Alternative autophagy dampens UVB-induced NLRP3 inflammasome activation in human keratinocytes.

Sunlight exposure results in an inflammatory reaction of the skin commonly known as sunburn, which increases skin cancer risk. In particular, the ultraviolet B (UVB) component of sunlight induces inflammasome activation in keratinocytes to instigate the cutaneous inflammatory responses. Here, we explore the intracellular machinery that maintains skin homeostasis by suppressing UVB-induced inflammasome activation in human keratinocytes. We found that pharmacological inhibition of autophagy promoted UVB-induced NLRP3 inflammasome activation. Unexpectedly, however, gene silencing of Atg5 or Atg7, which are critical for conventional autophagy, had no effect, whereas gene silencing of Beclin1, which is essential not only for conventional autophagy but also for Atg5/Atg7-independent alternative autophagy, promoted UVB-induced inflammasome activation, indicating an involvement of alternative autophagy. We found that damaged mitochondria were highly accumulated in UVB-irradiated keratinocytes when alternative autophagy was inhibited, and they appear to be recognized by NLRP3. Overall, our findings indicate that alternative autophagy, rather than conventional autophagy, suppresses UVB-induced NLRP3 inflammasome activation through the clearance of damaged mitochondria in human keratinocytes and illustrate a previously unknown involvement of alternative autophagy in inflammation. Alternative autophagy may be a new therapeutic target for sunburn and associated cutaneous disorders.

The effects of chest drainage on pressure-controlled ventilation.

BACKGROUND: The use of pressure-controlled ventilation (PCV) for anesthesia management is becoming more commonly used. Chest drainage is commonly performed after thoracic surgery, and the negative pressure it generates might affect the transpulmonary pressure (TPP). In the present study, we investigated how chest drainage could affect ventilating conditions during PCV. METHODS: We created a hand-made simple thoracic and lung model, which was connected to an anesthesia machine. The tidal volume (TV) was measured with positive end-expiratory pressure (PEEP) 0 and no chest drainage (baseline), followed by 10 cmH2O PEEP/no drainage, 10 cmH2O PEEP/drainage with - 10 cmH2O and 10 cmH2O PEEP/drainage with - 20 cmH2O. Finally, TV with 20 cmH2O and 30 cmH2O PEEP/no drainage was measured. Driving (inspiratory) pressure was maintained at 20 cmH2O during the whole experiment. RESULTS: TV was significantly increased by applying 10 cmH2O PEEP compared with baseline, further increased by applying - 10 cmH2O by drainage, similar to the value with PEEP 20 cmH2O with no drainage (end-tidal TPP of 20 cmH2O for both). TV decreased to < 50% of the baseline by applying 10 cmH2O PEEP with - 20 cmH2O by drainage, which was similar to that with 30 cmH2O PEEP with no drainage (end-tidal TPP of 30 cmH2O for both). CONCLUSIONS: TV was maintained at similar levels with the same TPP, regardless of PEEP or negative pressure by chest drainage change, suggesting that negative intrapleural pressure by the chest tube drainage system might mimic PEEP from the point of TV.

Evaluation of a Capillary Microsampling Device for Analyzing Plasma Lenvatinib Concentration in Patients With Hepatocellular Carcinoma.

BACKGROUND: The anticancer drug, Lenvima (lenvatinib), has severe side effects. Therapeutic drug monitoring helps ensure its efficacy and safety. Regular and optimally timed blood sampling is tough, especially when lenvatinib is self-medicated. Microsampling using the easy to handle Microsampling Wing (MSW) may help circumvent this problem. However, current lenvatinib detection methods are not sensitive enough to detect its concentrations in microsamples (<50-250 µL). Thus, the aim of this study was 2-fold (1) develop an analytic method to estimate plasma lenvatinib concentrations in microsamples and (2) verify whether this method works on micro (5.6 µL) blood plasma samples obtained clinically through MSW from patients with unresectable hepatocellular carcinoma (HCC). METHODS: A simple, highly sensitive, and specific liquid chromatography-electrospray ionization tandem mass spectrometry method was developed. Using this novel protocol, the trough blood plasma concentration of lenvatinib was measured for both blood sampled conventionally and that using MSW. Thirty-five venous whole blood samples were obtained from 11 patients with HCC. Furthermore, the stability of lenvatinib in MSW samples during storage was evaluated. RESULTS: The mean plasma lenvatinib concentration estimates were not significantly different between the MSW and conventional venous blood samples. CV for interday and intraday assays was low. Up to day 5, the lenvatinib concentration in the MSW samples was 85%-115% of the initial day concentration (when stored at 25°C or 4°C). The interference of endogenous matrix components in the human plasma was low. CONCLUSIONS: These results indicate that the novel mass spectrometry protocol accurately measures lenvatinib in human plasma and is reproducible. Thus, MSW could be a useful microsampling device for lenvatinib therapeutic drug monitoring in patients with HCC when used in combination with this novel liquid chromatography-electrospray ionization tandem mass spectrometry detection method.

Inflammasome Sensor NLRP1 Confers Acquired Drug Resistance to Temozolomide in Human Melanoma.

Cancer cells gain drug resistance through a complex mechanism, in which nuclear factor-κB (NF-κB) and interleukin-1ß (IL-1ß) are critical contributors. Because NACHT, LRR and PYD domains-containing protein (NLRP) inflammasomes mediate IL-1ß maturation and NF-κB activation, we investigated the role of inflammasome sensor NLRP1 in acquired drug resistance to temozolomide (TMZ) in melanoma. The sensitivity of melanoma cells to TMZ was negatively correlated with the expression levels of O6-methylguanine-DNA methyltransferase (MGMT), the enzyme to repair TMZ-induced DNA lesions. When MGMT-low human melanoma cells (1205Lu and HS294T) were treated with TMZ for over two months, MGMT was upregulated, and cells became resistant. However, the resistance mechanism was independent of MGMT, and the cells that acquired TMZ resistance showed increased NLRP1 expression, NLRP inflammasome activation, IL-1ß secretion, and NF-κB activity, which contributed to the acquired resistance to TMZ. Finally, blocking IL-1 receptor (IL-1R) signaling with IL-1R antagonist decreased TMZ-resistant 1205Lu tumor growth in vivo. Although inflammation has been associated with drug resistance in various cancers, our paper is the first to demonstrate the involvement of NLRP in the development of acquired drug resistance. Because drug-tolerant cancer cells become cross-tolerant to other classes of cancer drugs, NLRP1 might be a suitable therapeutic target in drug-resistant melanoma, as well as in other cancers.

A Simplified Gas Chromatographic Fatty-Acid Analysis by the Direct Saponification/Methylation Procedure and Its Application on Wild Tuna Larvae.

A method for the direct preparation of fatty-acid methyl esters (FAME) was simplified for fatty-acid analysis of a single fish larva using gas chromatography (GC). The method included the isolation of a larval trunk and drying in a glass vial, followed by saponification of all the contents without prior lipid extraction. Thereafter, the fatty acids released were methylated by trimethylsilyldiazomethane. This method has advantages over another method, direct acid-catalyzed transesterification, because both the saponification and methylation at room temperature can reduce loss of unsaturated fatty acids and formation of artifacts unavoidable in acidic reaction at high temperature. GC of the products showed that the simplified method can yield methyl esters without artifacts interfering analysis. More than 50 fatty acids were determined, which are twice as many as those previously analyzed using high-performance liquid chromatography. Observation of consistent small impurities in GC of blank tests allowed the accurate determination of fatty acids by correcting the peak areas. Dry matter weights (<3 mg) and the total fatty-acid contents displayed a linear relationship. Fatty-acid analysis of wild larvae of bluefin tuna, yellowfin tuna, and skipjack tuna collected from the waters around Japan (n = 100) revealed that the eicosapentaenoic acid (EPA) level in bluefin tuna collected from the Japan Sea was significantly higher than that in the three species collected from Nansei Islands. The simplified direct saponification/methylation method will be a powerful tool for investigating growth and survival of individual larval tuna and other fish species.

Quantification of autonomic nervous activity by heart rate variability and approximate entropy in high ultrafiltration rate during hemodialysis.

BACKGROUND: Few studies have focused on the imbalance of the autonomic nervous system in ultrafiltration rate (UFR) subjects without blood pressure variation during maintenance hemodialysis (HD), although the role of autonomic nervous system activation during HD has been proposed to be an important factor for the maintenance of blood pressure. METHODS: Variations over time in autonomic nervous activity due to differences in UFR were evaluated by measuring heart rate variability (HRV) and approximate entropy (ApEn) in 35 HD patients without blood pressure variations during HD session. The subjects were divided into 3 groups, those with UFR <10 ml/h/kg; ≥10 ml/h/kg but ≤15 ml/h/kg; and >15 ml/h/kg, and Holter ECG was recorded continuously during HD session using frequency analysis of RR intervals. High frequency (HF) and low frequency (LF) spectral components are found to be representative of the parasympathetic nervous system and sympathovagal balance, respectively, with the ratio of LF to HF of HRV providing a measure of sympathetic nervous system. RESULTS: In subjects with UFR >15 ml/h/kg, HF components were significantly lower, and LF/HF and ApEn values were significantly higher, in the latter half of an HD session than before starting HD. CONCLUSION: Removing water from these subjects would promote sustained sympathetic nervous overactivity. These findings indicate that the UFR during HD needs to be set at ≤15 ml/h/kg.

Antiedema effects of Siberian ginseng in humans and its molecular mechanism of lymphatic vascular function in vitro.

The lymphatic system in the skin plays a major role in tissue fluid homeostasis, in the afferent phase of the immune response, and in tumor metastasis. Although lymphangiogenic factors involved in embryonic development and the metastatic spread of tumor cells have been well studied, little is known about small-molecule compounds that activate lymphatic function, especially under physiological conditions. We hypothesized that the identification of a lymphatic-activating compound could provide a method for improving edema. Here, we show that Siberian ginseng (Eleutherococcus senticosus) and its component eleutheroside E induce phosphorylation of the endothelial-specific receptor Tie2 in vitro. The activation of Tie2 on lymphatic endothelial cells (LECs) is known to stabilize lymphatic vessels, so we examined the effects of Siberian ginseng on LECs. We found that Siberian ginseng induces the migration and cord formation of LECs. Permeability assays demonstrated that it stabilizes LECs by promoting the intercellular localization of vascular endothelial cadherin, which is an endothelium-specific cell-cell adhesion molecule involved in endothelial barrier function, and it induces the phosphorylation of endothelial nitric oxide synthase by LECs. These effects appear to be mediated by the activation of Tie2 in LECs. Finally, we investigated whether the consumption of Siberian ginseng powder improves edema in a 2-way, randomized, crossover study in 50 healthy female volunteers. Edema of the lower limbs was significantly attenuated at 2 and 4hours after ingestion as compared with the control group. Thus, we demonstrate that Siberian ginseng exerts its potent antiedema activity mainly by promoting lymphatic function.

Factors Predicting Adhesion between Renal Capsule and Perinephric Adipose Tissue in Partial Nephrectomy.

In minimally invasive partial nephrectomy (MIPN), it is important to preoperatively predict the degree of difficulty of tumor resection. When severe adhesions occur between the renal capsule and perinephric adipose tissue, detachment can be difficult. Preoperative prediction of adhesion is thought to be useful in the selection of surgical procedure. Subjects were 63 patients of a single surgeon who had received MIPN between April 2008 and August 2013 at Okayama University Hospital. Of these patients, this study followed 47 in whom the presence or absence of adhesions between the renal capsule and perinephric adipose tissue was confirmed using intraoperative videos. Data collected included: sex, BMI, CT finding (presence of fi broids in perinephric adipose tissue), comorbidities and lifestyle. Adhesion was observed in 7 patients (14.9%). The mean operative time was 291.6 min in the adhesion group, and 226.3 min in the group without. The increased time in the adhesions group was significant (p＜0.05). Predictive factors were a positive CT finding for fibroid structure and comorbidity of hypertension (p＜0.05). In MIPN, difficulty of surgery can be affected by the presence of adhesion of the perinephric adipose tissue. Predicting such adhesion from preoperative CT is thus important.

Activation-Induced Cytidine Deaminase Contributes to Pancreatic Tumorigenesis by Inducing Tumor-Related Gene Mutations.

Pancreatic ductal adenocarcinoma (PDAC) develops via an accumulation of various gene mutations. The mechanism underlying the mutations in PDAC development, however, is not fully understood. Recent insight into the close association between the mutation pattern of various cancers and specific mutagens led us to investigate the possible involvement of activation-induced cytidine deaminase (AID), a DNA editing enzyme, in pancreatic tumorigenesis. Our immunohistochemical findings revealed AID protein expression in human acinar ductal metaplasia, pancreatic intraepithelial neoplasia, and PDAC. Both the amount and intensity of the AID protein expression increased with the progression from precancerous to cancerous lesions in human PDAC tissues. To further assess the significance of ectopic epithelial AID expression in pancreatic tumorigenesis, we analyzed the phenotype of AID transgenic (AID Tg) mice. Consistent with our hypothesis that AID is involved in the mechanism of the mutations underlying pancreatic tumorigenesis, we found precancerous lesions developing in the pancreas of AID Tg mice. Using deep sequencing, we also detected Kras and c-Myc mutations in our analysis of the whole pancreas of AID Tg mice. In addition, Sanger sequencing confirmed the presence of Kras, c-Myc, and Smad4 mutations, with the typical mutational footprint of AID in precancerous lesions in AID Tg mice separated by laser capture microdissection. Taken together, our findings suggest that AID contributes to the development of pancreatic precancerous lesions by inducing tumor-related gene mutations. Our new mouse model without intentional manipulation of specific tumor-related genes provides a powerful system for analyzing the mutations involved in PDAC.

Hepatic inflammation facilitates transcription-associated mutagenesis via AID activity and enhances liver tumorigenesis.

Chronic inflammation triggers the aberrant expression of a DNA mutator enzyme, activation-induced cytidine deaminase (AID), and contributes to tumorigenesis through the accumulation of genetic aberrations. To gain further insight into the inflammation-mediated genotoxic events required for carcinogenesis, we examined the role of chronic inflammation in the emergence of genetic aberrations in the liver with constitutive AID expression. Treatment with thioacetamide (TAA) at low-dose concentrations caused minimal hepatic inflammation in both wild-type (WT) and AID transgenic (Tg) mice. None of the WT mice with low-dose TAA administration or AID Tg mice without hepatic inflammation developed cancers in their liver tissues over the 6 month study period. In contrast, all the AID Tg mice with TAA treatment developed multiple macroscopic hepatocellular carcinomas during the same observation period. Whole exome sequencing and additional deep-sequencing analyses revealed the enhanced accumulation of somatic mutations in various genes, including dual specificity phosphatase 6 (Dusp6), early growth response 1 (Egr1) and inhibitor of DNA binding 2 (Id2), which are putative tumor suppressors, in AID-expressing liver with TAA-mediated hepatic inflammation. Microarray and quantitative reverse transcription-polymerase chain reaction analyses showed the transcriptional upregulation of various genes including Dusp6, Egr1 and Id2 under hepatic inflammatory conditions. Together, these findings suggest that inflammation-mediated transcriptional upregulation of target genes, including putative tumor suppressor genes, enhances the opportunity for inflamed cells to acquire somatic mutations and contributes to the acceleration of tumorigenesis in the inflamed liver tissues.