Functional Analysis of a Novel HNF4A Variant Identified in a Patient With MODY1.

Context: HNF4A-maturity-onset diabetes of the young (MODY1) is a relatively rare subtype of monogenic diabetes caused by loss of function of the HNF4A gene, which encodes the transcription factor HNF4α. HNF4α is known to form heterodimers, and the various combinations of isoforms that make up these heterodimers have been reported to result in a diversity of targeted genes. However, the function of individual HNF4α variant isoforms and the heterodimers comprising both wild-type (WT) and variant HNF4α have not yet been assessed. Objective: In this study, we analyzed the functional consequence of the HNF4A D248Y variant in vitro. Methods: We investigated the case of a 12-year-old Japanese girl who developed diabetes at age 11 years. Genetic sequencing detected a novel heterozygous missense HNF4A variant (c.742G > T, p.Asp248Tyr; referred as "D248Y") in the patient and her relatives who presented with diabetes. Results: Although the WT HNF4α isoforms (HNF4α2, HNF4α3, HNF4α8, HNF4α9) enhanced the INS gene promoter activity in HepG2 cells, the promoter activity of D248Y was consistently low across all isoforms. The presence of D248Y in homodimers and heterodimers, comprising either HNF4α8 or HNF4α3 or a combination of both isoforms, also reduced the INS promoter activity in Panc-1 cells. Conclusion: We report the clinical course of a patient with HNF4A-MODY and the functional analysis of novel HNF4A variants, with a focus on the isoforms and heterodimers they form. Our results serve to improve the understanding of the dominant-negative effects of pathogenic HNF4A variants.

cccDNA-Targeted Drug Screen Reveals a Class of Antihistamines as Suppressors of HBV Genome Levels.

Chronic infection with hepatitis B virus (HBV) is incurable, as the current therapeutics cannot eliminate its persistent genomic material, cccDNA. Screening systems for cccDNA-targeting therapeutics are unavailable, as low copies of cccDNA in vitro complicate detection. To address this, cccDNA copies were massively increased to levels detectable via automated plate readers. This was achieved via continuous infection in a contact-free co-culture of an HBV generator (clone F881), which stably produced clinically relevant amounts of HBV, and HBV acceptors selected to carry high cccDNA loads. cccDNA-targeted therapeutics were then identified via reduced cccDNA-specific fluorescence, taking differences in the cell numbers and viability into account. Amongst the drugs tested, the H1 antihistamine Bilastine, HBVCP inhibitors and, surprisingly, current HBV therapeutics downregulated the cccDNA significantly, reflecting the assay's accuracy and sensitivity in identifying drugs that induce subtle changes in cccDNA levels, which take years to manifest in vivo. Bilastine was the only therapeutic that did not reduce HBV production from F881, indicating it to be a novel direct suppressor of cccDNA levels. When further assessed, only the structurally similar antihistamines Pitolisant and Nizatidine suppressed cccDNA levels when other H1 antihistamines could not. Taken together, our rapid fluorescence cccDNA-targeted drug screen successfully identified a class of molecules with the potential to treat hepatitis B.

Single institute experience of intraoperative radiation therapy in early-stage breast cancer.

ABSTRACT: Intraoperative radiation therapy (IORT) is an alternative to whole breast irradiation in selected early-stage breast cancer patients. In this single institute analysis, we report the preliminary results of IORT given by Axxent Electronic Brachytherapy (eBT) system.Patients treated with lumpectomy and eBT within a minimum follow-up period of 12 months were analyzed. Eligible criteria include being over the age of 45, having unifocal invasive ductal carcinoma (IDC) or ductal carcinoma in situ <3 cm in diameter, not exhibiting lymph node involvement on preoperative images, and negative sentinel lymph node biopsy. The eBT was given by preloaded radiation plans to deliver a single fraction of 20 Gray (Gy) right after lumpectomy.From January 2016 to April 2019, a total of 103 patients were collected. There were 78 patients with IDC and 25 with ductal carcinoma in situ. At a mean follow-up time of 31.1 months (range, 14.5-54.0 months), the local control rate was 98.1%. Two IDC patients had tumor recurrences (1 local and 1 regional failure). Post-IORT radiotherapy was given to 4 patients. There were no cancer related deaths, no distant metastases, and treatment side effects greater than grade 3 documented.We report the largest single institute analysis using the eBT system in Taiwan. The low recurrence and complication rates at a 31.1 month follow-up time support the use of the eBT system in selected early-stage breast cancer patients.

Intracellular interferon signalling pathways as potential regulators of covalently closed circular DNA in the treatment of chronic hepatitis B.

Infection with the hepatitis B virus (HBV) is still a major global health threat as 250 million people worldwide continue to be chronically infected with the virus. While patients may be treated with nucleoside/nucleotide analogues, this only suppresses HBV titre to sub-detection levels without eliminating the persistent HBV covalently closed circular DNA (cccDNA) genome. As a result, HBV infection cannot be cured, and the virus reactivates when conditions are favorable. Interferons (IFNs) are cytokines known to induce powerful antiviral mechanisms that clear viruses from infected cells. They have been shown to induce cccDNA clearance, but their use in the treatment of HBV infection is limited as HBV-targeting immune cells are exhausted and HBV has evolved multiple mechanisms to evade and suppress IFN signalling. Thus, to fully utilize IFN-mediated intracellular mechanisms to effectively eliminate HBV, instead of direct IFN administration, novel strategies to sustain IFN-mediated anti-cccDNA and antiviral mechanisms need to be developed. This review will consolidate what is known about how IFNs act to achieve its intracellular antiviral effects and highlight the critical interferon-stimulated gene targets and effector mechanisms with potent anti-cccDNA functions. These include cccDNA degradation by APOBECs and cccDNA silencing and transcription repression by epigenetic modifications. In addition, the mechanisms that HBV employs to disrupt IFN signalling will be discussed. Drugs that have been developed or are in the pipeline for components of the IFN signalling pathway and HBV targets that detract IFN signalling mechanisms will also be identified and discussed for utility in the treatment of HBV infections. Together, these will provide useful insights into design strategies that specifically target cccDNA for the eradication of HBV.

Axitinib in combination with radiotherapy for advanced hepatocellular carcinoma: a phase I clinical trial.

BACKGROUND: To investigate maximum tolerated dose (MTD) of axitinib, a selective vascular endothelial growth factor receptor 1-3 inhibitor, in combination with radiotherapy (RT) for advanced hepatocellular carcinoma (HCC). METHODS: This phase I study followed the rule of traditional 3 + 3 design. Major eligibility included: (1) patients with advanced HCC unsuitable for surgery, radiofrequency ablation or transarterial chemoembolization, or who failed after prior local-regional treatment; (2) failure on sorafenib or no grant for sorafenib from health insurance system. Eligible patients with advanced HCC received axitinib for total 8 weeks during and after RT. Three cohorts with axitinib dose escalation were planned: 1 mg twice daily (level I), 2 mg twice daily (level II) and 3 mg twice daily (level III). The prescribed doses of RT ranged from 37.5 to 67.5 Gy in 15 fractions to liver tumor(s) and were determined based on an upper limit of mean liver dose of 18 Gy (intended isotoxic RT for normal liver). The primary endpoint was MTD of axitinib in combination with RT. The secondary endpoints included overall response rate (ORR), RT in-field response rate, acute and late toxicities, overall survival (OS) and progression free survival (PFS). RESULTS: Total nine eligible patients received axitinib dose levels of 1 mg twice daily (n = 3), 2 mg twice daily (n = 3) and 3 mg twice daily (n = 3). Dose-limiting toxicity (DLT) did not occur in the 3 cohorts; the MTD was defined as 3 mg twice daily in this study. ORR was 66.7%, including 3 complete responses and 3 partial responses, at 3 months after treatment initiation. With a median follow-up of 16.6 months, median OS was not reached, 1-year OS was 66.7%, and median PFS was 7.4 months. CONCLUSIONS: Axitinib in combination with RT for advanced HCC was well tolerated with an axitinib MTD of 3 mg twice daily in this study. The outcome analysis should be interpreted with caution due to the small total cohort. Trial registration ClinicalTrials.gov (Identifier: NCT02814461), Registered June 27, 2016-Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT02814461.

Putative Abscopal Effect in Three Patients Treated by Combined Radiotherapy and Modulated Electrohyperthermia.

Purpose: True abscopal responses from radiation therapy are extremely rare; the combination of immune checkpoint inhibitors with radiation therapy has led to more reports of the abscopal effect, but even in this setting, the genuine magnitude remains unknown and is still considered generally uncommon. We report the occurrence of what appears to be putative, durable abscopal tumor responses with associated auto-immune systemic reactions resulting from the combination of local radiotherapy (RT) and modulated electrohyperthermia (mEHT). Materials and Methods: Data from advanced cancer patients treated palliatively with RT and mEHT between January and December 2017 were collected as part of a post-marketing safety monitoring program of mEHT therapy. We specified a minimum RT dose of 30 Gy and at least four mEHT treatments for reporting toxicities, which was the primary aim of the larger study. Results: Thirty-three patients treated with RT and mEHT, both applied to the same lesion, were included. The median RT dose was 45.5 Gy in 20 fractions (fxs) and the median number of mEHT treatments was 12 (range, 4-20). Most patients had subsequent systemic therapy after one course of RT and mEHT. Three patients (9.1%) developed autoimmune toxicities. Case number 1 received RT and mEHT only; case number 2 had two cycles of concurrent low dose chemotherapy during RT; and case number 3 received concurrent immune checkpoint inhibitors. None of the three patients received any further systemic treatment due to obvious treatment-related autoimmune reactions which occurred rapidly after RT; one had autoimmune hepatitis, one had dermatitis herpetiformis and the third developed severe myasthenia gravis. Interestingly, what we surmise to be long-lasting abscopal responses outside the irradiated area, were noted in all three patients. Conclusion: RT combined with mEHT could putatively result in enhancing immune responsiveness. These preliminary observational findings lead to the generation of a hypothesis that this combination induces both an in-situ, tumor-specific immune reaction and an anti-self-autoimmune reaction, in at least a small proportion of patients, and of those who experience the auto-immune response, tumor response is a concomitant finding. Mechanisms underlying this phenomenon need to be investigated further.

HNF4α Combinatorial Isoform Heterodimers Activate Distinct Gene Targets that Differ from Their Corresponding Homodimers.

Hepatocyte nuclear factor 4α (HNF4α) is a dimeric transcription factor that controls as much as 60% of all liver genes. However, how it achieves such broad functional diversity is unknown. Here, we show that inflammation and immune pathway genes are differentially regulated in an isoform-dependent manner, confirming that each isoform homodimer preferentially regulates a subset of HNF4α targets. With all 12 human HNF4α isoform clones, we tested combinatorial pairings to determine whether isoform heterodimers are functional. Indeed, synergistic and potent pairing combinations of isoform heterodimers were noted for HNF4α3-8, HNF4α6-12, and HNF4α5-8 that activated CYP7A1, IL6, and IL17A genes, respectively. Surprisingly, these genes are not at all activated by their corresponding isoform homodimers, suggesting that a particular heterodimer pair can regulate its own subset of target genes. Given the combinatorial possibility of 66 isoform heterodimers, our data provide the basis for a more detailed understanding of the diverse influence of HNF4α.

Comparing the Effectiveness of Combined External Beam Radiation and Hyperthermia Versus External Beam Radiation Alone in Treating Patients With Painful Bony Metastases: A Phase 3 Prospective, Randomized, Controlled Trial.

PURPOSE: To compare the response, duration of pain relief, and time to achieve complete pain relief after radiation therapy (RT) with or without hyperthermia (HT) in patients with painful bony metastases. METHODS AND MATERIALS: Cancer patients with bony metastases and pain score ≥4 on the Brief Pain Inventory (BPI) were randomized to RT of 30 Gy in 10 fractions combined with HT (RT + HT) versus RT alone. Hyperthermia was performed by the Thermotron RF-8, with maintenance of the target temperature for 40 minutes per treatment within 2 hours after RT, twice weekly for 2 weeks. Patients were stratified by lesion number (solitary or multiple), BPI score (4-6 vs 7-10), and primary site. The primary endpoint was complete response (CR) (BPI = 0 with no increase of analgesics) within 3 months after treatment. This study was registered with ClinicalTrials.gov. RESULTS: The study was terminated early after an interim analysis of 57 patients, 3 years after the first enrollment (November 2013 to November 2016): 29 patients in the RT + HT group and 28 patients in the RT-alone group. The CR rate at 3 months after treatment was 37.9% in the RT + HT group versus 7.1% in the RT-alone group (P=.006). The accumulated CR rate within 3 months after treatment was 58.6% in the RT + HT group versus 32.1% in the RT-alone group (P=.045). Median time to pain progression was 55 days in patients with CR (n=9) in the RT-alone group, whereas the endpoint was not reached during the 24-week follow-up in the RT + HT group (P<.01). CONCLUSIONS: The addition of HT to RT significantly increases the pain control rate and extends response duration compared with RT alone for painful bony metastases.

Identification of Slug and SOX7 as transcriptional repressors binding to the hepatitis B virus core promoter.

BACKGROUND & AIMS: The Hepatitis B Virus (HBV) may gain entry into non-liver cells but does not actively replicate in them. We investigated the possibility that these cells possess mechanisms that block HBV core promoter (HBVCP) transcription, specifically absent in liver cells, which together with other liver-specific mechanisms, such as sodium-taurocholate cotransporting polypeptide-mediated entry, enable liver cells to effectively produce HBV. METHODS: Liver and non-liver cell lines were screened for their capacity to activate the HBVCP and synthesize pre-genomic RNA (pgRNA). Transcription regulators differentially expressed between cells with active or inactive HBVCP were determined by human transcriptome array. Slug (SNAI2) and SRY-related HMG box 7 (SOX7) transcriptional repressors were identified and shown to bind specifically to the HBVCP by electrophoretic mobility shift assay. The resultant inhibitory effect on HBVCP transcription was validated using luciferase reporter and assays for pgRNA, HBcAg and cccDNA accumulation in cells with HBV replicon and HBV infection models. To further confirm their specific activity, short peptide mimetics generated from Slug zinc-finger domains and SOX7 HMG-box were generated. RESULTS: The HBVCP was found to be active in liver and selected non-liver cells. These cells have low/negligible expression of Slug and SOX7, which inhibit HBVCP transcription specifically by binding at the pgRNA initiator site and competitively displacing hepatocyte nuclear factor 4α, respectively. Overexpression of Slug and/or SOX7 specifically reduced HBVCP transcription, significantly diminishing pgRNA synthesis, HBcAg and cccDNA accumulation in HBV-infected primary human hepatocytes. Similar results were obtained with Slug and SOX7 stapled peptides individually, which were even more potent in combination. CONCLUSIONS: Slug and SOX7 are transcriptional repressors that bind specifically to the HBVCP. Their absence or weak expression in liver cells contribute to the favorable host environment for the active and efficient production of HBV. LAY SUMMARY: Hepatitis B virus (HBV) replication occurs efficiently in human liver because of the specificity of viral uptake receptors and presence of numerous liver-enriched transcription activators. Herein, we show that the specific lack of transcriptional inhibitory mechanisms in liver cells also contribute to effective HBV production. HBV replication is kept low in non-liver cells as transcriptional repressors Slug and SRY-related HMG box 7 (SOX7) actively bind to the transcriptional initiator and displace transcription activators, respectively, within the HBV core promoter.

The mediating effect of self-reflection and learning effectiveness on clinical nursing performance in nursing students: A follow-up study.

The effectiveness of simulation learning and the effects of anxiety in the simulated situation have been understudied. In addition, research on the association between learning effectiveness and students' clinical care performance in the hospital setting is very limited in Taiwan. The aim of this study is to examine the mediating effect of self-reflection and simulation learning effectiveness on the clinical nursing performance of nursing students. A Prospective, longitudinal, and correlational design was used. The study was conducted from December 2014 to July 2015. Participants were 293 nursing students in southern Taiwan. A structural model was specified and tested using partial least squares structural equation modeling to examine the relationships between the variables. The results revealed that the model was robust in terms of its measurement quality (reliability, validity, and goodness of fit), with the data's explaining 38.3% of variance in nursing competence. As self-reflection and learning effectiveness were added into the structural model, the effect of anxiety on nursing competence was still significant, but the regression coefficient (ß) estimate of -0.41 (p<0.05) changed to ß=-0.15 (p<0.050),indicating that self-reflection and learning effectiveness mediated the relationship between anxiety and nursing competence. Nursing competence was negatively affected by anxiety and positively affected by self-reflection (ß=0.49, p<0.05) and simulation learning effectiveness (ß=0.10, p<0.05). The teacher's encouraging learning can have a positive influence on students' self-reflection and learning effectiveness, which then decreases the effect of anxiety on nursing competence and further promotes students' clinical care ability.

Optimizing Survival of Patients With Marginally Operable Stage IIIA Non-Small-Cell Lung Cancer Receiving Chemoradiotherapy With or Without Surgery.

BACKGROUND: For marginally operable stage IIIA non-small-cell lung cancer (NSCLC), surgery might not be done as planned after neoadjuvant concurrent chemoradiotherapy (CCRT) for reasons (unresectable or medically inoperable conditions, or patient refusal). This study aims to investigate the outcomes of a phased CCRT protocol established to maximize the operability of marginally operable stage IIIA NSCLC and to care for reassessed inoperable patients, in comparison with continuous-course definitive CCRT. MATERIALS AND METHODS: Forty-seven patients with marginally operable stage IIIA NSCLC receiving CCRT were included. Twenty-eight patients were treated with our phased CCRT protocol, including neoadjuvant CCRT followed by surgery (group A, n = 16) or, for reassessed inoperable patients, maintenance chemotherapy and split-course CCRT boost (group B, n = 12). The other 19 were treated with continuous-course definitive CCRT (group C). Overall survival (OS) and progression-free survival (PFS) were analyzed. RESULTS: Among all, median OS and PFS were 35.6 and 12.8 months, respectively (median follow-up, 22.3 months). The median OS of group A (not reached) was better than that of group B (34.4 months) and group C (15.2 months) (P = .009). On multivariate analysis, performance status 0 to 1 (hazard ratio [HR], 0.026; P < .001), adenocarcinoma (HR, 0.156; P = .003), and group A (HR, 0.199; P = .033) were independent prognostic factors. The OS of group B (HR, 0.450; 95% confidence interval, 0.118-1.717; P = .243) was not statistically different from that of group C. CONCLUSIONS: For marginally operable stage IIIA NSCLC, our phased CCRT strategy may optimize survival by maximizing operability and maintain an acceptable survival for reassessed inoperable patients by split-course CCRT boost following maintenance chemotherapy.

Double autophagy modulators reduce 2-deoxyglucose uptake in sarcoma patients.

RATIONALE: According to the metabolic symbiosis model, cancer stromal fibroblasts could be hijacked by surrounding cancer cells into a state of autophagy with aerobic glycolysis to help provide recycled nutrients. The purpose of this study was to investigate whether combined treatment with the autophagy inhibitor: hydroxychloroquine (HCQ) and the autophagy inducer: sirolimus (rapamycin, Rapa) would reduce glucose utilization in sarcoma patients. METHODS: Ten sarcoma patients who failed first-line treatment were enrolled in this study. They were treated with 1 mg of Rapa and 200 mg of HCQ twice daily for two weeks. The standardized uptake values (SUV) from pretreatment and posttreatment [18F]-fluorodeoxyglucose positron emission tomography (FDG PET) scans were reviewed, and changes from the baseline SUVmax were evaluated. RESULTS: Based on FDG PET response criteria, six patients had a partial response; three had stable disease, and one had progressive disease. Nevertheless, none of them showed a reduction in tumor volume. The mean SUVmax reduction in the 34 lesions evaluated was - 19.6% (95% CI = -30.1% to -9.1%), while the mean volume change was +16.4% (95% CI = +5.8% to + 27%). Only grade 1 toxicities were observed. Elevated serum levels of lactate dehydrogenase were detected after treatment in most metabolic responders. CONCLUSIONS: The results of reduced SUVmax without tumor volume reduction after two weeks of Rapa and HCQ treatment may indicate that non-proliferative glycolysis occurred mainly in the cancer associated fibroblast compartment, and decreased glycolytic activity was evident from Rapa + HCQ double autophagy modulator treatment.

Addition of rapamycin and hydroxychloroquine to metronomic chemotherapy as a second line treatment results in high salvage rates for refractory metastatic solid tumors: a pilot safety and effectiveness analysis in a small patient cohort.

BACKGROUND: Autophagy is an important oncotarget that can be modulated during anti-cancer therapy. Enhancing autophagy using chemotherapy and rapamycin (Rapa) treatment and then inhibiting it using hydroxychloroquine (HCQ) could synergistically improve therapy outcome in cancer patients. It is still unclear whether addition of Rapa and HCQ to chemotherapy could be used for reversing drug resistance. PATIENTS AND METHODS: Twenty-five stage IV cancer patients were identified. They had no clinical response to first-line metronomic chemotherapy; the patients were salvaged by adding an autophagy inducer (Rapa, 2 mg/day) and an autophagosome inhibitor (HCQ, 400 mg/day) to their current metronomic chemotherapy for at least 3 months. Patients included 4 prostate, 4 bladder, 4 lung, 4 breast, 2 colon, and 3 head and neck cancer patients as well as 4 sarcoma patients. RESULTS: Chemotherapy was administered for a total of 137 months. The median duration of chemotherapy cycles per patient was 4 months (95% confidence interval, 3-7 months). The overall response rate to this treatment was of 40%, with an 84% disease control rate. The most frequent and clinically significant toxicities were myelotoxicities. Grade ≥3 leucopenia occurred in 6 patients (24%), grade ≥3 thrombocytopenia in 8 (32%), and anemia in 3 (12%). None of them developed febrile neutropenia. Non-hematologic toxicities were fatigue (total 32%, with 1 patient developing grade 3 fatigue), diarrhea (total 20%, 1 patient developed grade 3 fatigue), reversible grade 3 cardiotoxicity (1 patient), and grade V liver toxicity from hepatitis B reactivation (1 patient). CONCLUSION: Our results of Rapa, HCQ and chemotherapy triplet combination suggest autophagy is a promising oncotarget and warrants further investigation in phase II studies.

Trimodality bladder-sparing approach without neoadjuvant chemotherapy for node-negative localized muscle-invasive urinary bladder cancer resulted in comparable cystectomy-free survival.

BACKGROUND: To retrospectively review the efficacy and organ preservation experience for muscle-invasive bladder cancer by trimodality therapy at our institution. METHODS: Between July 2004 and February 2012, seventy patients (M/F = 55/15; median age = 69 years) of lymph node negative localized muscle-invasive bladder cancer were treated primarily with trimodality approach including transurethral resection of bladder tumor (TURBT) prior to combined chemotherapy and radiotherapy (CCRT). Radiotherapy consisted of initial large field size irradiation with 3D conformal technique (3D-CRT), followed by cone-down tumor bed boost with intensity modulated radiotherapy (IMRT) technique. The median total doses delivered to bladder tumor bed and whole bladder were 59.4Gy and 40.0Gy, respectively. No patient received neoadjuvant chemotherapy (NAC). Weekly cisplatin was administered during radiotherapy. Toxicity was scored according to the RTOG criteria. Tumor response was evaluated both cystoscopically and radiographically 3 months after treatment. RESULTS: The numbers of patients with T2, T3 and T4 lesions were 41, 16 and 13, respectively. Overall survival (OS) and progression-free survival (PFS) at 2 and 5 year were 65.7%, 51.9% and 50.8%, 39.9%, respectively, after a median follow-up time of 24 months. Local-regional control and distant metastasis free survival at 2 year were 69.8% and 73.5%, respectively. Complete response (CR) rate assessed three month after CCRT was 78.1%. Ten patients (20%) had local recurrence after initial CR (n = 50), 3 of them were superficial recurrence. One patient underwent radical cystectomy after recurrence. The overall 5-year bladder intact survival was 49.0% (95% CI, 35.5% to 62.5%). Acute toxicities were limited to grade 1-2. One patient developed late grade 3 GU toxicity. CONCLUSIONS: Our result suggested that trimodality bladder-sparing approach without NAC or dose-intensification could be well-tolerated with a high CR rate and bladder preserving rate for muscle-invasive bladder cancer.

Activating killer cell immunoglobulin-like receptor 2DS2 binds to HLA-A*11.

Inhibitory killer cell Ig-like receptors (KIRs) are known to recognize HLA ligands mainly of the HLA-C and Bw4 groups, but the ligands for KIRs are poorly understood. We report here the identification of the cognate ligand for the activating KIR 2DS2 as HLA-A*11:01. The crystal structure of the KIR2DS2-HLA-A*11:01 complex was solved at 2.5-Å resolution and revealed residue-binding characteristics distinct from those of inhibitory KIRs with HLA-C and the critical role of residues Tyr45 and Asp72 in shaping binding specificity to HLA-A*11:01. Using KIR2DS2 tetramers, binding to surface HLA-A*11:01 on live cells was demonstrated and, furthermore, that binding can be altered by residue changes at p8 of the peptide, indicating the influence of peptide sequence on KIR-HLA association. In addition, heteronuclear single quantum coherence NMR was used to map the involvement of critical residues in HLA binding at the interface of KIR and HLA, and validates the data observed in the crystal structure. Our data provide structural evidence of the recognition of A*11:01 by the activating KIR2DS2 and extend our understanding of the KIR-HLA binding spectrum.

Apolipoprotein C1 (APOC1) as a novel diagnostic and prognostic biomarker for lung cancer: A marker phase I trial.

BACKGROUND: Tumor cells continuously evolve over time in response to host pressures. However, explanations as to how tumor cells are influenced by the inflammatory tumor microenvironment over time are, to date, poorly defined. We hypothesized that prognostic biomarkers could be obtained by exploring the expression of inflammation-associated genes between early and late stage lung cancer tumor samples. METHODS: Candidate inflammation-associated genes, apolipoprotein C-1 (APOC1), MMP1, KMO)1, CXCL5, CXCL)7, IL-1α, IL-1ß, TNF-α and IL-6 were verified by real-time quantitative polymerase chain reaction. Gene expression profiles and immunofluorescence staining of 30 lung cancer tissues were compared. RESULTS: Expressions of APOC1 and IL-6 mRNA on tumor tissues in late stage disease were significantly higher than in early stage lung cancer samples. Immunofluorescence staining of tumor samples showed that the expression of APOC1 gradually increased from early to late stage in lung cancer patients. The expression levels of IL-6 and APOC1 in tumor samples were positively correlated; however, no prognostic value of APOC1 can be identified in serum samples. CONCLUSIONS: We found that the level of tumor APOC1 was highly expressed in late stage lung cancer. Further research is warranted to determine the molecular mechanisms underlying the cross talk of APOC1 and IL-6 in tumor progression. An expanded sample size marker phase II study may lead to the discovery of new lung cancer therapeutics targeting APOC1.

Effect of caring behavior on disposition toward critical thinking of nursing students.

The purpose of this study was to explore the relationship between caring behavior and the disposition toward critical thinking of nursing students in clinical practice. A structural equation model was used to test the hypothesized relationship between caring behavior and critical thinking skills. Caring is the core of nursing practice, and the disposition toward critical thinking is needed for competent nursing care. In a fast-paced and complex environment, however, "caring" may be lost. Because nursing students will become professional nurses, it is essential to explore their caring behaviors and critical thinking skills and to understand how to improve their critical thinking skills based on their caring behavior. A cross-sectional study was used, with convenience sampling of students who were participating in associate degree nursing programs at 3 colleges of nursing. The following instruments were used: critical thinking disposition inventory Chinese version and caring behaviors scale. The study found that individuals with a higher frequency of caring behaviors had a higher score on critical thinking about nursing practice (ß = .44, t = 5.14, P < .001). Specifically, caring behaviors accounted for 19.4% of the variance in students' critical thinking. The findings of this study revealed the importance of caring behavior and its relationship with the disposition toward critical thinking. Thus, it is recommended that nursing education should emphasize a curriculum related to caring behavior to improve the disposition toward critical thinking of nursing students.

hnRNP K suppresses apoptosis independent of p53 status by maintaining high levels of endogenous caspase inhibitors.

Hepatocellular carcinoma (HCC) is the third highest cause of cancer-related deaths globally. One of the cellular hallmarks of this disease is dysregulation of apoptosis, and a better understanding of this process is important if progress is to be made toward effectively treating HCC. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a RNA-binding protein that is implicated in apoptosis and is upregulated in various cancers, including HCC. In this study, we report new evidence for a crucial role of hnRNP K in suppressing apoptosis in HCC cells. We used the chemotherapeutic agent 5-fluorouracil to induce apoptosis in HCC cell lines and found that hnRNP K was downregulated, independent of both p53 and caspases. Prolonged downregulation of hnRNP K using small interfering RNA (siRNA) significantly decreased cell viability and increased apoptosis in HCC cell lines in a p53-independent manner. Moreover, enhanced tumor necrosis factor-related apoptosis-inducing ligand potency, independent of BH3-interacting domain death agonist (BID) cleavage, was also observed in hnRNP K siRNA-treated cells. Examination of the underlying mechanism revealed that hnRNP K suppresses the activity of various caspases through controlling transcription of the caspase inhibitor XIAP. Taken together, this study establishes that hnRNP K plays an antiapoptotic role in HCC cell lines, independent of p53 status, via the maintenance of high levels of endogenous caspase inhibitors, and also identifies hnRNP K as a possible therapeutic marker for cancer treatment.

Evaluating the accuracy of morphological identification of larval fishes by applying DNA barcoding.

Due to insufficient morphological diagnostic characters in larval fishes, it is easy to misidentify them and difficult to key to the genus or species level. The identification results from different laboratories are often inconsistent. This experiment aims to find out, by applying DNA barcoding, how inconsistent the identifications can be among larval fish taxonomists. One hundred morphotypes of larval fishes were chosen as test specimens. The fishes were collected with either larval fish nets or light traps in the northern, southern and northwestern waters of Taiwan. After their body lengths (SL) were measured and specimen photos were taken, all specimens were delivered, in turn, to five laboratories (A-E) in Taiwan to be identified independently. When all the results were collected, these specimens were then identified using COI barcoding. Out of a total of 100 specimens, 87 were identified to the family level, 79 to the genus level and 69 to the species level, based on the COI database currently available. The average accuracy rates of the five laboratories were quite low: 80.1% for the family level, 41.1% for the genus level, and 13.5% for the species level. If the results marked as "unidentified" were excluded from calculations, the rates went up to 75.4% and 43.7% for the genus and species levels, respectively. Thus, we suggest that larval fish identification should be more conservative; i.e., when in doubt, it is better to key only to the family and not to the genus or species level. As to the most misidentified families in our experiment, they were Sparidae, Scorpaenidae, Scombridae, Serranidae and Malacanthidae. On the other hand, Mene maculata and Microcanthus strigatus were all correctly identified to the species level because their larvae have distinct morphology. Nevertheless, barcoding remains one of the best methods to confirm species identification.