Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 Systems.

Visualizing the location and dynamics of RNAs in live cells is key to understanding their function. Here, we identify two endonuclease-deficient, single-component programmable RNA-guided and RNA-targeting Cas13 RNases (dCas13s) that allow robust real-time imaging and tracking of RNAs in live cells, even when using single 20- to 27-nt-long guide RNAs. Compared to the aptamer-based MS2-MCP strategy, an optimized dCas13 system is user friendly, does not require genetic manipulation, and achieves comparable RNA-labeling efficiency. We demonstrate that the dCas13 system is capable of labeling NEAT1, SatIII, MUC4, and GCN4 RNAs and allows the study of paraspeckle-associated NEAT1 dynamics. Applying orthogonal dCas13 proteins or combining dCas13 and MS2-MCP allows dual-color imaging of RNAs in single cells. Further combination of dCas13 and dCas9 systems allows simultaneous visualization of genomic DNA and RNA transcripts in living cells.

m6 A modification of HSATIII lncRNAs regulates temperature-dependent splicing.

Nuclear stress bodies (nSBs) are nuclear membraneless organelles formed around stress-inducible HSATIII architectural long noncoding RNAs (lncRNAs). nSBs repress splicing of hundreds of introns during thermal stress recovery, which are partly regulated by CLK1 kinase phosphorylation of temperature-dependent Ser/Arg-rich splicing factors (SRSFs). Here, we report a distinct mechanism for this splicing repression through protein sequestration by nSBs. Comprehensive identification of RNA-binding proteins revealed HSATIII association with proteins related to N6 -methyladenosine (m6 A) RNA modification. 11% of the first adenosine in the repetitive HSATIII sequence were m6 A-modified. nSBs sequester the m6 A writer complex to methylate HSATIII, leading to subsequent sequestration of the nuclear m6 A reader, YTHDC1. Sequestration of these factors from the nucleoplasm represses m6 A modification of pre-mRNAs, leading to repression of m6 A-dependent splicing during stress recovery phase. Thus, nSBs serve as a common platform for regulation of temperature-dependent splicing through dual mechanisms employing two distinct ribonucleoprotein modules with partially m6 A-modified architectural lncRNAs.

LncRNA-dependent nuclear stress bodies promote intron retention through SR protein phosphorylation.

A number of long noncoding RNAs (lncRNAs) are induced in response to specific stresses to construct membrane-less nuclear bodies; however, their function remains poorly understood. Here, we report the role of nuclear stress bodies (nSBs) formed on highly repetitive satellite III (HSATIII) lncRNAs derived from primate-specific satellite III repeats upon thermal stress exposure. A transcriptomic analysis revealed that depletion of HSATIII lncRNAs, resulting in elimination of nSBs, promoted splicing of 533 retained introns during thermal stress recovery. A HSATIII-Comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) analysis identified multiple splicing factors in nSBs, including serine and arginine-rich pre-mRNA splicing factors (SRSFs), the phosphorylation states of which affect splicing patterns. SRSFs are rapidly de-phosphorylated upon thermal stress exposure. During stress recovery, CDC like kinase 1 (CLK1) was recruited to nSBs and accelerated the re-phosphorylation of SRSF9, thereby promoting target intron retention. Our findings suggest that HSATIII-dependent nSBs serve as a conditional platform for phosphorylation of SRSFs by CLK1 to promote the rapid adaptation of gene expression through intron retention following thermal stress exposure.

Nucleolar- and Nuclear-Stress-Induced Membrane-Less Organelles: A Proteome Analysis through the Prism of Liquid-Liquid Phase Separation.

Radical changes in the idea of the organization of intracellular space that occurred in the early 2010s made it possible to consider the formation and functioning of so-called membrane-less organelles (MLOs) based on a single physical principle: the liquid-liquid phase separation (LLPS) of biopolymers. Weak non-specific inter- and intramolecular interactions of disordered polymers, primarily intrinsically disordered proteins, and RNA, play a central role in the initiation and regulation of these processes. On the other hand, in some cases, the "maturation" of MLOs can be accompanied by a "liquid-gel" phase transition, where other types of interactions can play a significant role in the reorganization of their structure. In this work, we conducted a bioinformatics analysis of the propensity of the proteomes of two membrane-less organelles, formed in response to stress in the same compartment, for spontaneous phase separation and examined their intrinsic disorder predispositions. These MLOs, amyloid bodies (A-bodies) formed in the response to acidosis and heat shock and nuclear stress bodies (nSBs), are characterized by a partially overlapping composition, but show different functional activities and morphologies. We show that the proteomes of these biocondensates are differently enriched in proteins, and many have high potential for spontaneous LLPS that correlates with the different morphology and function of these organelles. The results of these analyses allowed us to evaluate the role of weak interactions in the formation and functioning of these important organelles.

Diagnostic safety of a machine learning-based automatic patient selection algorithm for stress-only myocardial perfusion SPECT.

BACKGROUND: Stress-only myocardial perfusion imaging (MPI) markedly reduces radiation dose, scanning time, and cost. We developed an automated clinical algorithm to safely cancel unnecessary rest imaging with high sensitivity for obstructive coronary artery disease (CAD). METHODS AND RESULTS: Patients without known CAD undergoing both MPI and invasive coronary angiography from REFINE SPECT were studied. A machine learning score (MLS) for prediction of obstructive CAD was generated using stress-only MPI and pre-test clinical variables. An MLS threshold with a pre-defined sensitivity of 95% was applied to the automated patient selection algorithm. Obstructive CAD was present in 1309/2079 (63%) patients. MLS had higher area under the receiver operator characteristic curve (AUC) for prediction of CAD than reader diagnosis and TPD (0.84 vs 0.70 vs 0.78, P < .01). An MLS threshold of 0.29 had superior sensitivity than reader diagnosis and TPD for obstructive CAD (95% vs 87% vs 87%, P < .01) and high-risk CAD, defined as stenosis of the left main, proximal left anterior descending, or triple-vessel CAD (sensitivity 96% vs 89% vs 90%, P < .01). CONCLUSIONS: The MLS is highly sensitive for prediction of both obstructive and high-risk CAD from stress-only MPI and can be applied to a stress-first protocol for automatic cancellation of unnecessary rest imaging.

Inhibition of HSF1 and SAFB Granule Formation Enhances Apoptosis Induced by Heat Stress.

Stress resistance mechanisms include upregulation of heat shock proteins (HSPs) and formation of granules. Stress-induced granules are classified into stress granules and nuclear stress bodies (nSBs). The present study examined the involvement of nSB formation in thermal resistance. We used chemical compounds that inhibit heat shock transcription factor 1 (HSF1) and scaffold attachment factor B (SAFB) granule formation and determined their effect on granule formation and HSP expression in HeLa cells. We found that formation of HSF1 and SAFB granules was inhibited by 2,5-hexanediol. We also found that suppression of HSF1 and SAFB granule formation enhanced heat stress-induced apoptosis. In addition, the upregulation of HSP27 and HSP70 during heat stress recovery was suppressed by 2,5-hexanediol. Our results suggested that the formation of HSF1 and SAFB granules was likely to be involved in the upregulation of HSP27 and HSP70 during heat stress recovery. Thus, the formation of HSF1 and SAFB granules was involved in thermal resistance.

Prognostic significance of ischaemic electrocardiographic changes during stress testing in patients with normal nuclear myocardial perfusion imaging.

OBJECTIVE: To evaluate cardiovascular outcomes in patients with normal nuclear myocardial perfusion imaging, but ischaemic electrocardiogram changes during pharmacological or exercise stress tests. METHODS: The retrospective study was conducted at the Aga Khan University Hospital, Karachi, and comprised data of patients who underwent either pharmacological or exercise stress myocardial perfusion scan and had a normal scan with ischaemic electrocardiogram changes between January 2013 and December 2014. All cardiac events, including angina, myocardial infarction, heart failure, coronary revascularisation and cardiac death, as well as non-cardiac deaths were noted. Data was analysed using STATA 14.2. RESULTS: Of the 2770 patients whose data was initially checked, 296(10.6%) developed ischaemic electrocardiogram changes during the stress test but had normal myocardial perfusion scan. Of them, 181(61%) patients were male, and the overall mean age was 62±15 years. Follow-up data was available for 280(94.5%) of these patients, with a mean follow-up of 48±7 months. Of these patients, 8(2.8%) died, and 1(0.3%) of them died due to inferior wall myocardial infarction. Myocardial infarction was found in 2(0.7%) patients, and 1(0.3%) patient was hospitalised with heart failure. Also, 12(4.3%) patients underwent revascularization for stable angina, 9(3.2%) underwent percutaneous transluminal coronary angioplasty, and 3(1.07%) needed coronary artery bypass grafting. CONCLUSIONS: Ischaemic electrocardiographic changes during stress testing in patients with normal myocardial perfusion scan were not associated with adverse outcomes, and the risk of cardiovascular events was relatively low for an intermediate follow-up period.

Heat Shock Affects Mitotic Segregation of Human Chromosomes Bound to Stress-Induced Satellite III RNAs.

Heat shock activates the transcription of arrays of Satellite III (SatIII) DNA repeats in the pericentromeric heterochromatic domains of specific human chromosomes, the longest of which is on chromosome 9. Long non-coding SatIII RNAs remain associated with transcription sites where they form nuclear stress bodies or nSBs. The biology of SatIII RNAs is still poorly understood. Here, we show that SatIII RNAs and nSBs are detectable up to four days after thermal stress and are linked to defects in chromosome behavior during mitosis. Heat shock perturbs the execution of mitosis. Cells reaching mitosis during the first 3 h of recovery accumulate in pro-metaphase. During the ensuing 48 h, this block is no longer detectable; however, a significant fraction of mitoses shows chromosome segregation defects. Notably, most of lagging chromosomes and chromosomal bridges are bound to nSBs and contain arrays of SatIII DNA. Disappearance of mitotic defects at the end of day 2 coincides with the processing of long non-coding SatIII RNAs into a ladder of small RNAs associated with chromatin and ranging in size from 25 to 75 nt. The production of these molecules does not rely on DICER and Argonaute 2 components of the RNA interference apparatus. Thus, massive transcription of SatIII DNA may contribute to chromosomal instability.

Nuclear stress bodies: Interaction of its components in oncogenic regulation.

Oncogenesis involves continuous genetic alterations that lead to compromised cellular integrity and immortal cell fate. The cells remain under excessive stress due to endo- and exogenous influences. Human Satellite III long noncoding RNA (SatIII lncRNA) is a key regulator of the global cellular stress response, although its function is poorly explained in cancers. The principal regulator of cancer meshwork is tumor protein p53, which if altered may result in chemoresistance. The heat shock factor 1 (HSF1) being a common molecule between the oncogenic control and global cellular stress acts as an oncogene as well as transcribes SatIII upon heat shock. This prompted us to determine the structure of SatIII RNA and establish the association between SatIII-HSF1-p53. We determined the most stable structure of SatIII RNA with the least energy of - 115.7 kcal/mol. Also, we observed a possible interaction of p53 with SatIII and HSF1 using support vector machine (SVM) algorithm for predicting RNA-protein interaction (RPI). Further, we employ the STRING database to understand if p53 is an interacting component of the nuclear stress bodies (nSBs). A precise inference was drawn from molecular docking which confirmed the interaction of SatIII-HSF1-p53, where a mutated p53 resulted in an altered DNA-binding property with the SatIII molecule. This study being first of its kind infers p53 to be a possible integral component of the nSBs, which may regulate cellular stress response during cancer progression in the presence of HSF1 and SatIII. An extended research on the regulations of SatIII and p53 may open new avenues in the field of apoptosis in cancer and the early approach of molecular targeting.

Two distinct nuclear stress bodies containing different sets of RNA-binding proteins are formed with HSATIII architectural noncoding RNAs upon thermal stress exposure.

Nuclear stress bodies (nSBs) are thermal stress-inducible membrane-less nuclear bodies that are formed on highly repetitive satellite III architectural noncoding RNAs (HSATIII arcRNAs). Upon thermal stress exposure, HSATIII expression is induced to sequestrate specific sets of RNA-binding proteins and form nSBs. The major population of nSBs contain SAFB as a marker, whereas the minor population are SAFB-negative. Here, we found that HNRNPM, which was previously reported to localize in nuclear foci adjacent to SAFB-positive foci upon thermal stress, localizes in a minor population of HSATIII-dependent nSBs. Hence, we used the terms nSB-S and nSB-M to distinguish the SAFB foci and HNRNPM foci, respectively. Analysis of the components of the nSBs revealed that each set contains distinct RNA-binding proteins, including SLTM and NCO5A in nSB-Ss and HNRNPA1 and HNRNPH1 in nSB-Ms. Overall, our findings indicate that two sets of nSBs containing HSATIII arcRNAs and distinct sets of RNA-binding proteins are formed upon thermal stress exposure.

Prevalence and predictors of frailty in a high-income developing country: A cross-sectional study.

Background: Frailty is a state of vulnerability and a decreased physiological response to stressors. As the population ages, the prevalence of frailty is expected to increase. Thus, identifying tools and resources that efficiently predict frailty among the Saudi population is important. We aimed to describe the prevalence and predictors of frailty among Saudi patients referred for cardiac stress testing with nuclear imaging. Methods: We included 876 patients (mean age 60.3 ± 11 years, women 48%) who underwent clinically indicated cardiac nuclear stress testing between January and October 2016. Fried Clinical Frailty Scale was used to assess frailty. Patients were considered frail if they had a score of four or higher. Multivariate adjusted logistic regression models were used to determine the independent predictors of elderly frail patients. Results: In this cohort, the median age of the included patients was 61 years, and the prevalence of frailty was 40%. The frail patients were older, more frequently women, and had a higher body mass index. Additionally, frailty was associated with a higher prevalence of cardiovascular risk factors: hypertension (85% vs. 70%) and diabetes (75% vs. 60%). In a fully adjusted logistic regression model, women, hypertension, and obesity (BMI ≥ 30 kg/m2) were independent predictors of elderly frail patients. Conclusions: With the aging of the Saudi population, frailty prevalence is expected to increase. Elderly, obesity, hypertension, and female gender are risk factors of frailty. Interventions to reduce frailty should be focused on this high-risk population.

Age-dependent neurodegeneration and organelle transport deficiencies in mutant TDP43 patient-derived neurons are independent of TDP43 aggregation.

TAR DNA-binding protein 43 (TDP43) plays a significant role in familiar and sporadic amyotrophic lateral sclerosis (ALS). The diverse postulated mechanisms by which TDP43 mutations cause the disease are not fully understood. Human wildtype and TDP43 S393L and G294V mutant spinal motor neuron cultures were differentiated from patient-derived iPSCs. Mutant hTDP43 and wildtype motor neuron cultures did not differ in neuron differentiation capacity during early maturation stage. During aging we detected a dramatic neurodegeneration including neuron loss and pathological neurofilament abnormalities only in TDP43 mutant cultures. Additionally mitochondria and lysosomes of aging spinal motor neurons revealed robust TDP43 mutation dependent abnormal phenotypes in size, shape, speed and motility which all appeared without TDP43 mislocalization or aggregation formation. Furthermore, D-sorbitol - known to induce stress granules and cytoplasmic mislocalization of TDP43 - rescued axonal trafficking phenotypes without signs of TDP43 mislocalization or aggregation formation. Our data indicate TDP43 mutation-dependent but cytosolic aggregation-independent mechanisms of motor neuron degeneration in TDP43 ALS.

Human satellite-III non-coding RNAs modulate heat-shock-induced transcriptional repression.

The heat shock response is a conserved defense mechanism that protects cells from physiological stress, including thermal stress. Besides the activation of heat-shock-protein genes, the heat shock response is also known to bring about global suppression of transcription; however, the mechanism by which this occurs is poorly understood. One of the intriguing aspects of the heat shock response in human cells is the transcription of satellite-III (Sat3) long non-coding RNAs and their association with nuclear stress bodies (nSBs) of unknown function. Besides association with the Sat3 transcript, the nSBs are also known to recruit the transcription factors HSF1 and CREBBP, and several RNA-binding proteins, including the splicing factor SRSF1. We demonstrate here that the recruitment of CREBBP and SRSF1 to nSBs is Sat3-dependent, and that loss of Sat3 transcripts relieves the heat-shock-induced transcriptional repression of a few target genes. Conversely, forced expression of Sat3 transcripts results in the formation of nSBs and transcriptional repression even without a heat shock. Our results thus provide a novel insight into the regulatory role for the Sat3 transcripts in heat-shock-dependent transcriptional repression.

Artificial neural network-based model enhances risk stratification and reduces non-invasive cardiac stress imaging compared to Diamond-Forrester and Morise risk assessment models: A prospective study.

BACKGROUND: Coronary artery disease (CAD) accounts for more than half of all cardiovascular events. Stress testing remains the cornerstone for non-invasive assessment of patients with possible or known CAD. Clinical utilization reviews show that most patients presenting for evaluation of stable CAD by stress testing are categorized as low risk prior to the test. Attempts to enhance risk stratification of individuals who are sent for stress testing seem to be more in need today. The present study compares artificial neural networks (ANN)-based prediction models to the other risk models being used in practice (the Diamond-Forrester and the Morise models). METHODS: In our study, we prospectively recruited patients who were 19 years of age or older, and were being evaluated for coronary artery disease with imaging-based stress tests. For ANN, the network architecture employed a systematic method, where the number of neurons is changed incrementally, and bootstrapping was performed to evaluate the accuracy of the models. RESULTS: We prospectively enrolled 486 patients. The mean age of patients undergoing stress test was 55.2 ± 11.2 years, 35% were women, and 12% had a positive stress test for ischemic heart disease. When compared to Diamond-Forrester and Morise risk models, the ANN model for predicting ischemia provided higher discriminatory power (DP)(1.61), had a negative predictive value of 98%, Sensitivity 91% [81%-97%], Specificity 65% [60%-79%], positive predictive value 26%, and a potential 59% reduction of non-invasive imaging. CONCLUSION: The ANN models improved risk stratification when compared to the other risk scores (Diamond-Forrester and Morise) with a 98% negative predictive value and a significant potential reduction in non-invasive imaging tests.

Reducing radiation to patients and improving image quality in a real-world nuclear cardiology laboratory.

In part because of aging equipment and reduced reimbursement for imaging services in the last several years, nuclear cardiologists who remain in private practice face challenges in maintaining high quality and in reducing radiation exposure to patients. We review patient-centered approaches and affordable software solutions employed in our practice combined with supine-prone myocardial perfusion imaging to achieve increased interpretive confidence with reduced radiation exposure to patients.

Segmentals weigh much more in comprehensibility than nuclear stress in read speech.

This study investigates the impact of segmental accuracy and nucleus placement on the comprehensibility of English as an International Language (EIL), with the aim of informing phonological norms and teaching models. Speech samples from 59 EIL speakers with varying levels of segmental accuracy were collected during a reading task, involving reading a passage in three different versions of speech, each version lasting approximately 30 to 40 s. To directly compare the impact of nuclear stress placement on comprehensibility, based on these samples, two versions of stimuli were created, each differing only in their placement of nuclear stress - either correct or incorrect. The correctness of placements was determined by seven native speakers of English. Eight native English speakers, aged 19-24, and eight EIL speakers, aged 20-24 with an upper-intermediate to advanced proficiency level, rated the comprehensibility of the two versions of speech. Results suggest that while correct nucleus placement enhances comprehensibility for native English listeners, it has little influence on EIL listeners. Segmental accuracy in EIL speech impacts comprehensibility substantially more than nucleus placement on both native and EIL listeners, indicating that English language teaching should focus on minimizing segmental errors to improve comprehensibility for EIL speakers, despite the benefits of correct nucleus placement.

Multivessel Coronary Artery Disease Presenting as a False-Negative Nuclear Stress Test: A Case of Balanced Ischemia.

Myocardial perfusion imaging (MPI) is fundamental to comparing coronary vessel perfusion levels and guides in identifying ischemic areas. However, false negatives, such as balanced ischemia, are important considerations in interpreting these results. In this case report, we describe a 77-year-old female who presented with cardiac chest pain with normal laboratory results, electrocardiogram, and imaging. However, given her history and risk factors, left heart catheterization was performed, which showed triple vessel coronary artery disease.

Myocardial Bridging of Mid-left Anterior Descending Artery (LAD) Presenting As Transient Left Bundle Branch Block During Nuclear Stress Test.

Transient left bundle branch block occurring during a nuclear stress test in the setting of myocardial bridging is a relatively rare finding. We report a case of a 75-year-old male who presented with typical stable angina. Serial troponins were negative, and the electrocardiogram revealed normal sinus rhythm with left ventricular hypertrophy and T-wave inversions in the lateral leads. The nuclear stress test was non-ischemic but showed a transient left bundle branch block associated with chest pain and shortness of breath that occurred right after the administration of regadenoson. Coronary angiography revealed non-obstructive coronary artery disease and a mid-LAD myocardial bridge.

P53: A key player in diverse cellular processes including nuclear stress and ribosome biogenesis, highlighting potential therapeutic compounds.

The tumor suppressor proteins are key transcription factors involved in the regulation of various cellular processes, such as apoptosis, DNA repair, cell cycle, senescence, and metabolism. The tumor suppressor protein p53 responds to different type of stress signaling, such as hypoxia, DNA damage, nutrient deprivation, oncogene activation, by activating or repressing the expression of different genes that target processes mentioned earlier. p53 has the ability to modulate the activity of many other proteins and signaling pathway through protein-protein interaction, post-translational modifications, or non-coding RNAs. In many cancers the p53 is found to be mutated or inactivated, resulting in the loss of its tumor suppressor function and acquisition of new oncogenic properties. The tumor suppressor protein p53 also plays a role in the development of other metabolic disorders such as diabetes, obesity, and fatty liver disease. In this review, we will summarize the current data and knowledge on the molecular mechanisms and the functions of p53 in different pathways and processes at the cellular level and discuss the its implications for human health and disease.

CDK7/CDK9 mediates transcriptional activation to prime paraptosis in cancer cells.

BACKGROUND: Paraptosis is a programmed cell death characterized by cytoplasmic vacuolation, which has been explored as an alternative method for cancer treatment and is associated with cancer resistance. However, the mechanisms underlying the progression of paraptosis in cancer cells remain largely unknown. METHODS: Paraptosis-inducing agents, CPYPP, cyclosporin A, and curcumin, were utilized to investigate the underlying mechanism of paraptosis. Next-generation sequencing and liquid chromatography-mass spectrometry analysis revealed significant changes in gene and protein expressions. Pharmacological and genetic approaches were employed to elucidate the transcriptional events related to paraptosis. Xenograft mouse models were employed to evaluate the potential of paraptosis as an anti-cancer strategy. RESULTS: CPYPP, cyclosporin A, and curcumin induced cytoplasmic vacuolization and triggered paraptosis in cancer cells. The paraptotic program involved reactive oxygen species (ROS) provocation and the activation of proteostatic dynamics, leading to transcriptional activation associated with redox homeostasis and proteostasis. Both pharmacological and genetic approaches suggested that cyclin-dependent kinase (CDK) 7/9 drive paraptotic progression in a mutually-dependent manner with heat shock proteins (HSPs). Proteostatic stress, such as accumulated cysteine-thiols, HSPs, ubiquitin-proteasome system, endoplasmic reticulum stress, and unfolded protein response, as well as ROS provocation primarily within the nucleus, enforced CDK7/CDK9-Rpb1 (RNAPII subunit B1) activation by potentiating its interaction with HSPs and protein kinase R in a forward loop, amplifying transcriptional regulation and thereby exacerbating proteotoxicity leading to initiate paraptosis. The xenograft mouse models of MDA-MB-231 breast cancer and docetaxel-resistant OECM-1 head and neck cancer cells further confirmed the induction of paraptosis against tumor growth. CONCLUSIONS: We propose a novel regulatory paradigm in which the activation of CDK7/CDK9-Rpb1 by nuclear proteostatic stress mediates transcriptional regulation to prime cancer cell paraptosis.