MARK2/MARK3 kinases are catalytic co-dependencies of YAP/TAZ in human cancer.

The Hippo signaling pathway is commonly dysregulated in human cancer, which leads to a powerful tumor dependency on the YAP/TAZ transcriptional coactivators. Here, we used paralog co-targeting CRISPR screens to identify the kinases MARK2/3 as absolute catalytic requirements for YAP/TAZ function in diverse carcinoma and sarcoma contexts. Underlying this observation is direct MARK2/3-dependent phosphorylation of NF2 and YAP/TAZ, which effectively reverses the tumor suppressive activity of the Hippo module kinases LATS1/2. To simulate targeting of MARK2/3, we adapted the CagA protein from H. pylori as a catalytic inhibitor of MARK2/3, which we show can regress established tumors in vivo. Together, these findings reveal MARK2/3 as powerful co-dependencies of YAP/TAZ in human cancer; targets that may allow for pharmacology that restores Hippo pathway-mediated tumor suppression.

Association between increased screen time, sedentary behavior, and insomnia among Indian adults: A cross-sectional nationwide online survey.

Context: During the "coronavirus disease 2019 (COVID-19)" pandemic, screen time saw a notable increase, ranging from 2.5 to 7.5 hours per day. Scientific evidence has demonstrated a strong correlation between heightened digital media usage and heightened levels of stress, anxiety, and depression. Research indicates that engaging in screen time for four hours or more daily can elevate the likelihood of experiencing symptoms of depression among children and adolescents by 46-80%. Despite this, there remains a paucity of medical evidence elucidating the intricate interplay between screen time, physical inactivity, and insomnia in adults. Aim: The aim of the study was to estimate the prevalence of increased screen time, insomnia, and physical inactivity among adults and their association during the COVID-19 pandemic. Settings and Design: A cross-sectional study through an online Google Form questionnaire was conducted among the Indian population. Methods and Materials: The study was conducted between June and August 2020 and comprised 658 participants aged 18 and above. Participants were recruited using a chain sampling procedure, with the majority being female, accounting for 54% (355 individuals). Statistical Analysis: The mean and standard deviation were calculated for numerical variables, while percentages and proportions were determined for categorical variables. The Chi-square test was employed to examine associations between variables. For assessing the predictors of screen time, a multivariate logistic regression analysis was conducted. Results: The majority of participants reported screen time exceeding 2 hours per day (85%), clinical insomnia symptoms (59%), moderate to high physical activity levels (92.8%), and low levels of sedentary behavior (60.5%). Variables such as gender, age, and screen time demonstrated significant associations with insomnia and physical activity. The odds ratio for insomnia in relation to screen time was 2.84 (95% confidence interval: 1.78-4.58) with a P value of 0.001. Multivariate logistic regression analysis indicated that increasing age was significantly associated with lower levels of screen time. Conclusions: Screen time showed a significant association with insomnia. Less screen time was reported with increased age.

Establishment and Culture of Patient-Derived Breast Organoids.

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

Single Oligonucleotide Capture of RNA And Temperature Elution Series ( SOCRATES ) for Identification of RNA-binding Proteins.

The importance of studying the mechanistic aspects of long non-coding RNAs is being increasingly emphasized as more and more regulatory RNAs are being discovered. Non-coding RNA sequences directly associate with generic RNA-binding proteins as well as specific proteins, which cooperate in the downstream functions of the RNA and can also be dysregulated in various physiologic states and/or diseases. While current methods exist for identifying RNA-protein interactions, these methods require high quantities of input cells or use pooled capture reagents that may increase non-specific binding. We have developed a method to efficiently capture specific RNAs using less than one million input cells. One single oligonucleotide is used to pull down the target RNA of choice and oligonucleotide selection is driven by sequence accessibility. We perform thermal elution to specifically elute the target RNA and its associated proteins, which are identified by mass spectrometry. Ultimately, two target and control oligonucleotides are used to create an enrichment map of interacting proteins of interest. This protocol was validated in: eLife (2021), DOI: 10.7554/eLife.68263.

MALAT1 Long Non-Coding RNA: Functional Implications.

The mammalian genome is pervasively transcribed and the functional significance of many long non-coding RNA (lncRNA) transcripts are gradually being elucidated. Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) is one of the most well-studied lncRNAs. MALAT1 is a highly conserved nuclear retained lncRNA that is abundantly expressed in cells and tissues and has been shown to play a role in regulating genes at both the transcriptional and post-transcriptional levels in a context-dependent manner. However, Malat1 has been shown to be dispensable for normal development and viability in mice. Interestingly, accumulating evidence suggests that MALAT1 plays an important role in numerous diseases including cancer. Here, we discuss the current state-of-knowledge in regard to MALAT1 with respect to its function, role in diseases, and the potential therapeutic opportunities for targeting MALAT1 using antisense oligonucleotides and small molecules.