Chromosomal instability induced in cancer can enhance macrophage-initiated immune responses that include anti-tumor IgG.

Solid tumors generally exhibit chromosome copy number variation, which is typically caused by chromosomal instability (CIN) in mitosis. The resulting aneuploidy can drive evolution and associates with poor prognosis in various cancer types as well as poor response to T-cell checkpoint blockade in melanoma. Macrophages and the SIRPα-CD47 checkpoint are understudied in such contexts. Here, CIN is induced in poorly immunogenic B16F10 mouse melanoma cells using spindle assembly checkpoint MPS1 inhibitors that generate persistent micronuclei and diverse aneuploidy while skewing macrophages toward a tumoricidal 'M1-like' phenotype based on markers and short-term anti-tumor studies. Mice bearing CIN-afflicted tumors with wild-type CD47 levels succumb similar to controls, but long-term survival is maximized by SIRPα blockade on adoptively transferred myeloid cells plus anti-tumor monoclonal IgG. Such cells are the initiating effector cells, and survivors make de novo anti-cancer IgG that not only promote phagocytosis of CD47-null cells but also suppress tumor growth. CIN does not affect the IgG response, but pairing CIN with maximal macrophage anti-cancer activity increases durable cures that possess a vaccination-like response against recurrence.

Lipid Nanoparticle-Associated Inflammation is Triggered by Sensing of Endosomal Damage: Engineering Endosomal Escape Without Side Effects.

Lipid nanoparticles (LNPs) have emerged as the dominant platform for RNA delivery, based on their success in the COVID-19 vaccines and late-stage clinical studies in other indications. However, we and others have shown that LNPs induce severe inflammation, and massively aggravate pre-existing inflammation. Here, using structure-function screening of lipids and analyses of signaling pathways, we elucidate the mechanisms of LNP-associated inflammation and demonstrate solutions. We show that LNPs' hallmark feature, endosomal escape, which is necessary for RNA expression, also directly triggers inflammation by causing endosomal membrane damage. Large, irreparable, endosomal holes are recognized by cytosolic proteins called galectins, which bind to sugars on the inner endosomal membrane and then regulate downstream inflammation. We find that inhibition of galectins abrogates LNP-associated inflammation, both in vitro and in vivo . We show that rapidly biodegradable ionizable lipids can preferentially create endosomal holes that are smaller in size and reparable by the endosomal sorting complex required for transport (ESCRT) pathway. Ionizable lipids producing such ESCRT-recruiting endosomal holes can produce high expression from cargo mRNA with minimal inflammation. Finally, we show that both routes to non-inflammatory LNPs, either galectin inhibition or ESCRT-recruiting ionizable lipids, are compatible with therapeutic mRNAs that ameliorate inflammation in disease models. LNPs without galectin inhibition or biodegradable ionizable lipids lead to severe exacerbation of inflammation in these models. In summary, endosomal escape induces endosomal membrane damage that can lead to inflammation. However, the inflammation can be controlled by inhibiting galectins (large hole detectors) or by using biodegradable lipids, which create smaller holes that are reparable by the ESCRT pathway. These strategies should lead to generally safer LNPs that can be used to treat inflammatory diseases.

Chromosomal instability can favor macrophage-mediated immune response and induce a broad, vaccination-like anti-tumor IgG response.

Chromosomal instability (CIN), a state in which cells undergo mitotic aberrations that generate chromosome copy number variations, generates aneuploidy and is thought to drive cancer evolution. Although associated with poor prognosis and reduced immune response, CIN generates aneuploidy-induced stresses that could be exploited for immunotherapies. In such contexts, macrophages and the CD47-SIRPα checkpoint are understudied. Here, CIN is induced pharmacologically induced in poorly immunogenic B16F10 mouse melanoma cells, generating persistent micronuclei and diverse aneuploidy while skewing macrophages towards an anti-cancer M1-like phenotype, based on RNA-sequencing profiling, surface marker expression and short-term antitumor studies. These results further translate to in vivo efficacy: Mice bearing CIN-afflicted tumors with wild-type CD47 levels survive only slightly longer relative to chromosomally stable controls, but long-term survival is maximized when combining macrophage-stimulating anti-tumor IgG opsonization and some form of disruption of the CD47-SIRPα checkpoint. Survivors make multi-epitope, de novo anti-cancer IgG that promote macrophage-mediated phagocytosis of CD47 knockout B16F10 cells and suppress tumoroids in vitro and growth of tumors in vivo . CIN does not greatly affect the level of the IgG response compared to previous studies but does significantly increase survival. These results highlight an unexpected therapeutic benefit from CIN when paired with maximal macrophage anti-cancer activity: an anti-cancer vaccination-like antibody response that can lead to more durable cures and further potentiate cell-mediated acquired immunity.

Differences in cell shape, motility, and growth reflect chromosomal number variations that can be visualized with live-cell ChReporters.

Chromosome numbers often change dynamically in tumors and cultured cells, which complicates therapy as well as understanding genotype-mechanotype relationships. Here we use a live-cell "ChReporter" method to identify cells with a single chromosomal loss in efforts to better understand differences in cell shape, motility, and growth. We focus on a standard cancer line and first show clonal populations that retain the ChReporter exhibit large differences in cell and nuclear morphology as well as motility. Phenotype metrics follow simple rules, including migratory persistence scaling with speed, and cytoskeletal differences are evident from drug responses, imaging, and single-cell RNA sequencing. However, mechanotype-genotype relationships between fluorescent ChReporter-positive clones proved complex and motivated comparisons of clones that differ only in loss or retention of a Chromosome-5 ChReporter. When lost, fluorescence-null cells show low expression of Chromosome-5 genes, including a key tumor suppressor APC that regulates microtubules and proliferation. Colonies are compact, nuclei are rounded, and cells proliferate more, with drug results implicating APC, and patient survival data indicating an association in multiple tumor-types. Visual identification of genotype with ChReporters can thus help clarify mechanotype and mechano-evolution.

Confinement plus myosin-II suppression maximizes heritable loss of chromosomes, as revealed by live-cell ChReporters.

The mechanical environment of a cell can have many effects, but whether it impacts the DNA sequence of a cell has remained unexamined. To investigate this, we developed a live-cell method to measure changes in chromosome numbers. We edited constitutive genes with GFP or RFP tags on single alleles and discovered that cells that lose Chromosome reporters (ChReporters) become non-fluorescent. We applied our new tools to confined mitosis and to inhibition of the putative tumor suppressor myosin-II. We quantified compression of mitotic chromatin in vivo and demonstrated that similar compression in vitro resulted in cell death, but also rare and heritable ChReptorter loss. Myosin-II suppression rescued lethal multipolar divisions and maximized ChReporter loss during three-dimensional (3D) compression and two-dimensional (2D) lateral confinement, but not in standard 2D culture. ChReporter loss was associated with chromosome mis-segregation, rather than just the number of divisions, and loss in vitro and in mice was selected against in subsequent 2D cultures. Inhibition of the spindle assembly checkpoint (SAC) caused ChReporter loss in 2D culture, as expected, but not during 3D compression, suggesting a SAC perturbation. Thus, ChReporters enable diverse studies of viable genetic changes, and show that confinement and myosin-II affect DNA sequence and mechano-evolution.

Small lipid droplets are rigid enough to indent a nucleus, dilute the lamina, and cause rupture.

The nucleus in many cell types is a stiff organelle, but fat-filled lipid droplets (FDs) in cytoplasm are seen to indent and displace the nucleus. FDs are phase-separated liquids with a poorly understood interfacial tension γ that determines how FDs interact with other organelles. Here, micron-sized FDs remain spherical as they indent peri-nuclear actomyosin and the nucleus, while causing local dilution of Lamin-B1 independent of Lamin-A,C and sometimes triggering nuclear rupture. Focal accumulation of the cytosolic DNA sensor cGAS at the rupture site is accompanied by sustained mislocalization of DNA repair factors to cytoplasm, increased DNA damage, and delayed cell cycle. Macrophages show FDs and engulfed rigid beads cause similar indentation dilution. Spherical shapes of small FDs indicate a high γ, which we measure for FDs mechanically isolated from fresh adipose tissue as ∼40 mN/m. This value is far higher than that of protein condensates, but typical of oils in water and sufficiently rigid to perturb cell structures including nuclei.

Rare Adult-onset Citrullinemia Type 1 in the Postpartum Period: A Case Report.

INTRODUCTION: Citrullinemia type 1 (CTLN1) is a urea cycle disorder caused by defective argininosuccinate synthetase leading to impaired ammonia elimination. Urea cycle disorders are typically diagnosed on neonatal screening but rarely can lie dormant until a metabolic stressor causes initial onset of symptoms in adulthood. CASE REPORT: A 23-year-old female presented four days postpartum to the emergency department (ED) obtunded and declined to the point of requiring intubation. Labs revealed hyperammonemia, and she was subsequently found to have CTLN1. CONCLUSION: Urea cycle disorders presenting in adulthood are a rare etiology for the common ED complaint of altered mental status. The low incidence makes these treatable disorders easy to overlook leading to potentially significant morbidity and mortality. Therefore, it is important to recognize the risk factors that can trigger an acute metabolic derangement. This case highlights common risk factors for metabolic stress, possible presenting symptoms, and the positive outcome achievable when recognized and treated in a timely fashion.

CD47-SIRPα Checkpoint Disruption in Metastases Requires Tumor-Targeting Antibody for Molecular and Engineered Macrophage Therapies.

The macrophage checkpoint interaction CD47-SIRPα is an emerging target for cancer therapy, but clinical trials of monoclonal anti-CD47 show efficacy only in liquid tumors when combined with tumor-opsonizing IgG. Here, in challenging metastatic solid tumors, CD47 deletion shows no effect on tumor growth unless combined with otherwise ineffective tumor-opsonization, and we likewise show wild-type metastases are suppressed by SIRPα-blocked macrophages plus tumor-opsonization. Lung tumor nodules of syngeneic B16F10 melanoma cells with CD47 deletion show opsonization drives macrophage phagocytosis of B16F10s, consistent with growth versus phagocytosis calculus for exponential suppression of cancer. Wild-type CD47 levels on metastases in lungs of immunocompetent mice and on human metastases in livers of immunodeficient mice show that systemic injection of antibody-engineered macrophages also suppresses growth. Such in vivo functionality can be modulated by particle pre-loading of the macrophages. Thus, even though CD47-SIRPα disruption and tumor-opsonizing IgG are separately ineffective against established metastatic solid tumors, their combination in molecular and cellular therapies prolongs survival.

Suppressing or Enhancing Macrophage Engulfment through the Use of CD47 and Related Peptides.

Foreign particles and microbes are rapidly cleared by macrophages in vivo, although many key aspects of uptake mechanisms remain unclear. "Self" cells express CD47 which functions as an anti-phagocytic ligand for SIRPα on macrophages, particularly when pro-phagocytic ligands such as antibodies are displayed in parallel. Here, we review CD47 and related "Self" peptides as modulators of macrophage uptake. Nanoparticles conjugated with either CD47 or peptides derived from its SIRPα binding site can suppress phagocytic uptake by macrophages in vitro and in vivo, with similar findings for CD47-displaying viruses. Drugs, dyes, and genes as payloads thus show increased delivery to targeted cells. On the other hand, CD47 expression by cancer cells enables such cells to evade macrophages and immune surveillance. This has motivated development of soluble antagonists to CD47-SIRPα, ranging from blocking antibodies in the clinic to synthetic peptides in preclinical models. CD47 and peptides are thus emerging as dual-use phagocytosis modulators against diseases.

Gaussian curvature dilutes the nuclear lamina, favoring nuclear rupture, especially at high strain rate.

Nuclear rupture has long been associated with deficits or defects in lamins, with recent results also indicating a role for actomyosin stress, but key physical determinants of rupture remain unclear. Here, lamin-B filaments stably interact with the nuclear membrane at sites of low Gaussian curvature yet dilute at high curvature to favor rupture, whereas lamin-A depletion requires high strain-rates. Live-cell imaging of lamin-B1 gene-edited cancer cells is complemented by fixed-cell imaging of rupture in: iPS-derived progeria patients cells, cells within beating chick embryo hearts, and cancer cells with multi-site rupture after migration through small pores. Data fit a model of stiff filaments that detach from a curved surface.Rupture is modestly suppressed by inhibiting myosin-II and by hypotonic stress, which slow the strain-rates. Lamin-A dilution and rupture probability indeed increase above a threshold rate of nuclear pulling. Curvature-sensing mechanisms of proteins at plasma membranes, including Piezo1, might thus apply at nuclear membranes.Summary statement: High nuclear curvature drives lamina dilution and nuclear envelope rupture even when myosin stress is inhibited. Stiff filaments generally dilute from sites of high Gaussian curvature, providing mathematical fits of experiments.

New Versus Old, The i-View Video Laryngoscope Versus the GlideScope: A Prospective, Randomized, Crossover Trial.

BACKGROUND: A novel video laryngoscope device, the i-view, may extend intubation capability to the lowest echelons of deployed military medicine. The i-view is a one-time use, disposable laryngoscope. We compared time to completion of endotracheal intubation (ETI) between the i-view and GlideScope among military emergency medicine providers in a simulation setting. METHODS: We conducted a prospective, randomized, crossover trial. We randomized participants to i-view or GlideScope first before they performed 2 ETI-1 with each device. The primary outcome was time to completion of ETI. Secondary outcomes included first-pass success, optimal glottic view, and end-user appraisal. We used a Laerdal Airway Management Trainer for all intubations. RESULTS: Thirty-three emergency medicine providers participated. ETI time was less with GlideScope than i-view (22.2 +/- 9.0 seconds versus 30.2 +/- 24.0 seconds; p=0.048). Optimal glottic views, using the Cormack-Lehan scale, also favored the GlideScope (2 [1,2] versus 2[2,2]; p=0.044). There was no difference in first-pass success rates (100% versus 100%). More participants preferred the GlideScope (24 versus 9; p=0.165); however, participants agreed that the i-view would be easier to use than the GlideScope in an austere environment (4[4,5]). CONCLUSIONS: We found the GlideScope outperformed the i-view with respect to procedural completion time. Participants preferred the GlideScope over i-view, but indicated the i-view would be easier to use than the GlideScope in an austere setting. Our findings suggest the i-view may be a suitable alternative to GlideScope for US military providers, especially for those in the prehospital setting.

Optimal allocation in annual plants with density-dependent fitness.

We study optimal two-sector (vegetative and reproductive) allocation models of annual plants in temporally variable environments that incorporate effects of density-dependent lifetime variability and juvenile mortality in a fitness function whose expected value is maximized. Only special cases of arithmetic and geometric mean maximizers have previously been considered in the literature, and we also allow a wider range of production functions with diminishing returns. The model predicts that the time of maturity is pushed to an earlier date as the correlation between individual lifetimes increases, and while optimal schedules are bang-bang at the extremes, the transition is mediated by schedules where vegetative growth is mixed with reproduction for a wide intermediate range. The mixed growth lasts longer when the production function is less concave allowing for better leveraging of plant size when generating seeds. Analytic estimates are obtained for the power means that interpolate between arithmetic and geometric mean and correspond to partially correlated lifetime distributions.

The inhibitory impact of ammonia on thermally hydrolyzed sludge fed anaerobic digestion.

This study evaluated the impact of ammonia on mesophilic anaerobic digestion (AD) with thermal hydrolysis pretreatment (THP) treating a mixture of primary sludge and waste activated sludge and operated under constant organic loading rate of 9 kg COD/m3 /d. Free ammonia concentrations in the digesters were varied between 37 and 966 mg NH3 -N/L, while maintaining all other operational conditions constant. A decrease in volatile solids reduction from 54 ± 5% (at <554 mg NH3 -N/L) to 35 ± 6% at the maximum free ammonia concentration of 966 mg NH3 -N/L was observed at steady-state conditions. No impact of free ammonia on final dewaterability was detected. Free ammonia thus mostly limited methanogenesis. A free ammonia Monod inhibition constant of 847 ± 222 mg NH3 -N/L for methanogens was estimated based on the digester steady-state methane rates dynamics. This study showed that current THP AD digesters (typically 110-260 mg NH3 -N/L) operate under 12%-18% ammonia inhibition for methanogenesis. Operation under SRT of 15 days, about 2 times more than needed to retain methanogens, can compensate for lower methanogens rates and avoid performance impacts. The later shows a good potential to operate under higher free and total ammonia concentration without jeopardizing performance. PRACTITIONER POINTS: Only from a free ammonia concentration above 554 mg NH3 -N/L, decreased volatile solids reduction and biogas yield were observed. A volatile solids reduction of 35 ± 6% at maximum free ammonia concentration of 966 mg NH3 -N/L was still achieved. A Monod inhibition constant for methanogens of 847 ± 222 mg NH3 -N/L was estimated. It was estimated that current THP AD systems (110-260 mg NH3 -N/L) operate under 12%-18% NH3 inhibition for methanogenesis.

Starch storage capacity of sapwood is related to dehydration avoidance during drought.

PREMISE: The xylem tissue of plants performs three principal functions: transport of water, support of the plant body, and nutrient storage. Tradeoffs may arise because different structural requirements are associated with different functions or because suites of traits are under selection that relate to resource acquisition, use, and turnover. The structural and functional basis of xylem storage is not well established. We hypothesized that greater starch storage would be associated with greater sapwood parenchyma and reduced fibers, which would compromise resistance to xylem tensions during dehydration. METHODS: We measured cavitation resistance, minimum water potential, starch content, and sapwood parenchyma and fiber area in 30 species of southern California chaparral shrubs (evergreen and deciduous). RESULTS: We found that species storing greater starch within their xylem tended to avoid dehydration and were less cavitation resistant, and this was supported by phylogenetic independent contrasts. Greater sapwood starch was associated with greater parenchyma area and reduced fiber area. For species without living fibers, the associations with parenchyma were stronger, suggesting that living fibers may expand starch storage capacity while also contributing to the support function of the vascular tissue. Drought-deciduous species were associated with greater dehydration avoidance than evergreens. CONCLUSIONS: Evolutionary forces have led to an association between starch storage and dehydration resistance as part of an adaptive suite of traits. We found evidence for a tradeoff between tissue mechanical traits and starch storage; moreover, the evolution of novel strategies, such as starch-storing living fibers, may mitigate the strength of this tradeoff.

Canalicular system reorganization during mouse platelet activation as revealed by 3D ultrastructural analysis.

The canalicular system (CS) has been defined as: 1) an inward, invaginated membrane connector that supports entry into and exit from the platelet; 2) a static structure stable during platelet isolation; and 3) the major source of plasma membrane (PM) for surface area expansion during activation. Recent analysis from STEM tomography and serial block face electron microscopy has challenged the relative importance of CS as the route for granule secretion. Here, We used 3D ultrastructural imaging to reexamine the CS in mouse platelets by generating high-resolution 3D reconstructions to test assumptions 2 and 3. Qualitative and quantitative analysis of whole platelet reconstructions, obtained from immediately fixed or washed platelets fixed post-washing, indicated that CS, even in the presence of activation inhibitors, reorganized during platelet isolation to generate a more interconnected network. Further, CS redistribution into the PM at different times, post-activation, appeared to account for only about half the PM expansion seen in thrombin-activated platelets, in vitro, suggesting that CS reorganization is not sufficient to serve as a dominant membrane reservoir for activated platelets. In sum, our analysis highlights the need to revisit past assumptions about the platelet CS to better understand how this membrane system contributes to platelet function.

Unusual Case of Acute Decompensated Maple Syrup Urine Disease in the Emergency Department.

A case report of an army soldier presenting to the emergency department with acute metabolic derangement resulting in encephalopathy, cerebral edema, and death is presented. The patient had no medical diagnoses before entering military service and was triaged in the emergency department with the common complaint of presyncope. However, as encephalopathy worsened, the patient experienced altered mental status, lethargy, emesis, and seizure. This patient ultimately died because of rapid decompensation. Maple syrup urine disease pathophysiology and treatment recommendations are reviewed.

Structural analysis of resting mouse platelets by 3D-EM reveals an unexpected variation in α-granule shape.

Mice and mouse platelets are major experimental models for hemostasis and thrombosis; however, important physiological data from this model has received little to no quantitative, 3D ultrastructural analysis. We used state-of-the-art, serial block imaging scanning electron microscopy (SBF-SEM, nominal Z-step size was 35 nm) to image resting platelets from C57BL/6 mice. α-Granules were identified morphologically and rendered in 3D space. The quantitative analysis revealed that mouse α-granules typically had a variable, elongated, rod shape, different from the round/ovoid shape of human α-granules. This variation in length was confirmed qualitatively by higher-resolution, focused ion beam (FIB) SEM at a nominal 5 nm Z-step size. The unexpected α-granule shape raises novel questions regarding α-granule biogenesis and dynamics. Does the variation arise at the level of the megakaryocyte and α-granule biogenesis or from differences in α-granule dynamics and organelle fusion/fission events within circulating platelets? Further quantitative analysis revealed that the two major organelles in circulating platelets, α-granules and mitochondria, displayed a stronger linear relationship between organelle number/volume and platelet size, i.e., a scaling in number and volume to platelet size, than found in human platelets suggestive of a tighter mechanistic regulation of their inclusion during platelet biogenesis. In conclusion, the overall spatial arrangement of organelles within mouse platelets was similar to that of resting human platelets, with mouse α-granules clustered closely together with little space for interdigitation of other organelles.

Utility of Nonspecific Laboratory Testing for Psychiatric Patients Undergoing Medical Screening in a Military Emergency Department.

INTRODUCTION: Psychiatric complaints account for a sizable and increasing portion of emergency department (ED) visits. Compared with other medical patients, these patients often require substantial resources because of limited specialized resources and prolonged boarding times, which can be detrimental to the safety and satisfaction of other patients. This can prompt early and indiscriminate laboratory testing to expedite early requests for admission consideration. Numerous emergency medicine literature and clinical policies already recommend against indiscriminate screening labs for these patients, yet many psychiatric services require these tests. This study further evidences the limited clinical utility and high associated costs of mandatory protocol screening labs for psychiatric patients evaluated in military EDs. MATERIALS AND METHODS: A retrospective chart review of 441 active duty military patients and their families presenting to Madigan Army Medical Center's ED who received psychiatric diagnoses underwent analysis. A 3-physician review panel evaluated each identified patient case to confirm eligibility and determine whether or not laboratory studies led to a change in patient disposition that was not identified by history, review of systems, physical exam, and known past medical history. The review was approved by the hospital's institutional review board. Contemporary laboratory tests ordered in the evaluation of these patients included complete blood count with differential, complete metabolic panel, thyroid-stimulating hormone, serum ethanol, serum acetaminophen, serum salicylates, urine drug screening, urinalysis, urine human chorionic gonadotropin, and electrocardiograms. RESULTS: Broad screening labs may have altered dispositions for 0.9% (4) of patients. In total, 93% (202) of admitted patients were dispositioned to a psychiatric service. Of the 15 patients admitted to a medical service, 10 involved overdoses or intoxication. One patient had anemia in addition to opioid use disorder as diagnoses and was dispositioned to a medicine service. One pediatric patient was admitted for observation only. The remaining patients had diagnoses based on physical exam and history requiring medical service admission. In total, 7 patients had unknown dispositions, of which 4 carried solely psychiatric diagnoses. CONCLUSIONS: The cumulative reimbursement costs of broad testing in the studied population were estimated at $36,325.17 and rarely altered patient disposition. Further testing does not increase the incidence of disposition altering diagnoses and is associated with increased costs. When individual state laws and the clinical assessment by the responsible emergency physician are considered, future standardized ED lab screening evaluations of psychiatric patients in military EDs may be concentrated to breathalyzer alcohol level, urine drug screen, serum salicylates, serum acetaminophen, and urine human chorionic gonadotropin.

Recuperative thickening for sludge retention time and throughput management in anaerobic digestion with thermal hydrolysis pretreatment.

This study evaluated the application of recuperative thickening (RT) to enhance anaerobic digestion (AD) performance for AD systems with thermal hydrolysis pretreatment (THP). RT was applied for two different reasons: (a) for increasing the sludge retention time (SRT) to degrade slowly hydrolyzable materials more efficiently and (b) for maintaining SRT at decreased hydraulic retention time (HRT) thus showing potential for increased AD throughput rates. A SRT increase from 15 to 30 days by RT application did not improve AD performance or hydrolysis rates significantly as 15-day SRT was already a factor 2 higher than the estimated washout SRT. When applying RT to increase throughput rates (HRT of 7 days) while maintaining 15-day SRT, no negative impact on biogas production or hydrolysis kinetics was observed. It was estimated that RT application on THP digesters can increase digester throughput by 100% and thus show clear potential for further AD intensification. PRACTITIONER POINTS: Increased SRT from 15 to 30 days through recuperative thickening application did not improve biogas production. A lower required minimum SRT (6-7 days) was estimated in THP-AD systems compared to conventional AD. Operation at decreased HRT by RT application resulted in similar AD performance under constant organic loading rates. A 100% increase in throughput rates can be applied using RT without decreasing AD performance.

Rescue of DNA damage after constricted migration reveals a mechano-regulated threshold for cell cycle.

Migration through 3D constrictions can cause nuclear rupture and mislocalization of nuclear proteins, but damage to DNA remains uncertain, as does any effect on cell cycle. Here, myosin II inhibition rescues rupture and partially rescues the DNA damage marker γH2AX, but an apparent block in cell cycle appears unaffected. Co-overexpression of multiple DNA repair factors or antioxidant inhibition of break formation also exert partial effects, independently of rupture. Combined treatments completely rescue cell cycle suppression by DNA damage, revealing a sigmoidal dependence of cell cycle on excess DNA damage. Migration through custom-etched pores yields the same damage threshold, with ∼4-µm pores causing intermediate levels of both damage and cell cycle suppression. High curvature imposed rapidly by pores or probes or else by small micronuclei consistently associates nuclear rupture with dilution of stiff lamin-B filaments, loss of repair factors, and entry from cytoplasm of chromatin-binding cGAS (cyclic GMP-AMP synthase). The cell cycle block caused by constricted migration is nonetheless reversible, with a potential for DNA misrepair and genome variation.