Comprehensive mapping of cell fates in microsatellite unstable cancer cells supports dual targeting of WRN and ATR.

Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN-knowledge that would be helpful for informing clinical development of WRN targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system in which the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We found that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we found no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low-dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provide the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggest that dual targeting of WRN and ATR might be a useful strategy for treating MSI-H cancers.

Loss of CDK4/6 activity in S/G2 phase leads to cell cycle reversal.

In mammalian cells, the decision to proliferate is thought to be irreversibly made at the restriction point of the cell cycle1,2, when mitogen signalling engages a positive feedback loop between cyclin A2/cyclin-dependent kinase 2 (CDK2) and the retinoblastoma protein3-5. Contrary to this textbook model, here we show that the decision to proliferate is actually fully reversible. Instead, we find that all cycling cells will exit the cell cycle in the absence of mitogens unless they make it to mitosis and divide first. This temporal competition between two fates, mitosis and cell cycle exit, arises because cyclin A2/CDK2 activity depends upon CDK4/6 activity throughout the cell cycle, not just in G1 phase. Without mitogens, mitosis is only observed when the half-life of cyclin A2 protein is long enough to sustain CDK2 activity throughout G2/M. Thus, cells are dependent on mitogens and CDK4/6 activity to maintain CDK2 activity and retinoblastoma protein phosphorylation throughout interphase. Consequently, even a 2-h delay in a cell's progression towards mitosis can induce cell cycle exit if mitogen signalling is lost. Our results uncover the molecular mechanism underlying the restriction point phenomenon, reveal an unexpected role for CDK4/6 activity in S and G2 phases and explain the behaviour of all cells following loss of mitogen signalling.

Rescue of expression and function of long QT syndrome-causing mutant hERG channels by enhancing channel stability in the plasma membrane.

The human ether-g-go-go-related gene (hERG) encodes the Kv11.1 (or hERG) channel that conducts the rapidly activating delayed rectifier potassium current (IKr). Naturally occurring mutations in hERG impair the channel function and cause long QT syndrome type 2 (LQT2). Many missense hERG mutations lead to a lack of channel expression on the cell surface, representing a major mechanism for the loss-of-function of mutant channels. While it is generally thought that a trafficking defect underlies the lack of channel expression on the cell surface, in the present study, we demonstrate that the trafficking defective mutant hERG G601S can reach the plasma membrane but is unstable and quickly degrades, which is akin to Wild Type (WT) hERG channels under low K+ conditions. We previously showed that Serine (S) residue at 624 in the innermost position of the selectivity filter of hERG is involved in hERG membrane stability such that substitution of Serine 624 with Threonine (S624T) enhances hERG stability and renders hERG insensitive to low K+ culture. Here, we report that the intragenic addition of S624T substitution to trafficking defective hERG mutants G601S, N470D and P596R led to a complete rescue of the function of these otherwise loss-of-function mutant channels to a level similar to the WT channel, representing the most effective rescue means for the function of mutant hERG channels. These findings not only provide novel insights into hERG mutation-mediated channel dysfunction, but also point to the critical role of S624 in hERG stability on the plasma membrane.

Ubiquitination is involved in PKC-mediated degradation of cell surface Kv1.5 channels.

The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid delayed rectifier K+ current (IKur) in human cells, plays important roles in the repolarization of atrial action potentials and regulation of the vascular tone. We previously reported that activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) induces endocytic degradation of cell-surface Kv1.5 channels, and a point mutation removing the phosphorylation site, T15A, in the N terminus of Kv1.5 abolished the PMA-effect. In the present study, using mutagenesis, patch clamp recording, Western blot analysis and immunocytochemical staining, we demonstrate that ubiquitination is involved in PMA-mediated degradation of mature Kv1.5 channels. Since the expression of Kv1.4 channel is unaffected by PMA treatment, we swapped the N- and/or C-termini between Kv1.5 and Kv1.4. We found that N-terminus alone did not, but both N- and C-termini of Kv1.5 did confer PMA sensitivity to mature Kv1.4 channels, suggesting the involvement of Kv1.5 C-terminus in the channel ubiquitination. Removal of each of the potential ubiquitination residue Lysine at position 536, 565, and 591 by Arginine substitution (K536R, K565R, and K591R) had little effect, but removal of all three Lysine residues with Arginine substitution (3K-R) partially reduced PMA-mediated Kv1.5 degradation. Furthermore, removing the cysteine residue at position 604 by Serine substitution (C604S) drastically reduced PMA-induced channel degradation. Removal of the three Lysines and Cys604 with a quadruple mutation (3K-R/C604S) or a truncation mutation (Δ536) completely abolished the PKC activation-mediated degradation of Kv1.5 channels. These results provide mechanistic insight into PKC activation-mediated Kv1.5 degradation.

Dispositional mindfulness moderates the links between potentially morally injurious event exposure and symptoms of anxiety and depression but not suicidal ideation.

Exposure to potentially morally injurious events (PMIEs) is a pervasive threat for military service members and may be associated with symptoms of anxiety, depression, and suicidal ideation. However, coping mechanisms, such as mindfulness, may ameliorate symptoms and improve recovery. Two studies were conducted to test dispositional mindfulness as a moderator of the links between PMIEs, as assessed using the Moral Injury Events Scale (i.e., total score and Self-Transgression, Other-Transgression, and Betrayal subscale scores), and symptoms of anxiety, depression, and suicidal ideation among different samples of active-duty soldiers in garrison. In Sample 1 (N = 310), mindfulness buffered the links between PMIE exposure and symptoms of both anxiety, ∆R2 = .02, and depression, ∆R2 = .03. In Sample 2 (N = 669), mindfulness moderated the link between the MIES Betrayal subscale and anxiety symptoms, ∆R2 = .01. The results suggest that dispositional mindfulness may be a protective factor against some of the negative impacts of PMIE exposure. Further implications are discussed.

The top sports medicine influencers on X (formerly Twitter).

BACKGROUND: With increasing sports medicine content on social media, we sought to identify the top sports medicine influencers on X (formerly Twitter) and analyse their common characteristics. METHODS: X influence scores for "Sports Medicine" were collected in November 2021 from Right Relevance. Accounts were then screened in a blind, duplicate manner for personal and X profile characteristics. RESULTS: Physical therapists/physiotherapists made up 48% of the top sports science and medicine influencers. Locations in the U.S. and U.K. were listed for half of the influencers. The mean h-index was 30.2 (95% CI = [24.8-35.6]) with a median of 22.0 (range = 1-101). Most individuals reported multiple practice settings (63%), with 60% associated with an academic setting. Professional (62%) and Olympic (49%) level athletics were most frequently mentioned, with soccer (48%) and rugby (30%) as the most common sports. Among 76 profiles with URLs, most were linked to personal websites (57.9%). CONCLUSION: The top influencers on X consisted of accredited sports science and medicine professionals across various locations and occupations, providing ample networking and collaboration opportunities. The relatively high h-index in this study suggests sports science and medicine influencers on X are notable contributors to academic literature.

Potential Hydrodynamic Cytoplasmic Transfer between Mammalian Cells: Cell-Projection Pumping.

We earlier reported cytoplasmic fluorescence exchange between cultured human fibroblasts (Fibs) and malignant cells (MCs). Others report similar transfer via either tunneling nanotubes (TNTs) or shed membrane vesicles, and this changes the phenotype of recipient cells. Our time-lapse microscopy showed most exchange was from Fibs into MCs, with less in the reverse direction. Although TNTs were seen, we were surprised transfer was not via TNTs but was instead via fine and often branching cell projections that defied direct visual resolution because of their size and rapid movement. Their structure was revealed nonetheless by their organellar cargo and the grooves they formed indenting MCs, which was consistent with holotomography. Discrete, rapid, and highly localized transfer events evidenced against a role for shed vesicles. Transfer coincided with rapid retraction of the cell projections, suggesting a hydrodynamic mechanism. Increased hydrodynamic pressure in retracting cell projections normally returns cytoplasm to the cell body. We hypothesize "cell-projection pumping" (CPP), in which cytoplasm in retracting cell projections partially equilibrates into adjacent recipient cells via microfusions that form temporary intercellular cytoplasmic continuities. We tested plausibility for CPP by combined mathematical modeling, comparison of predictions from the model with experimental results, and then computer simulations based on experimental data. The mathematical model predicted preferential CPP into cells with lower cell stiffness, expected from equilibration of pressure toward least resistance. Predictions from the model were satisfied when Fibs were cocultured with MCs and fluorescence exchange was related to cell stiffness by atomic force microscopy. When transfer into 5000 simulated recipient MCs or Fibs was studied in computer simulations, inputting experimental cell stiffness and donor cell fluorescence values generated transfers to simulated recipient cells similar to those seen by experiment. We propose CPP as a potentially novel mechanism in mammalian intercellular cytoplasmic transfer and communication.

Repeating the past: prevention focus motivates repetition, even for unethical decisions.

Prevention-focused individuals are motivated to maintain the status quo. Given this, we predicted that individuals with a strong prevention focus, either as a chronic predisposition or situationally induced, would treat their initial decision on how to behave on a first task as the status quo and would thus be motivated to repeat that decision on a subsequent task-even for decisions that were ethically questionable. Results from five studies supported this prediction in multiple ethical domains: whether or not to overstate performance (Studies 1, 2a, and 2b), whether or not to disclose disadvantageous facts (Study 3), and whether or not to pledge a donation (Study 4). The prevention-repetition effect was observed both when the initial and subsequent decisions were in the same domain (Studies 1-3) and when they were in different domains (Study 4). Alternative accounts for this effect, such as justification for the initial decision and preference for consistency, were ruled out (Study 2b).

Abrogation of the G2/M checkpoint as a chemosensitization approach for alkylating agents.

BACKGROUND: The cell cycle is tightly regulated by checkpoints, which play a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anticancer treatments, allowing for DNA repair prior to cell division. Manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies. METHODS: In this study, we conducted a forward genome-wide CRISPR/Cas9 screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress. RESULTS: Our findings revealed that canonical DNA repair pathways, including the Ataxia-telangiectasia mutated (ATM)/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of PKMYT1, in ensuring cell survival. Depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between alkylating agents and a Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice. CONCLUSIONS: Collectively, our findings highlight the potential of targeting cell cycle timing through Myt1 inhibition as an effective strategy to enhance the efficacy of current standard cancer therapies, potentially leading to improved disease outcomes.

MEF2 in cardiac hypertrophy in response to hypertension.

Hypertension is a globally prevalent pathological condition and an underlying risk factor for the development of cardiac hypertrophy leading to heart failure. Myocyte enhancer factor 2 (Mef2) has been identified as one of the primary effectors of morphological changes in the hypertensive heart, as part of a complex network of molecular signaling controlling cardiac gene expression. Experimental chronic pressure-overload models that mimic hypertension in the mammalian heart lead to the activation of various pathological mechanisms that result in structural changes leading to debilitating cardiac hypertrophy and ultimately heart failure. The purpose here is to survey the literature implicating Mef2 in hypertension induced cardiac hypertrophy, towards illuminating points of interest for understanding and potentially treating heart failure.

Comprehensive mapping of cell fates in microsatellite unstable cancer cells support dual targe6ng of WRN and ATR.

Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN, knowledge that would be helpful for informing clinical development of WRN-targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system wherein the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We find that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we find no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provided the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggested a potential therapeutical rationale for dual targeting of WRN and ATR.

Irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation.

Cells can irreversibly exit the cell cycle and become senescent to safeguard against uncontrolled proliferation. While the p53-p21 and p16-Rb pathways are thought to mediate senescence, they also mediate reversible cell cycle arrest (quiescence), raising the question of whether senescence is actually reversible or whether alternative mechanisms underly the irreversibility associated with senescence. Here, we show that senescence is irreversible and that commitment to and maintenance of senescence are mediated by irreversible MYC degradation. Senescent cells start dividing when a non-degradable MYC mutant is expressed, and quiescent cells convert to senescence when MYC is knocked down. In early oral carcinogenesis, epithelial cells exhibit MYC loss and become senescent as a safeguard against malignant transformation. Later stages of oral premalignant lesions exhibit elevated MYC levels and cellular dysplasia. Thus, irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation, but bypassing this degradation may allow tumor cells to escape during cancer initiation.

Toxicity of Orthodontic Brackets Examined by Single Cell Tracking.

Subtle toxic effects may be masked in traditional assays that average or summate the response of thousands of cells. We overcome this by using the recent method of single cell tracking in time-lapse recordings. This follows the fate and behavior of individual cells and their progeny and provides unambiguous results for multiple simultaneous biological responses. Further, single cell tracking permits correlation between progeny relationships and cell behavior that is not otherwise possible, including disruption by toxins and toxicants of similarity between paired sister cells. Notably, single cell tracking seems not to have been previously used to study biomaterials toxicity. The culture medium was pre-conditioned by 79 days incubation with orthodontic brackets from seven separate commercial sources. Metal levels were determined by Inductively Coupled Plasma Mass Spectrometry. Metal levels varied amongst conditioned media, with elevated Cr, Mn, Ni, and Cu and often Mo, Pb, Zn, Pd, and Ag were occasionally found. The effect on human dermal fibroblasts was determined by single cell tracking. All bracket-conditioned media reduced cell division (p < 0.05), while some reduced cell migration (p < 0.05). Most bracket-conditioned media increased the rate of asynchronous sister cell division (p < 0.05), a seemingly novel measure for toxicity. No clear effect on cell morphology was seen. We conclude that orthodontic brackets have cytotoxic effects, and that single cell tracking is effective for the study of subtle biomaterials cytotoxicity.

Revealing ß-TrCP activity dynamics in live cells with a genetically encoded biosensor.

The F-box protein beta-transducin repeat containing protein (ß-TrCP) acts as a substrate adapter for the SCF E3 ubiquitin ligase complex, plays a crucial role in cell physiology, and is often deregulated in many types of cancers. Here, we develop a fluorescent biosensor to quantitatively measure ß-TrCP activity in live, single cells in real-time. We find ß-TrCP remains constitutively active throughout the cell cycle and functions to maintain discreet steady-state levels of its substrates. We find no correlation between expression levels of ß-TrCP and ß-TrCP activity, indicating post-transcriptional regulation. A high throughput screen of small-molecules using our reporter identifies receptor-tyrosine kinase signaling as a key axis for regulating ß-TrCP activity by inhibiting binding between ß-TrCP and the core SCF complex. Our study introduces a method to monitor ß-TrCP activity in live cells and identifies a key signaling network that regulates ß-TrCP activity throughout the cell cycle.

Cell-Projection Pumping of Fibroblast Contents into Osteosarcoma SAOS-2 Cells Correlates with Increased SAOS-2 Proliferation and Migration, as well as Altered Morphology.

We earlier reported that cell-projection pumping transfers fibroblast contents to cancer cells and this alters the cancer cell phenotype. Here, we report on single-cell tracking of time lapse recordings from co-cultured fluorescent fibroblasts and SAOS-2 osteosarcoma cells, tracking 5201 cells across 7 experiments. The fluorescent lipophilic marker DiD was used to label fibroblast organelles and to trace the transfer of fibroblast cytoplasm into SAOS-2 cells. We related SAOS-2 phenotypic change to levels of fluorescence transfer from fibroblasts to SAOS-2 cells, as well as what we term 'compensated fluorescence', that numerically projects mother cell fluorescence post-mitosis into daughter cells. The comparison of absolute with compensated fluorescence allowed us to deduct if the phenotypic effects in mother SAOS-2 cells were inherited by their daughters. SAOS-2 receipt of fibroblast fluorescence correlated by Kendall's tau with cell-profile area and without evidence of persistence in daughter cells (median tau = 0.51, p < 0.016); negatively and weakly with cell circularity and with evidence of persistence (median tau = -0.19, p < 0.05); and very weakly with cell migration velocity and without evidence of persistence (median tau = 0.01, p < 0.016). In addition, mitotic SAOS-2 cells had higher rates of prior fluorescence uptake (median = 64.9 units/day) than non-dividing cells (median = 35.6 units/day, p < 0.016) and there was no evidence of persistence post-mitosis. We conclude that there was an appreciable impact of cell-projection pumping on cancer cell phenotype relevant to cancer histopathological diagnosis, clinical spread and growth, with most effects being 'reset' by cancer cell mitosis.

Cell cycle inertia underlies a bifurcation in cell fates after DNA damage.

The G1-S checkpoint is thought to prevent cells with damaged DNA from entering S phase and replicating their DNA and efficiently arrests cells at the G1-S transition. Here, using time-lapse imaging and single-cell tracking, we instead find that DNA damage leads to highly variable and divergent fate outcomes. Contrary to the textbook model that cells arrest at the G1-S transition, cells triggering the DNA damage checkpoint in G1 phase route back to quiescence, and this cellular rerouting can be initiated at any point in G1 phase. Furthermore, we find that most of the cells receiving damage in G1 phase actually fail to arrest and proceed through the G1-S transition due to persistent cyclin-dependent kinase (CDK) activity in the interval between DNA damage and induction of the CDK inhibitor p21. These observations necessitate a revised model of DNA damage response in G1 phase and indicate that cells have a G1 checkpoint.

Cognitive and antismoking effects of varenicline in patients with schizophrenia or schizoaffective disorder.

OBJECTIVE: Varenicline has been shown to be an effective anti-smoking treatment in smokers without identified psychiatric illness, and the drug's pharmacology suggests possibilities of pro-cognitive effects. However, recent reports suggest varenicline may have the potential for important psychiatric side-effects in some people. We present the first prospective quantitative data on the effects of varenicline on cognitive function, cigarette smoking, and psychopathology in a small sample of schizophrenic patients. METHOD: Fourteen schizophrenic smokers were enrolled in an open-label study of varenicline with a pre-post design. Measures of cognitive function (RBANS, Virtual Water-Maze Task), cigarette smoking (cotinine levels, CO levels, self-reported smoking and smoking urges), and psychopathology (PANSS) were evaluated prior to and during treatment with varenicline. Data on psychopathology changes among schizophrenic smokers in another drug study, in which patients were not receiving varenicline, were used for comparison. RESULTS: 12 patients completed the study, and 2 patients terminated in the first two weeks of active varenicline because of complaints of nausea or shaking. Varenicline produced significant improvements in some cognitive test scores, primarily associated with verbal learning and memory, but not in scores on visual-spatial learning or memory, or attention. Varenicline significantly decreased all indices of smoking, but did not produce complete smoking abstinence in most patients. During treatment with varenicline there were no significant increases in psychopathology scores and no patient developed signs of clinical depression or suicidal ideation. CONCLUSIONS: Our small prospective study suggests that treatment with varenicline appears to have some beneficial cognitive effects which need to be confirmed in larger studies with additional neuropsychological tests. Varenicline appears to have some anti-smoking efficacy in schizophrenia but longer studies are needed to determine whether it will produce rates of smoking abstinence similar to those found in control smokers. Treatment with varenicline may not increase psychopathology or depression in most patients with schizophrenia, but we cannot accurately estimate the absolute risk of a potentially rare side-effect from this small sample.