Whole Genome Amplification in Preimplantation Genetic Testing in the Era of Massively Parallel Sequencing

Successful whole genome amplification (WGA) is a cornerstone of contemporary preimplantation genetic testing (PGT). Choosing the most suitable WGA technique for PGT can be particularly challenging because each WGA technique performs differently in combination with different downstream processing and detection methods. The aim of this review is to provide insight into the performance and drawbacks of DOP-PCR, MDA and MALBAC, as well as the hybrid WGA techniques most widely used in PGT. As the field of PGT is moving towards a wide adaptation of comprehensive massively parallel sequencing (MPS)-based approaches, we especially focus our review on MPS parameters and detection opportunities of WGA-amplified material, i.e., mappability of reads, uniformity of coverage and its influence on copy number variation analysis, and genomic coverage and its influence on single nucleotide variation calling. The ability of MDA-based WGA solutions to better cover the targeted genome and the ability of PCR-based solutions to provide better uniformity of coverage are highlighted. While numerous comprehensive PGT solutions exploiting different WGA types and adjusted bioinformatic pipelines to detect copy number and single nucleotide changes are available, the ones exploiting MDA appear more advantageous. The opportunity to fully analyse the targeted genome is influenced by the MPS parameters themselves rather than the solely chosen WGA.     

Molecular Drivers of Developmental Arrest in the Human Preimplantation Embryo: A Systematic Review and Critical Analysis Leading to Mapping Future Research

Developmental arrest of the preimplantation embryo is a multifactorial condition, characterized by lack of cellular division for at least 24 hours, hindering the in vitro fertilization cycle outcome. This systematic review aims to present the molecular drivers of developmental arrest, focusing on embryonic and parental factors. A systematic search in PubMed/Medline, Embase and Cochrane-Central-Database was performed in January 2021. A total of 76 studies were included. The identified embryonic factors associated with arrest included gene variations, mitochondrial DNA copy number, methylation patterns, chromosomal abnormalities, metabolic profile and morphological features. Parental factors included, gene variation, protein expression levels and infertility etiology. A valuable conclusion emerging through critical analysis indicated that genetic origins of developmental arrest analyzed from the perspective of parental infertility etiology and the embryo itself, share common ground. This is a unique and long-overdue contribution to literature that for the first time presents an all-inclusive methodological report on the molecular drivers leading to preimplantation embryos’ arrested development. The variety and heterogeneity of developmental arrest drivers, along with their inevitable intertwining relationships does not allow for prioritization on the factors playing a more definitive role in arrested development. This systematic review provides the basis for further research in the field.     

Update on the Diagnostic Pitfalls of Autopsy and Post-Mortem Genetic Testing in Cardiomyopathies

Inherited cardiomyopathies are frequent causes of sudden cardiac death (SCD), especially in young patients. Despite at the autopsy they usually have distinctive microscopic and/or macroscopic diagnostic features, their phenotypes may be mild or ambiguous, possibly leading to misdiagnoses or missed diagnoses. In this review, the main differential diagnoses of hypertrophic cardiomyopathy (e.g., athlete’s heart, idiopathic left ventricular hypertrophy), arrhythmogenic cardiomyopathy (e.g., adipositas cordis, myocarditis) and dilated cardiomyopathy (e.g., acquired forms of dilated cardiomyopathy, left ventricular noncompaction) are discussed. Moreover, the diagnostic issues in SCD victims affected by phenotype-negative hypertrophic cardiomyopathy and the relationship between myocardial bridging and hypertrophic cardiomyopathy are analyzed. Finally, the applications/limits of virtopsy and post-mortem genetic testing in this field are discussed, with particular attention to the issues related to the assessment of the significance of the genetic variants.     

Review Article: A Review of the Development and Operational Characteristics of the TALSPEAK Process

Abstract

The separation of trivalent transplutonium actinides from fission product lanthanide ions represents arguably the most challenging aspect of advanced nuclear fuel partitioning schemes. A considerable amount of effort has been dedicated to the development of effective methods for accomplishing this separation, essential for transmutation of the actinides heavier than Pu. Among the methods currently considered to be ready for technological deployment is the TALSPEAK (Trivalent Actinide ‐ Lanthanide Separation by Phosphorus reagent Extraction from Aqueous Komplexes) Process, developed in the late 1960s at Oak Ridge National Laboratory. This process is based on the partitioning of lanthanides and actinides between an acidic organophosphorus extractant ((RO)₂PO₂H) solution and an aqueous phase containing a high concentration of a carboxylic acid buffer and a polyaminopolycarboxylate complexant. The latter reagent is principally responsible for holding back the trivalent actinides, allowing the selective transfer of the lanthanides into the organic phase. Several combinations of different extractants and aqueous complexants have been investigated, as have the effect of diluent, temperature and p[H⁺] on separation efficiency. In this report, the prior literature is examined to help provide guidance for potential deployment of the technology in advanced nuclear fuel cycles and to identify opportunities for fine‐tuning the process.     

Molecular Epidemiology of Mitochondrial Cardiomyopathy: A Search Among Mitochondrial and Nuclear Genes

Mitochondrial Cardiomyopathy (MCM) is a common manifestation of multi-organ Mitochondrial Diseases (MDs), occasionally present in non-syndromic cases. Diagnosis of MCM is complex because of wide clinical and genetic heterogeneity and requires medical, laboratory, and neuroimaging investigations. Currently, the molecular screening for MCM is fundamental part of MDs management and allows achieving the definitive diagnosis. In this article, we review the current genetic knowledge associated with MDs, focusing on diagnosis of MCM and MDs showing cardiac involvement. We searched for publications on mitochondrial and nuclear genes involved in MCM, mainly focusing on genetic screening based on targeted gene panels for the molecular diagnosis of the MCM, by using Next Generation Sequencing. Here we report twelve case reports, four case-control studies, eleven retrospective studies, and two prospective studies, for a total of twenty-nine papers concerning the evaluation of cardiac manifestations in mitochondrial diseases. From the analysis of published causal mutations, we identified 130 genes to be associated with mitochondrial heart diseases. A large proportion of these genes (34.3%) encode for key proteins involved in the oxidative phosphorylation system (OXPHOS), either as directly OXPHOS subunits (22.8%), and as OXPHOS assembly factors (11.5%). Mutations in several mitochondrial tRNA genes have been also reported in multi-organ or isolated MCM (15.3%). This review highlights the main disease-genes, identified by extensive genetic analysis, which could be included as target genes in next generation panels for the molecular diagnosis of patients with clinical suspect of mitochondrial cardiomyopathies.     

Effects of the Escherichia coli Bacterial Toxin Cytotoxic Necrotizing Factor 1 on Different Human and Animal Cells: A Systematic Review

Cytotoxic necrotizing factor 1 (CNF1) is a bacterial virulence factor, the target of which is represented by Rho GTPases, small proteins involved in a huge number of crucial cellular processes. CNF1, due to its ability to modulate the activity of Rho GTPases, represents a widely used tool to unravel the role played by these regulatory proteins in different biological processes. In this review, we summarized the data available in the scientific literature concerning the observed in vitro effects induced by CNF1. An article search was performed on electronic bibliographic resources. Screenings were performed of titles, abstracts, and full-texts according to PRISMA guidelines, whereas eligibility criteria were defined for in vitro studies. We identified a total of 299 records by electronic article search and included 76 original peer-reviewed scientific articles reporting morphological or biochemical modifications induced in vitro by soluble CNF1, either recombinant or from pathogenic Escherichia coli extracts highly purified with chromatographic methods. Most of the described CNF1-induced effects on cultured cells are ascribable to the modulating activity of the toxin on Rho GTPases and the consequent effects on actin cytoskeleton organization. All in all, the present review could be a prospectus about the CNF1-induced effects on cultured cells reported so far.     

The Protective Effects of the Autophagic and Lysosomal Machinery in Vascular and Valvular Calcification: A Systematic Review

Background: Autophagy is a highly conserved catabolic homeostatic process, crucial for cell survival. It has been shown that autophagy can modulate different cardiovascular pathologies, including vascular calcification (VCN). Objective: To assess how modulation of autophagy, either through induction or inhibition, affects vascular and valvular calcification and to determine the therapeutic applicability of inducing autophagy. Data sources: A systematic review of English language articles using MEDLINE/PubMed, Web of Science (WoS) and the Cochrane library. The search terms included autophagy, autolysosome, mitophagy, endoplasmic reticulum (ER)-phagy, lysosomal, calcification and calcinosis. Study characteristics: Thirty-seven articles were selected based on pre-defined eligibility criteria. Thirty-three studies (89%) studied vascular smooth muscle cell (VSMC) calcification of which 27 (82%) studies investigated autophagy and six (18%) studies lysosomal function in VCN. Four studies (11%) studied aortic valve calcification (AVCN). Thirty-four studies were published in the time period 2015–2020 (92%). Conclusion: There is compelling evidence that both autophagy and lysosomal function are critical regulators of VCN, which opens new perspectives for treatment strategies. However, there are still challenges to overcome, such as the development of more selective pharmacological agents and standardization of methods to measure autophagic flux.     

Characteristics of the Protocols Used in Electrical Pulse Stimulation of Cultured Cells for Mimicking In Vivo Exercise: A Systematic Review,Meta-Analysis,and Meta-Regression

While exercise benefits a wide spectrum of diseases and affects most tissues and organs, many aspects of its underlying mechanistic effects remain unsolved. In vitro exercise, mimicking neuronal signals leading to muscle contraction in vitro, can be a valuable tool to address this issue. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines for this systematic review and meta-analysis, we searched EMBASE and PubMed (from database inception to 4 February 2022) for relevant studies assessing in vitro exercise using electrical pulse stimulation to mimic exercise. Meta-analyses of mean differences and meta-regression analyses were conducted. Of 985 reports identified, 41 were eligible for analysis. We observed variability among existing protocols of in vitro exercise and heterogeneity among protocols of the same type of exercise. Our analyses showed that AMPK, Akt, IL-6, and PGC1a levels and glucose uptake increased in stimulated compared to non-stimulated cells, following the patterns of in vivo exercise, and that these effects correlated with the duration of stimulation. We conclude that in vitro exercise follows motifs of exercise in humans, allowing biological parameters, such as the aforementioned, to be valuable tools in defining the types of in vitro exercise. It might be useful in transferring obtained knowledge to human research.     

Epigenetic and Genetic Factors Related to Curve Progression in Adolescent Idiopathic Scoliosis: A Systematic Scoping Review of the Current Literature

Adolescent idiopathic scoliosis (AIS) is a progressive deformity of the spine. Scoliotic curves progress until skeletal maturity leading, in rare cases, to a severe deformity. While the Cobb angle is a straightforward tool in initial curve magnitude measurement, assessing the risk of curve progression at the time of diagnosis may be more challenging. Epigenetic and genetic markers are potential prognostic tools to predict curve progression. The aim of this study is to review the available literature regarding the epigenetic and genetic factors associated with the risk of AIS curve progression. This review was carried out in accordance with Preferential Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. The search was carried out in January 2022. Only peer-reviewed articles were considered for inclusion. Forty studies were included; fifteen genes were reported as having SNPs with significant association with progressive AIS, but none showed sufficient power to sustain clinical applications. In contrast, nine studies reporting epigenetic modifications showed promising results in terms of reliable markers. Prognostic testing for AIS has the potential to significantly modify disease management. Most recent evidence suggests epigenetics as a more promising field for the identification of factors associated with AIS progression, offering a rationale for further investigation in this field.     

FLT3 Tyrosine Kinase Inhibitors for the Treatment of Fit and Unfit Patients with FLT3-Mutated AML: A Systematic Review

FLT3-mutated acute myeloid leukemia accounts for around 30% of acute myeloid leukemia (AML). The mutation carried a poor prognosis until the rise of tyrosine kinase inhibitors (TKIs). New potent and specific inhibitors have successfully altered the course of the disease, increasing the complete response rate and the survival of patients with FLT3-mutated AML. The aim of this article is to review all the current knowledge on these game-changing drugs as well as the unsolved issues raised by their use for fit and unfit FLT3-mutated AML patients. To this end, we analyzed the results of phase I, II, III clinical trials evaluating FLT3-TKI both in the first-line, relapse monotherapy or in combination referenced in the PubMed, the American Society of Hematology, the European Hematology Association, and the Clinicaltrials.gov databases, as well as basic science reports on TKI resistance from the same databases. The review follows a chronological presentation of the different trials that allowed the development of first- and second-generation TKI and ends with a review of the current lines of evidence on leukemic blasts resistance mechanisms that allow them to escape TKI.     

Malignant Transformation of Giant Cell Tumour of Bone: A Review of Literature and the Experience of a Referral Centre

Giant cell tumour of bone (GCTB) is a benign, locally aggressive primary bone neoplasm that represents 5% of all bone tumours. The principal treatment approach is surgery. Although generally GCTB is considered only a locally aggressive disease, it can metastasise, and lung metastases occur in 1–9% of patients. To date, only the use of denosumab has been approved as medical treatment for GCTB. Even more rarely, GCTB undergoes sarcomatous transformation into a malignant tumour (4% of all GCTB), but history of this malignant transformation is unclear and unpredictable. Considering the rarity of the event, the data in the literature are few. In this review, we summarise published data of GCTB malignant transformation and we analyse three cases of malignant transformation of GCTB, evaluating histopathology, genetics, and radiological aspects. Despite the rarity of this event, we conclude that a strict follow up is recommended to detect early malignant transformation.     

Senescence Marker Protein 30 (SMP30): A Novel Pan-Species Diagnostic Marker for the Histopathological Diagnosis of Breast Cancer in Humans and Animals

Senescence marker protein 30 (SMP30) is a cell survival factor playing an important role in vitamin C synthesis and antiapoptosis. Moreover, its cytoprotective role suggests a possibility to be related to cancer cell survival. Mammary carcinoma is a common cancer in both humans and animals. Because of its histopathological diversity, especially in the early stage, histopathological diagnosis may be complicated; therefore, a diagnostic marker is helpful for confirmation. The present study analyzed the expression pattern of SMP30 in mammary carcinoma in humans, dogs, and cats. Immunohistochemistry, immunofluorescence, and western blot analysis were used to investigate SMP30 expression patterns. The expression was specifically observed in neoplastic glandular epithelial cells. The expression increased with the malignancy of glandular epithelial cells with a highly proliferative status. However, SMP30 expression was low in normal mammary gland tissues or well-differentiated adenoma tissues. The patterns were consistently reproduced in canine primary mammary carcinoma cells and MCF-7 and MDA-MB-231 human carcinoma cell lines. This study provides useful information to understand SMP30 expression in various stages of mammary carcinoma and to suggest its utility as a pan-species diagnostic marker, thereby helping to establish strategies for diagnosing mammary carcinoma in several species.     

How to Attain Ultralow Interfacial Tension and Three-Phase Behavior with Surfactant Formulation for Enhanced Oil Recovery: A Review. Part 4: Robustness of the Optimum Formulation Zone Through the Insensibility to Some Variables and the Occurrence of Complex Artifacts

In enhanced oil recovery, not only the low-tension performance, but also the robustness at optimum formulation is an important issue. The fourth part of our review series is dedicated to robustness, defined as the width of the zone exhibiting three-phase behavior around the optimum formulation, whatever the scanned variable. It is first corroborated from a screening of the available data in the literature that the tension minimum is inversely proportional to the square of the three-phase range in the HLD scale. However, since there is still an inaccuracy of about a factor 10 in the tension minimum, some significant improvement can be attained in some cases by increasing the three-phase behavior width in two ways. The first approach consists of finding systems that are insensitive to some formulation variable such as temperature, surfactant mixture composition or concentration, and water-to-oil ratio. The second way is to produce an artifact through which the optimum formulation is produced twice in a scan. If the distance between the two events in the scan is reduced down to be zero, their corresponding three-phase behavior zones merge and result in a wider WIII region with a low tension. Several cases of such events are reported: alkaline scans, anionic-nonionic and anionic-cationic mixture changes, linear change in composition in three-surfactant mixture, partial precipitation from a surfactant mixture in a salinity scan, and excessive partitioning of polyethoxylated nonionics. More complex transitions with three effects in a single scan or three concomitantly scanned variables show even more possibilities in practice.