Distinct Lymphocyte Immunophenotyping and Quantitative Anti-Interferon Gamma Autoantibodies in Taiwanese HIV-Negative Patients with Non-Tuberculous Mycobacterial Infections.

PURPOSE: The presence of anti-interferon-γ autoantibodies (AutoAbs-IFN-γ) is not rare in patients suffering from persistent non-tuberculous mycobacterial (NTM) infections that are characteristic of adult-onset immunodeficiency syndrome. The immune disturbances in this distinct disorder remain to be elucidated. METHODS: Patients with NTM infections but without effective response over 3 months' treatment were referred to our institute to quantify their level of AutoAbs-IFN-γ after excluding defective IL12/23-IFN-γ circuit and reactive oxygen species production. The AutoAbs-IFN-γ and percentage of lymphocyte subpopulations most relevant to T and B cell pools were assessed and compared with age-matched healthy controls. RESULTS: A total of 31 patients were enrolled during the 15-year study period (2008-2022), 20 patients with > 50% suppression of IFN-γ detection at 1:100 serum dilution were classified into the Auto-NTM group. The remaining 11 with negligible suppression were assigned to the No Auto-NTM group. Mycobacterium chimaera-intracellulare group (MAC), M. kansasii, and M. abscessus were the most common pathogens. Pneumonia (19 vs 7), lymphadenitis (11 vs 5), Salmonella sepsis (6 vs 2), osteomyelitis (5 vs 1), and cutaneous herpes zoster (4 vs 4) were the main manifestations in both the Auto-NTM and No Auto-NTM groups who had similar onset-age (55.3 vs 53.6 years; p = 0.73) and follow-up duration (71.9 vs 54.6 months; p = 0.45). The Auto-NTM group had significantly higher transitional (IgM + + CD38 + +), CD19 + CD21-low, and plasmablast (IgM-CD38 + +) in the B cell pool, with higher effector memory (CD4 + /CD8 + CD45RO + CCR7 -), senescent CD8 + CD57 + , and Th17 cells, but lower naïve (CD4 + /CD8 + CD45RO - CCR7 +) and Treg cells in the T cell pool when compared to the No Auto-NTM and healthy groups. NTM patients with/without AutoAbs-IFN-γ had lower Th1-like Tfh (CD4 + CXCR5 + CXCR3 + CCR6 -) cells. All Auto-NTM patients still had non-remitted mycobacterial infections and higher AutoAbs-IFN-γ despite anti-CD20 therapy in 3 patients. CONCLUSION: In patients with suspected adult-onset immunodeficiency syndrome, two thirds (20/31) were recognized as having significantly inhibitory AutoAbs-IFN-γ with higher antibody-enhancing transitional, CD19 + CD21-low and plasmablast B cells; as well as higher effector memory, senescent CD8 + CD57 + and Th17 cells, but lower naïve T and Treg cells in contrast to those with negligible AutoAbs-IFN-γ. Such immunophenotyping disturbances might correlate with the presence of AutoAbs-IFN-γ. However, the mutual mechanisms need to be further clarified.

Flow Cytometry Analysis Reveals a Wide Cytologic and Immunophenotypic Spectrum of Peripheral B Lymphocytes in Angioimmunoblastic T-Cell Lymphoma.

INTRODUCTION: Angioimmunoblastic T-cell lymphoma (AITL) is an aggressive T-cell lymphoma commonly associated with B-cell dysregulation. Correlations involving B-cell dysregulation and clinicopathological features remain unclear. METHODS: We prospectively collected blood samples from 11 AITL patients and 17 healthy controls. The percentages of B-cell subpopulations and lymphocytes with IL-21 production were assessed using flow cytometry. Peripheral blood lymphocyte morphology was evaluated microscopically. RESULTS: Six of 11 (54.5%) patients presented with polyclonal hypergammaglobulinemia. Three of 11 (27.3%) tumor biopsies showed monoclonal immunoglobulin gene rearrangement. The patients exhibited significantly lower levels of naive (p < 0.001) and class-switched (p < 0.001) B cells than controls. The percentages of IgD-CD27- B cells (p = 0.007) and antibody-secreting cells (ASCs) (p = 0.001) were increased. Blood smears revealed atypical lymphocytes and immature plasma cells with morphological diversity. In comparison to normal controls, IL-21 production significantly increased in CD4+ (p < 0.001) and CD8+ (p = 0.020) T cells. B-cell clonality, RHOA G17V mutation, and the presence of sheets of clear cells and immature/mature plasma cells in lymph nodes were significantly associated with percentages of class-switched B cells and ASCs. The patients with circulating EBV DNA had a lower percentage of naive B cells (p = 0.009). CONCLUSIONS: Our results demonstrated a wide spectrum of peripheral B-cell morphologies and immunophenotypes of peripheral B cells in AITL. These findings correspond to dysregulated B-cell immunity and heterogeneous clinicopathological features.

FT895 Impairs Mitochondrial Function in Malignant Peripheral Nerve Sheath Tumor Cells.

Neurofibromatosis type 1 (NF1) stands as a prevalent neurocutaneous disorder. Approximately a quarter of NF1 patients experience the development of plexiform neurofibromas, potentially progressing into malignant peripheral nerve sheath tumors (MPNST). FT895, an HDAC11 inhibitor, exhibits potent anti-tumor effects on MPNST cells and enhances the cytotoxicity of cordycepin against MPNST. The study aims to investigate the molecular mechanism underlying FT895's efficacy against MPNST cells. Initially, our study unveiled that FT895 disrupts mitochondrial biogenesis and function. Post-FT895 treatment, reactive oxygen species (ROS) in MPNST notably increased, while mitochondrial DNA copy numbers decreased significantly. Seahorse analysis indicated a considerable decrease in basal, maximal, and ATP-production-coupled respiration following FT895 treatment. Immunostaining highlighted FT895's role in promoting mitochondrial aggregation without triggering mitophagy, possibly due to reduced levels of XBP1, Parkin, and PINK1 proteins. Moreover, the study using CHIP-qPCR analysis revealed a significant reduction in the copy numbers of promoters of the MPV17L2, POLG, TFAM, PINK1, and Parkin genes. The RNA-seq analysis underscored the prominent role of the HIF-1α signaling pathway post-FT895 treatment, aligning with the observed impairment in mitochondrial respiration. In summary, the study pioneers the revelation that FT895 induces mitochondrial respiratory damage in MPNST cells.

Clinicopathological Manifestations and Immune Phenotypes in Adult-Onset Immunodeficiency with Anti-interferon-γ Autoantibodies.

PURPOSE: Anti-interferon (IFN)-γ autoantibodies (anti-IFN-γ Abs) is an emerging adult-onset immunodeficiency syndrome. Immune dysfunction in this distinct disorder remains to be clarified. METHODS: We prospectively collected blood samples of 20 patients with anti-IFN-γ Abs and 40 healthy normal subjects. The percentages of lymphocyte subpopulations, most relevant to T, B, and NK cells, and the percentages of stimulated lymphocytes with cytokine production were assessed using eight-color flow cytometry. The results were adjusted to age and absolute lymphocyte counts. RESULTS: Most (85%) patients presented nontuberculous mycobacterial infection. Skin lesions were predominantly manifested by neutrophilic dermatoses. The involved lymph nodes had granulomatous inflammation, except 22.2% showing atypical lymphoid hyperplasia without granuloma formation. The percentages of CD4 + T cells and nonactivated subpopulations (recent thymic emigrants and naïve subtypes) decreased significantly with increased expression of activation markers and polarization to differentiated cells. The percentage of NK cells increased, but that of two major NK subpopulations, CD161 + CD56bright and CD161 + CD56 + CD16 + subsets, decreased. Increased CD161dim, CD161 + CD56 - CD16 + , and CD57 + NK cell subsets coupled with the decreased expression of NKp30 and NKp46 indicate reconfiguration of the NK cell population and acquisition of adaptive features. Intracellular cytokine production of the lymphocyte subpopulations was significantly low in the patients compared with the control group. CONCLUSION: We conclude that the immune system in patients with anti-IFN-γ Abs could be exhausted in T cells and be adaptive in NK cells, contributing to the distinct clinicopathologic features.

Exposure to tris(2-butoxyethyl) phosphate induces abnormal sperm morphology and testicular histopathology in male rats.

Tris(2-butoxyethyl) phosphate (TBEP) is one of the most abundant organophosphate flame retardants in the environment. This study aimed to evaluate the effect of TBEP exposure during adolescence on male reproductive function in adult rats. Male Sprague-Dawley rats were treated with 20 and 200 mg/kg body weight of TBEP or corn oil from postnatal day (PND) 42 to PND 105. A significant increase in the proportion of sperm with abnormal morphology (flattened head and bent tail) and superoxide anion (O2-.) production in the sperm of the 200 mg/kg treated group was observed (p < 0.05). Excessive production of sperm hydrogen peroxide (H2O2) was found in both the 20 and 200 mg/kg treatment groups (p < 0.05). Disruption of testicular structure was observed in the 20 and 200 mg/kg treated groups and seminiferous tubule degeneration was observed in the 200 mg/kg treated group. Our study demonstrated the adverse effects of TBEP on male reproductive function in rats.

Application of the electronic book to promote self-directed learning in medical technologist continuing education: a cross-sectional study.

BACKGROUND: Continuing education (CE) is essential for health professionals to improve competence in clinical practice, yet many medical technologists still experience barriers to learning in complex clinical settings. To better manage CE and address medical technologists' learning needs, we developed a learner-centred electronic book (e-book) to promote self-directed learning for medical technologists. METHODS: A cross-sectional study was conducted to explore the acceptability and learning impacts of the e-book as CE material for medical technologists in two medical centres in Taiwan. We designed the learner-centred context in the e-book based on medical technologists' practice requirements and learning needs. Moreover, we adopted The New World Kirkpatrick Model with four levels (reactions, learning, behaviours and results) to evaluate the e-book's learning impacts on medical technologists. A total of 280 medical technologists were invited to complete a questionnaire and a post-test, providing learning patterns as well as their satisfaction with the e-book and their learning outcomes after using it. RESULTS: Most readers had positive learning experiences and better learning outcomes, including knowledge acquisition and behavioural change, after reading the e-book. The e-book became a new CE activity and reached medical technologists in various types of laboratories. CONCLUSIONS: The low-cost and learner-centred e-book effectively overcame CE learning barriers for medical technologists. The interactivity and flexibility of e-learning particularly helped learners to engage in clinical scenarios in laboratory medicine. This study could pave the way for medical educators to build a high-quality e-learning model in CE.

Arsenic Induces M2 Macrophage Polarization and Shifts M1/M2 Cytokine Production via Mitophagy.

Arsenic is an environmental factor associated with epithelial-mesenchymal transition (EMT). Since macrophages play a crucial role in regulating EMT, we studied the effects of arsenic on macrophage polarization. We first determined the arsenic concentrations to be used by cell viability assays in conjunction with previous studies. In our results, arsenic treatment increased the alternatively activated (M2) macrophage markers, including arginase 1 (ARG-1) gene expression, chemokine (C-C motif) ligand 16 (CCL16), transforming growth factor-ß1 (TGF-ß1), and the cluster of differentiation 206 (CD206) surface marker. Arsenic-treated macrophages promoted A549 lung epithelial cell invasion and migration in a cell co-culture model and a 3D gel cell co-culture model, confirming that arsenic treatment promoted EMT in lung epithelial cells. We confirmed that arsenic induced autophagy/mitophagy by microtubule-associated protein 1 light-chain 3-II (LC3 II) and phosphor-Parkin (p-Parkin) protein markers. The autophagy inhibitor chloroquine (CQ) recovered the expression of the inducible nitric oxide synthase (iNOS) gene in arsenic-treated M1 macrophages, which represents a confirmation that arsenic indeed induced the repolarization of classically activated (M1) macrophage to M2 macrophages through the autophagy/mitophagy pathway. Next, we verified that arsenic increased M2 cell markers in mouse blood and lungs. This study suggests that mitophagy is involved in the arsenic-induced M1 macrophage switch to an M2-like phenotype.

HSPA4 Is a Biomarker of Placenta Accreta and Enhances the Angiogenesis Ability of Vessel Endothelial Cells.

Placenta accreta spectrum (PAS) accounts for 7% of maternal mortality and is associated with intraoperative and postoperative morbidity caused by massive blood loss, infection, and adjacent organ damage. The aims of this study were to identify the protein biomarkers of PAS and to further explore their pathogenetic roles in PAS. For this purpose, we collected five placentas from pregnant subjects with PAS complications and another five placentas from normal pregnancy (NP) cases. Then, we enriched protein samples by specifically isolating the trophoblast villous, deeply invading into the uterine muscle layer in the PAS patients. Next, fluorescence-based two-dimensional difference gel electrophoresis (2D-DIGE) and MALDI-TOF/MS were used to identify the proteins differentially abundant between PAS and NP placenta tissues. As a result, nineteen spots were determined as differentially abundant proteins, ten and nine of which were more abundant in PAS and NP placenta tissues, respectively. Then, specific validation with western blot assay and immunohisto/cytochemistry (IHC) assay confirmed that heat shock 70 kDa protein 4 (HSPA4) and chorionic somatomammotropin hormone (CSH) were PAS protein biomarkers. Further tube formation assays demonstrated that HSPA4 promoted the in vitro angiogenesis ability of vessel endothelial cells, which is consistent with the in vivo scenario of PAS complications. In this study, we not only identified PAS protein biomarkers but also connected the promoted angiogenesis with placenta invasion, investigating the pathogenetic mechanism of PAS.

Analysis of latent T-cell epitopes in Epstein-Barr virus isolated from extranodal nasal-type natural killer/T-cell lymphoma in Taiwanese population.

Extranodal nasal-type natural killer (NK)/T-cell lymphoma (NKTCL) is an aggressive lymphoma that is prevalent among East Asian and South American populations. Although Epstein-Barr virus (EBV) is commonly detected in NKTCL, there are limited studies that have analyzed the EBV genomic variations in NKTCL. In this study, 8 EBV latent genes were analyzed using targeted gene sequencing in 23 formalin-fixed paraffin-embedded tissues derived from 18 patients with NKTCL. Five cases with paired samples were comparatively analyzed. The consistency of EBV sequencing data between tissue samples was high (96.3%-98.7%), whereas that of variant calling among the tissue samples and plasma samples (74.3%-79.2%) was low. The highest densities of non-synonymous variants were detected in the EBNA3B gene. Among the 74 known T-cell epitopes, 363 non-synonymous variants were identified in 32 (43.2%) epitopes. Additionally, the AVFDRKSDAK (A1S/P and V2F/M/L) and YHLIVDTDSL (I4L and L10R/V/G/H) epitopes were associated with 5 patterns of amino acid changes in EBNA3B and EBNA-2, respectively. The frequency of variation in the human leukocyte antigen (HLA)-restricted epitopes with corresponding HLA types common among Taiwanese population was significantly low (P = 0.011), whereas that in anchor residues was significantly high (P = 0.012). In conclusion, this study demonstrated the genomic diversity of EBV in NKTCL and its correlation with the HLA-restricted epitope variations in Taiwanese population. The findings of this study provide useful insights for the development of novel therapeutic strategies for NKTCL.

Incidence, characteristics and outcomes among inpatient, outpatient and emergency department with reported high critical serum potassium values.

OBJECTIVES: Severe hyperkalemia can cause life-threatening arrhythmia, cardiac arrest, or death. This study aimed to investigate the incidence and the associated factors relevant to critical hyperkalemia (≥6 mmol/L) among inpatients, outpatients, and emergency department. Their clinical outcomes were also analyzed. METHODS: All patients whose high serum potassium values had been reported as critical laboratory values in 2016 were enrolled. Their demographic data, comorbidities, clinical symptoms, biochemical data, and outcomes were reviewed and collected. The Charlson comorbidity score (CCS) and glomerular filtration rate (GFR) were computed to assess the comorbidity burden and renal function. Patients were divided into groups according to different settings, potassium and GFR levels, and their survival. RESULTS: Of the 293,830 total serum potassium tests, 1,382 (0.47%) reports were listed as critical laboratory values. The average reply time was 6.3 min. Their mean age was 67.2 years, while the average GFR was 12.2 mL/min/1.73 m2. The overall mortality rate was 34%. Patients in the emergency department had the highest incidence (0.92%), while inpatients had the worst outcome (51% mortality). The leading cause of mortality was septic shock. The fatal group had higher rates of clinical symptoms, higher potassium values, CCS, and eGFR (all p<0.05). CONCLUSIONS: Most of the responses for the reports were obtained within a short period of time. Patients with reported high critical serum potassium values were characterized by high rates of comorbidity, reduced eGFR, and mortality. The incidence, clinical manifestations, and outcomes varied in the different clinical settings.

Effects of a quasi-experimental study of using flipped classroom approach to teach evidence-based medicine to medical technology students.

BACKGROUND: Flipped classroom is known to improve learning efficiency and to develop one's ability to apply high-level knowledge. To investigate the effect of flipped classroom approach on teaching evidence-based medicine to medical technology students, we conducted a tailor-made six flipped classroom based EBM courses for medical technology students. METHODS: This study adopted a qusai-experimental design with 62 medical technology interns as the research object. Students in the experimental group attended the flipped classroom course, while students in the control group attended the traditional course. The learning outcomes were evaluated by Fresno test in both groups. Furthermore, to understand student's perceptions on the flipped classroom approach, students in the experimental group were required to fill in a satisfaction survey and answer some open-ended questions. RESULTS: The Fresno test scores of the experimental group were significantly higher than that of the control group. From the results of the satisfaction survey, we know that students were satisfied with this course format. Students claimed that the flipped classroom approach could improve their learning efficiency and the interactions with teacher could help them to think more deeply. CONCLUSIONS: To conclude, most students showed positive attitudes and views on flipped classroom strategy. Moreover, students' questions were solved more effectively during class resulting in an improvement of effectiveness of evidence-based medicine trainings.

RHOA G17V mutation in angioimmunoblastic T-cell lymphoma: A potential biomarker for cytological assessment.

BACKGROUND: The World Health Organization, in a 2016 revision, introduced recurrent genetic abnormalities for classifying mature T- and NK-cell neoplasms. However, the role of genetic analyses from lymph node aspiration cytology is still not elucidated. We hypothesize that the use of genetic analyses may increase the accuracy of diagnosis from cytological preparations. METHODS: Fifty-seven formalin-fixed paraffin-embedded (FFPE) samples were collected for next-generation sequencing (NGS) targeting potential driver mutations including TET2, DMN3TA, IDH2, RHOA, STAT3, and STAT5B. Competitive allele-specific TaqMan polymerase chain reaction (cast-PCR) was performed to validate the mutation status by using FFPE and preoperative fine needle aspiration cytology (FNAC) samples. RESULTS: Among these six candidate genes, only IDH2 and RHOA mutations were significantly more frequent in nodal subtypes, angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) (P = .002 and <0.001, respectively). All genes exhibited different mutation patterns except RHOA with a hotspot mutation involving the Gly17 residue. The RHOA G17V mutation was found in 15 (75%) of 20 AITL and two (22%) of nine PTCL, NOS. Cast-PCR using FFPE samples showed 100% concordance with NGS. Among 12 lymph node aspirates, the preliminary diagnoses were suspicious for lymphoma (3, 25%), atypical lymphoid cells (3, 25%) and benign/negative (6, 50%). Cast-PCR detected the RHOA G17V mutation in six (75%) of eight RHOA-mutated aspirates and revealed negative results in all (100%) of four wild-type aspirates, with an 83.3% (10/12) concordance comparing to FFPE samples. CONCLUSIONS: The RHOA G17V mutation serves as a useful biomarker for cytological assessment in AITL. The use of cast-PCR is valuable in the diagnosis of malignant lymphomas from cytological preparations, and thus avoiding the potential risks of invasive procedures.

Bacterial detection and identification from human synovial fluids on an integrated microfluidic system.

Periprosthetic joint infections (PJIs) are among the most severe complications emerging from prosthetic joint replacement surgeries. In order to possess a rapid means of diagnosing PJIs, an integrated microfluidic system was developed herein for detecting and identifying bacteria in human synovial fluid (HSF). The entire molecular diagnostic process, including (1) sample treatment, (2) bacterial isolation, (3) bacterial lysis, (4) nucleic acid amplification (via polymerase chain reaction (PCR)), and (5) optical detection, could be automated on a single chip. First, N-acetyl-l-cysteine was used to decrease the viscosity of HSF samples and consequently enhance bacterial isolation with vancomycin-coated nano-magnetic beads. Then, a universal 16S ribosomal ribonucleic acid PCR primer set and four species-specific primer sets were used for PCR-based detection and identification of four common bacteria previously associated with PJIs, including Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli, and Acinetobacter baumannii. With this approach, the limit of detection was as low as 100 colony forming units (CFUs) per milliliter (or 20 CFUs per reaction), which is suitable for clinical diagnostics and for making informed decisions regarding post-operative antibiotic administration. More importantly, bacterial detection and identification data could be acquired within 90 minutes, representing a significant improvement over traditional culture-based methods (3-7 days). The developed microfluidic system may therefore serve as a promising tool for rapid diagnosis of PJIs.

Correction: Bacterial detection and identification from human synovial fluids on an integrated microfluidic system.

Correction for 'Bacterial detection and identification from human synovial fluids on an integrated microfluidic system' by Ting-Hang Liu et al., Analyst, 2019, 144, 1210-1222.

An Omics Approach to Diagnosing or Investigating Fungal Keratitis.

Fungal keratitis (FK) is one of the most severe corneal infectious diseases. FK often leads to poor visual prognosis and thus requires accurate diagnosis. Conventional approaches, including clinical diagnoses, smears, and cultures, often fail to provide reliable diagnostic value. Omics approaches, such as those using genomic, metagenomic, and tear proteomic data sources, provide promising features for improving the diagnosis and monitoring the progression of FK. Genomic approaches are based mainly on detecting amplicons of ribosomal RNA genes, and internal transcribed spacers are gradually gaining popularity in clinical practices. A metagenomic approach based on 16S rRNA genes may help monitor the dynamic change of conjunctival microbiota associated with an FK event, whereas that based on shot-gun and 18S rRNA target enrichment sequencing could have the potential to diagnose FK using clinical samples. A tear proteomic approach may provide comprehensive information about ocular surface defense and injury during FK. Representative up- and down-regulated proteins during FK could also be used as biomarkers to determine the clinical course and develop a treatment strategy in different stages of FK. Consequently, a personalized tear proteomic approach will soon play a key role in FK management.

Anti-Cancerous Effect of Inonotus taiwanensis Polysaccharide Extract on Human Acute Monocytic Leukemia Cells through ROS-Independent Intrinsic Mitochondrial Pathway.

Acute leukemia is one of the commonly diagnosed neoplasms and causes human death. However, the treatment for acute leukemia is not yet satisfactory. Studies have shown that mushroom-derived polysaccharides display low toxicity and have been used clinically for cancer therapy. Therefore, we set out to evaluate the anti-cancerous efficacy of a water-soluble polysaccharide extract from Inonotus taiwanensis (WSPIS) on human acute monocytic leukemia THP-1 and U937 cell lines in vitro. Under our experimental conditions, WSPIS elicited dose-dependent growth retardation and induced apoptotic cell death. Further analysis showed that WSPIS-induced apoptosis was associated with a mitochondrial apoptotic pathway, such as the disruption of mitochondrial membrane potential (MMP), followed by the activation of caspase-9, caspase-3, and PARP (poly(ADP-ribose) polymerase) cleavage. However, a broad caspase inhibitor, Z-VAD.fmk, could not prevent WSPIS-induced apoptosis. These data imply that mechanism(s) other than caspase might be involved. Thus, the involvement of endonuclease G (endoG), a mediator arbitrating caspase-independent oligonucleosomal DNA fragmentation, was examined. Western blotting demonstrated that WSPIS could elicit nuclear translocation of endoG. MMP disruption after WSPIS treatment was accompanied by intracellular reactive oxygen species (ROS) generation. However, pretreatment with N-acetyl-l-cysteine (NAC) could not attenuate WSPIS-induced apoptosis. In addition, our data also show that WSPIS could inhibit autophagy. Activation of autophagy by rapamycin decreased WSPIS-induced apoptosis and cell death. Taken together, our findings suggest that cell cycle arrest, endonuclease G-mediated apoptosis, and autophagy inhibition contribute to the anti-cancerous effect of WSPIS on human acute monocytic leukemia cells.

CUL4A overexpression as an independent adverse prognosticator in intrahepatic cholangiocarcinoma.

BACKGROUND: CUL4A has been known for its oncogenic properties in various human cancers. However, its role in intrahepatic cholangiocarcinoma (iCCA) has not been explored. METHODS: We retrospectively investigated 105 iCCA cases from a single medical institution. Tissue microarrays were used for immunohistochemical analysis of CUL4A expression. CUL4A expression vectors were introduced in cell lines. Cell migration and invasion assays were used to compare the mobility potential of iCCA cells under basal conditions and after manipulation. Then we evaluated the effects of CUL4A on the cell growth by proliferation assay, and further checked the susceptibility to cisplatin in iCCA cells with or without CUL4A overexpression. RESULTS: CUL4A overexpression was detected in 34 cases (32.4%). Patients with CUL4A-overexpressing tumors exhibited shortened disease-free survival (mean, 27.7 versus 90.4 months; P = 0.011). In the multivariate analysis model, CUL4A overexpression was shown to be an independent unfavorable predictor for disease-free survival (P = 0.045). Moreover, stably transfected CUL4A-overexpressing iCCA cell lines displayed an increased mobility potential and enhanced cell growth without impact on susceptibility to cisplatin. CONCLUSIONS: Our data demonstrate that overexpression of CUL4A plays an oncogenic role in iCCA and adversely affects disease-free survival. Thus, it may prove to be a powerful prognostic factor and a potential therapeutic target.

Mutations of candidate tumor suppressor genes at chromosome 3p in intrahepatic cholangiocarcinoma.

The genetic status of candidate tumor suppressor genes (TSGs) at chromosome 3p has not yet been elucidated in intrahepatic cholangiocarcinoma (iCCA). Herein, we retrospectively investigated 32 fresh iCCA case samples from a single medical institution to clarify mutations of 11 TSGs by next-generation sequencing. Validation of the mutations was performed on the MassARRAY platform or by high-resolution melting curve analysis. We then integrated the gene mutations into copy number alterations at chromosome 3p that had been generated in a previous study using the same fresh iCCA samples, and correlated the integration results with the clinicopathologic features. Nine of the 32 (28.1%) iCCA patients had gene mutations at chromosome 3p, totaling 11 mutations across five genes. Those included five (15.6%) BAP1 mutations, two each (6.3%) of CACNA2D3 and RASSF1 mutations, and one each (3.1%) of ATG7 and PLCD1 mutations. Six (18.8%) cases had concurrent loss of chromosome 3p and gene mutations. Patients with TSG mutations had shorter disease-free and survival times than those without the mutations. Our data showed that iCCA patients with TSG mutations at chromosome 3p faced an adverse prognosis. BAP1 was the common target of mutational inactivation and may be a principal driver of 3p21 losses.

Simultaneous detection of respiratory syncytial virus and human metapneumovirus by one-step multiplex real-time RT-PCR in patients with respiratory symptoms.

BACKGROUND: Both respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are important viral pathogens causing respiratory tract infection (RTI) in the pediatric population. However, the clinical manifestations of RSV and hMPV infections are similar. Therefore, a reliable and rapid diagnostic tool is needed for diagnostic performance. METHODS: In order to optimize diagnosis efficiency of RTI, the aim of this study is to establish a rapid and advanced method for simultaneous detecting RSV and hMPV in nasopharyngeal aspirates specimens from patients. We designed a one-step triplex real-time RT-PCR (qRT-PCR) protocol using TaqMan probes for detecting RSV and hMPV. The plasmid clones containing RSV nucleoprotein gene and hMPV fusion gene were established as reference standards. We used virus culture supernatants from 86 known pediatric RTI patient to test the specificity and sensitivity of our assay. Then we used total 222 nasopharyngeal aspirates specimens from pediatric patients hospitalized with respiratory symptoms to evaluate our assay. RESULTS: Our one-step triplex qRT-PCR assay showed 100% sensitivity and specificity in testing RSV and hMPV in 86 known virus culture supernatants, with excellent linearity (R2 > 0.99) and reliable reproducibility (CV lower than 1.04%). This assay has a wide dynamic range 102-109copies/reaction (limit of detection; LOD = 100 copies/reaction). A total of 222 patients hospitalized with respiratory symptoms were enrolled for clinical evaluation. In these samples, our qRT-PCR assay detected 68 RSV positive and 18 hMPV positive cases. However, standard virus culture only detected 8 RSV positive cases and 0 hMPV cases. Based on this improved triplex qRT-PCR assay, we found that RSV infection was associated with severe inflammation by chest X-ray and occurrence of pneumonia which were not observed previously. CONCLUSIONS: In summary, we have developed a highly specific and sensitive one-step triplex qRT-PCR assay to detect hMPV and RSV simultaneously. This assay offers a valuable tool for routine diagnosis.

TMPRSS6 rs855791 polymorphism influences the susceptibility to iron deficiency anemia in women at reproductive age.

BACKGROUND: Genome-wide-association studies have identified the TMPRSS6 polymorphism rs855791 has the strongest association with red blood cell indices or iron parameters in general population. Whether this genetic variant influences the susceptibility of iron deficiency anemia (IDA) in women with menstruation has not been well studied. METHODS: In this case-control study, we enrolled 67 women with IDA and 107 healthy volunteers, and analyzed their complete blood counts, rs855791 genotypes, and menstrual amounts. Menstrual blood loss was evaluated with a pictorial blood-loss assessment chart. RESULTS: There were significantly fewer rs855791 C homozygotes in the IDA group than in the healthy group (11.9% vs. 25.2%, p = 0.03). The odds ratio (OR) of C homozygotes having IDA versus non-CC subjects having IDA was 0.4 (95% CI, 0.17 - 0.95, p = 0.04). When the analysis was confined to study subjects with menorrhagia, this difference became more prominent (9.6% vs. 28.6%, p = 0.01; OR, 0.27, 95% CI, 0.09 - 0.77, p = 0.01). For women with non-CC genotypes, there was an inverse correlation between hemoglobin levels and menstrual loss (p < 0.001); however, this association was not found for those with genotypes CC (p = 0.15). CONCLUSIONS: Our study suggests homozygosity for TMPRSS6 rs855791 C genotype has a protective role against IDA in women at reproductive age, especially in those with menorrhagia.