Specificity of cancer-related chromosomal translocations is linked to proximity after the DNA double-strand break and subsequent selection.

Most cancer-related chromosomal translocations appear to be cell type specific. It is currently unknown why different chromosomal translocations occur in different cells. This can be due to either the occurrence of particular translocations in specific cell types or adaptive survival advantage conferred by translocations only in specific cells. We experimentally addressed this question by double-strand break (DSB) induction at MYC, IGH, AML and ETO loci in the same cell to generate chromosomal translocations in different cell lineages. Our results show that any translocation can potentially arise in any cell type. We have analyzed different factors that could affect the frequency of the translocations, and only the spatial proximity between gene loci after the DSB induction correlated with the resulting translocation frequency, supporting the 'breakage-first' model. Furthermore, upon long-term culture of cells with the generated chromosomal translocations, only oncogenic MYC-IGH and AML-ETO translocations persisted over a 60-day period. Overall, the results suggest that chromosomal translocation can be generated after DSB induction in any type of cell, but whether the cell with the translocation would persist in a cell population depends on the cell type-specific selective survival advantage that the chromosomal translocation confers to the cell.

Epstein-Barr virus reactivation induces MYC-IGH spatial proximity and t(8;14) in B cells.

Burkitt lymphoma (BL) is a B cell malignancy associated with the Epstein-Barr virus (EBV). Most BL cases are characterized by a t(8;14) chromosomal translocation involving the MYC oncogene and the immunoglobulin heavy chain gene (IGH). The role of EBV in promoting this translocation remains largely unknown. Here we provide the experimental evidence that EBV reactivation from latency leads to an increase in the proximity between the MYC and IGH loci, otherwise located far away in the nuclear space both in B-lymphoblastoid cell lines and in patients' B-cells. Specific DNA damage within the MYC locus, followed by the MRE11-dependent DNA repair plays a role in this process. Using a CRISPR/Cas9-based B cell model to induce specific DNA double strand breaks in MYC and IGH loci, we have shown that the MYC-IGH proximity induced by EBV reactivation leads to an increased t(8;14) translocation frequency.

Easy and robust electrotransfection protocol for efficient ectopic gene expression and genome editing in human B cells.

B-cell lines and primary PBMCs are notoriously hard to transfect, thus making genome editing, ectopic gene expression, or gene silencing experiments particularly tedious. Here we propose a novel efficient and reproducible protocol for electrotransfection of lymphoblastoid, B-cell lymphoma, leukemia cell lines, and B cells from PBMCs. The proposed protocol requires neither costly equipment nor expensive reagents; it can be used with small or large plasmids. Transfection and viability rates of about 79% and 58%, respectively, have been routinely achieved by optimizing the salt concentration in the electrotransfection medium and the amount of plasmid used. A validation of the protocol was obtained via the generation of a TP53-/- RPMI8866 lymphoblastoid cell line which should prove useful in future hematological and blood cancer studies.

Factors That Affect the Formation of Chromosomal Translocations in Cells.

Chromosomal translocations are products of the illegitimate repair of DNA double-strand breaks (DSBs). Their formation can bring about significant structural and molecular changes in the cell that can be physiologically and pathologically relevant. The induced changes may lead to serious and life-threatening diseases such as cancer. As a growing body of evidence suggests, the formation of chromosomal translocation is not only affected by the mere close spatial proximity of gene loci as potential translocation partners. Several factors may affect formation of chromosomal translocations, including chromatin motion to the potential sources of DSBs in the cell. While these can be apparently random events, certain chromosomal translocations appear to be cell-type-specific. In this review, we discuss how chromosomal translocations are formed and explore how different cellular factors contribute to their formation.

Biosafety Capacity Building During the COVID-19 Pandemic: Results, Insights, and Lessons Learned from an Online Approach in the Philippines.

Introduction: COVID-19 diagnosis was one of the most pressing needs during the early stages of the pandemic. Its entire procedure has inherent biosafety risks that if not properly managed and mitigated can be life threatening. Cognizant of this vital aspect, the Department of Health (DOH) imposed a biosafety training requirement to all laboratories and institutions before they could perform COVID-19 diagnostic testing. But with the mandatory lockdown, conventional face-to-face training could not be conducted. To address this need, the Biosafety Education and Awareness Training COVID-19 Online Program was offered by the National Training Center for Biosafety and Biosecurity of the University of the Philippines Manila. Methods and Materials: This online training program implemented a distance learning approach made available through the Canvas Learning Management System. It consisted of seven modules on biosafety that were sufficient enough to capacitate the participants with information for them to effectively implement a biorisk management system. The participants were evaluated based on quiz, examination, and case analysis. Certificates of completion were awarded to participants who passed all evaluation methods. Results: A total of 3371 trainees from various medical professions passed and obtained the certificate. This resulted in >100 DOH-accredited COVID-19 testing laboratories by the end of 2020. Discussion and Conclusion: The online availability of this program proved to be an effective innovative solution to a unique problem. Therefore, this training program demonstrated that biosafety training can be effectively conducted online and in a distance learning approach.

Ca2+ Transportome and the Interorganelle Communication in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a type of liver cancer with a poor prognosis for survival given the complications it bears on the patient. Though damages to the liver are acknowledged prodromic factors, the precise molecular aetiology remains ill-defined. However, many genes coding for proteins involved in calcium (Ca2+) homeostasis emerge as either mutated or deregulated. Ca2+ is a versatile signalling messenger that regulates functions that prime and drive oncogenesis, favouring metabolic reprogramming and gene expression. Ca2+ is present in cell compartments, between which it is trafficked through a network of transporters and exchangers, known as the Ca2+ transportome. The latter regulates and controls Ca2+ dynamics and tonicity. In HCC, the deregulation of the Ca2+ transportome contributes to tumorigenesis, the formation of metastasizing cells, and evasion of cell death. In this review, we reflect on these aspects by summarizing the current knowledge of the Ca2+ transportome and overviewing its composition in the plasma membrane, endoplasmic reticulum, and the mitochondria.

DNA barcoding of fishes of Laguna de Bay, Philippines.

Laguna de Bay, the largest lake in the Philippines, is an important part of the country's fisheries industry. It is also home to a number of endemic fishes including Gobiopterus lacustris (Herre 1927) of family Gobiidae, Leiopotherapon plumbeus (Kner 1864) of family Terapontidae, Zenarchopterus philippinus (Peters 1868) of family Hemiramphidae and Arius manillensis Valenciennes 1840 of family Ariidae. Over the years, a steady decline has been observed in the abundance and diversity of native fishes in the lake due to anthropogenic disturbances. In this study, a total of 71 specimens of 18 different species belonging to 18 genera, 16 families, and seven orders were DNA barcoded using the mitochondrial cytochrome c oxidase subunit I (COI) gene. All of the fish species were discriminated by their COI sequences and one endemic species G. lacustris, showing deep genetic divergence, was highlighted for further taxonomic investigation. Average Kimura 2-parameter genetic distances within species, family, and order were 1.33%, 18.91%, and 24.22%, respectively. These values show that COI divergence increases as taxa become less exclusive. All of the COI sequences obtained were grouped together according to their species designation in the Neighbor-joining tree that was constructed. This study demonstrated that DNA barcoding has great potential as a tool for fast and accurate species identification and also for highlighting species that warrant further taxonomic investigation.