NFTest: automated testing of Nextflow pipelines.

MOTIVATION: The ongoing expansion in the volume of biomedical data has contributed to a growing complexity in the tools and technologies used in research with an increased reliance on complex workflows written in orchestration languages such as Nextflow to integrate algorithms into processing pipelines. The growing use of workflows involving various tools and algorithms has led to increased scrutiny of software development practices to avoid errors in individual tools and in the connections between them. RESULTS: To facilitate test-driven development of Nextflow pipelines, we created NFTest, a framework for automated pipeline testing and validation with customizability options for Nextflow features. It is open-source, easy to initialize and use, and customizable to allow for testing of complex workflows with test success configurable through a broad range of assertions. NFTest simplifies the testing burden on developers by automating tests once defined and providing a flexible interface for running tests to validate workflows. This reduces the barrier to rigorous biomedical workflow testing and paves the way toward reducing computational errors in biomedicine. AVAILABILITY AND IMPLEMENTATION: NFTest is an open-source Python framework under the GPLv2 license and is freely available at https://github.com/uclahs-cds/tool-NFTest. The call-sSNV Nextflow pipeline is available at: https://github.com/uclahs-cds/pipeline-call-sSNV.

The increase of Nrf2 m6A modification induced by FTO downregulation promotes hippocampal neuron injury and aggravates the progression of epilepsy in a rat model.

Epilepsy is a common chronic neurological disorder characterized by widespread neuronal death. The purpose of this study was to investigate the role of nuclear factor erythroid 2-related factor 2 (Nrf2) m6A methylation in epilepsy. To create epileptic models, the rats were given Lithium chloride and pilocarpine, and isolated primary rat hippocampal neurons were cultured in an Mg2+ -free medium. The frequency of seizures was recorded in the epilepsy group of rats. The functional tests included TUNEL, MTT, and flow cytometry. Mechanistically, RNA degradation assay, RNA immunoprecipitation, and methylated RNA immunoprecipitation were performed. In epileptic models, Nrf2 and fat mass and obesity-associated (FTO) levels were downregulated, whereas YT521-B homology (YTH) domain family protein 2 (YTHDF2) was upregulated. Additionally, in epileptic models, there was a rise in the m6A methylation level of Nrf2 mRNA. Overexpressing FTO increased cell viability and reduced apoptosis, but Nrf2 interference reversed these effects. Meanwhile, FTO overexpression decreased the m6A methylation of Nrf2 mRNA. Moreover, YTHDF2 bound to Nrf2 mRNA and decreased its stability. Furthermore, FTO overexpression reduced seizure frequency in rats and inhibited hippocampal neuron apoptosis via lowering the m6A methylation level of Nrf2 mRNA. Overexpressing FTO reduced m6A methylation of Nrf2 mRNA, increased cell viability, suppressed apoptosis, and slowed the progression of epileptic diseases, which is linked to YTHDF2 binding to m6A-modified Nrf2 and promoting its degradation, as well as downregulating Nrf2 expression in hippocampal neurons.

Detection and Quantification of Candidatus Methanoperedens-Like Archaea in Freshwater Wetland Soils.

Candidatus Methanoperedens-like archaea, which can use multiple electron acceptors (nitrate, iron, manganese, and sulfate) for anaerobic methane oxidation, could play an important role in reducing methane emissions from freshwater wetlands. Currently, very little is known about the distribution and community composition of Methanoperedens-like archaea in freshwater wetlands, particularly based on their alpha subunit of methyl-coenzyme M reductase (mcrA) genes. Here, the community composition, diversity, and abundance of Methanoperedens-like archaea were investigated in a freshwater wetland through high-throughput sequencing and quantitative PCR on their mcrA genes. A large number of Methanoperedens-like mcrA gene sequences (119,250) were recovered, and a total of 31 operational taxonomic units (OTUs) were generated based on 95% sequence similarity cut-off. The majority of Methanoperedens-like sequences can be grouped into three distinct clusters that were closely associated with the known Methanoperedens species which can couple anaerobic methane oxidation to nitrate or iron reduction. The community composition of Methanoperedens-like archaea differed significantly among different sampling sites, and their mcrA gene abundance was 1.49 × 106 ~ 4.62 × 106 copies g-1 dry soil in the examined wetland. In addition, the community composition of Methanoperedens-like archaea was significantly affected by the soil water content, and the archaeal abundance was significantly positively correlated with the water content. Our results suggest that the mcrA gene is a good biomarker for detection and quantification of Methanoperedens-like archaea, and provide new insights into the distribution and environmental regulation of these archaea in freshwater wetlands.

A novel UV-resistant bacterium Sphingomonas endolithica sp. nov., and genomic analysis, isolated from the north slope of Mount Everest.

Gram-stain-negative, aerobic, rod-shaped, non-motile bacterium strain ZFBP2030T was isolated from a rock on the North slope of Mount Everest. This strain contained a unique ubiquinone-10 (Q-10) as a predominant respiratory quinone. Among the tested fatty acids, the strain contained summed feature 8, C14:0 2OH, and C16:0, as major cellular fatty acids. The polar lipid profile contained phosphatidyl glycerol, phosphatidyl ethanolamine, three unidentified phospholipids, two unidentified aminolipids, and six unidentified lipids. The cell-wall peptidoglycan was a meso-diaminopimelic acid, and cell-wall sugars were ribose and galactose. Phylogenetic analyses based on 16S rRNA gene sequence revealed that strain ZFBP2030T was a member of the genus Sphingomonas, exhibiting high sequence similarity to the 16S rRNA gene sequences of Sphingomonas aliaeris DH-S5T (97.9%), Sphingomonas alpina DSM 22537T (97.3%) and Sphingomonas hylomeconis CCTCC AB 2013304T (97.0%). The 16S rRNA gene sequence similarity between ZFBP2030T and other typical strains was less than 97.0%. The average amino acid identity values, average nucleotide identity, and digital DNA-DNA hybridization values between strain ZFBP2030T and its highest sequence similarity strains were 56.9-79.9%, 65.1-82.2%, and 19.3-25.8%, respectively. The whole-genome size of the novel strain ZFBP2030T was 4.1 Mbp, annotated with 3838 protein-coding genes and 54 RNA genes. Moreover, DNA G + C content was 64.7 mol%. Stress-related functions predicted in the subsystem classification of the strain ZFBP2030T genome included osmotic, oxidative, cold/heat shock, detoxification, and periplasmic stress responses. The overall results of this study clearly showed that strain ZFBP2030T is a novel species of the genus Sphingomonas, for which the name Sphingomonas endolithica sp. nov. is proposed. The type of strain is ZFBP2030T (= EE 013T = GDMCC 1.3123T = JCM 35386T).

CD40LG-associated X-linked Hyper-IgM Syndrome (XHIGM) with pulmonary alveolar proteinosis: a case report.

BACKGROUND: D40LG-associated X-linked hyper-IgM syndrome with pulmonary alveolar proteinosis has rarely been reported, and its genotype-phenotypic correlation remains elusive. CASE PRESENTATION: We describe a five-month-old boy with CD40LG mutation (c.516T > A, p.Tyr172Ter) X-linked hyper-IgM syndrome with pulmonary alveolar proteinosis as the first manifestation. The patient completely recovered after immunotherapy and allogeneic hematopoietic stem cell transplantation. In addition, four previously reported patients with CD40LG mutation with pulmonary alveolar proteinosis were also analyzed. All of these patients presented with early onset of pulmonary infections and a good response to immunotherapy. The structural model of CD40LG indicated that all mutations caused the X-linked hyper-IgM syndrome with pulmonary alveolar proteinosis to be located within the tumor necrosis factor homology domain. CONCLUSIONS: A case was presented, and the characteristics of four cases of CD40LG-associated X-linked hyper-IgM syndrome with pulmonary alveolar proteinosis were summarized. The variant locations may explain the phenotypic heterogeneity of patients with the CD40LG mutation.

Effect of gradual increase of atmospheric CO2 concentration on nitrification potential and communities of ammonia oxidizers in paddy fields.

Currently, the influence of elevated atmospheric CO2 concentration (eCO2) on ammonia oxidation to nitrite, the rate-limiting step of nitrification in paddy soil, is poorly known. Previous studies that simulate the effect of eCO2 on nitrification are primarily based on an abrupt increase of atmospheric CO2 concentration. However, paddy ecosystems are experiencing a gradual increase of CO2 concentration. To better understand how the nitrification potential, abundance and communities of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) respond to eCO2 in paddy ecosystems, a field experiment was conducted using the following two treatments: a gradual increase of CO2 (EC, increase of 40 ppm per year until 200 ppm above ambient) and ambient CO2 (CK). The results demonstrated that the EC treatment significantly (P < 0.05) stimulated the soil potential nitrification rate (PNR) at the jointing and milky stages, which increased by 127.83% and 27.35%, respectively, compared with CK. Furthermore, the EC treatment significantly (P < 0.05) stimulated the AOA and AOB abundance by 56.60% and 133.84%, respectively, at the jointing stage. Correlation analysis showed that the PNR correlated well with the abundance of AOB (R2 = 0.7389, P < 0.001). In addition, the EC treatment significantly (P < 0.05) altered the community structure of AOB, while it had little effect on that of AOA. A significant difference in the proportion of Nitrosospira was observed between CO2 treatments. In conclusion, the gradual increase of CO2 positively influenced the PNR and abundance of ammonia oxidizers, and AOB could be more important than AOA in nitrification under eCO2.

Stable Lithium Plating and Stripping Enabled by a LiPON Nanolayer on PP Separator.

The practical application of the Li metal anode (LMA) is hindered by its low coulombic efficiency and dendrite formation. Although solid-state electrolytes hold promise as ideal partners for LMA, their effectiveness is limited by the poor workability and ionic conductivity. Herein, a modified separator combining the rapid Li+ transport of a liquid electrolyte and the interfacial stability of a solid-state electrolyte is explored to realize stable cycling of the LMA. A conformal nanolayer of LiPON is coated on a polypropylene separator by a scalable magnetron sputtering method, which is compatible with current Li-ion battery production lines and promising for the practical applications. The resulting LMA-electrolyte/separator interface is Li+ -conductive, electron-insulating, mechanically and chemically stable. Consequently, Li|Li cells maintain stable dendrite-free cycling with overpotentials of 10 and 40 mV over 2000 h at 1 and 5 mA cm-2 , respectively. Additionally, the Li|LiFePO4 full cells achieve a capacity retention of 92% after 550 cycles, confirming its application potential.

Arthrobacter antioxidans sp. nov., a blue pigment-producing species isolated from Mount Everest.

Bacteria in the genus Arthrobacter have been found in extreme environments, e.g. glaciers, brine and mural paintings. Here, we report the discovery of a novel pink-coloured bacterium, strain QL17T, capable of producing an extracellular water-soluble blue pigment. The bacterium was isolated from the soil of the East Rongbuk Glacier of Mt. Everest, China. 16S rRNA gene sequence analysis showed that strain QL17T was most closely related to the species Arthrobacter bussei KR32 T. However, compared to A.bussei KR32T and the next closest relatives, the new species demonstrates considerable phylogenetic distance at the whole-genome level, with an average nucleotide identity of <85 % and inferred DNA-DNA hybridization of <30 %. Polyphasic taxonomy results support our conclusion that strain QL17T represents a novel species of the genus Arthrobacter. Strain QL17T had the highest tolerance to hydrogen peroxide at 400 mM. Whole-genome sequencing of strain QL17T revealed the presence of numerous cold-adaptation, antioxidation and UV resistance-associated genes, which are related to adaptation to the extreme environment of Mt. Everest. Results of this study characterized a novel psychrotolerant Arthrobacter species, for which the name Arthrobacter antioxidans sp. nov. is proposed. The type strain is QL17T (GDMCC 1.2948T=JCM 35246T).

Detection of hemoglobin H disease by long molecule sequencing.

BACKGROUND: Hemoglobin H (Hb H) disease is a moderate-to-severe form of α-thalassemia (α-thal), and parts of patients may require intermittent transfusion therapy, especially during intercurrent illness. However, rare Hb H diseases remain undetected using routine methods being outside of the testing scope. In this study, we present an approach to detecting Hb H disease by long molecule sequencing (LMS). METHODS: A total of 206 known genotype samples were collected and carried to blind detected by LMS on the PacBio Sequel platform. Circular consensus sequencing reads were aligned to the hg19 reference genome using Free-Bayes finished LMS. LMS accuracy would be compared with routine methods, including Gap-PCR and PCR-Reverse dot blot hybridization (PCR-RDB). RESULTS: The assay could detect carriers of both deletion and point mutations. It had an overall accuracy of 100% when compared with routine methods. In addition, LMS detected six mutations based on routine methods and corrected three case results. Hb H diseases were identified using LMS, whether a common or rare genotype, a deletion or non-deletion genotype. However, two cases of Hb H disease were misdiagnosed using routine methods. CONCLUSIONS: Long molecule sequencing can be suggested as a rapid and reliable assay to detect probable carriers of hemoglobinopathies. LMS accurately identified the common and rare genotypes of Hb H disease.

Weakness in both lower limbs for 1 week and blepharoptosis for 3 days in a boy aged 1 year and 7 months. / ç”·æ€§å¹¼å„¿åŒä¸‹è‚¢æ— åŠ›1周伴眼睑下垂3 d.

A boy, aged 1 year and 7 months, was hospitalized due to weakness in both lower limbs and blepharoptosis, which showed progressive aggravation and developed into irregular breathing. Neurological examinations showed lethargy, blepharoptosis, grade 4 muscle strength of both upper limbs, grade 3 muscle strength of both lower limbs, and disappearance of tendon reflex. Laboratory tests revealed albuminocytological dissociation in cerebrospinal fluid, disappearance of H reflex, and positive serum anti-GD1b IgG. The boy was finally diagnosed with Guillain-Barré syndrome (GBS) overlapping with Miller-Fisher syndrome and Bickerstaff brainstem encephalitis. He recovered and was discharged after treatment including immunoglobulin, plasma exchange, and respiratory support. The GBS overlap syndromes in children have strong clinical heterogeneity due to the injury of both peripheral nerve and brainstem, among which anti-GD1b antibody-related GBS overlap syndromes have special clinical manifestations and complex neuroelectrophysiological changes and are thus difficult to diagnose. Nerve conduction velocity tests, especially H reflex test, should be performed for children with weakness in both lower limbs and blepharoptosis.

[Progressive psychomotor regression for 2.5 years in a boy aged 5 years]. / 5岁男童进行性智力运动功能倒退2.5年.

A boy, aged 5 years, attended the hospital due to progressive psychomotor regression for 2.5 years. Motor function regression was the main manifestation in the early stage, and brain MRI and whole-exome sequencing (WES) of the family showed no abnormalities. After the age of 4 years and 9 months, the boy developed cognitive function regression, and brain MRI showed cerebellar atrophy. The reanalysis of WES results revealed a compound heterozygous mutation, [NM_000520, c.784C>T(p.His262Tyr]), c.1412C>T(p.Pro471Leu)], in the HEXA gene. The enzyme activity detection showed a significant reduction in the level of ß-hexosaminidase encoded by this gene. The boy was diagnosed with juvenile Tay-Sachs disease (TSD). TSD has strong clinical heterogeneity, and cerebellar atrophy may be an important clue for the diagnosis of juvenile TSD. The reanalysis of genetic data when appropriate based on disease evolution may improve the positive rate of WES.

BIM and Computer Vision-Based Framework for Fire Emergency Evacuation Considering Local Safety Performance.

Fire hazard in public buildings may result in serious casualties due to the difficulty of evacuation caused by intricate interior space and unpredictable development of fire situations. It is essential to provide safe and reliable indoor navigation for people trapped in the fire. Distinguished from the global shortest rescue route planning, a framework focusing on the local safety performance is proposed for emergency evacuation navigation. Sufficiently utilizing the information from Building Information Modeling (BIM), this framework automatically constructs geometry network model (GNM) through Industry Foundation Classes (IFC) and integrates computer vision for indoor positioning. Considering the available local egress time (ALET), a back propagation (BP) neural network is applied for adjusting the rescue route according to the fire situation, improving the local safety performance of evacuation. A campus building is taken as an example for proving the feasibility of the framework proposed. The result indicates that the rescue route generated by proposed framework is secure and reasonable. The proposed framework provides an idea for using real-time images only to implement the automatic generation of rescue route when a fire hazard occurs, which is passive, cheap, and convenient.

Using a Nomogram to Preoperatively Predict Distant Metastasis of Pancreatic Neuroendocrine Tumor in Elderly Patients.

Objective To establish a nomogram for predicting the distant metastasis risk of pancreatic neuroendocrine tumors (pNETs) in elderly patients. Methods We extracted data of patients with diagnosis of pNETs at age ≥65 years old between 1973 and 2015 from the Surveillance, Epidemiology, and End Results (SEER) database. All eligible patients were divided randomly into a training cohort and validation cohort. Uni- and multivariate logistic regression analyses were performed on the training cohort to identify independent factors for distant metastasis. A nomogram was developed based on the independent risk factors using rms packages of R software, and was validated internally by the training cohort and externally by the validation cohort using C-index and calibration curves. Results A total of 411 elderly patients were identified, of which 260 were assigned to training cohort and 151 to validation cohort. Univariate and multivariate logistic regression analyses indicated the tumor site (body/tail of pancreas: odds ratio [OR]=2.282; 95% confidence interval [CI]: 1.174 - 4.436, P<0.05), histological grade (poorly differentiated/undifferentiated: OR=2.600, 95% CI: 1.266-5.339, P<0.05), T stage (T2: OR=8.913, 95% CI: 1.985-40.010, P<0.05; T3: OR=11.830, 95% CI: 2.530-55.350, P<0.05; T4: OR=68.650, 95% CI: 8.020-587.600, P<0.05), and N stage (N1: OR=3.480, 95% CI: 1.807-6.703, P<0.05) were identified as independent risk factors for distant metastasis of pNETs in elderly. The nomogram exhibited good predicting accuracy, with a C-index of 0.809 (95% CI: 0.757 - 0.861) in internal validation and 0.795 (95% CI: 0.723 - 0.867) in external validation, respectively. The predicted distant metastasis rates were in satisfactory agreement with the observed values by the calibration curves. Conclusion The nomogram we established showed high discriminative ability and accuracy in evaluation of distant metastasis risk in elderly pNETs patients, and could provide a reference for individualized tumor evaluation and treatment decision in elderly pNETs patients.

Does Prior Cancer Have an Influence on the Survival Outcomes of Patients with Localized Pancreatic Neuroendocrine Tumors?

Objective To investigate the impact of prior non-pancreatic cancer on the survival outcomes of patients with localized pancreatic neuroendocrine tumors (PanNETs). Methods We reviewed the Surveillance, Epidemiology, and End Results database and selected patients with localized PanNETs diagnosed between 1973 and 2015. We divided the patients into two groups according to the presence or absence of prior non-pancreatic malignancy. Before and after propensity score matching, we compared the clinicopathological characteristics and studied the overall survival and cancer-specific survival. Results A total of 357 (12.9%) of 2778 patients with localized PanNETs had prior cancer. A total of 1211 cases with only a localized PanNET and 133 cases with a localized PanNET and prior cancer had complete data and met the inclusion criteria of the current study. Patients with prior cancer were associated with advanced age (>65 years, 57.9% prior cancer vs. 31.0% no prior cancer, P<0.001), later year of diagnosis (87.2% vs. 80.2%, P=0.049), a higher proportion of poorly differentiated/undifferentiated grade tumors (4.5% vs. 1.5%, P=0.025), and a higher proportion of no primary site surgery (19.5% vs. 10.4%, P=0.003). Prostate (29.32%), breast (18.05%), other genitourinary and retroperitoneal (16.54%), and gastrointestinal (12.78%) cancers were the most common prior cancer types. Most of the prior cancers (95.49%) were localized and regional, and only 4.51% of the prior cancers were distant. Patients with interval periods between the prior cancer and PanNET of ≤36 months, 36-60 months, 60-120 months, and >120 months accounted for 33.08%, 13.53%, 24.06%, and 29.32% of all cases with prior cancers, respectively. Univariate and multivariate Cox proportional hazards analyses were performed. The presence/absence of prior cancers did not impact survival outcomes of patients with localized PanNETs before and after propensity score matching (PSM). Further subgroups analysis showed that, patients with localized PanNETs and prior distant cancer had worse cancer-specific survival than patients with prior local/regional cancer or patients without prior cancer (P<0.001). No significant differences in cancer-specific survival were observed in terms of the different sites of the prior cancers and the different interval periods of prior cancers and PanNETs (P<0.05). Conclusions Patients with localized PanNETs and a history of prior cancer had survival outcomes that were comparable to those of patients with no history of prior cancer. Patients with localized PanNETs and prior cancer could be candidates for clinical trials if they satisfy all other conditions; aggressive and potentially curative therapies should be offered to these patients.

[Fever for 2 months and disturbance of consciousness for 1 week in a preschool-aged girl].

Rapid-onset obesity with hypothalamic dysfunction, hypoventilation, and autonomic dysregulation syndrome (ROHHADS) is a rare multi-system disease, and delayed diagnosis and treatment may lead to catastrophic cardiopulmonary complications. As far as we know, no patient with ROHHADS has been reported in China, and this article reports a child with ROHHADS to improve the awareness of this disease among clinicians. A girl, aged 3 years, had the clinical manifestations of rapid weight gain, fever, disturbance of consciousness, and convulsion. The physical examination showed a body weight of 20 kg, somnolence, irregular breathing, and stiff neck. She had increased blood levels of prolactin and follicle-stimulating hormone and hyponatremia. The lumbar puncture showed an increased intracranial pressure. The brain MRI and magnetic resonance venography showed symmetrical lesions in the periventricular region and venous thrombosis in the right transverse sinus and the superior sagittal sinus. The sleep monitoring showed hypopnea. The girl was finally diagnosed with ROHHADS and intracranial venous thrombosis. She recovered after symptomatic treatment including decreasing intracranial pressure, anticoagulation, and respiratory support. The possibility of ROHHADS should be considered for patients with unexplained obesity, fever, and hypoventilation, with or without central nervous system symptoms. Early diagnosis and standardized follow-up can improve the prognosis of children with ROHHADS.

Electrophoresis features and genotypes of Hb bart's hydrops fetalis.

Hydrops fetalis syndrome (Hb Bart's disease) is one of the common fetal development abnormalities and the most severe form of α-thalassemia. It causes fetal death during the third trimester or shortly after birth. The aim of this study is to investigate the feature of Hb Bart's disease by Capillary electrophoresis (CE) and its genotypes. A total of 38 cases of suspected fetal Hb Bart's disease were analyzed by B-ultrasound and CE, including 17 with 24-28 weeks of pregnancy, 9 with 29-32 weeks of pregnancy, and 12 with 33-38 weeks of pregnancy. All cases were then identified by DNA analysis. The minimum concentration of Hb Bart's was 72.2% and the highest 90.9%. Hb Bart's fraction increased while that of Hb Portland decreased with the duration of pregnancy. In order to avoid serious maternal morbidity, it is necessary to diagnose Hb Bart's disease as soon as possible and CE can be used as a method of rapid diagnosis.

C-reactive protein for predicting cardiovascular and all-cause mortality in type 2 diabetic patients: A meta-analysis.

AIMS: There is interest in using blood C-reactive protein (CRP) to predict adverse prognosis outcomes patients with type 2 diabetes. This meta-analysis aimed to investigate the association between elevated baseline CRP level and unfavorable outcomes in type 2 diabetes patients. MATERIALS/METHODS: PubMed and Embase databases were systematically searched for studies on the association of elevated baseline CRP level with cardiovascular mortality and all-cause mortality from their inception to July 2018. Pooled risk ratio (RR) with 95% confidence intervals (CI) was calculated for the highest versus the lowest CRP level. RESULTS: Six prospective cohort studies and two post hoc analyses of randomized controlled trials involving 22,322 type 2 diabetes patients were included. Meta-analysis indicated that type 2 diabetes patients with the highest CRP level had a greater risk of all-cause mortality (RR 2.03; 95% CI 1.49-2.75) and cardiovascular mortality (RR 1.76; 95% CI 1.46-2.13). Subgroups analysis indicated that the increased cardiovascular and all-cause mortality risk was consistently found in different study design, follow-up duration or patients with or without cardiovascular risk/established cardiovascular disease subgroups. CONCLUSIONS: This meta-analysis indicates that elevated baseline serum CRP level is independently associated with future cardiovascular and all-cause mortality in type 2 diabetes patients.

Detection of Hb H disease caused by a novel mutation and --SEA deletion using capillary electrophoresis.

BACKGROUND: Hb H disease is a serious type of α-thalassemia which cause moderate anemia while misdiagnosis by routine genetic analysis in a rare or novel Hb H disease. METHODS: The study was done on three patients and one fetus in a suspected Hb H disease family. Hb analysis was carried out using capillary electrophoresis (CE), and hematological analysis was conducted with an automated cell counter. Common α- and ß-thalassemia mutations were detected by routine genetic analysis (gap-PCR and RDB-PCR). Novel mutation diagnostic methods were based on DNA sequencing. RESULTS: Capillary electrophoresis revealed clinical feature of classic Hb H disease in the proband, and hematology analysis showed moderate anemia (Hb 87 g/L). But routine genetic analysis was found that it was only a heterozygote for the --SEA deletion. DNA sequencing of α-globin genes (α1 and α2) identified the breakpoints between nts 34162 and 34171 at α2 gene, named CD 90-93 (-AGCTTCGG) mutation. The genotype of proband and fetus was the same --SEA /-αCD90-93 . His father was homozygous for the novel mutation (-αCD90-93 /-αCD90-93 ), and his mother was heterozygote for the --SEA deletion. CONCLUSIONS: Our study for the first time described the novel mutation CD 90-93 (-AGCTTCGG). CE is a way to avoid misdiagnosis of rare or novel Hb H disease.

Capillary Electrophoresis Resolves Inconclusive HPLC Analysis for Hemoglobin Variants: a Study of Two Cases.

BACKGROUND: Hb I and Hb Shaare Zedek are rare hemoglobin variants in the Chinese population. High-performance liquid chromatography (HPLC) is a widely used technique for screening of thalassemias and hemoglobin variants in Chinese primary hospitals. However, some rare hemoglobin variants cannot be effectively separated by HPLC. Here, we report one case of Hb I and one case of Hb Shaare Zedek which could not be detected by HPLC but required capillary electrophoresis (CE) in our hospital. METHODS: Two blood samples with high Hb F level were analyzed by HPLC as part of routine screening, and then globin genes were analyzed using Gap-PCR, PCR-Reverse dot-blot (RDB), and DNA sequencing. Subsequently, samples were analyzed by CE and results compared to HPLC. RESULTS: In case 1, results were as follows: Hb F 16.9%, Hb A0 72.5% (failed to show the value of Hb A in HPLC) and Hb A2 2.2% for HPLC. No mutations were detected using Gap-PCR and PCR-RDB, but there was a mutation of codon (CD) 16 in the α2 globin gene (AAG>GAG, corresponds to Hb I) by DNA sequencing. CE showed Hb A2 1.9%, Hb A 74%, Hb I 24.1%. In case 2, results were as follows: Hb F 11.0%, Hb A0 76.6%, and Hb A2 4.7% for HPLC. It showed a CD 41 - 42 mutation of the ß-globin gene (-TTCT) using PCR-RDB and the CD 56 mutation in α2/α1 globin gene (AAG>GAG, corresponding to Hb Shaare Zedek) with DNA sequencing. CE displayed Hb A2 4.5%, Hb A 84.6%, Hb F 0.4%, Hb Shaare Zedek 10.5%. CONCLUSIONS: Hb I and Hb Shaare Zedek cannot be separated by HPLC because of co-elution with Hb F. However, they were identified and quantified using CE. As a general precaution, therefore, and owing to the existence of rare variants that may co-elute with normal hemoglobin fractions, it is recommended to use at least two complementary methods for the diagnostic detection of hemoglobin species.

[Establishing Glioma-associated Oncogene Homolog 1 Genetic Lineage-tracing Mice for Studies on Hepatic Fibrosis].

OBJECTIVE: To establish Gli1-CreERt2; tdTomato genetic lineage-tracing mice for studies on hepatic fibrosis. METHODS: Offspring of ROSA26 td Tomato (tdTomato) mice and Gli1-CreERt2 mice (Gli1 mice) were obtained, with Gli1-CreERt2; tdTomato genotype being identified by PCR. The mice model of hepatic fibrosis was induced with CCl4. Their liver samples were taken. The formalin-fixed and paraffin-embedded samples were prepared for HE staining and Masson staining. The expression of tdTomato was observed under immunofluorescent microscope. RESULTS: An ideal number of Gli1-CreERt2; tdTomato genetic lineage-tracing mice were harvested. The differences in fertility between the parental and the offspring mice were not significant (P>0.05). Pseudolobular formation occurred in the CCl4-induced hepatic fibrosis model mice. Enhanced red fluoresce was observed in the model mice. CONCLUSION: Gli1-CreERt2; tdTomato genetic lineage-tracing mice can be used to monitor the cell source of fibrous tissues, its transition as well as the underlying mechanism of pathogenesis of hepatic fibrosis.