred due to an increase in internal pressure in the residual intestinal tract after Hartmann's surgery without blood flow disorder. We believe that further attention is required to the management of residual intestinal tract at the blind end for the obstructive colorectal cancer.

Effects of carbohydrate-electrolyte dissolved alkaline electrolyzed water on physiological responses during exercise under heat stress in physically active men.

Purpose: This study investigated the effects of 1400 mL intake of alkaline electrolyzed water (AEW) or purified water (PW) into which carbohydrate-electrolyte (CE) was dissolved on improving physiological responses during exercise under heat stress. Methods: This double-blinded, crossover randomized controlled trial included 10 male participants who completed two exercise trials in a hot environment (35 °C, ambient temperature, and 50% relative humidity) after consuming CE-dissolved PW (P-CE) or CE-dissolved AEW (A-CE). The exercise trial consisted of running for 30 min on a treadmill (at an intensity corresponding to 65% of heart rate reserve adjusted for heat stress conditions) and repeated sprint cycling (10 × 7-s maximal sprint cycling), with a 35-min rest interval between the two exercises, followed by a 30-min post-exercise recovery period. Before and after running, and after cycling, the participants drank P-CE (hydrogen concentration of 0 ppm, pH 3.8) or A-CE (0.3 ppm, pH 4.1). Blood samples were obtained before, during (rest interval between running and cycling), and post-exercise. Results: Repeated sprint performance and oxidative stress response did not differ between the P-CE and A-CE trials. A-CE consumption significantly attenuated the increase in blood lactate concentration during the running exercise but not during repeated sprint cycling under heat stress conditions. Conclusion: Our findings suggested that A-CE did not significantly affect repeated sprint performance; however, the attenuated elevation in blood lactate by A-CE ingestion implies a partial enhancement of endurance performance during submaximal exercise under heat stress.

Improved survival in real-world patients with advanced urothelial carcinoma: A multicenter propensity score-matched cohort study comparing a period before the introduction of pembrolizumab (2003-2011) and a more recent period (2016-2020).

OBJECTIVES: Although the treatment strategy for advanced urothelial carcinoma (aUC) has drastically changed since pembrolizumab was introduced in 2017, studies revealing current survival rates in aUC are lacking. This study aimed to assess (1) the improvement in survival among real-world patients with aUC after the introduction of pembrolizumab and (2) the direct survival-prolonging effect of pembrolizumab. METHODS: This multicenter retrospective study included 531 patients with aUC undergoing salvage chemotherapy, including 200 patients treated in the pre-pembrolizumab era (2003-2011; earlier era) and 331 patients treated in a recent 5-year period (2016-2020; recent era). Using propensity score matching (PSM), cancer-specific survival (CSS) and overall survival (OS) were compared between the earlier and recent eras, in addition to between the recent era, both with and without pembrolizumab use, and the earlier era. RESULTS: After PSM, the recent era cohort had significantly longer CSS (21 months) and OS (19 months) than the earlier era cohort (CSS and OS: 12 months). In secondary analyses using PSM, patients treated with pembrolizumab had significantly longer CSS (25 months) and OS (24 months) than those in the earlier era cohort (CSS and OS: 11 months), whereas patients who did not receive pembrolizumab in the recent era had similar outcomes (CSS and OS: 14 months) as the earlier era cohort (CSS and OS: 12 months). CONCLUSIONS: Patients with aUC treated in the recent era exhibited significantly longer survival than those treated before the introduction of pembrolizumab. The improved survival was primarily attributable to the use of pembrolizumab.

Analysis of fall kinematics and injury risks in ground impact in car-pedestrian collisions using impulse.

In vehicle-to-pedestrian collisions, pedestrian injuries occur due to contact with the car and the ground. Previous studies investigated pedestrian kinematic behavior using a parameter study or through statistical analysis although the force interaction between the pedestrian and the vehicle has not been considered. In this study, multibody analyses were conducted for vehicle-pedestrian collisions for adult and child pedestrian with various vehicle shapes. The impulse and impulse moment acting on the pedestrian from the vehicle were introduced, and the kinematic behavior, rotation and ground impact of the pedestrian model were examined. It was found that if an impulse moment acts on the pedestrian when the pedestrian re-contacts with the hood of the car, the angular velocity of the pedestrian's torso changes in the opposite direction (away from the car), and the torso angle prior to the ground contact decreases to less than 90°. This re-contact between the pedestrian and the vehicle was more likely to occur for cases where the collision involves an adult pedestrian, lower hood leading edge (HLE), longer hood length, and lower collision velocity. When the pedestrian torso angle in contact with the ground was less than 90°, the head vertical impact velocity with respect to the ground became less than 2.9 m/s which corresponds to the injury threshold of the head. This study demonstrated that pedestrian-vehicle re-contact is crucial for reducing ground injury. The vehicle shape, pedestrian size, and collision velocity can determine whether re-contact of the pedestrian with the vehicle occurs. This can then explain the factors affecting pedestrian ground impact injury (e.g., higher HLE, higher risk of ground head injury for children) that were shown in previous studies. A strategy to mitigate ground injury is to apply enough impulse moment onto the pedestrian's upper body from the hood in order to change the torso angular velocity during re-contact, thus making the torso angle less than 90°prior to the ground contact.

Significance of alveolar nitric oxide concentration in the airway of patients with organizing pneumonia after allogeneic hematopoietic stem cell transplantation.

Organizing pneumonia (OP) is a complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and a manifestation of peripheral airway/alveolar inflammation. Recently, alveolar nitric oxide concentration (Calv) has been revealed as a noninvasive marker of peripheral airway inflammation; however, whether Calv levels are associated with OP and peripheral airway in patients after allo-HSCT remains unclear. Herein, we evaluated whether Calv levels could reflect the presence of OP and structural airway changes in patients after allo-HSCT. We measured the eNO levels of 38 patients (6 with OP and 32 without OP) who underwent allo-HSCT. Three-dimensional computed tomography (CT) analysis of the airway was performed in 19 patients. We found that in patients with OP, Calv levels were significantly higher than in those without OP (10.6 vs. 5.5 ppb, p < 0.01). Receiver-operating characteristic analyses revealed a Calv cut-off value for OP detection of 10.2 ppb. No significant differences in the patient characteristics, except for the presence of OP (p < 0.01), were noted between the two groups stratified by the Calv cut-off value. Three-dimensional CT images of the airway revealed gradually increasing positive correlations between Calv levels and airway wall area of the third-, fourth-, and fifth-generation bronchi (r = 0.20, 0.31, 0.38; p = 0.42, 0.19, 0.038, respectively), indicating that Calv levels are strongly correlated with the wall thickness of the distal bronchi. Our results suggest that the Calv level may be a useful noninvasive detectable marker for OP after an allo-HSCT.

Abemaciclib and Vacuolin-1 induce vacuole-like autolysosome formation - A new tool to study autophagosome-lysosome fusion.

Macroautophagy (hereafter autophagy) is a conserved cellular degradation system, impairments in which have been implicated in the development of a wide range of diseases, including cancer and neurodegenerative diseases. Autophagy is mainly comprised of two processes: the formation of autophagosomes and autolysosomes. A detailed understanding of the formation of autophagosomes has been obtained in the past several decades. However, limited information is currently available on the formation of autolysosomes, which may partially be attributed to fewer methods to study the formation of autolysosomes than that of autophagosomes. Abemaciclib (Abe) and vacuolin-1 (Vac) are drugs that suppress the progression of breast cancer and induce characteristic vacuole formation in cells. Since Abe-induced vacuoles have the appearance of autolysosomes, they may be used to examine the formation of autolysosomes. However, it remains unknown whether Abe-/Vac-induced vacuoles are regulated by autophagosome-lysosome fusion. Markers for endosomes, lysosomes, and autophagosomes (Rab7, LAMP1, and mRFP-GFP-LC3, respectively) indicated that Abe-/Vac-induced vacuoles were autolysosomes. Abe and Vac failed to induce vacuolation in ATG16L1-deficient autophagy-null cells. Furthermore, Abe-/Vac-induced vacuolation was suppressed by bafilomycin A1, an inhibitor of autophagosome-lysosome fusion, whereas it was facilitated by rapamycin and the overexpression of Beclin-1, inducers of autophagosome-lysosome fusion. Moreover, vacuole formation was inhibited by the knockdown of progranulin (PGRN), a regulator of autophagosome-lysosome fusion, and promoted by its overexpression. The present results suggest the potential of Abe-/Vac-induced vacuole-like autolysosomes as a tool for evaluating autophagosome-lysosome fusion and examining the effects of PGRN in autophagy.

Overexpression of progranulin increases pathological protein accumulation by suppressing autophagic flux.

Progranulin (PGRN) haploinsufficiency from autosomal dominant mutations in the PGRN gene causes frontotemporal lobar degeneration, which is characterized by cytoplasmic inclusions predominantly containing TDP-43 (FTLD-TDP). PGRN supplementation for patients with a PGRN gene mutation has recently been proposed as a therapeutic strategy to suppress FTLD-TDP. However, it currently remains unclear whether excessive amounts of PGRN are beneficial or harmful. We herein report the effects of PGRN overexpression on autophagic flux in a cultured cell model. PGRN overexpression increased the level of an autophagosome marker without promoting autophagosome formation and decreased the signal intensity of an autolysosome marker, indicating the suppression of autophagic flux due to reductions in the formation of autolysosomes. Assessments of lysosome numbers and biogenesis using LysoTracker and cells stably expressing TFEB-GFP, respectively, indicated that PGRN overexpression increased the lysosome numbers without lysosomal biogenesis. These results suggest that PGRN overexpression suppressed autophagic flux by inhibiting autophagosome-lysosome fusion. Moreover, PGRN overexpression enhanced polyglutamine aggregation and aggregate-prone TDP-43 accumulation, indicating that the suppression of autophagic flux by excessive amounts of PGRN worsens the pathology of neurodegenerative diseases.

Possible roles of N- and C-terminal unstructured tails of CPI-17 in regulating Ca2+ sensitization force of smooth muscle.

CPI-17 regulates the myosin phosphatase and mediates the agonist-induced contraction of smooth muscle. PKC and ROCK phosphorylate CPI-17 at Thr38 leading to a conformational change of the central inhibitory domain (PHIN domain). The N- and C-terminal tails of CPI-17 are predicted as unstructured loops and their sequences are conserved among mammals. Here we characterized CPI-17 N- and C-terminal unstructured tails using recombinant proteins that lack the potions. Recombinant CPI-17 proteins at a physiologic level (10 µM) were doped into beta-escin-permeabilized smooth muscle strips for Ca2+ sensitization force measurement. The ectopic full-length CPI-17 augmented the PDBu-induced Ca2+ sensitization force at pCa6.3, indicating myosin phosphatase inhibition. Deletion of N- and C-terminal tails of CPI-17 attenuated the extent of PDBu-induced Ca2+-sensitization force. The N-terminal deletion dampened phosphorylation at Thr38 by protein kinase C (PKC), and the C-terminal truncation lowered the affinity to the myosin phosphatase. Under the physiologic conditions, PKC and myosin phosphatase may recognize CPI-17 N-/C-terminal unstructured tails inducing Ca2+ sensitization force in smooth muscle cells.

[Surgical Outcomes of the Patients with Gastric Cancer in Aged 85 and Older at Our Hospital].

We investigated the surgical outcomes of the patients with gastric cancer in aged 85 and older. There were 9 males and 8 females, with a median age of 86 years. All had comorbidities and 7 had double cancers. Type of surgery was distal gastrectomy in 14 and total gastrectomy in 3, respectively. Postoperative complications occurred in 8 cases, and case with adhesion ileus or mesenteric bleeding performed reoperation. The postoperative hospital stay was 15 days. The cause of death was recurrent diseases in 2 cases and other diseases in 4. The overall survival rate was 63.9% for 3 years and 42.6% for 5 years, respectively. Elderly patients with gastric cancer may be increase in Japan, but they have large individual differences about tolerance of surgical intervention. Therefore, it is important to evaluate the detail of general condition in such patients.

Precision cancer genome testing needs proficiency testing involving all stakeholders.

To implement precision oncology, analytical validity as well as clinical validity and utility are important. However, proficiency testing (PT) to assess validity has not yet been systematically performed in Japan. To investigate the quality of next-generation sequencing (NGS) platforms and cancer genome testing prevalent in laboratories, we performed pilot PT using patient samples. We prepared genomic DNA from the cancer tissue and peripheral blood of 5 cancer patients and distributed these to 15 laboratories. Most participating laboratories successfully identified the pathogenic variants, except for two closely located KRAS variants and 25 bp delins in EGFR. Conversely, the EGFR L858R variant was successfully identified, and the allele frequency was similar for all the laboratories. A high DNA integrity number led to excellent depth and reliable NGS results. By conducting this pilot study using patient samples, we were able to obtain a glimpse of the current status of cancer genome testing at participating laboratories. To enhance domestic cancer genome testing, it is important to conduct local PT and to involve the parties concerned as organizers and participants.