The first clinical implementation of real-time 6 degree-of-freedom image-guided radiotherapy for liver SABR patients.

PURPOSE: Multiple survey results have identified a demand for improved motion management for liver cancer IGRT. Until now, real-time IGRT for liver has been the domain of dedicated and expensive cancer radiotherapy systems. The purpose of this study was to clinically implement and characterise the performance of a novel real-time 6 degree-of-freedom (DoF) IGRT system, Kilovoltage Intrafraction Monitoring (KIM) for liver SABR patients. METHODS/MATERIALS: The KIM technology segmented gold fiducial markers in intra-fraction x-ray images as a surrogate for the liver tumour and converted the 2D segmented marker positions into a real-time 6DoF tumour position. Fifteen liver SABR patients were recruited and treated with KIM combined with external surrogate guidance at three radiotherapy centres in the TROG 17.03 LARK multi-institutional prospective clinical trial. Patients were either treated in breath-hold or in free breathing using the gating method. The KIM localisation accuracy and dosimetric accuracy achieved with KIM + external surrogate were measured and the results were compared to those with the estimated external surrogate alone. RESULTS: The KIM localisation accuracy was 0.2±0.9 mm (left-right), 0.3±0.6 mm (superior-inferior) and 1.2±0.8 mm (anterior-posterior) for translations and -0.1◦±0.8◦ (left-right), 0.6◦±1.2◦ (superior-inferior) and 0.1◦±0.9◦ (anterior-posterior) for rotations. The cumulative dose to the GTV with KIM + external surrogate was always within 5% of the plan. In 2 out of 15 patients, >5% dose error would have occurred to the GTV and an organ-at-risk with external surrogate alone. CONCLUSIONS: This work demonstrates that real-time 6DoF IGRT for liver can be implemented on standard radiotherapy systems to improve treatment accuracy and safety. The observations made during the treatments highlight the potential false assurance of using traditional external surrogates to assess tumour motion in patients and the need for ongoing improvement of IGRT technologies.

Changes of antioxidant enzymes in the kidney after cardiac arrest in the rat model.

Globally, cardiac arrest (CA) is a leading cause of death and disability. Asphyxial CA (ACA)-induced kidney damage is a crucial factor in reducing the survival rate. The purpose of this study was to investigate the role of antioxidant enzymes in histopathological renal damage in an ACA rat model at different time points. A total of 88 rats were divided into five groups and exposed to ACA except for the sham group. To evaluate glomerular function and oxidative stress, serum levels of blood urea nitrogen (BUN) and creatinine (Crtn) and malondialdehyde (MDA) levels in renal tissues were measured. To determine histopathological damage, hematoxylin and eosin staining, periodic acid-Schiff staining, and Masson's trichrome staining were performed. Expression levels of antioxidant enzymes including superoxide dismutase-1 (SOD-1), superoxide dismutase-2 (SOD-2), catalase (CAT), and glutathione peroxidase (GPx) were measured by immunohistochemistry (IHC). Survival rate of the experimental rats was reduced to 80% at 6 h, 55% at 12 h, 42.9% at 1 day, and 33% at 2 days after return of spontaneous circulation. Levels of BUN, Crtn, and MDA started to increase significantly in the early period of CA induction. Renal histopathological damage increased markedly from 6 h until two days post-CA. Additionally, expression levels of antioxidant enzymes were significantly decreased at 6 h, 12 h, 1 day, and 2 days after CA. CA-induced oxidative stress and decreased levels of antioxidant enzymes (SOD-1, SOD-2, CAT, GPx) from 6 h to two days could be possible mediators of severe renal tissue damage and increased mortality rate.

Changes of antioxidant enzymes in the kidney after cardiac arrest in the rat model

Globally, cardiac arrest (CA) is a leading cause of death and disability. Asphyxial CA (ACA)-induced kidney damage is a crucial factor in reducing the survival rate. The purpose of this study was to investigate the role of antioxidant enzymes in histopathological renal damage in an ACA rat model at different time points. A total of 88 rats were divided into five groups and exposed to ACA except for the sham group. To evaluate glomerular function and oxidative stress, serum levels of blood urea nitrogen (BUN) and creatinine (Crtn) and malondialdehyde (MDA) levels in renal tissues were measured. To determine histopathological damage, hematoxylin and eosin staining, periodic acid-Schiff staining, and Masson's trichrome staining were performed. Expression levels of antioxidant enzymes including superoxide dismutase-1 (SOD-1), superoxide dismutase-2 (SOD-2), catalase (CAT), and glutathione peroxidase (GPx) were measured by immunohistochemistry (IHC). Survival rate of the experimental rats was reduced to 80% at 6 h, 55% at 12 h, 42.9% at 1 day, and 33% at 2 days after return of spontaneous circulation. Levels of BUN, Crtn, and MDA started to increase significantly in the early period of CA induction. Renal histopathological damage increased markedly from 6 h until two days post-CA. Additionally, expression levels of antioxidant enzymes were significantly decreased at 6 h, 12 h, 1 day, and 2 days after CA. CA-induced oxidative stress and decreased levels of antioxidant enzymes (SOD-1, SOD-2, CAT, GPx) from 6 h to two days could be possible mediators of severe renal tissue damage and increased mortality rate.

Diagnosis and management of primary central nervous system lymphoma: a consensus practice statement from the Australasian Lymphoma Alliance.

Primary central nervous system lymphoma is a clinicopathological disease entity that accounts for 1% of all non-Hodgkin lymphoma (NHL). Advanced patient age, adverse disease biology and complexities of diagnosis and treatment render outcomes markedly inferior to systemic NHL. Despite this, an increasing evidence base, including limited randomised controlled clinical trial data, is informing optimal therapeutic strategies with methotrexate-based induction chemotherapy schedules and intensified consolidation in selected patients. This practice statement represents an evidence-based review of the literature and has been devised to assist healthcare professionals in the diagnosis and management of this disease.

The role of intraoperative frozen section analysis in joint arthroplasty with CD66b immunohistochemical staining.

The preoperative diagnosis of infection during joint arthroplasty is important for clinical management. However, the evaluation of polymorphonuclear leukocytes (PMNs) during frozen section analysis is sometimes difficult due to frozen artifacts. In the present study, we sought to investigate the utility of intraoperative fresh frozen section (FFS) examination for diagnosis of infection and to evaluate whether the neutrophil-specific surface marker CD66b helps to improve the diagnostic accuracy of infection. A consecutive series of 65 original frozen sections at the time of resection arthroplasty was retrospectively reviewed compared with corresponding permanent sections. The presence of PMNs was determined using intraoperative FFS and permanent sections. Furthermore, CD66b staining was performed to identify PMNs clearly. The ratio of male to female patients was 21:42. The mean age was 70 years. Postoperatively, 25 of 65 cases were histologically diagnosed with infection (25/65; 39%). The sensitivity and specificity of intraoperative FFS relative to permanent section histology were 100% (25/25) and 95% (38/40), respectively. Among 40 patients without infection, two showed false-positive results during intraoperative FFS diagnosis (2/40, 5%). In addition, on CD66b staining, six cases (9%) experienced changes in results, which altered the sensitivity and specificity of intraoperative FFS compared with permanent histology only to 87% and 87%, respectively. In conclusion, the diagnostic performance of intraoperative FFS is high and comparable to yields of permanent section histology. Therefore, intraoperative FFS is highly suitable diagnostic method for detection of infection during joint arthroplasty. And CD66b immunostaining facilitates delicate identification of PMNs, especially in equivocal cases.

Maxillary molar root and canal morphology of Neolithic and modern Chinese.

OBJECTIVE: This study aimed to characterize Neolithic human maxillary molars from archeological remains at the Jiaojia site, Shandong, China, and compare their ultrastructural features with sex and age-matched modern locals. DESIGN: Maxillary first (n = 86) and second (n = 80) molars in 5000-year-old individuals (n = 50) from the Jiaojia site were scanned by cone-beam computed tomography (CBCT). Sex and age-matched control groups were assigned from oral surgical patients at Shandong University. Images were analyzed for crown size, root length, root morphology, canal inter-orifice distances, mesiobuccal canal morphology, and second mesiobuccal (MB2) canal prevalence and location. Neolithic and modern values were compared statistically using Chi-squared and Mann-Whitney test at p < .05. RESULTS: Crown and root size were smaller, and canal inter-orifice distances were shorter in Neolithic maxillary molars than their modern counterparts. For mesiobuccal roots, Weine's Type I single canals were the most prevalent in Neolithic and modern first and second molars. MB2 canal prevalence were not significantly different (p > .05) in Neolithic (53.3%) or modern (60.5%) first molars, and Neolithic (11.3%) or modern (21.3%) second molars. But, MB2 prevalence was significantly higher for modern than ancient male first (p = .032) and second (p = .005) molars. Additionally, MB2 were located more mesially and closer to MB1 in Neolithic than modern molars. CONCLUSIONS: Maxillary molar root and canal morphology of ancient 5000-year-old remains at the Jiaojia site resemble that of local patients. A trend towards larger tooth size, and more dispersed MB2 canals over this short evolutionary period warrants additional investigation.

A therapeutic intervention for Alzheimer's disease using ginsenoside Rg3: its role in M2 microglial activation and non-amyloidogenesis.

Previously, we have reported that ginsenoside Rg3 has typical activities for neuroprotection and Aß42 clearance by modulating microglia. In this study, we determined the pivotal role of ginsenoside Rg3 in microglia and neuronal cells. In human microglia, Rg3 and its stereoisomers significantly restored inflammatory M1 to normal M0 state and promoted M2 activation by up-regulating acute cytokines such as interleukin-10 and Arginase 1. Moreover, scavenger receptor type A (SRA) was significantly elevated in the presence of ginsenoside Rg3 and 20(S)-Rg3. This indicated that ginsenoside Rg3 could play a crucial role in Aß uptake and clearance under activated M2 state. We also observed that soluble amyloid precursor protein-alpha (sAPPα) and ADAM10 levels were increased in APP swe-transfected Nuro-2a neuronal cells, whereas sAPPß was not processed, suggesting that ginsenoside Rg3 was involved in non-amyloidogenic processing. In immunocytochemistry, SRA and a disintegrin and metalloproteinase 10 (desintegrin and metalloproteinase-containing protein 10, ADAM10) were coincidently upregulated in the presence of ginsenoside Rg3 and its stereoisomers compared to those in normal control. Taken together, these results suggested that ginsenoside Rg3 could boost acute activation of microglia, promote Aß uptake, and elevate the sAPPα processing under activated M2 state. Although in vivo studies need to be performed, it is certain that ginsenoside Rg3 is highly involved in ameliorating the pathogenesis of neurodegeneration and can be a promising candidate for treating Alzheimer's disease as a new therapeutic intervention.

The correlation between amylin and insulin by treatment with 2-deoxy-D-glucose and/or mannose in rat insulinoma ins-1E cells.

Amylin or islet amyloid polypeptide (IAPP) is a peptide synthesized and secreted with insulin by the pancreatic ß-cells. A role for amylin in the pathogenesis of type 2 diabetes (T2D) by causing insulin resistance or inhibiting insulin synthesis and secretion has been suggested by in vitro and in vivo studies. These studies are consistent with the effect of endogenous amylin on pancreatic ßcells to modulate and/or restrain insulin secretion. Here, we reported the correlation between amylin and insulin in rat insulinoma INS-1E cells by treating 2-deoxy-D-glucose (2-DG) and/or mannose. Cell viability was not affected by 24 h treatment with 2-DG and/or mannose, but it was significantly decreased by 48 h treatment with 5 and 10 mM 2-DG. in the 24 h treatment, the synthesis of insulin in the cells and the secretion of insulin into the media showed a significant inverse association. in the 48-h treatment, amylin synthesis vs. the secretion and insulin synthesis vs. the secretion showed a significant inverse relation. The synthesis of amylin vs. insulin and the secretion of amylin vs. insulin showed a significant inverse relationship. The p-ERK, antioxidant enzymes (Cu/Zn-superoxide dismutase (SOD), Mn-SOD, and catalase), and endoplasmic reticulum (ER) stress markers (cleaved caspase-12, CHOP, p-SAPK/JNK, and BiP/GRP78) were significantly increased or decreased by the 24 h and 48 h treatments. These data suggest the relative correlation to the synthesis of amylin by cells vs. the secretion into the media, the synthesis of amylin vs. insulin, and the secretion of amylin vs. insulin under 2-DG and/or mannose in rat insulinoma INS-1E cells. Therefore, these results can provide primary data for the hypothesis that the amylin-insulin relationships may be involved with the human amylin toxicity in pancreatic beta cells.

Potassium-dependent sodium/calcium exchanger 3 (Nckx3) depletion leads to abnormal motor function and social behavior in mice.

Transcellular calcium transport is an essential activity in mineralized tissue formation, including that in nervous systems. Dysregulation of Ca2+ homeostasis can induce excitotoxicity and neurodegeneration in the central nervous system. Nckx3, a potassium-dependent Na+/Ca2+ exchanger, is most abundant in the brain and has a critical role in the transport of intracellular calcium across the cell membrane. However, the roles of Nckx3 in neuron development and function remain unreported. Herein, we examined the behaviors of Nckx3-knock-out mice at the age of six weeks. Detailed behavioral analyses showed Nckx3-/- mice exhibited an increase in moving distances in the open field test. Additionally, the rotarod test revealed motor learning defects in Nckx3-/- mice. Both Nckx3+/- and Nckx-/- mice also exhibited deficits in sociability and social novelty preference. Furthermore, Nckx-/- mice displayed increased depression-related behavior. However, there was no significant change in cognition function detected in Nckx-/- mice. This study demonstrates that NCKX3 is involved in behavior and neuronal function.

Histone methyltransferase DOT1L coordinates AR and MYC stability in prostate cancer.

The histone methyltransferase DOT1L methylates lysine 79 (K79) on histone H3 and is involved in Mixed Lineage Leukemia (MLL) fusion leukemogenesis; however, its role in prostate cancer (PCa) is undefined. Here we show that DOT1L is overexpressed in PCa and is associated with poor outcome. Genetic and chemical inhibition of DOT1L selectively impaired the viability of androgen receptor (AR)-positive PCa cells and organoids, including castration-resistant and enzalutamide-resistant cells. The sensitivity of AR-positive cells is due to a distal K79 methylation-marked enhancer in the MYC gene bound by AR and DOT1L not present in AR-negative cells. DOT1L inhibition leads to reduced MYC expression and upregulation of MYC-regulated E3 ubiquitin ligases HECTD4 and MYCBP2, which promote AR and MYC degradation. This leads to further repression of MYC in a negative feed forward manner. Thus DOT1L selectively regulates the tumorigenicity of AR-positive prostate cancer cells and is a promising therapeutic target for PCa.

Second-phase validation study of an alternative developmental toxicity test using mouse embryonic stem cell-derived embryoid bodies.

The embryoid body test (EBT) is a developmental toxicity test method that measures the size of embryoid bodies (EBs) and the viability of mouse embryonic stem cells (mESCs) and fibroblasts (3T3 cells). The previous pre-validation study confirmed the high accuracy (above 80%) of EBT using 26 coded test chemicals. This second-phase validation study assessed the inter-laboratory reproducibility (5 chemicals in common) and predictive capacity (10 chemicals in each laboratory) test using the coded test chemicals at three laboratories. For the prediction model, the accuracy is increased when more data is accumulated. Therefore, we updated the prediction model and analyzed the results of the second year with the newly created-prediction model. Statistical analysis of the inter-laboratory reproducibility test results indicated that accuracy, sensitivity, and specificity were 87%, 78%, and 100%, respectively. The results of the statistical analysis of the predictive capacity test showed an accuracy of 80%, sensitivity of 78%, and specificity of 81%. In conclusion, the EBT can accurately classify various embryotoxicants within a short period and with relatively little effort. Therefore, EBT can be used as a good way to test developmental toxicity.

[Melatonin Induces Apoptotic Cell Death in 3T3-L1 Preadipocytes].

Obesity is a major disease that causes significant complications. Inhibition of preadipocyte proliferation has the potential to prevent obesity and metabolic diseases. Melatonin is a pineal gland hormone that has various effects on cells and tissues. In this research, we investigated whether melatonin induces apoptosis in 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were cultured until confluence and then treated with 0, 10, 100, and 1000 µM melatonin for 1, 3, and 5 days. A cell viability assay kit was used for determining cell viability. Cell death marker proteins were assessed by Western blot analysis using GAPDH for control. Apoptotic morphological changes with nuclei fragmentation were observed using DAPI staining. Melatonin treatment decreased the phosphorylated extracellular signal-regulated kinases (p-ERK) activation while increasing the activation of caspase-3, 8, and 9. Furthermore, melatonin not only increased Bcl-2-associated X protein (Bax) but decreased B-cell lymphoma 2 (Bcl-2) expression as dose increases from 0 to 1000 µM. The melatonin treatment also suppressed the growth of preadipocytes with increasing concentration. These effects were attenuated by luzindole, a melatonin receptor antagonist and U0126, an inhibitor of p-ERK activation. In conclusion, melatonin can induce apoptosis of 3T3-L1 preadipocytes via p-ERK decrease.

Correlating results from high resolution EBSD with TEM- and ECCI-based dislocation microscopy: Approaching single dislocation sensitivity via noise reduction.

High resolution electron backscatter diffraction (HREBSD), an SEM-based diffraction technique, may be used to measure the lattice distortion of a crystalline material and to infer the geometrically necessary dislocation content. Uncertainty in the image correlation process used to compare diffraction patterns leads to an uneven distribution of measurement noise in terms of the lattice distortion, which results in erroneous identification of dislocation type and density. This work presents a method of reducing noise in HREBSD dislocation measurements by removing the effect of the most problematic components of the measured distortion. The method is then validated by comparing with TEM analysis of dislocation pile-ups near a twin boundary in austenitic stainless steel and with ECCI analysis near a nano-indentation on a tantalum oligocrystal. The HREBSD dislocation microscopy technique is able to resolve individual dislocations visible in TEM and ECCI and correctly identify their Burgers vectors.

Nanomolar melatonin influences insulin synthesis and secretion in rat insulinoma INS-1E cells.

Pancreatic beta-cell dysfunction results in reductions of insulin synthesis/secretion, cell survival, and insulin sensitivity thereby inducing diabetes mellitus. In this study, how nanomolar melatonin regulates insulin synthesis and secretion in rat insulinoma INS-1E cells was investigated. At melatonin concentrations of 10 - 100 nM for 48 hours, melatonin significantly increased the insulin protein level in INS-1E cells above the level in control cells without melatonin or glucose treatments and decreased the insulin level in media with glucose: increases in insulin synthesis and decreases in insulin secretion occurred in dose-dependent manners. Luzindole or 4-phenyl-2-propionamidotetralin (4P-PDOT), melatonin receptor antagonists, inhibited the melatonin-induced insulin level in cells and media. Levels of membrane vesicle trafficking-related proteins including Rab5, GOPC, phospho-caveolin-1, EEA1, and clathrin proteins significantly increased with melatonin treatment above that in control cells without melatonin or glucose treatments, whereas expressions of APPL1 and syntaxin-6 proteins significantly decreased with melatonin treatment. The increases in the phosphorylation of mammalian target of rapamycin (p-mTOR), raptor protein, and mTOR complex 1 (mTORC1) levels were consistent with the increments in the expressions of p-Akt (Ser473, Thr308) and stress-induced IRE1α/p-eIF2α proteins in the endoplasmic reticulum following melatonin treatment. also, expression levels of Bcl-2 and Bcl-xl proteins were significantly increased compared to those in control cells without melatonin or glucose treatments, whereas the Bax protein level decreased. These results indicate that nanomolar melatonin regulates insulin synthesis and secretion associated with membrane vesicle trafficking-related proteins, including Rab5, GOPC, p-Caveolin-1, EEA1, and clathrin, through the Akt/mTOR pathway.

Cigarette smoke extraxt influences intracellular calcium concentration in A549 cells.

Cigarette smoking is a major risk factor for pulmonary diseases, including chronic obstructive pulmonary disease (COPD) and cancer. Cigarette smoke is reported to contain over 4,000 chemical compounds. Therefore, it needs to study the effects of cigarette smoke extract (CSE) administration on intracellular calcium concentration. In this study, we investigated how CSE influences intracellular calcium concentration in human lung adenocarcinoma A549 cells. The CSE concentrations used (0.4, 2, 3%) did not influence cell viability. However, at these CSE concentrations, calcium influx transient receptor potential vanilloid 4 (TRPV4) and transient receptor potential vanilloid 6 (TRPV6) proteins significantly increased, whereas calcium efflux sodium-calcium exchanger (NCX1) and plasma membrane Ca2+ ATPase (PMCA1) proteins significantly decreased from those of the control cells. The 3% CSE treatment produced an intracellular calcium concentration higher than that of the control treatment through methods of co-transfection of pGP-CMV-GCaMP6f/CMV-R-GECO1.2 and Rhod-4 Assay. CSE induced concentration-dependent increments in hypoxia-inducible factor (HIF)-1α and HIF-2α protein levels. Moreover, phosphorylation of ERK and Akt was induced by CSE treatment. Also, mitochondrial marker B-cell lymphoma 2 (Bcl-2) protein level decreased and Bcl-2-associated X (Bax) protein level increased following CSE treatment. Also, endoplasmic reticulum (ER) stress markers BiP, CHOP, p-SAPK, and p-eIF2α levels were increased by CSE treatment. These results suggest that CSE may increase the concentration of intracellular calcium, thus increasing mitochondrial and ER stress.

Inhibitory effect of octyl-phenol and bisphenol A on calcium signaling in cardiomyocyte differentiation of mouse embryonic stem cells.

Endocrine-disrupting chemicals (EDCs) have structures similar to steroid hormones and can interfere with hormone synthesis and normal physiological functions of reproductive organs. For example, sex steroid hormones influence calcium signaling of the cardiac muscle in early embryo development. To confirm the effect of progesterone (P4), octyl-phenol (OP), and bisphenol A (BPA) on early differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes, mESCs were treated with P4, OP, and BPA two days after attachment and media were replaced every two days. In addition, cells were treated with mifepristone (RU486), a synthetic steroid that has an affinity for progesterone receptor (Pgr), for one day starting on day 11. Beating ratio was decreased with P4, OP, and BPA treatment. The Pgr mRNA level was significantly increased in the P4-, OP- and BPA-treated groups. However, the mRNA level of the calcium channel gene (Trpv2), contraction-related genes (Ryr2, Cam2, and Mylk3) and cardiac development and morphogenesis genes (Rbp4, Ly6e, and Gata4) were significantly decreased in the P4-, OP-, and BPA-treated groups. Interestingly, treatment with RU486 rescued the altered calcium channel gene, contraction-related genes, and cardiac development and morphogenesis genes. P4, OP, and BPA treatments reduced the intracellular calcium level. Taken together, these results indicate that EDCs (OP and BPA) has a structure similar to that of endogenous steroid hormones such as progesterone and estrogen, and OP and BPA act like progesterone to inhibit and disrupt cardiomyocyte differentiation of mESCs.

Regulatory effect of dexamethasone on tracheal calcium processing proteins and mucosal secretion.

Dexamethasone inhibits mucin secretion considering the primary option for treating acute asthma exacerbation. However, the mechanism underlying dexamethasone-induced decreased in mucosecretion is unclear. Recent studies have reported that dexamethasone exerts an inhibitory effect on mucosecretion in the lung by modulating the expression of calcium processing genes. However, the expression of the calcium processing genes in the trachea is not examined yet. Thus, the present study is the first to report the localization of calcium processing proteins such as transient receptor potential vanilloid-4 (Trpv4), transient receptor potential vanilloid-6 (Trpv6), calbindin-D9k (CaBP-9k) and plasma membrane Ca2+-ATPase 1 (Pmca1) in the mouse trachea and their glucocorticoid-induced response. In this study, mice were subcutaneously injected with dexamethasone for 5 days, and their tracheal samples were collected by dividing the trachea into the cervical, and thoracic sections based on its anatomical structure. The localization of TRPV4, TRPV6, CaBP-9k, and PMCA1 proteins was detected in the tracheal epithelium, submucosal glands, cartilages and muscles. Dexamethasone treatment downregulated the mRNA expression of the four calcium processing genes and mucin producing genes. The dexamethasone-induced decrease in the secretion of mucosubstances in the trachea was determined by performing Alcian blue-periodic acid-Schiff staining. Thus, the findings of the present study suggest that glucocorticoids simultaneously can regulate the expression of calcium processing genes and tracheal mucosecretion.

Melatonin influences the expression and oligomerization of amylin in rat INS-1E cells.

The present study investigated whether melatonin influences the expression/oligomerization of amylin with endoplasmic reticulum (ER) stress in rat insulinoma INS-1E cells. No change in cell survival after exposure to thapsigargin- and tunicamycin-combined melatonin treatment or melatonin-only treatment was observed when compared with the normal control cells. With thapsigargin-only or combined tunicamycin-melatonin treatments, phosphorylation of extracellular signal-regulated kinase (ERK) was significantly increased compared with control and melatonin-only treatments. A significant increase was observed in the levels of ER stress markers, namely, phosphorylated inositol-requiring protein 1α (p-IRE1α), CCAAT enhancer binding proteins (C/EBP)-homologous protein, p-eukaryotic translation initiation factor 2α and cleaved caspase-12, in the thapsigargin-combined melatonin-treated cells as compared with the tunicamycin-combined or only melatonin treatment. The melatonin-only treatment resulted in increased levels of amylin expression/oligomerization in 15-25 kDa and insulin proteins, compared with the thapsigargin- and tunicamycin-combined melatonin treatments. Treatment with ER stress inhibitor 4-phenylbutyric acid (4-PBA) did not suppress amylin expression/oligomerization or insulin production with thapsigargin or tunicamycin treatment. Levels of cleaved caspase-12 were significantly decreased in the thapsigargin- or tunicamycin-4-PBA combination treatments. Therefore, whether melatonin regulates the amylin expression/oligomerization in thapsigargin- or tunicamycin-combined with Bafilomycin A1 (autophagy inhibitor) or MG132 (proteasome inhibitor) treatments were investigated. Amylin expression/oligomerization with melatonin treatment was significantly decreased in the thapsigargin- or tunicamycin-combined Bafilomycin A1 or MG132 treatments. Since these outcomes are involved in cell viability, they indicate that increased cell death leads to decreased amylin expression/oligomerization, however, the effects of melatonin treatment on amylin expression/oligomerization induce proliferation of pancreatic ß cells and improve the cellular functions of pancreatic ß cells.

Effects of coat length and faecal hair removal on measured nutrient digestibility in cats.

OBJECTIVES: This experiment was conducted to evaluate the effect of coat length and faecal hair removal on measured nutrient digestibility in longhair and shorthair cats. METHODS: A total of 14 adult domestic cats, with a mean ± SD body weight of 4.5 ± 1.21 kg and a mean ± SD age of 3.3 ± 1.38 years, were used for a nutrient digestibility trial. The nutrient digestibility of cats was measured by hair-included faeces and hair-removed faeces. Food was provided twice daily (09:00 h and 16:00 h) and water was provided ad libitum. Cats were adapted to a steel cage and diet for 16 days before a 10 day collection period. During the collection period, food offered, food refused and faecal output were measured daily and used for digestibility analysis. RESULTS: The digestibility of dry matter, crude protein and neutral detergent fibre (NDF) of longhair cats was underestimated by 8% ( P = 0.02), 9% ( P = 0.04) and 14% ( P = 0.04), respectively. In shorthair cats, the digestibility of dry matter, crude protein and crude ash increased by 4% ( P = 0.01), 5% ( P = 0.02) and 15% ( P <0.01), respectively, with hair-removed faeces. The nutrient digestibility between longhair and shorthair cats showed no difference when hair-included faeces were used. However, when using hair-removed faeces, the digestibility of NDF and amino acids in longhair cats were about 4-8% higher than in shorthair cats ( P <0.05). CONCLUSIONS AND RELEVANCE: This study showed that the nutrient digestibility values were affected by whether the hair in faeces was removed or not. Removing hair from faeces is a more precise method with which to evaluate the apparent nutrient digestibility of domestic cats.

Evaluation of grooming behaviour and apparent digestibility method in cats.

OBJECTIVES: This experiment was conducted to evaluate the behavioural time budget for grooming and grooming patterns for shorthair and longhair cats, and to assess the effect of grooming behaviour on apparent digestibility of nutrients in domestic cats ( Felis catus) by comparing hair-included faeces and hair-removed faeces. METHODS: A total of 10 adult domestic cats, with a mean ± SD body weight of 4.3 ± 0.89 kg and a mean ± SD age of 3.5 ± 1.38 years, were used for behavioural observation. Cats were housed individually in stainless steel cages at the animal hospital. The cats' behaviour was recorded on a webcam videotaping system for one 24 h period; then, faecal samples were collected and analysed to measure apparent digestibility. RESULTS: There was no significant difference between longhair and shorthair cats in behavioural time budget for grooming and grooming patterns. The apparent digestibility of dry matter, crude protein, crude ash, acid detergent fibre (ADF) and neutral detergent fibre (NDF) of hair-removed faeces was significantly higher than that of hair-included faeces: about 6% ( P <0.01), 7% ( P <0.01), 14% ( P <0.01), 12% ( P = 0.01) and 10% ( P <0.01), respectively. CONCLUSIONS AND RELEVANCE: There was no difference in grooming patterns between longhair cats and shorthair cats. Also, the digestibility of dry matter, crude protein, crude ash, ADF and NDF has been underestimated by approximately 6%, 7%, 14%, 12%, and 10%, respectively, when they have been calculated using the conventional digestibility method for domestic cats.