The Rab27 effector granuphilin plays an indispensable role in stable docking of secretory granules to the plasma membrane by interacting with the complex of Munc18-1 and the fusion-incompetent, ...closed form of syntaxins-1~3. Although this process prevents spontaneous granule exocytosis, those docked granules actively fuse in parallel with other undocked granules after stimulation. Therefore, it is postulated that the closed form of syntaxins must be converted into the fusion-competent open form in a stimulus-dependent manner. Although Munc13 family proteins are generally thought to prime docked vesicles by facilitating conformational change in syntaxins, it is unknown which isoform acts in granuphilin-mediated, docked granule exocytosis. In the present study, we show that, although both Munc13a and Munc13b are expressed in mouse pancreatic islets and their beta-cell line MIN6, the silencing of Munc13b, but not that of Munc13a, severely affects glucose-induced insulin secretion. Furthermore, Munc13b accumulates on a subset of granules beneath the plasma membrane just prior to fusion during stimulation, whereas Munc13a is translocated to the plasma membrane where granules do not exist. When fluorescently labeled granuphilin was introduced to discriminate between molecularly docked granules and other undocked granules in living cells, Munc13b downregulation was observed to preferentially decrease the fusion of granuphilin-positive granules immobilized to the plasma membrane. These findings suggest that Munc13b promotes insulin exocytosis by clustering on molecularly docked granules in a stimulus-dependent manner.Key words: docking, insulin, live cell imaging, priming, TIRF microscopy
Background
The impact of prehabilitation on physical fitness and postoperative course after hepato-pancreato-biliary (HPB) surgeries for malignancy is unknown. The current study aimed to investigate ...the effect of preoperative exercise and nutritional therapies on nutritional status, physical fitness, and postoperative outcomes of patients undergoing an invasive HPB surgery for malignancy.
Methods
Patients who underwent open abdominal surgeries for HPB malignancies (major hepatectomy, pancreatoduodenectomy, or hepato-pancreatoduodenectomy) between 2016 and 2017 were subjected to prehabilitation. Patients before the introduction of prehabilitation were included as historical control subjects for 1:1 propensity score-matching (no-prehabilitation group). The preoperative nutritional status and postoperative course were compared between the two groups.
Results
The prehabilitation group consisted of 76 patients scheduled to undergo HPB surgeries for malignancy. An identical number of patients were selected as the no-prehabilitation group after propensity score-matching. During the waiting period, serum albumin levels were significantly deteriorated in the no-prehabilitation group, whereas this index did not deteriorate or even improved in the prehabilitation group. By performing prehabilitation, a 6-min walk distance and total muscle/fat ratio were significantly increased during the waiting period. Although the overall incidence of postoperative complications did not differ between the two groups, the postoperative hospital stay was shorter in the prehabilitation group than in the no-prehabilitation group (median, 23 vs 30 days;
p
= 0.045).
Conclusion
The introduction of prehabilitation prevented nutritional deterioration, improved physical fitness before surgery, and shortened the postoperative hospital stay for the patients undergoing HPB surgeries for malignancy.
Anterior cervical disc replacement (ACDR) using cervical artificial disc (CAD) has the advantage of maintaining the range of motion (ROM) at the surgical level, subsequently reducing the ...postoperative risk of adjacent disc disease. Following the approval for the clinical use in Japan, a post-marketing surveillance (PMS) study was conducted for two different types of CAD, namely, Mobi-C (metal-on-plastic design) and Prestige LP (metal-on-metal design). The objective of this prospective observational multicenter study was to analyze the first 2-year surgical results of the PMS study of 1-level ACDR in Japan. A total of 54 patients were registered (Mobi-C, n = 24, MC group; Prestige LP, n = 30, PLP group). Preoperative neurological assessment revealed radiculopathy in 31 patients (57.4%) and myelopathy in 15 patients (27.8%). Preoperative radiological assessment classified the disease category as disc herniation in 15 patients (27.8%), osteophyte in 6 patients (11.1%), and both in 33 patients (61.1%). The postoperative follow-up rates at 6 weeks, 6 months, 1 year, and 2 years after ACDR were 92.6%, 87.0%, 83.3%, and 79.6%, respectively. In both groups, patients' neurological condition improved significantly after surgery. Radiographic assessment revealed loss of mobility at the surgical level in 9.5% of patients in the MC group and in 9.1% of patients in the PLP group. No secondary surgeries at the initial surgical level and no serious adverse events were observed in either group. The present results suggest that 1-level ACDR is safe, although medium- to long-term follow-up is mandatory to further verify the validity of ACDR for Japanese patients.
The Rab27 effector granuphilin plays an indispensable role in stable docking of secretory granules to the plasma membrane by interacting with the complex of Munc18-1 and the fusion-incompetent, ...closed form of syntaxins-1~3. Although this process prevents spontaneous granule exocytosis, those docked granules actively fuse in parallel with other undocked granules after stimulation. Therefore, it is postulated that the closed form of syntaxins must be converted into the fusion-competent open form in a stimulus-dependent manner. Although Munc13 family proteins are generally thought to prime docked vesicles by facilitating conformational change in syntaxins, it is unknown which isoform acts in granuphilin-mediated, docked granule exocytosis. In the present study, we show that, although both Munc13a and Munc13b are expressed in mouse pancreatic islets and their beta-cell line MIN6, the silencing of Munc13b, but not that of Munc13a, severely affects glucose-induced insulin secretion. Furthermore, Munc13b accumulates on a subset of granules beneath the plasma membrane just prior to fusion during stimulation, whereas Munc13a is translocated to the plasma membrane where granules do not exist. When fluorescently labeled granuphilin was introduced to discriminate between molecularly docked granules and other undocked granules in living cells, Munc13b downregulation was observed to preferentially decrease the fusion of granuphilin-positive granules immobilized to the plasma membrane. These findings suggest that Munc13b promotes insulin exocytosis by clustering on molecularly docked granules in a stimulus-dependent manner. Key words: docking; insulin; live cell imaging; priming; TIRF microscopy
Impediments to DNA replication are known to induce gross chromosomal rearrangements (GCRs) and copy-number variations (CNVs). GCRs and CNVs underlie human genomic disorders and are a feature of ...cancer. During cancer development, environmental factors and oncogene-driven proliferation promote replication stress. Resulting GCRs and CNVs are proposed to contribute to cancer development and therapy resistance. When stress arrests replication, the replisome remains associated with the fork DNA (stalled fork) and is protected by the inter-S-phase checkpoint. Stalled forks efficiently resume when the stress is relieved. However, if the polymerases dissociate from the fork (fork collapse) or the fork structure breaks (broken fork), replication restart can proceed either by homologous recombination or microhomology-primed re-initiation. Here we ascertain the consequences of replication with a fork restarted by homologous recombination in fission yeast. We identify a new mechanism of chromosomal rearrangement through the observation that recombination-restarted forks have a considerably high propensity to execute a U-turn at small inverted repeats (up to 1 in 40 replication events). We propose that the error-prone nature of restarted forks contributes to the generation of GCRs and gene amplification in cancer, and to non-recurrent CNVs in genomic disorders.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The junction between the epithelium and the underlying connective tissue undulates, constituting of rete ridges, which lack currently available soft tissue constructs. In this study, using a micro ...electro mechanical systems process and soft lithography, fifteen negative molds, with different dimensions and aspect ratios in grid- and pillar-type configurations, were designed and fabricated to create three-dimensional micropatterns and replicated onto fish-scale type I collagen scaffolds treated with chemical crosslinking. Image analyses showed the micropatterns were well-transferred onto the scaffold surfaces, showing the versatility of our manufacturing system. With the help of rheological test, the collagen scaffold manufactured in this study was confirmed to be an ideal gel and have visco-elastic features. As compared with our previous study, its mechanical and handling properties were improved by chemical cross-linking, which is beneficial for grafting and suturing into the complex structures of oral cavity. Histologic evaluation of a tissue-engineered oral mucosa showed the topographical microstructures of grid-type were well-preserved, rather than pillar-type, a well-stratified epithelial layer was regenerated on all scaffolds and the epithelial rete ridge-like structure was developed. As this three-dimensional microstructure is valuable for maintaining epithelial integrity, our micropatterned collagen scaffolds can be used not only intraorally but extraorally as a graft material for human use.
Mutations in several genes encoding components of the SWI/SNF chromatin remodeling complex cause neurodevelopmental disorders (NDDs). Here, we report on five individuals with mutations in SMARCD1; ...the individuals present with developmental delay, intellectual disability, hypotonia, feeding difficulties, and small hands and feet. Trio exome sequencing proved the mutations to be de novo in four of the five individuals. Mutations in other SWI/SNF components cause Coffin-Siris syndrome, Nicolaides-Baraitser syndrome, or other syndromic and non-syndromic NDDs. Although the individuals presented here have dysmorphisms and some clinical overlap with these syndromes, they lack their typical facial dysmorphisms. To gain insight into the function of SMARCD1 in neurons, we investigated the Drosophila ortholog Bap60 in postmitotic memory-forming neurons of the adult Drosophila mushroom body (MB). Targeted knockdown of Bap60 in the MB of adult flies causes defects in long-term memory. Mushroom-body-specific transcriptome analysis revealed that Bap60 is required for context-dependent expression of genes involved in neuron function and development in juvenile flies when synaptic connections are actively being formed in response to experience. Taken together, we identify an NDD caused by SMARCD1 mutations and establish a role for the SMARCD1 ortholog Bap60 in the regulation of neurodevelopmental genes during a critical time window of juvenile adult brain development when neuronal circuits that are required for learning and memory are formed.
Oxidative stress plays pivotal roles in aging, neurodegenerative disease, and pathological conditions such as ischemia. We investigated the effect of sulforaphane and 6-(methysulfinyl) hexyl ...isothiocyanate (6-HITC), a naturally occurring isothiocyanate, on oxidative stress–induced cytotoxicity using primary neuronal cultures of rat striatum. Pretreatment with sulforaphane and 6-HITC significantly protected against H2O2- and paraquat-induced cytotoxicity in a concentration-dependent manner. Sulforaphane and 6-HITC induced the translocation of nuclear factor E2–related factor 2 (Nrf2) into the nucleus and increased the expression of γ-glutamylcysteine synthetase (γ-GCS), a rate-limiting enzyme in glutathione synthesis, and the intracellular glutathione content. Treatment with reduced glutathione (GSH) and N-acetyl-l-cysteine, a substance for glutathione synthesis, significantly prevented the cytotoxicity induced by H2O2 and paraquat. Moreover, exposure to l-buthionine-sulfoximine, an irreversible inhibitor of γ-GCS, suppressed the protective effects of sulforaphane and 6-HITC. In contrast, sulforaphane and 6-HITC increased heme oxygenase-1 (HO-1) expression in neurons. However, zinc-protophorphyrin IX, a competitive inhibitor of HO-1, did not influence the protective effects of sulforaphane and 6-HITC. These results suggest that sulforaphane and 6-HITC prevent oxidative stress-induced cytotoxicity in rat striatal cultures by raising the intracellular glutathione content via an increase in γ-GCS expression induced by the activation of the Nrf2-antioxidant response element pathway.
Nicotinamide-N-methyltransferase (NNMT) is an enzyme that consumes S-adenosyl-methionine (SAM) and nicotinamide (NAM) to produce S-adenosyl-homocysteine (SAH) and 1-methylnicotinamide (MNAM). How ...much NNMT contributes to the quantity regulation of these four metabolites depends on whether NNMT is a major consumer or producer of these metabolites, which varies among various cellular contexts. Yet, whether NNMT critically regulates these metabolites in the AML12 hepatocyte cell line has been unexplored. To address this, we knockdown Nnmt in AML12 cells and investigate the effects of Nnmt RNAi on metabolism and gene expression. We find that Nnmt RNAi accumulates SAM and SAH, whereas it reduces MNAM with NAM being unaltered. These results indicate that NNMT is a significant consumer of SAM and critical for MNAM production in this cell line. Moreover, transcriptome analyses reveal that altered SAM and MNAM homeostasis is accompanied by various detrimental molecular phenotypes, as exemplified by the down-regulations of lipogenic genes, such as Srebf1. Consistent with this, oil-red O-staining experiments demonstrate the decrease of total neutral lipids upon Nnmt RNAi. Treating Nnmt RNAi AML12 cells with cycloleucine, an inhibitor of SAM biogenesis suppresses SAM accumulation and rescues the decrease of neutral lipids. MNAM also shows activity to elevate neutral lipids. These results suggest that NNMT contributes to lipid metabolism by maintaining proper SAM and MNAM homeostasis. This study provides an additional example where NNMT plays a critical role in regulating SAM and MNAM metabolism.
Previous genetic studies in mice have shown that functional loss of activin receptor-like kinase 7 (ALK7), a type I transforming growth factor-β receptor, increases lipolysis to resist fat ...accumulation in adipocytes. Although growth/differentiation factor 3 (GDF3) has been suggested to function as a ligand of ALK7 under nutrient-excess conditions, it is unknown how GDF3 production is regulated. Here, we show that a physiologically low level of insulin converts CD11c
adipose tissue macrophages (ATMs) into GDF3-producing CD11c
macrophages ex vivo and directs ALK7-dependent accumulation of fat in vivo. Depletion of ATMs by clodronate upregulates adipose lipases and reduces fat mass in ALK7-intact obese mice, but not in their ALK7-deficient counterparts. Furthermore, depletion of ATMs or transplantation of GDF3-deficient bone marrow negates the in vivo effects of insulin on both lipolysis and fat accumulation in ALK7-intact mice. The GDF3-ALK7 axis between ATMs and adipocytes represents a previously unrecognized mechanism by which insulin regulates both fat metabolism and mass.
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