small-molecule-screening-toxicity-testing-early-stage-zebrafish

Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, ...

University of Malta

The extent to which low-frequency (minor allele frequency (MAF) between 1-5%) and rare (MAF ≤ 1%) variants contribute to complex traits and disease in the general population is mainly unknown. Bone mineral density (BMD) is highly heritable, a major predictor of osteoporotic fractures, and has been previously associated with common genetic variants, as well as rare, population-specific, coding variants. Here we identify novel non-coding genetic variants with large effects on BMD (ntotal = 53,236) and fracture (ntotal = 508,253) in individuals of European ancestry from the general population. Associations for BMD were derived from whole-genome sequencing (n = 2,882 from UK10K (ref. 10); a population-based genome sequencing consortium), whole-exome sequencing (n = 3,549), deep imputation of genotyped samples using a combined UK10K/1000 Genomes reference panel (n = 26,534), and de novo replication genotyping (n = 20,271). We identified a low-frequency non-coding variant near a novel locus, EN1, with an effect size fourfold larger than the mean of previously reported common variants for lumbar spine BMD (rs11692564(T), MAF = 1.6%, replication effect size = +0.20 s.d., Pmeta = 2 × 10(-14)), which was also associated with a decreased risk of fracture (odds ratio = 0.85; P = 2 × 10(-11); ncases = 98,742 and ncontrols = 409,511). Using an En1(cre/flox) mouse model, we observed that conditional loss of En1 results in low bone mass, probably as a consequence of high bone turnover. We also identified a novel low-frequency non-coding variant with large effects on BMD near WNT16 (rs148771817(T), MAF = 1.2%, replication effect size = +0.41 s.d., Pmeta = 1 × 10(-11)). In general, there was an excess of association signals arising from deleterious coding and conserved non-coding variants. These findings provide evidence that low-frequency non-coding variants have large effects on BMD and fracture, thereby providing rationale for whole-genome sequencing and improved imputation reference panels to study the genetic architecture of complex traits and disease in the general population.

Whole-genome sequencing identifies EN1 as a determinant of bone density and fracture.

Osteoporosis and fractures are complex conditions influenced by an interplay of genetic and environmental factors. The aim of the study was to investigate three biochemical parameters including total serum calcium, total serum alkaline phosphatase (sALP) and albumin in relation to bone mineral density (BMD) at the lumbar spine and femoral neck (FN), and with all-type of low-trauma fractures in Maltese postmenopausal women. Levels were also correlated with age and physical activity. A case-control study of 1045 women was performed. Women who suffered a fracture were classified as cases whereas women without a fracture history were included as controls subdivided into normal, osteopenic, or osteoporotic according to their BMD measurements. Blood specimens were collected following good standard practice and testing was performed by spectrophotometry. Calcium and sALP levels were weakly correlated with FN BMD levels (calcium: r = -0.111, p = 0.002; sALP: r = 0.089, p = 0.013). Fracture cases had the lowest serum levels of calcium, sALP and albumin relative to all other control groups, which decreased with increasing age, possibly increasing fracture risk. Biochemical levels were lowest in women who sustained a hip fracture and more than one fracture. Biochemical parameters decreased with reduced physical activity; however, this was most evident for fracture cases. Reduced physical activity was associated with lower BMD levels at the hip, and to a lower extent at the spine. In conclusion, results suggest that levels of serum calcium and albumin could be indicative of fracture risk, whereas calcium levels and to lower extent sALP levels could be indicators of hip BMD.

Biochemical Predictors of Low Bone Mineral Density and Fracture Susceptibility in Maltese Postmenopausal Women.

Current anti-cancer drugs can cause many undesirable side effects to patients. Thus, there is a constant need to develop alternative therapeutic drugs. Bioactive compounds derived from natural products including animals, plants and microorganisms are being actively studied as sources for anticancer treatments. Freshwater planarians are important models for stem cell research and regeneration. However, to date, no studies on the biological activities of planaria extracts on cancer have been published. The aim of this study was to examine the potential antitumoral activity of the extract from planaria species-Malta (PSM) on human acute myeloid leukemia (AML) HL-60 cells. Antiproliferative activity was studied in terms of proliferation, apoptosis and differentiation. The expression of genes involved in the regulation of these important cellular processes was also analyzed using real-time PCR. PSM extract exhibited a selective cytotoxic effect on HL-60 cells when compared to normal lymphocytes. Furthermore, cell cycle analysis and Annexin V/PI assay showed that the extract induced apoptosis in HL-60 cells. The PSM extract induced myeloid differentiation with HL-60 cells showing a decreased nucleo/cytoplasmic ratio, an increase in nitroblue tetrazolium-positive cells, and CD11b- and CD14-positive cells. Finally, we also found that the PSM extract increased the expression of CEBPA, CEBPB, CEBPE, SPI1, BAX, CDKN1A and CDKN2C; whereas it reduced the expression of c-MYC and BCL2. This is the first study to reveal the antiproliferative, cytotoxic, and differentiation potential of PSM on HL-60 cells and suggests that it may have considerable potential for development as a novel natural product-based anticancer agent against AML.

Anticancer effects of an extract from a local planarian species on human acute myeloid leukemia HL-60 cells in vitro.

Acute myeloid leukemia is the most common form of acute leukemia in adults, constituting about 80% of cases. Although remarkable progress has been made in the therapeutic scenario for patients with acute myeloid leukemia, research and development of new and effective anticancer agents to improve patient outcome and minimize toxicity is needed. In this study, the antitumor activity of axolotl (AXO)  crude extract was assessed in vitro on the human acute myeloid leukemia HL-60 cell line. The anticancer activity was evaluated in terms of ability to influence proliferative activity, cell viability, cell cycle arrest, and differentiation. Moreover, gene expression analysis was performed to evaluate the genes involved in the regulation of these processes. The AXO crude extract exhibited antiproliferative but not cytotoxic activities on HL-60 cells, with cell cycle arrest in the G0/G1 phase. Furthermore, the AXO-treated HL-60 cells showed an increase in both the percentage of nitroblue tetrazolium positive cells and the expression of CD11b, whereas the proportion of CD14-positive cells did not change, suggesting that extract is able to induce differentiation toward the granulocytic lineage. Finally, the treatment with AXO extract caused upregulation of , and , and downregulation of . Our data clearly show the potential anticancer activity of  on HL-60 cells and suggest that it could help develop promising therapeutic agents for the treatment of acute myeloid leukemia.

Axolotl  extract induces cell cycle arrest and differentiation in human acute myeloid leukemia HL-60 cells.

Recent studies have shown that β-blocker (BB) users have a decreased risk of fracture and higher bone mineral density (BMD) compared to nonusers, likely due to the suppression of adrenergic signaling in osteoblasts, leading to increased BMD. There is also variability in the effect size of BB use on BMD in humans, which may be due to pharmacogenomic effects.

Pharmacogenomic Effects of β-Blocker Use on Femoral Neck Bone Mineral Density.

The Ehlers-Danlos syndromes (EDS) are a collection of rare hereditary connective tissue disorders with heterogeneous phenotypes, usually diagnosed following clinical examination and confirmatory genetic testing. Diagnosis of the commonest subtype, hypermobile Ehlers-Danlos Syndrome (hEDS), relies solely on a clinical diagnosis since its molecular aetiology remains unknown. We performed an up-to-date literature search and selected 11 out of 304 publications according to a set of established criteria. Studies reporting variants affecting collagen proteins were found to be hindered by cohort misclassification and subsequent lack of reproducibility of these genetic findings. The role of the described variants affecting Tenascin-X and LZTS1 is yet to be demonstrated in the majority of hEDS cases, while the functional implication of associated signaling pathways and genes requires further elucidation. The available literature on the genetics of hEDS is scant, dispersed and conflicting due to out-dated nosology terminology. Recent literature has suggested the role of several promising candidate mechanisms which may be linked to the underlying molecular aetiology. Knowledge of the molecular genetic basis of hEDS is expected to increase in the near future through the mainstream use of high-throughput sequencing combined with the updated classification of EDS, and the upcoming Hypermobile Ehlers-Danlos Genetic Evaluation (HEDGE) study.

Hypermobile Ehlers-Danlos syndrome: A review and a critical appraisal of published genetic research to date.

Musculoskeletal research has been enriched in the past ten years with a great wealth of new discoveries arising from genome wide association studies (GWAS). In addition to the novel factors identified by GWAS, the advent of whole-genome and whole-exome sequencing efforts in family based studies has also identified new genes and pathways. However, the function and the mechanisms by which such genes influence clinical traits remain largely unknown. There is imperative need to bring multidisciplinary expertise together that will allow translating these genomic discoveries into useful clinical applications with the potential of improving patient care. Therefore "GEnomics of MusculoSkeletal traits TranslatiOnal NEtwork" (GEMSTONE) aims to set the ground for the: 1) functional characterization of discovered genes and pathways; 2) understanding of the correspondence between molecular and clinical assessments; and 3) implementation of novel methodological approaches. This research network is funded by  (COST). GEMSTONE includes six working groups (WG), each with specific objectives: WG1- creating, maintaining and updating an inventory of experts and resources (studies and datasets) participating in the network, helping to assemble focus groups defined by phenotype, functional and methodological expertise. WG2- describe ways to decompose the phenotypes of the different functional studies into meaningful components that will aid the interpretation of identified biological pathways. WG3  makes an inventory of genes underlying musculoskeletal monogenic conditions that aids the assignment of genes to GWAS signals and prioritizing GWAS genes as candidates responsible for monogenic presentations, through biological plausibility. WG4 : creating a roadmap of genes and pathways to be prioritized for functional assessment in cell and organism models of the musculoskeletal system. WG5  seeks the integration of the knowledge derived from the distinct efforts, with particular emphasis on systems biology and artificial intelligence applications. Finally, WG6 : makes a synopsis of the knowledge derived from the distinct efforts, allowing to prioritize factors within biological pathways, use refined disease trait definitions and/or improve study design of future investigations in a potential therapeutic context (e.g. clinical trials) for musculoskeletal diseases.

The "GEnomics of Musculo Skeletal Traits TranslatiOnal NEtwork": Origins, Rationale, Organization, and Prospects.

Genetic disorders of the skeleton encompass a diverse group of bone diseases differing in clinical characteristics, severity, incidence and molecular etiology. Of particular interest are the monogenic rare bone mass disorders, with the underlying genetic defect contributing to either low or high bone mass phenotype. Extensive, deep phenotyping coupled with high-throughput, cost-effective genotyping is crucial in the characterization and diagnosis of affected individuals. Massive parallel sequencing efforts have been instrumental in the discovery of novel causal genes that merit functional validation using  and  cell-based techniques, and  models, mainly mice and zebrafish. These translational models also serve as an excellent platform for therapeutic discovery, bridging the gap between basic science research and the clinic. Altogether, genetic studies of monogenic rare bone mass disorders have broadened our knowledge on molecular signaling pathways coordinating bone development and metabolism, disease inheritance patterns, development of new and improved bone biomarkers, and identification of novel drug targets. In this comprehensive review we describe approaches to further enhance the innovative processes taking discoveries from clinic to bench, and then back to clinic in rare bone mass disorders. We highlight the importance of cross laboratory collaboration to perform functional validation in multiple model systems after identification of a novel disease gene. We describe the monogenic forms of rare low and high rare bone mass disorders known to date, provide a roadmap to unravel the genetic determinants of monogenic rare bone mass disorders using proper phenotyping and genotyping methods, and describe different genetic validation approaches paving the way for future treatments.

A Roadmap to Gene Discoveries and Novel Therapies in Monogenic Low and High Bone Mass Disorders.

The availability of large human datasets for genome-wide association studies (GWAS) and the advancement of sequencing technologies have boosted the identification of genetic variants in complex and rare diseases in the skeletal field. Yet, interpreting results from human association studies remains a challenge. To bridge the gap between genetic association and causality, a systematic functional investigation is necessary. Multiple unknowns exist for putative causal genes, including cellular localization of the molecular function. Intermediate traits ("endophenotypes"), e.g. molecular quantitative trait loci (molQTLs), are needed to identify mechanisms of underlying associations. Furthermore, index variants often reside in non-coding regions of the genome, therefore challenging for interpretation. Knowledge of non-coding variance (e.g. ncRNAs), repetitive sequences, and regulatory interactions between enhancers and their target genes is central for understanding causal genes in skeletal conditions. Animal models with deep skeletal phenotyping and cell culture models have already facilitated fine mapping of some association signals, elucidated gene mechanisms, and revealed disease-relevant biology. However, to accelerate research towards bridging the current gap between association and causality in skeletal diseases, alternative  platforms need to be used and developed in parallel with the current -omics and traditional  resources. Therefore, we argue that as a field we need to establish resource-sharing standards to collectively address complex research questions. These standards will promote data integration from various -omics technologies and functional dissection of human complex traits. In this mission statement, we review the current available resources and as a group propose a consensus to facilitate resource sharing using existing and future resources. Such coordination efforts will maximize the acquisition of knowledge from different approaches and thus reduce redundancy and duplication of resources. These measures will help to understand the pathogenesis of osteoporosis and other skeletal diseases towards defining new and more efficient therapeutic targets.

Perspective of the GEMSTONE Consortium on Current and Future Approaches to Functional Validation for Skeletal Genetic Disease Using Cellular, Molecular and Animal-Modeling Techniques.

Osteoporosis is a skeletal disease with a strong genetic background. The study aimed to identify the genetic determinants of early-onset familial osteoporosis and low bone mineral density (BMD) in a two-generation Maltese family.

Whole Genome Sequencing Unravels New Genetic Determinants of Early-Onset Familial Osteoporosis and Low BMD in Malta.

Monogenic high bone mass (HBM) disorders are characterized by an increased amount of bone in general, or at specific sites in the skeleton. Here, we describe 59 HBM disorders with 50 known disease-causing genes from the literature, and we provide an overview of the signaling pathways and mechanisms involved in the pathogenesis of these disorders. Based on this, we classify the known HBM genes into HBM (sub)groups according to uniform Gene Ontology (GO) terminology. This classification system may aid in hypothesis generation, for both wet lab experimental design and clinical genetic screening strategies. We discuss how functional genomics can shape discovery of novel HBM genes and/or mechanisms in the future, through implementation of omics assessments in existing and future model systems. Finally, we address strategies to improve gene identification in unsolved HBM cases and highlight the importance for cross-laboratory collaborations encompassing multidisciplinary efforts to transfer knowledge generated at the bench to the clinic. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

High Bone Mass Disorders: New Insights From Connecting the Clinic and the Bench.

Adipose tissue is the source of a broad array of signalling molecules (adipokines), which mediate interorgan communication and regulate metabolic homeostasis. Alterations in adipokine levels have been causally implicated in various metabolic disorders, including changes in bone mass. Osteoporosis is the commonest progressive metabolic bone disease, characterized by elevated risk of fragility fractures as a result of a reduced bone mass and microarchitectural deterioration. The effects of different adipokines on bone mass have been studied in an attempt to identify novel modulators of bone mass or diagnostic biomarkers of osteoporosis.

The relationship between adipokine levels and bone mass-A systematic review.

Osteoporosis is a multifactorial bone disease characterised by reduced bone mass and increased fracture risk. Family studies have made significant contribution in unravelling the genetics of osteoporosis. Yet, most of the underlying molecular and biological mechanisms remain unknown prompting the need for further studies. This review outlines the proper phenotyping and advanced genetic techniques in the form of high-throughput DNA sequencing used to identify genetic factors underlying monogenic osteoporosis in a family-based setting. The steps related to variant filtering prioritisation and curation are also described. From an evolutionary perspective, deleterious risk variants with higher penetrance tend to be rare as a result of negative selection. High-throughput sequencing (HTS) can identify rare variants with large effect sizes which are likely to be missed by candidate gene analysis or genome-wide association studies (GWAS) wherein common variants with small to moderate effect sizes are identified. We also describe the importance of replicating implicated genes, and possibly variants, identified following HTS to confirm their causality. Replication of the gene in other families, singletons or independent cohorts confirms that the shortlisted genes and/or variants are indeed causal. Furthermore, novel genes and/or variants implicated in monogenic osteoporosis require a thorough validation by means of  and  assessment. Therefore, analyses of families can continue to elucidate the genetic architecture of osteoporosis, paving the way for improved diagnostic and therapeutic strategies.

Melissa M. Formosa

Department of Applied Biomedical Science,
Faculty of Health Sciences,
Centre for Molecular Medicine and Biobanking,
Department of Applied Biomedical Science, Faculty of Health Sciences

University of Malta

Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae

Melissa M. Formosa

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Applied Biomedical Science,Faculty of Health Sciences,Centre for Molecular Medicine and Biobanking,Department of Applied Biomedical Science, Faculty of Health Sciences

University of Malta

Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae

Department of Applied Biomedical Science,
Faculty of Health Sciences,
Centre for Molecular Medicine and Biobanking,
Department of Applied Biomedical Science, Faculty of Health Sciences