Cathepsin L prevents the accumulation of alpha-synuclein fibrils in the cell.

The deposition of α-synuclein (α-Syn) fibrils in neuronal cells has been implicated as a causative factor in Parkinson's disease (PD) and dementia with Lewy Bodies (DLB). α-Syn can be degraded by autophagy, proteasome, and chaperone-mediated autophagy, and previous studies have suggested the potency of certain cathepsins, lysosomal proteases, for α-Syn degradation. However, no studies have comprehensively evaluated all cathepsins. Here, we evaluated the efficacy of all 15 cathepsins using a cell model of α-Syn fibril propagation and found that overexpression of cathepsin L (CTSL) was the most effective in preventing the accumulation of α-Syn aggregates. CTSL-mediated degradation of α-Syn aggregates was dependent on the autophagy machinery, and CTSL itself promoted autophagy flux. Interestingly, CTSL was effective in autophagic degradation of wild-type (WT) α-Syn, but not in the case of A53T and E46K missense mutations, which are causative for familial PD. These results suggest that CTSL is a potential therapeutic strategy for sporadic PD pathology in WT α-Syn.

A novel immunocomplex capture fluorescence assay (ICFA) using fluorescent beads and transfected cells for specific identification of human neutrophil antigen (HNA)-1a and -1b antibodies.

BACKGROUND: To identify specific human neutrophil antigen (HNA) antibodies, assays using neutrophils such as monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) are recommended. However, these assays are limited by labor-intensive neutrophil preparation and varying antigen expression levels. METHODS: We evaluated a newly developed immunocomplex capture fluorescence assay (ICFA) for identifying HNA-1 antibodies and compared it to MAIGA and LABScreen Multi (LABM), which utilizes recombinant HNA-coated Luminex beads. For ICFA, HNA-1a or HNA-1b transfected cells replaced neutrophils. Cells incubated with serum were lysed, and immune complexes were captured using five CD16 monoclonal antibody-conjugated Luminex beads. Nine antisera with known specificity and 26 samples suspected of containing HNA antibodies were analyzed by ICFA and MAIGA using neutrophils or transfected cells (ICFA-N or ICFA-T, and MAIGA-N or MAIGA-T, respectively). RESULTS: ICFA-T and MAIGA-N accurately determined the specificity of all antibodies in the nine antiserum samples. The ICFA-T detection limit was 2048-fold for anti-HNA-1a and 256-fold for anti-HNA-1b; the limits of MAIGA-T, MAIGA-N, and LABM were 32-, 4 ~ 64-, and 128-fold for anti-HNA-1a and 64-, 16 ~ 64-, and 32-fold for anti-HNA-1b, respectively. Twelve and 7 of the remaining 26 samples tested negative and positive, respectively, in both ICFA-T and MAIGA-N. Antibody specificity against HNA-1a or HNA-1b determined using ICFA-T agreed with that determined using MAIGA-N and LABM. Another seven samples tested positive in ICFA-T but negative in MAIGA-N. CONCLUSION: The novel ICFA is highly sensitive and exhibits specificity similar to MAIGA and LABM for detecting HNA-1 antibodies.

Roles of Stress Response in Autophagy Processes and Aging-Related Diseases.

The heat shock factor 1 (HSF1)-mediated stress response pathway and autophagy processes play important roles in the maintenance of proteostasis. Autophagy processes are subdivided into three subtypes: macroautophagy, chaperone-mediated autophagy (CMA), and microautophagy. Recently, molecular chaperones and co-factors were shown to be involved in the selective degradation of substrates by these three autophagy processes. This evidence suggests that autophagy processes are regulated in a coordinated manner by the HSF1-mediated stress response pathway. Recently, various studies have demonstrated that proteostasis pathways including HSF1 and autophagy are implicated in longevity. Furthermore, they serve as therapeutic targets for aging-related diseases such as cancer and neurodegenerative diseases. In the future, these studies will underpin the development of therapies against various diseases.

The Role of Neuropeptide Y in the Nucleus Accumbens.

Neuropeptide Y (NPY), an abundant peptide in the central nervous system, is expressed in neurons of various regions throughout the brain. The physiological and behavioral effects of NPY are mainly mediated through Y1, Y2, and Y5 receptor subtypes, which are expressed in regions regulating food intake, fear and anxiety, learning and memory, depression, and posttraumatic stress. In particular, the nucleus accumbens (NAc) has one of the highest NPY concentrations in the brain. In this review, we summarize the role of NPY in the NAc. NPY is expressed principally in medium-sized aspiny neurons, and numerous NPY immunoreactive fibers are observed in the NAc. Alterations in NPY expression under certain conditions through intra-NAc injections of NPY or receptor agonists/antagonists revealed NPY to be involved in the characteristic functions of the NAc, such as alcohol intake and drug addiction. In addition, control of mesolimbic dopaminergic release via NPY receptors may take part in these functions. NPY in the NAc also participates in fat intake and emotional behavior. Accumbal NPY neurons and fibers may exert physiological and pathophysiological actions partly through neuroendocrine mechanisms and the autonomic nervous system.

[Injection of High Concentration Glucose Solution for Pleural Coating Reduces Postoperative Recurrence of Spontaneous Pneumothorax;A Short-term Retrospective Study].

BACKGROUND: Video-assisted thoracoscopic surgery (VATS) is the standard treatment for patients with spontaneous pneumothorax (SP). However, postoperative recurrence is not infrequent even with an absorbable covering sheet used to reinforce the visceral pleura. Recent reports suggest that intraoperative injection of a highly concentrated glucose solution into the thoracic cavity provides effective prophylaxis against postoperative SP recurrence. Since September 2015, we have been injecting 50 ml of 50 % glucose solution intraoperatively for pleural coating (GPC) around an absorbable sheet to prevent postoperative SP recurrence. METHODS: We evaluated 340 patients who underwent VATS between February 2011 and June 2017(88 patients:GPC group, 252:non-GPC group), and we retrospectively analyzed the efficacy of GPC in preventing postoperative SP recurrence. RESULTS: One year postoperative recurrence rates of GPC and non-GPC groups were 9.0 and 17.9%,respectively. The log-rank test revealed GPC as a significant factor in preventing postoperative recurrence (p=0.020). No severe adverse events occurred in either group. Minor postoperative complications, viz., high blood sugar, high volume of chest tube drainage occurred in the GPC group. CONCLUSION: Application of GPC is beneficial in reducing postoperative recurrence of SP.

Involvement of serotonin 2C receptor RNA editing in accumbal neuropeptide Y expression and behavioural despair.

Serotonin 2C receptors (5-HT2 C Rs) are widely expressed in the central nervous system, and are associated with various neurological disorders. 5-HT2 C R mRNA undergoes adenosine-to-inosine RNA editing at five sites within its coding sequence, resulting in expression of 24 different isoforms. Several edited isoforms show reduced activity, suggesting that RNA editing modulates serotonergic systems in the brain with causative relevance to neuropsychiatric disorders. Transgenic mice solely expressing the non-edited 5-HT2 C R INI-isoform (INI) or the fully edited VGV-isoform exhibit various phenotypes including metabolic abnormalities, aggressive behaviour, anxiety-like behaviour, and depression-like behaviour. Here, we examined the behavioural phenotype and molecular changes of INI mice on a C57BL/6J background. INI mice showed an enhanced behavioural despair in the forced swimming test, elevated sensitivity to the tricyclic antidepressant desipramine, and significantly decreased serotonin in the nucleus accumbens (NAc), amygdala, and striatum. They also showed reduced expression of neuropeptide Y (NPY) mRNA in the NAc. In addition, by stereotactic injection of adeno-associated virus encoding NPY into the NAc, we demonstrated that accumbal NPY overexpression relieved behavioural despair. Our results suggest that accumbal NPY expression may be regulated by 5-HT2 C R RNA editing, and its impairment may be linked to mood disorders.

Congenital haptoglobin deficiency discovered on the occasion of anaphylaxis induced by platelet concentrate transfusion.

A 77-year-old man with myelodysplastic syndrome suffered from duodenal perforation after undergoing endoscopic submucosal dissection (ESD) for treatment of duodenal cancer. He presented with hemorrhagic shock, peritonitis and disseminated intravascular coagulation (DIC), and received transfusions of red blood cells (RBC), fresh frozen plasma (FFP), γ-globulin and albumin (Alb). One month after the last RBC transfusion, prolonged thrombocytopenia was observed, and platelet concentrate (PC) was transfused. However, immediately after starting PC transfusion, he developed dyspnea, hypotension and rash, and was thus diagnosed as being in anaphylactic shock. Analysis of the patient's serum revealed absence of haptoglobin (Hp) and the presence of anti-Hp antibody. Further studies, using PCR detected Hpdel, yielded a diagnosis of congenital Hp deficiency. Thus, the anaphylactic shock was considered to have been induced by Hp in the transfused PC reacting with pre-existing anti-Hp antibodies. Thereafter, transfusions were safely carried out with the use of washed PC. Congenital Hp deficiency is relatively prevalent, and in such cases transfusions should be carried out using washed RBC, washed PC and congenital Hp deficiency donor derived FFP to avoid anaphylactic transfusion reactions. Transfusions would be even safer if production of congenital Hp deficiency donor derived PC were to be made available in the future.

[Propensity-score-matched Comparison of Postoperative Pain Between Young and Elderly Patients Who Underwent Video-assisted Thoracoscopic Surgery for Spontaneous Pneumothorax].

Video-assisted thoracoscopic surgery (VATS) is the standard treatment for spontaneous pneumothorax(SP). Although VATS has decreased the postoperative pain in comparison with conventional thoracotomy, the procedure still often requires sufficient postoperative pain management especially for young patients, and the present study on the postoperative pain management focused on the age difference was designed. Using the numerical rating scale, we compared postoperative pain between the young group(36 patients) and the elderly group (36 patients) selected by propensity score matching in order to adjust for the patients' backgrounds. Although the young group had significantly stronger pain than the elderly group immediately after surgery(4.9±2.5 vs.3.2±2.4, p=0.002), it improved promptly. Moreover, the young group required significantly more frequent continuous infusions of opioids after surgery( p=0.001). In conclusion, it is considered that the postoperative pain management in the pneumothorax surgery should be customized according to the age.

Lysosomal enzyme cathepsin B enhances the aggregate forming activity of exogenous α-synuclein fibrils.

The formation of intracellular aggregates containing α-synuclein (α-Syn) is one of the key steps in the progression of Parkinson's disease and dementia with Lewy bodies. Recently, it was reported that pathological α-Syn fibrils can undergo cell-to-cell transmission and form Lewy body-like aggregates. However, little is known about how they form α-Syn aggregates from fibril seeds. Here, we developed an assay to study the process of aggregate formation using fluorescent protein-tagged α-Syn-expressing cells and examined the aggregate forming activity of exogenous α-Syn fibrils. α-Syn fibril-induced formation of intracellular aggregates was suppressed by a cathepsin B specific inhibitor, but not by a cathepsin D inhibitor. α-Syn fibrils pretreated with cathepsin B in vitro enhanced seeding activity in cells. Knockdown of cathepsin B also reduced fibril-induced aggregate formation. Moreover, using LAMP-1 immunocytochemistry and live-cell imaging, we observed that these aggregates initially occurred in the lysosome. They then rapidly grew larger and moved outside the boundary of the lysosome within one day. These results suggest that the lysosomal protease cathepsin B is involved in triggering intracellular aggregate formation by α-Syn fibrils.

Single-incision thoracoscopic surgery using a chest wall pulley for lung excision in patients with primary spontaneous pneumothorax.

PURPOSE: The aim of the study was to evaluate the feasibility and compare the outcomes of single-incision thoracoscopic surgery using a chest wall pulley for lung excision (PulLE) vs. those of conventional video-assisted thoracic surgery (cVATS) in patients with primary spontaneous pneumothorax (PSP). METHODS: Sixty-nine patients who underwent PulLE (n = 34) or cVATS (n = 35) between January 2009 and December 2013 were enrolled in this study. PulLE was performed as follows. After making a 17- to 25-mm single incision in the 6th intercostal space (6ICS) at the median axillary line, the visceral pleura near the bulla was sutured for traction. The parietal pleura at 3ICS was then sutured from the thoracic cavity to serve as the chest wall pulley and a traction thread was passed through the pulley. By manipulating the traction thread, it was possible to move the lesion to an arbitrary site for excision. The postoperative scar was nearly invisible. RESULTS: The operative time, duration of postoperative drainage, and postoperative hospital stay were equivalent for PulLE vs. cVATS. There was no significant difference in postoperative recurrence rates. CONCLUSIONS: PulLE has cosmetic benefits over cVATS and is easy to perform. We believe our novel procedure has the potential to become the standard operative treatment for PSP.

Enhancement of alcohol drinking in mice depends on alterations in RNA editing of serotonin 2C receptors.

Serotonin 2C receptors (5-HT(2C)R) are G-protein-coupled receptors with various actions, including involvement in drug addiction. 5-HT2CR undergoes mRNA editing, converting genomically encoded adenosine residues to inosines via adenosine deaminases acting on RNA (ADARs). Here we show that enhanced alcohol drinking behaviour in mice is associated with the degree of 5-HT(2C)R mRNA editing in the nucleus accumbens and dorsal raphe nuceus, brain regions important for reward and addiction. Following chronic alcohol vapour exposure, voluntary alcohol intake increased in C57BL/6J mice, but remained unchanged in C3H/HeJ and DBA/2J mice. 5-HT(2C)R mRNA editing frequency in both regions increased significantly in C57BL/6J mice, as did expressions of 5-HT(2C)R, ADAR1 and ADAR2, but not in other strains. Moreover, mice that exclusively express the unedited isoform (INI) of 5-HT(2C)R mRNA on a C57BL/6J background did not exhibit increased alcohol intake compared with wild-type mice. Our results indicate that alterations in 5-HT(2C)R mRNA editing underlie alcohol preference in mice.

Experimental studies of remarkable monoamine releases and neural resistance to the transient ischemia and reperfusion.

INTRODUCTION: The literature described that neural damage caused by ischemia definitely occurs in brain areas. However, few studies have shown real-time changes of extracellular monoamine levels at the time of transient ischemia. METHODS: We examined changes in the responses of dopamine (DA) and serotonin (5-HT) release in the nucleus accumbens (ACC) of rats treated with four-vessel occlusion (4VO) in experiment 1. In the second experiment, we investigated the selective neural vulnerabilities among the ACC, lateral hypothalamus (LH), and frontal cortex (FC) of rats treated with 4VO and four days of reperfusion. RESULTS: The extracellular levels of DA and 5-HT were remarkably increased 200- and 20-fold upon the 10-min clipping of both common carotid arteries in transient cerebral ischemia, respectively. Each increased monoamine release returned to the baseline levels immediately. The release of DA in the ACC and FC was significantly decreased in the rats treated with the coagulation of bilateral vertebral arteries (2VO), compared with that of sham-operated rats. K(+)-induced DA release in the ACC and FC of 4VO-treated rats was increased without alteration of DA content. DISCUSSION: Surviving dopaminergic neurons in the ACC and FC showed neural hyperfunction associated with the monoamine release, serotonergic neurons in particular these areas exhibiting functional resistance to the transient ischemic change. CONCLUSION: It is suggested that the remarkable extracellular release of DA and 5-HT was not the cause of the ischemic delayed neural degeneration in each brain area, and that the functions of neurotransmitter release involved remarkable resistance to the transient ischemia.

p62/SQSTM1 in autophagic clearance of a non-ubiquitylated substrate.

Proteolytic systems and the aggresome pathway contribute to preventing accumulation of cytotoxic aggregation-prone proteins. Although polyubiquitylation is usually required for degradation or aggresome formation, several substrates are processed independently of ubiquitin through a poorly understood mechanism. Here, we found that p62/SQSTM1, a multifunctional adaptor protein, was involved in the selective autophagic clearance of a non-ubiquitylated substrate, namely an aggregation-prone isoform of STAT5A (STAT5A_ΔE18). By using a cell line that stably expressed STAT5A_ΔE18, we investigated the properties of its aggregation and degradation. We found that STAT5A_ΔE18 formed non-ubiquitylated aggresomes and/or aggregates by impairment of proteasome functioning or autophagy. Transport of these aggregates to the perinuclear region was inhibited by trichostatin A or tubacin, inhibitors of histone deacetylase (HDAC), indicating that the non-ubiquitylated aggregates of STAT5A_ΔE18 were sequestered into aggresomes in an HDAC6-dependent manner. Moreover, p62 was bound to STAT5A_ΔE18 through its PB1 domain, and the oligomerization of p62 was required for this interaction. In p62-knockdown experiments, we found that p62 was required for autophagic clearance of STAT5A_ΔE18 but not for its aggregate formation, suggesting that the binding of p62 to non-ubiquitylated substrates might trigger their autophagic clearance.

[Evaluation of 2 ports video-assisted thoracoscopic surgery (VATS) using endo-close for primary spontaneous pneumothorax].

We aimed to assess the perioperative outcomes of 2 ports video-assisted thoracoscopic surgery(VATS) using Endo-Close (2 ports VATS) in patient for the primary spontaneous pneumothorax(PSP) compared to conventional 3 ports VATS in our hospital. 31 consecutive patients(11;2 ports VATS and 20;3 ports VATS) since 2009 were enrolled in this study. Endo-Close is a device that for pulling the anchoring thread by puncture 1.5 cm incision at the level of the 7th intercostal space( ICS) at the median axillary line( MAL) for camera port, a 1.5 cm incision at the level of the 4th ICS at the anterior axillary line for working port, and 2 mm puncture at the level of the 5th ICS at the MAL using the Endo-Close were placed for this procedure. Compared with 3 ports VATS, equivalent results were obtained by the present procedure;operation time(58.6±18.3 minutes vs 63.0±15.1 minutes, NS), duration of drainage after operation(1.0±0 days vs 1.3±0.5 days, NS), postoperative hospital stay(3.0±1.5 days vs 3.7±1.4 days, NS) significantly.Blood loss was minimal in both cases. In conclusion, 2 port VATS using Endo-Close proved to be feasible method in surgical treatment for PSP.

Odontogenic Cutaneous Sinus Tract in a 10-Year-Old Girl: A Case Report of a Rare Entity.

Odontogenic cutaneous sinus tract (OCST) is defined as pulp necrosis caused by dental caries or trauma that forms a fistula on the body surface as a drainage channel for the infected pulp. OCST can be difficult to diagnose because subjective symptoms, such as pain in the affected tooth, may be minimal. In addition, lesions in the cervical region are extremely rare. In this report, we discuss the case of a 10-year-old girl who presented with inflammation, edema, and purulent exudation on the right neck. Her symptoms resembled those of lateral cervical cysts and fistulas. However, upon evaluation, she was diagnosed with OCST. Although OCST is an important differential diagnosis for head and neck lesions, it is often overlooked. OCST should be considered in the differential diagnosis of neck masses and fistulas.

Serotonin2C receptors in the nucleus accumbens are involved in enhanced alcohol-drinking behavior.

Dopamine and serotonin (5-HT) in the nucleus accumbens (ACC) and ventral tegmental area of the mesoaccumbens reward pathways have been implicated in the mechanisms underlying development of alcohol dependence. We used a C57BL/6J mouse model with increased voluntary alcohol-drinking behavior by exposing the mice to alcohol vapor for 20 consecutive days. In the alcohol-exposed mice, the expression of 5-HT(2C) receptor mRNA increased in the ACC, caudate nucleus and putamen, dorsal raphe nucleus (DRN), hippocampus and lateral hypothalamus, while the protein level of 5-HT(2C) receptor significantly increased in the ACC. The expression of 5-HT(7) receptor mRNA increased in the ACC and DRN. Contents of 5-HT decreased in the ACC shell (ACC(S) ) and DRN of the alcohol-exposed mice. The basal extracellular releases of dopamine (DA) and 5-HT in the ACC(S) increased more in the alcohol-exposed mice than in alcohol-naïve mice. The magnitude of the alcohol-induced ACC(S) DA and 5-HT release in the alcohol-exposed mice was increased compared with the control mice. Intraperitoneal (i.p.) administration or local injection into ACC(S) of the 5-HT(2C) receptor antagonist, SB-242084, suppressed voluntary alcohol-drinking behavior in the alcohol-exposed mice. But the i.p. administration of the 5-HT(7) receptor antagonist, SB-258719, did not have significant effects on alcohol-drinking behavior in the alcohol-exposed mice. The effects of the 5-HT(2C) receptor antagonist were not observed in the air-exposed control mice. These results suggest that adaptations of the 5-HT system, especially the upregulation of 5-HT(2C) receptors in the ACC(S) , are involved in the development of enhanced voluntary alcohol-drinking behavior.

[Pleuroperitoneal communication with liver cirrhosis and chronic kidney disease needed hemodialysis successfully treated by video-assisted thoracoscopic surgery; report of a case].

A 59-year-old man treated with hemodialysis for liver cirhhosis and chronic kidney disease developed right pleural effusion and ascites. Ascites always decreased after thoracocentesis for pleural effusion. In spite of repeated treatment with chest tube drainage, massive pleural effusion reappeared. Under the diagnosis of pleuroperitoneal communication, surgical repair of the diaphragm by video assisted thoracoscopic surgery (VATS) were performed. Bulla and pin hole were found and they were resected and sutured. Surgery was safely and successfully accomplished though the patient had Child-Pugh B liver cirhhosis. Pleural effusion disappeared after surgery.

[A case of sarcomatoid carcinoma (undifferentiated carcinoma, sarcomatoid type) of the duodenum].

A 98-year-old woman with aspiration pneumonia caused by vomiting was admitted to our hospital. Abdominal computed tomography and ultrasonography showed common bile duct dilation and occlusion of the gastrointestinal tract due to a large tumor in the duodenum. Aspiration pneumonia and hepatic failure led to the patient's death. Pathological autopsy showed an 11 × 6 × 2.5-cm polypoid tumor originating from the descending part of the duodenum and involving the common bile duct. Histological examination of the tumor showed mainly spindle cells but also some adenocarcinoma cells. Tumor cells, including spindle cells, were strongly positive for cytokeratin 7; therefore, sarcomatoid carcinoma originating from the duodenum was diagnosed. To the best of our knowledge, only 5 cases of sarcomatoid carcinoma originating from the duodenum have been reported, and they often show polypoid growth. The possibility of sarcomatoid carcinoma should be considered if such findings are noted.

Synthesis and cytotoxicity of the depsipeptides analogues of callipeltin B.

Solid phase synthesis of the cyclic depsipeptides of callipeltin B analogues and evaluation of cytotoxicity of synthetic peptides are described. We attempted to synthesize cyclic depsipeptides via the esterification or the amide bond formation for cyclization steps. In the assay of synthetic peptides, the linear peptide (10) exhibited modest cytotoxicity against HeLa cells.

Identification of characteristic proteins at late-stage erythroid differentiation in vitro.

The production of red blood cells in vitro, which is useful for basic or clinical research, has been improved. Further optimization of culture protocols may facilitate erythroid differentiation from hematopoietic stem cells to red blood cells. However, the details of erythropoiesis, particularly regarding the behaviors of differentiation-related proteins, remain unclear. Here, we performed erythroid differentiation using two independent bone marrow- or cord blood-derived CD34+ cell sources and identified proteins showing reproducible differential expression in all groups. Notably, most of the proteins expressed at the early stage were downregulated during erythroid differentiation. However, seven proteins showed upregulated expression in both bone marrow cells and cord blood cells. These proteins included alpha-synuclein and selenium-binding protein 1, the roles of which have not been clarified in erythropoiesis. There is a possibility that these factors contribute to erythroid differentiation as they maintained a high expression level. These findings provide a foundation for further mechanistic studies on erythropoiesis.