DomeTree: a canonical toolkit for mitochondrial DNA analyses in domesticated animals

Mitochondrial DNA (mtDNA) is widely used in various genetic studies of domesticated animals. Many applications require comprehensive knowledge about the phylogeny of mtDNA variants. Herein, we provide the most up‐to‐date mtDNA phylogeny (i.e. haplogroup tree or matrilineal genealogy) and a standardized hierarchical haplogroup nomenclature system for domesticated cattle, dogs, goats, horses, pigs, sheep, yaks and chickens. These high‐resolution mtDNA haplogroup trees based on 1240 complete or near‐complete mtDNA genome sequences are available in open resource DomeTree ( http://www.dometree.org ). In addition, we offer the software MitoToolPy ( http://www.mitotool.org/mp.html ) to facilitate the mtDNA data analyses. We will continuously and regularly update DomeTree and MitoToolPy.     

A novel approach for metabolic pathway optimization: Oligo-linker mediated assembly (OLMA) method

Background

Imbalances in gene expression of a metabolic pathway can result in less-yield of the desired products. Several targets were intensively investigated to balance the gene expression, such as promoter, ribosome binding site (RBS), the order of genes, as well as the species of the enzymes. However, the capability of simultaneous manipulation of multiple targets still needs to be explored.

Results

We reported a new DNA assembling method to vary all the above types of regulatory targets simultaneously, named oligo-linker mediated assembly (OLMA) method, which can incorporate up to 8 targets in a single assembly step. Two experimental cases were used to demonstrate the capability of the method: (1) assembly of multiple pieces of lacZ expression cassette; (2) optimization of four enzymes in lycopene biosynthetic pathway. Our results indicated that the OLMA method not only exploited larger combinatorial space, but also reduced the inefficient mutants.

Conclusions

The unique feature of oligo-linker mediated assembly (OLMA) method is inclusion of a set of chemically synthetic double-stranded DNA oligo library, which can be designed as promoters and RBSs, or designed with different overhang to bridge the genes in different orders. The inclusion of the oligos resulted in a PCR-free and zipcode-free DNA assembly reaction for OLMA.

     

Scoliosis Related Information on the Internet in China: Can Patients Benefit from This Information?

BackgroundThere has been an increasing popularity of searching health related information online in recent years. Despite that considerable amount of scoliosis patients have shown interest in obtaining scoliosis information through Internet, previous studies have demonstrated poor quality of online information. However, this conclusion may vary depending on region and culture. Since China has a restricted Internet access outside of its borders, the aim of this study is to evaluate the quality of scoliosis information available online using recognized scoring systems and to analyze the Internet as a source of health information in China.MethodsA survey-based questionnaire was distributed to 280 respondents at outpatient clinics. Information on demographics and Internet use was collected. Binary logistic analysis was performed to identify possible predictors for the use of Internet. In addition, the top 60 scoliosis related websites assessed through 4 search engines were reviewed by a surgeon and the quality of online information was evaluated using DISCERN score and JAMA benchmark.ResultsUse of the Internet as a source for scoliosis related information was confirmed in 87.8% of the respondents. College education, Internet access at home and urban residence were identified as potential predictors for Internet use. However, the quality of online scoliosis related information was poor with an average DISCERN score of 27.9±11.7 and may be misleading for scoliosis patients.ConclusionThe study outlines the profile of scoliosis patients who use the Internet as a source of health information. It was shown that 87.8% of the scoliosis patients in outpatient clinics have searched for scoliosis related information on Internet. Urban patients, higher education and Internet access at home were identified as potential predictors for Internet search. However, the overall quality of online scoliosis related information was poor and confusing. Physician based websites seemed to contain more reliable information.     

Establishment of a Patient-Derived Orthotopic Xenograft (PDOX) Model of HER-2-Positive Cervical Cancer Expressing the Clinical Metastatic Pattern

Squamous cell carcinoma of the cervix, highly prevalent in the developing world, is often metastatic and treatment resistant with no standard treatment protocol. Our laboratory pioneered the patient-derived orthotopic xenograft (PDOX) nude mouse model with the technique of surgical orthotopic implantation (SOI). Unlike subcutaneous transplant patient-derived xenograft (PDX) models, PDOX models metastasize. Most importantly, the metastasis pattern correlates to the patient. In the present report, we describe the development of a PDOX model of HER-2-positive cervical cancer. Metastasis after SOI in nude mice included peritoneal dissemination, liver metastasis, lung metastasis as well as lymph node metastasis reflecting the metastatic pattern in the donor patient. Metastasis was detected in 4 of 6 nude mice with primary tumors. Primary tumors and metastases in the nude mice had histological structures similar to the original tumor and were stained by an anti-HER-2 antibody in the same pattern as the patient’s cancer. The metastatic pattern, histology and HER-2 tumor expression of the patient were thus preserved in the PDOX model. In contrast, subcutaneous transplantation of the patient’s cervical tumors resulted in primary growth but not metastasis.     

MicroRNA-486 as a Biomarker for Early Diagnosis and Recurrence of Non-Small Cell Lung Cancer

Background

Non-small cell lung cancer (NSCLC) is a leading cause of cancer death worldwide. Early diagnosis is essential for improvements of prognosis and survival of the patients. Currently, there is no effective biomarker available in clinical settings for early detection of lung cancer. Altered expressions in many cancer types including NSCLC and stable existence in plasma make microRNAs (miRNAs) a group of potentially useful biomarkers for clinical assessments of patients with NSCLC.

Objectives

To evaluate the potential values of miRNAs as blood-based biomarkers for early diagnosis and prognosis in NSCLC patients.

Methods

Peripheral blood samples from healthy volunteers and early-staged NSCLC patients before and after surgery were collected, and plasma was separated. Expression of ten miRNAs in the plasma and tumor sections of the patients was detected by quantitative real-time polymerase chain reaction.

Results

MiRNA (miR)-486 and miR-150 were found to significantly distinguish lung cancer patients from healthy volunteers. Area under curve of miR-486 and miR-150 were 0.926 (sensitivity, 0.909; specificity, 0.818) and 0.752 (sensitivity, 0.818; specificity, 0.818), respectively. In response to therapy, patients with down-regulated miR-486 expression showed prolonged recurrence-free survival than those with un-reduced miR-486 expression (median, unreached vs. 19 months; hazard ratio, 0.1053; 95% confidence interval, 0.01045 to 1.060; P=0.056).

Conclusions

The results suggest that miR-486 and miR-150 could be potential blood-based biomarkers for early diagnosis of NSCLC. Monitoring change of miR-486 expression in plasma might be an effective and non-invasive method for recurrence prediction of early-staged NSCLC patients.     

Embryonic Lethality in Homozygous Human Her-2 Transgenic Mice Due to Disruption of the Pds5b Gene

The development of antigen-targeted therapeutics is dependent on the preferential expression of tumor-associated antigens (TAA) at targetable levels on the tumor. Tumor-associated antigens can be generated de novo or can arise from altered expression of normal basal proteins, such as the up-regulation of human epidermal growth factor receptor 2 (Her2/ErbB2). To properly assess the development of Her2 therapeutics in an immune tolerant model, we previously generated a transgenic mouse model in which expression of the human Her2 protein was present in both the brain and mammary tissue. This mouse model has facilitated the development of Her2 targeted therapies in a clinically relevant and suitable model. While heterozygous Her2^+/- mice appear to develop in a similar manner to wild type mice (Her2^-/-), it has proven difficult to generate homozygous Her2^+/+ mice, potentially due to embryonic lethality. In this study, we performed whole genome sequencing to determine if the integration site of the Her2 transgene was responsible for this lethality. Indeed, we report that the Her2 transgene had integrated into the Pds5b (precocious dissociation of sisters) gene on chromosome 5, as a 162 copy concatemer. Furthermore, our findings demonstrate that Her2^+/+ mice, similar to Pds5b^-/- mice, are embryonic lethal and confirm the necessity for Pds5b in embryonic development. This study confirms the value of whole genome sequencing in determining the integration site of transgenes to gain insight into associated phenotypes.     

Impact of HIV-1 Subtype on the Time to CD4+ T-Cell Recovery in Combination Antiretroviral Therapy (cART)-Experienced Patients

Human immunodeficiency virus type 1 (HIV-1) subtypes have been shown to differ in the rate of clinical progression. We studied the association between HIV-1 subtypes and the rate of CD4+ T-cell recovery in a longitudinal cohort of patients on combination antiretroviral therapy (cART). We studied 103 patients infected with CRF01_AE (69%) and subtype B (31%) who initiated cART between 2006 and 2013. Demographic data, CD4+ T-cell counts and HIV-1 viral load were abstracted from patient medical charts. Kaplan-Meier was used to estimate the time to CD4+ T-cell count increase to ≥350 between subtypes and effects of covariates were analysed using Cox proportional hazards. An 87% of the study population were male adults (mean age of 38.7 years old). Baseline CD4+ T-cell counts and viral loads, age at cART initiation, sex, ethnicity and co-infection did not differ significantly between subtypes. A shorter median time for CD4+ T-cell count increase to ≥350 cells/μL was observed for CRF01_AE (546 days; 95% confidence interval [CI], 186–906 days; P = .502) compared to subtype B (987 days; 95% CI, 894–1079 days). In multivariate analysis, female sex was significantly associated with a 2.7 times higher chance of achieving CD4+ T-cell recovery (adjusted hazard ratio [HR], 2.75; 95% CI, 1.21–6.22; P = .025) and both baseline CD4+ T-cell count (P = .001) and viral load (P = .001) were important predictors for CD4+ T-cell recovery. Immunological recovery correlated significantly with female sex, baseline CD4+ T-cell counts and viral load but not subtype.     

Finite Element Method (FEM) Modeling of Freeze-drying: Monitoring Pharmaceutical Product Robustness During Lyophilization

Lyophilization is an approach commonly undertaken to formulate drugs that are unstable to be commercialized as ready to use (RTU) solutions. One of the important aspects of commercializing a lyophilized product is to transfer the process parameters that are developed in lab scale lyophilizer to commercial scale without a loss in product quality. This process is often accomplished by costly engineering runs or through an iterative process at the commercial scale. Here, we are highlighting a combination of computational and experimental approach to predict commercial process parameters for the primary drying phase of lyophilization. Heat and mass transfer coefficients are determined experimentally either by manometric temperature measurement (MTM) or sublimation tests and used as inputs for the finite element model (FEM)-based software called PASSAGE, which computes various primary drying parameters such as primary drying time and product temperature. The heat and mass transfer coefficients will vary at different lyophilization scales; hence, we present an approach to use appropriate factors while scaling-up from lab scale to commercial scale. As a result, one can predict commercial scale primary drying time based on these parameters. Additionally, the model-based approach presented in this study provides a process to monitor pharmaceutical product robustness and accidental process deviations during Lyophilization to support commercial supply chain continuity. The approach presented here provides a robust lyophilization scale-up strategy; and because of the simple and minimalistic approach, it will also be less capital intensive path with minimal use of expensive drug substance/active material.