Enhanced Collateral Growth by Double Transplantation of Gene-Nucleofected Fibroblasts in Ischemic Hindlimb of Rats

BACKGROUND: Induction of neovascularization by releasing therapeutic growth factors is a promising application of cell-based gene therapy to treat ischemia-related problems. In the present study, we have developed a new strategy based on nucleofection with alternative solution and cuvette to promote collateral growth and re-establishment of circulation in ischemic limbs using double transplantation of gene nucleofected primary cultures of fibroblasts, which were isolated from rat receiving such therapy. METHODS AND RESULTS: Rat dermal fibroblasts were nucleofected ex vivo to release bFGF or VEGF165 in a hindlimb ischemia model in vivo. After femoral artery ligation, gene-modified cells were injected intramuscularly. One week post injection, local confined plasmid expression and transient distributions of the plasmids in other organs were detected by quantitative PCR. Quantitative micro-CT analyses showed improvements of vascularization in the ischemic zone (No. of collateral vessels via micro CT: 6.8±2.3 vs. 10.1±2.6; p<0.05). Moreover, improved collateral proliferation (BrdU incorporation: 0.48±0.05 vs. 0.57±0.05; p<0.05) and increase in blood perfusion (microspheres ratio: gastrocnemius: 0.41±0.10 vs. 0.50±0.11; p<0.05; soleus ratio: soleus: 0.42±0.08 vs. 0.60±0.08; p<0.01) in the lower hindlimb were also observed. CONCLUSIONS: These results demonstrate the feasibility and effectiveness of double transplantation of gene nucleofected primary fibroblasts in producing growth factors and promoting the formation of collateral circulation in ischemic hindlimb, suggesting that isolation and preparation of gene nucleofected cells from individual accepting gene therapy may be an alternative strategy for treating limb ischemia related diseases

Genomic insights into rapid speciation within the world's largest tree genus Syzygium

Acknowledgements Y.W.L. was supported by a postgraduate scholarship research grant from the Ministry of National Development, Singapore awarded through the National Parks Board, Singapore (NParks; NParks’ Garden City Fund). Principal research funding from NParks and the School of Biological Sciences (SBS), Nanyang Technological University (NTU), Singapore, is acknowledged. We thank Peter Preiser, Associate Vice President for Biomedical and Life Sciences, for facilitating NTU support, and Kenneth Er, CEO of NParks, for facilitating research funding through that organisation. V.A.A. and C.L. were funded by SBS, NTU for a one-year research leave. V.A.A. and C.L. also acknowledge support from the United States National Science Foundation (grants 2030871 and 1854550, respectively). S.R. was supported by a postdoctoral research fellowship under the NTU Strategic Plant Programme. S.R. and N.R.W.C. acknowledge funding from NTU start-up and the Academy of Finland (decisions 318288, 319947) grants to J.S. Fieldwork conducted by Y.W.L. was supported by an Indonesian Government RISTEK research permit (Application ID: 1517217008) and an Access License from the Sabah State government [JKM/MBS.1000-2/2JLD.7(84)]. T.N.C.V. is grateful to the Assemblée de la Province Nord and Assemblée de la Province Sud (New Caledonia) for facilitating relevant collection permits. A.N. was partly supported by the Research Project Promotion Grant (Strategic Research Grant No. 17SP01302) from the University of the Ryukyus, and partly by the Environment Research and Technology Development Fund (JPMEERF20204003) from the Environmental Restoration and Conservation Agency of Japan. Fieldwork in Fiji conducted by R.B. was hosted and facilitated by Elina Nabubuniyaka-Young (The Pacific Community’s Centre for Pacific Crops and Trees, Fiji). We thank the NTU-Smithsonian Partnership for tree data obtained for the Bukit Timah Nature Reserve (BTNR) long-term forest dynamics plots. Administrative support provided by Mui Hwang Khoo-Woon and Peter Ang at the molecular laboratory of the Singapore Botanic Gardens (SBG) is acknowledged. Rosie Woods and Imalka Kahandawala (DNA and Tissue Bank, Royal Botanic Gardens, Kew) facilitated additional DNA samples. Daniel Thomas (SBG) and Yan Yu (Sichuan University) commented on biogeographical analyses. NovogeneAIT in Singapore is acknowledged for personalised sequencing service.Peer reviewedPublisher PD

Genomic insights into rapid speciation within the world's largest tree genus Syzygium

The relative importance of the mechanisms underlying species radiation remains unclear. Here, the authors combine reference genome assembly and population genetics analyses to show that neutral forces have contributed to the radiation of the most species-rich tree genus Syzygium. Species radiations, despite immense phenotypic variation, can be difficult to resolve phylogenetically when genetic change poorly matches the rapidity of diversification. Genomic potential furnished by palaeopolyploidy, and relative roles for adaptation, random drift and hybridisation in the apportionment of genetic variation, remain poorly understood factors. Here, we study these aspects in a model radiation, Syzygium, the most species-rich tree genus worldwide. Genomes of 182 distinct species and 58 unidentified taxa are compared against a chromosome-level reference genome of the sea apple, Syzygium grande. We show that while Syzygium shares an ancient genome doubling event with other Myrtales, little evidence exists for recent polyploidy events. Phylogenomics confirms that Syzygium originated in Australia-New Guinea and diversified in multiple migrations, eastward to the Pacific and westward to India and Africa, in bursts of speciation visible as poorly resolved branches on phylogenies. Furthermore, some sublineages demonstrate genomic clines that recapitulate cladogenetic events, suggesting that stepwise geographic speciation, a neutral process, has been important in Syzygium diversification.Peer reviewe

Mitochondrial Diabetes in Children: Seek and You Will Find It

Maternally Inherited Diabetes and Deafness (MIDD) is a rare form of diabetes due to defects in mitochondrial DNA (mtDNA). 3243 A>G is the mutation most frequently associated with this condition, but other mtDNA variants have been linked with a diabetic phenotype suggestive of MIDD. From 1989 to 2009, we clinically diagnosed mitochondrial diabetes in 11 diabetic children. Diagnosis was based on the presence of one or more of the following criteria: 1) maculopathy; 2) hearing impairment; 3) maternal heritability of diabetes/impaired fasting glucose and/or hearing impairment and/or maculopathy in three consecutive generations (or in two generations if 2 or 3 members of a family were affected). We sequenced the mtDNA in the 11 probands, in their mothers and in 80 controls. We identified 33 diabetes-suspected mutations, 1/33 was 3243A>G. Most patients (91%) and their mothers had mutations in complex I and/or IV of the respiratory chain. We measured the activity of these two enzymes and found that they were less active in mutated patients and their mothers than in the healthy control pool. The prevalence of hearing loss (36% vs 75–98%) and macular dystrophy (54% vs 86%) was lower in our mitochondrial diabetic adolescents than reported in adults. Moreover, we found a hitherto unknown association between mitochondrial diabetes and celiac disease. In conclusion, mitochondrial diabetes should be considered a complex syndrome with several phenotypic variants. Moreover, deafness is not an essential component of the disease in children. The whole mtDNA should be screened because the 3243A>G variant is not as frequent in children as in adults. In fact, 91% of our patients were mutated in the complex I and/or IV genes. The enzymatic assay may be a useful tool with which to confirm the pathogenic significance of detected variants

Carboxylic ester hydrolases from hyperthermophiles

Carboxylic ester hydrolyzing enzymes constitute a large group of enzymes that are able to catalyze the hydrolysis, synthesis or transesterification of an ester bond. They can be found in all three domains of life, including the group of hyperthermophilic bacteria and archaea. Esterases from the latter group often exhibit a high intrinsic stability, which makes them of interest them for various biotechnological applications. In this review, we aim to give an overview of all characterized carboxylic ester hydrolases from hyperthermophilic microorganisms and provide details on their substrate specificity, kinetics, optimal catalytic conditions, and stability. Approaches for the discovery of new carboxylic ester hydrolases are described. Special attention is given to the currently characterized hyperthermophilic enzymes with respect to their biochemical properties, 3D structure, and classification

Pan-cancer analysis of whole genomes

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale(1-3). Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter(4); identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation(5,6); analyses timings and patterns of tumour evolution(7); describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity(8,9); and evaluates a range of more-specialized features of cancer genomes(8,10-18).Peer reviewe

Targeting Huntington’s disease through histone deacetylases

Huntington’s disease (HD) is a debilitating neurodegenerative condition with significant burdens on both patient and healthcare costs. Despite extensive research, treatment options for patients with this condition remain limited. Aberrant post-translational modification (PTM) of proteins is emerging as an important element in the pathogenesis of HD. These PTMs include acetylation, phosphorylation, methylation, sumoylation and ubiquitination. Several families of proteins are involved with the regulation of these PTMs. In this review, I discuss the current evidence linking aberrant PTMs and/or aberrant regulation of the cellular machinery regulating these PTMs to HD pathogenesis. Finally, I discuss the evidence suggesting that pharmacologically targeting one of these protein families the histone deacetylases may be of potential therapeutic benefit in the treatment of HD

SRA Regulates Adipogenesis by Modulating p38/JNK Phosphorylation and Stimulating Insulin Receptor Gene Expression and Downstream Signaling

The Steroid Receptor RNA Activator (SRA) enhances adipogenesis and increases both glucose uptake and phosphorylation of Akt and FOXO1 in response to insulin. To assess the mechanism, we differentiated ST2 mesenchymal precursor cells that did or did not overexpress SRA into adipocytes using combinations of methylisobutylxanthine, dexamethasone and insulin. These studies showed that SRA overexpression promotes full adipogenesis in part by stimulation of insulin/insulin-like growth factor-1 (IGF-1) signaling. SRA overexpression inhibited phosphorylation of p38 mitogen activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) in the early differentiation of ST2 cells. Conversely, knockdown of endogenous SRA in 3T3-L1 cells increased phosphorylation of JNK. Knockdown of SRA in mature 3T3-L1 adipocytes reduced insulin receptor (IR) mRNA and protein levels, which led to decreased autophosphorylation of IRβ and decreased phosphorylation of insulin receptor substrate-1 (IRS-1) and Akt. This likely reflects a stimulatory role of SRA on IR transcription, as transfection studies showed that SRA increased expression of an IR promoter-luciferase reporter construct

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe