Protocol for mapping physiological DSBs using in-suspension break labeling in situ and sequencing.

Physiological double-stranded breaks (DSBs) are a major source of genomic instability. Here, we present a protocol for mapping physiological DSBs by in-suspension break labeling in situ and sequencing (sBLISS) in a single-nucleotide resolution. We describe steps for cell fixation, labeling of DSBs, DNA isolation followed by in vitro transcription (IVT), reverse transcription, and library preparation.

Unveiling the relationship between WWOX and BRCA1 in mammary tumorigenicity and in DNA repair pathway selection.

Breast cancer is the leading cause of cancer-related deaths in women worldwide, with the basal-like or triple-negative breast cancer (TNBC) subtype being particularly aggressive and challenging to treat. Understanding the molecular mechanisms driving the development and progression of TNBC is essential. We previously showed that WW domain-containing oxidoreductase (WWOX) is commonly inactivated in TNBC and is implicated in the DNA damage response (DDR) through ATM and ATR activation.

TOP1 and R-loops facilitate transcriptional DSBs at hypertranscribed cancer driver genes.

DNA double-stranded breaks (DSBs) pose a significant threat to genomic integrity, and their generation during essential cellular processes like transcription remains poorly understood. In this study, we employ several techniques to map DSBs, R-loops, and topoisomerase 1 cleavage complex (TOP1cc) to comprehensively investigate the interplay between transcription, DSBs, topoisomerase 1 (TOP1), and R-loops. Our findings reveal the presence of DSBs at highly expressed genes enriched with TOP1 and R-loops.

Apoptotic cell death in disease-Current understanding of the NCCD 2023.

Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions.

Loss of tumor suppressor WWOX accelerates pancreatic cancer development through promotion of TGFβ/BMP2 signaling.

Pancreatic cancer is one of the most lethal cancers, owing to its late diagnosis and resistance to chemotherapy. The tumor suppressor WW domain-containing oxidoreductase (WWOX), one of the most active fragile sites in the human genome (FRA16D), is commonly altered in pancreatic cancer. However, the direct contribution of WWOX loss to pancreatic cancer development and progression remains largely unknown.

S6K1 phosphorylates Cdk1 and MSH6 to regulate DNA repair.

The mTORC1 substrate, S6 Kinase 1 (S6K1), is involved in the regulation of cell growth, ribosome biogenesis, glucose homeostasis, and adipogenesis. Accumulating evidence has suggested a role for mTORC1 signaling in the DNA damage response. This is mostly based on the findings that mTORC1 inhibitors sensitized cells to DNA damage.

WWOX-Related Neurodevelopmental Disorders: Models and Future Perspectives.

The WW domain-containing oxidoreductase () gene was originally discovered as a putative tumor suppressor spanning the common fragile site FRA16D, but as time has progressed the extent of its pleiotropic function has become apparent. At present, WWOX is a major source of interest in the context of neurological disorders, and more specifically developmental and epileptic encephalopathies (DEEs). This review article aims to introduce the many model systems used through the years to study its function and roles in neuropathies.

Neonatal neuronal WWOX gene therapy rescues Wwox null phenotypes.

WW domain-containing oxidoreductase (WWOX) is an emerging neural gene-regulating homeostasis of the central nervous system. Germline biallelic mutations in WWOX cause WWOX-related epileptic encephalopathy (WOREE) syndrome and spinocerebellar ataxia and autosomal recessive 12 (SCAR12), two devastating neurodevelopmental disorders with highly heterogenous clinical outcomes, the most common being severe epileptic encephalopathy and profound global developmental delay. We recently demonstrated that neuronal ablation of murine Wwox recapitulates phenotypes of Wwox-null mice leading to intractable epilepsy, hypomyelination, and postnatal lethality.

Altered neocortical oscillations and cellular excitability in an in vitro Wwox knockout mouse model of epileptic encephalopathy.

Loss of function mutations of the WW domain-containing oxidoreductase (WWOX) gene are associated with severe and fatal drug-resistant pediatric epileptic encephalopathy. Epileptic seizures are typically characterized by neuronal hyperexcitability; however, the specific contribution of WWOX to that hyperexcitability has yet to be investigated. Using a mouse model of neuronal Wwox-deletion that exhibit spontaneous seizures, in vitro whole-cell and field potential electrophysiological characterization identified spontaneous bursting activity in the neocortex, a marker of the underlying network hyperexcitability.

Real-Time Non-Invasive and Direct Determination of Lactate Dehydrogenase Activity in Cerebral Organoids-A New Method to Characterize the Metabolism of Brain Organoids?

Organoids are a powerful tool in the quest to understand human diseases. As the developing brain is extremely inaccessible in mammals, cerebral organoids (COs) provide a unique way to investigate neural development and related disorders. The aim of this study was to utilize hyperpolarized C NMR to investigate the metabolism of COs in real-time, in a non-destructive manner.

Modeling genetic epileptic encephalopathies using brain organoids.

Developmental and epileptic encephalopathies (DEE) are a group of disorders associated with intractable seizures, brain development, and functional abnormalities, and in some cases, premature death. Pathogenic human germline biallelic mutations in tumor suppressor WW domain-containing oxidoreductase (WWOX) are associated with a relatively mild autosomal recessive spinocerebellar ataxia-12 (SCAR12) and a more severe early infantile WWOX-related epileptic encephalopathy (WOREE). In this study, we generated an in vitro model for DEEs, using the devastating WOREE syndrome as a prototype, by establishing brain organoids from CRISPR-engineered human ES cells and from patient-derived iPSCs.

Neurological Disorders Associated with WWOX Germline Mutations-A Comprehensive Overview.

The transcriptional regulator WW domain-containing oxidoreductase (WWOX) is a key player in a number of cellular and biological processes including tumor suppression. Recent evidence has emerged associating WWOX with non-cancer disorders. Patients harboring pathogenic germline bi-allelic WWOX variants have been described with the rare devastating neurological syndromes autosomal recessive spinocerebellar ataxia 12 (SCAR12) (6 patients) and WWOX-related epileptic encephalopathy (DEE28 or WOREE syndrome) (56 patients).

Neuronal deletion of Wwox, associated with WOREE syndrome, causes epilepsy and myelin defects.

WWOX-related epileptic encephalopathy (WOREE) syndrome caused by human germline bi-allelic mutations in WWOX is a neurodevelopmental disorder characterized by intractable epilepsy, severe developmental delay, ataxia and premature death at the age of 2-4 years. The underlying mechanisms of WWOX actions are poorly understood. In the current study, we show that specific neuronal deletion of murine Wwox produces phenotypes typical of the Wwox-null mutation leading to brain hyperexcitability, intractable epilepsy, ataxia and postnatal lethality.

Programmed DNA Damage and Physiological DSBs: Mapping, Biological Significance and Perturbations in Disease States.

DNA double strand breaks (DSBs) are known to be the most toxic and threatening of the various types of breaks that may occur to the DNA. However, growing evidence continuously sheds light on the regulatory roles of programmed DSBs. Emerging studies demonstrate the roles of DSBs in processes such as T and B cell development, meiosis, transcription and replication.

Twist1-induced miR-199a-3p promotes liver fibrosis by suppressing caveolin-2 and activating TGF-β pathway.

The activation of hepatic stellate cells (HSCs) participates in liver fibrosis, and emerging evidences indicate that microRNAs (miRNAs) are abnormally expressed during HSC activation. However, the potential roles of miRNAs in liver fibrosis still remain elusive. Therefore, this study aimed to investigate the role of miR-199a-3p in liver fibrosis and its underlying mechanism.

Mapping the breakome reveals tight regulation on oncogenic super-enhancers.

DNA double-strand breaks (DSBs) could be deleterious and lead to age-related diseases, such as cancer. Recent evidence, however, associates DSBs with vital cellular processes. As discussed here, genome-wide mapping of DSBs revealed an unforeseen coupling mechanism between transcription and DNA repair at super-enhancers, as means of hypertranscription of oncogenic drivers.