NUCLEOCYTOSKELETAL CONNECTIONS IN MECHANOTRANSDUCTION

Cells continuously sense and respond to their physical surroundings through their cytoskeleton. The perinuclear actin cap (or actin cap) is a recently-characterized cytoskeletal organelle composed of thick, parallel, and highly contractile actomyosin filaments that are specifically anchored to the apical surface of the interphase nucleus. The actin cap is present in many types of adherent eukaryotic cells but is disrupted in several human disease models, including laminopathies and cancer. Through its large terminating focal adhesions and anchorage to the nuclear lamina and nuclear envelope through LINC (Linkers of the Nucleoskeleton to the Cytoskeleton) complexes, the perinuclear actin cap plays a critical role in mechanotransduction, the ability of cells to sense and respond to mechanical forces. In this work, I demonstrate that only fibers of the actin cap, not conventional basal actin stress fibers, form in response to low physiological mechanical stresses in adherent fibroblasts. While conventional basal stress fibers form only past a threshold shear stress of 0.5 dyn/cm2, actin-cap fibers form at shear stresses 50 times lower and orders-of-magnitude faster than biochemical stimulation. This fast differential response is uniquely mediated by focal adhesion protein zyxin at low shear stress and actomyosin contractility of the actin cap. I identify additional roles for lamin A/C of the nuclear lamina and LINC molecules nesprin2giant and nesprin3, which anchor actin cap fibers to the nucleus. I briefly explore mechanotransduction of interstitial fluid flow within a three-dimensional culture system. Next, I seek to characterize the extent of mechanotransduction in the nucleus through a novel microscopy assay that rapidly quantifies global acetylation on histone H3 and measures several cell and nuclear properties, including cell and nuclear morphology descriptors, cell-cycle phase, and filamentous-actin content of thousands of cells simultaneously, without cell detachment from the substrate, at single-cell resolution. These measurements reveal that isogenic, isotypic cells of identical DNA content and the same cell-cycle phase can still display large variations in H3 acetylation and that these variations correlate with specific phenotypic variations, in particular, nuclear size and actin cytoskeleton content, but not cell shape. The dependence of cell and nuclear properties on cell-cycle phase is assessed without artifact-prone cell synchronization. To further demonstrate the versatility of this assay, I quantify the complex interplay among cell cycle, epigenetic modifications, and phenotypic variations following pharmacological treatments targeting DNA integrity, cell cycle, and chromatin-modifying enzymes. Finally, recent literature suggests that the actin filament network regulates epigenetics that determine DNA packing in the nucleus and ultimately gene transcription, especially by way of histone modifications. However, a molecular mechanism underlying these cytoskeleton-based histone modifications is missing. Here, I hypothesize that the LINC complex proteins that physically connect the cytoskeleton to the nuclear lamina at the nuclear envelope are key mediators of histone modifications. Using the newly established high-throughput single-cell phenotyping method, I quantitatively examine actin filament content and organization in the cytoplasm, nuclear morphology, nuclear lamina and LINC protein expression and organization at the nuclear membrane, and histone acetylation and methylation of specific residues in the same individual cells simultaneously. I conclude that LINC complex proteins nesprin2giant and nesprin3, as well as lamin A/C of the nuclear lamina, regulate these histone modifications in a very complex manner. Taken together, all of these results suggest an interconnected pathway for mechanotransduction that physically connects the extracellular milieu to the nucleus

NUCLEOCYTOSKELETAL CONNECTIONS IN MECHANOTRANSDUCTION

Cells continuously sense and respond to their physical surroundings through their cytoskeleton. The perinuclear actin cap (or actin cap) is a recently-characterized cytoskeletal organelle composed of thick, parallel, and highly contractile actomyosin filaments that are specifically anchored to the apical surface of the interphase nucleus. The actin cap is present in many types of adherent eukaryotic cells but is disrupted in several human disease models, including laminopathies and cancer. Through its large terminating focal adhesions and anchorage to the nuclear lamina and nuclear envelope through LINC (Linkers of the Nucleoskeleton to the Cytoskeleton) complexes, the perinuclear actin cap plays a critical role in mechanotransduction, the ability of cells to sense and respond to mechanical forces. In this work, I demonstrate that only fibers of the actin cap, not conventional basal actin stress fibers, form in response to low physiological mechanical stresses in adherent fibroblasts. While conventional basal stress fibers form only past a threshold shear stress of 0.5 dyn/cm2, actin-cap fibers form at shear stresses 50 times lower and orders-of-magnitude faster than biochemical stimulation. This fast differential response is uniquely mediated by focal adhesion protein zyxin at low shear stress and actomyosin contractility of the actin cap. I identify additional roles for lamin A/C of the nuclear lamina and LINC molecules nesprin2giant and nesprin3, which anchor actin cap fibers to the nucleus. I briefly explore mechanotransduction of interstitial fluid flow within a three-dimensional culture system. Next, I seek to characterize the extent of mechanotransduction in the nucleus through a novel microscopy assay that rapidly quantifies global acetylation on histone H3 and measures several cell and nuclear properties, including cell and nuclear morphology descriptors, cell-cycle phase, and filamentous-actin content of thousands of cells simultaneously, without cell detachment from the substrate, at single-cell resolution. These measurements reveal that isogenic, isotypic cells of identical DNA content and the same cell-cycle phase can still display large variations in H3 acetylation and that these variations correlate with specific phenotypic variations, in particular, nuclear size and actin cytoskeleton content, but not cell shape. The dependence of cell and nuclear properties on cell-cycle phase is assessed without artifact-prone cell synchronization. To further demonstrate the versatility of this assay, I quantify the complex interplay among cell cycle, epigenetic modifications, and phenotypic variations following pharmacological treatments targeting DNA integrity, cell cycle, and chromatin-modifying enzymes. Finally, recent literature suggests that the actin filament network regulates epigenetics that determine DNA packing in the nucleus and ultimately gene transcription, especially by way of histone modifications. However, a molecular mechanism underlying these cytoskeleton-based histone modifications is missing. Here, I hypothesize that the LINC complex proteins that physically connect the cytoskeleton to the nuclear lamina at the nuclear envelope are key mediators of histone modifications. Using the newly established high-throughput single-cell phenotyping method, I quantitatively examine actin filament content and organization in the cytoplasm, nuclear morphology, nuclear lamina and LINC protein expression and organization at the nuclear membrane, and histone acetylation and methylation of specific residues in the same individual cells simultaneously. I conclude that LINC complex proteins nesprin2giant and nesprin3, as well as lamin A/C of the nuclear lamina, regulate these histone modifications in a very complex manner. Taken together, all of these results suggest an interconnected pathway for mechanotransduction that physically connects the extracellular milieu to the nucleus

Immune biomarkers associated with COVID-19 disease severity in an urban, hospitalized population

Objectives: We sought to identify immune biomarkers associated with severe Coronavirus disease 2019 (COVID-19) in patients admitted to a large urban hospital during the early phase of the SARS-CoV-2 pandemic. Design: The study population consisted of SARS-CoV-2 positive subjects admitted for COVID-19 (n = 58) or controls (n = 14) at the Los Angeles County University of Southern California Medical Center between April 2020 through December 2020. Immunologic markers including chemokine/cytokines (IL-6, IL-8, IL-10, IP-10, MCP-1, TNF-α) and serologic markers against SARS-CoV-2 antigens (including spike subunits S1 and S2, receptor binding domain, and nucleocapsid) were assessed in serum collected on the day of admission using bead-based multiplex immunoassay panels. Results: We observed that body mass index (BMI) and SARS-CoV-2 antibodies were significantly elevated in patients with the highest COVID-19 disease severity. IP-10 was significantly elevated in COVID-19 patients and was associated with increased SARS-CoV-2 antibodies. Interactions among all available variables on COVID-19 disease severity were explored using a linear support vector machine model which supported the importance of BMI and SARS-CoV-2 antibodies. Conclusions: Our results confirm the known adverse association of BMI on COVID-19 severity and suggest that IP-10 and SARS-CoV-2 antibodies could be useful to identify patients most likely to experience the most severe forms of the disease

Side-effects of COVID-19 on patient care: An INR story

BackgroundNumerous studies have documented reduced access to patient care due to the COVID-19 pandemic, including access to diagnostic or screening tests, prescription medications, and treatment for an ongoing condition. In the context of clinical management for venous thromboembolism, this could result in suboptimal therapy with warfarin. We aimed to determine the impact of the pandemic on utilization of International Normalized Ratio (INR) testing and the percentage of high and low results.MethodsINR data from 11 institutions were extracted to compare testing volume and the percentage of INR results ≥3.5 and ≤1.5 between a pre-pandemic period (January-June 2019, period 1) and a portion of the COVID-19 pandemic period (January-June 2020, period 2). The analysis was performed for inpatient and outpatient cohorts.ResultsTesting volumes showed relatively little change in January and February, followed by a significant decrease in March, April, and May, and then returned to baseline in June. Outpatient testing showed a larger percentage decrease in testing volume compared to inpatient testing. At 10 of the 11 study sites, we observed an increase in the percentage of abnormal high INR results as test volumes decreased, primarily among outpatients.ConclusionThe COVID-19 pandemic impacted INR testing among outpatients which may be attributable to several factors. Increased supratherapeutic INR results during the pandemic period when there was reduced laboratory utilization and access to care is concerning because of the risk of adverse bleeding events in this group of patients. This could be mitigated in the future by offering drive-through testing and/or widespread implementation of home INR monitoring

Limited Evidence for Use of a Black Race Modifier in eGFR Calculations: A Systematic Review.

BACKGROUND Commonly used estimated glomerular filtration rate (eGFR) equations include a Black race modifier (BRM) that was incorporated during equation derivation. Race is a social construct, and a poorly characterized variable that is applied inconsistently in clinical settings. The BRM results in higher eGFR for any creatinine concentration, implying fundamental differences in creatinine production or excretion in Black individuals compared to other populations. Equations without inclusion of the BRM have the potential to detect kidney disease earlier in patients at the greatest risk of chronic kidney disease (CKD), but also has the potential to over-diagnose CKD or impact downstream clinical interventions. The purpose of this study was to use an evidence-based approach to systematically evaluate the literature relevant to the performance of the eGFR equations with and without the BRM and to examine the clinical impact of the use or removal. CONTENT PubMed and Embase databases were searched for studies comparing measured GFR to eGFR in racially diverse adult populations using the Modification of Diet in Renal Disease or the 2009-Chronic Kidney Disease Epidemiology Collaboration-creatinine equations based on standardized creatinine measurements. Additionally, we searched for studies comparing clinical use of eGFR calculated with and without the BRM. Here, 8632 unique publications were identified; an additional 3 studies were added post hoc. In total, 96 studies were subjected to further analysis and 44 studies were used to make a final assessment. SUMMARY There is limited published evidence to support the use of a BRM in eGFR equations

The effect of the Covid-19 shutdown on glycemic testing and control

BackgroundThe coronavirus disease 2019 (COVID-19) pandemic caused a halt to in-person ambulatory care. We evaluated how the reduction in access to care affected HbA1c testing and patient HbA1c levels.MethodsHbA1c data from 11 institutions were extracted to compare testing volume and the percentage of abnormal results between a pre-pandemic period (January-June 2019, period 1) and a portion of the COVID-19 pandemic period (Jan-June 2020, period 2). HbA1c results greater than 6.4% were categorized as abnormal.ResultsHbA1C testing volumes decreased in March, April and May by 23, 61 and 40% relative to the corresponding months in 2019. The percentage of abnormal results increased in April, May and June (25, 23, 9%). On average, we found that the frequency of abnormal results increased by 0.31% for every 1% decrease in testing volume (p < 0.0005).ConclusionHbA1c testing volume for outpatients decreased by up to 70% during the early months of the pandemic. The decrease in testing was associated with an increase in abnormal HbA1c results

The effect of the Covid-19 shutdown on glycemic testing and control

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic caused a halt to in-person ambulatory care. We evaluated how the reduction in access to care affected HbA1c testing and patient HbA1c levels. METHODS: HbA1c data from 11 institutions were extracted to compare testing volume and the percentage of abnormal results between a pre-pandemic period (January-June 2019, period 1) and a portion of the COVID-19 pandemic period (Jan-June 2020, period 2). HbA1c results greater than 6.4% were categorized as abnormal. RESULTS: HbA1C testing volumes decreased in March, April and May by 23, 61 and 40% relative to the corresponding months in 2019. The percentage of abnormal results increased in April, May and June (25, 23, 9%). On average, we found that the frequency of abnormal results increased by 0.31% for every 1% decrease in testing volume (p \u3c 0.0005). CONCLUSION: HbA1c testing volume for outpatients decreased by up to 70% during the early months of the pandemic. The decrease in testing was associated with an increase in abnormal HbA1c results

Cannabis positivity rates in 17 emergency departments across the United States with varying degrees of marijuana legalization.

BACKGROUND: Many states in the United States have progressed towards legalization of marijuana including decriminalization, medicinal and/or recreational use. We studied the impact of legalization on cannabis-related emergency department visits in states with varying degrees of legalization. METHODS: Seventeen healthcare institutions in fifteen states (California, Colorado, Connecticut, Florida, Iowa, Kentucky, Maryland, Massachusetts, Missouri, New Hampshire, Oregon, South Carolina, Tennessee, Texas, Washington) participated. Cannabinoid immunoassay results and cannabis-related International Classification of Diseases (ninth and tenth versions) codes were obtained for emergency department visits over a 3- to 8-year period during various stages of legalization: no state laws, decriminalized, medical approval before dispensaries, medical dispensaries available, recreational approval before dispensaries and recreational dispensaries available. Trends and monthly rates of cannabinoid immunoassay and cannabis-related International Classification of Diseases code positivity were determined during these legalization periods. RESULTS: For most states, there was a significant increase in both cannabinoid immunoassay and International Classification of Diseases code positivity as legalization progressed; however, positivity rates differed. The availability of dispensaries may impact positivity in states with medical and/or recreational approval. In most states with no laws, there was a significant but smaller increase in cannabinoid immunoassay positivity rates. CONCLUSIONS: States may experience an increase in cannabis-related emergency department visits with progression toward marijuana legalization. The differences between states, including those in which no impact was seen, are likely multifactorial and include cultural norms, attitudes of local law enforcement, differing patient populations, legalization in surrounding states, availability of dispensaries, various ordering protocols in the emergency department, and the prevalence of non-regulated cannabis products