Search

Background: The severity classification of functional mitral regurgitation (FMR) remains controversial despite adverse prognosis and rapidly evolving interventions. Furthermore, it is unclear if quantitative assessment with cardiac magnetic resonance can provide incremental risk stratification for patients with ischemic cardiomyopathy (ICM) or non-ICM (NICM) in terms of FMR and late gadolinium enhancement (LGE). We evaluated the impact of quantitative cardiac magnetic resonance parameters on event-free survival separately for ICM and NICM, to assess prognostic FMR thresholds and interactions with LGE quantification., Methods: Patients (n=1414) undergoing cardiac magnetic resonance for cardiomyopathy (ejection fraction<50%) assessment from April 1, 2001 to December 31, 2017 were evaluated. The primary end point was all-cause death, heart transplant, or left ventricular assist device implantation during follow-up. Multivariable Cox analyses were conducted to determine the impact of FMR, LGE, and their interactions with event-free survival., Results: There were 510 primary end points, 395/782 (50.5%) in ICM and 114/632 (18.0%) in NICM. Mitral regurgitation-fraction per 5% increase was independently associated with the primary end point, hazards ratios (95% CIs) of 1.04 (1.01-1.07; P =0.034) in ICM and 1.09 (1.02-1.16; P =0.011) in NICM. Optimal mitral regurgitation-fraction threshold for moderate and severe FMR were ≥20% and ≥35%, respectively, in both ICM and NICM, based on the prediction of the primary outcome. Similarly, optimal LGE thresholds were ≥5% in ICM and ≥2% in NICM. Mitral regurgitation-fraction×LGE emerged as a significant interaction for the primary end point in ICM ( P =0.006), but not in NICM ( P =0.971)., Conclusions: Mitral regurgitation-fraction and LGE are key quantitative cardiac magnetic resonance biomarkers with differential associations with adverse outcomes in ICM and NICM. Optimal prognostic thresholds may provide important clinical risk prognostication and may further facilitate the ability to derive selection criteria to guide therapeutic decision-making., Competing Interests: Disclosures None.

Autophagy is a cellular catabolic pathway generally thought to be neuroprotective. However, autophagy and in particular its upstream regulator, the ULK1 kinase, can also promote axonal degeneration. We examined the role and the mechanisms of autophagy in axonal degeneration using a mouse model of contusive spinal cord injury (SCI). Consistent with activation of autophagy during axonal degeneration following SCI, autophagosome marker LC3, ULK1 kinase, and ULK1 target, phospho-ATG13, accumulated in the axonal bulbs and injured axons. SARM1, a TIR NADase with a pivotal role in axonal degeneration, colocalized with ULK1 within 1 h after SCI, suggesting possible interaction between autophagy and SARM1-mediated axonal degeneration. In our in vitro experiments, inhibition of autophagy, including Ulk1 knockdown and ULK1 inhibitor, attenuated neurite fragmentation and reduced accumulation of SARM1 puncta in neurites of primary cortical neurons subjected to glutamate excitotoxicity. Immunoprecipitation data demonstrated that ULK1 physically interacted with SARM1 in vitro and in vivo and that SAM domains of SARM1 were necessary for ULK1-SARM1 complex formation. Consistent with a role in regulation of axonal degeneration, in primary cortical neurons ULK1-SARM1 interaction increased upon neurite damage. Supporting a role for autophagy and ULK1 in regulation of SARM1 in axonal degeneration in vivo, axonal ULK1 activation and accumulation of SARM1 were both decreased after SCI in Becn1 +/- autophagy hypomorph mice compared to wild-type (WT) controls. These findings suggest a regulatory crosstalk between autophagy and axonal degeneration pathways, which is mediated through ULK1-SARM1 interaction and contributes to the ability of SARM1 to accumulate in injured axons.

Autophagy is a catabolic process that degrades cytoplasmic constituents and organelles in the lysosome, thus serving an important role in cellular homeostasis and protection against insults. We previously reported that defects in autophagy contribute to neuronal cell damage in traumatic spinal cord injury (SCI). Recent data from other inflammatory models implicate autophagy in regulation of immune and inflammatory responses, with low levels of autophagic flux associated with pro-inflammatory phenotypes. In the present study, we examined the effects of genetically or pharmacologically manipulating autophagy on posttraumatic neuroinflammation and motor function after SCI in mice. Methods: Young adult male C57BL/6, CX3CR1-GFP, autophagy hypomorph Becn1 +/- mice, and their wildtype (WT) littermates were subjected to moderate thoracic spinal cord contusion. Neuroinflammation and autophagic flux in the injured spinal cord were assessed using flow cytometry, immunohistochemistry, and NanoString gene expression analysis. Motor function was evaluated with the Basso Mouse Scale and horizontal ladder test. Lesion volume and spared white matter were evaluated by unbiased stereology. To stimulate autophagy, disaccharide trehalose, or sucrose control, was administered in the drinking water immediately after injury and for up to 6 weeks after SCI. Results: Flow cytometry demonstrated dysregulation of autophagic function in both microglia and infiltrating myeloid cells from the injured spinal cord at 3 days post-injury. Transgenic CX3CR1-GFP mice revealed increased autophagosome formation and inhibition of autophagic flux specifically in activated microglia/macrophages. NanoString analysis using the neuroinflammation panel demonstrated increased expression of proinflammatory genes and decreased expression of genes related to neuroprotection in Becn1 +/- mice as compared to WT controls at 3 days post-SCI. These findings were further validated by qPCR, wherein we observed significantly higher expression of proinflammatory cytokines. Western blot analysis confirmed higher protein expression of the microglia/macrophage marker IBA-1, inflammasome marker, NLRP3, and innate immune response markers cGAS and STING in Becn1 +/- mice at 3 day after SCI. Flow cytometry demonstrated that autophagy deficit did not affect either microglial or myeloid counts at 3 days post-injury, instead resulting in increased microglial production of proinflammatory cytokines. Finally, locomotor function showed significantly worse impairments in Becn1 +/- mice up to 6 weeks after SCI, which was accompanied by worsening tissue damage. Conversely, treatment with a naturally occurring autophagy inducer trehalose, reduced protein levels of p62, an adaptor protein targeting cargo to autophagosomes as well as the NLRP3, STING, and IBA-1 at 3 days post-injury. Six weeks of trehalose treatment after SCI led to improved motor function recovery as compared to control group, which was accompanied by reduced tissue damage. Conclusions: Our data indicate that inhibition of autophagy after SCI potentiates pro-inflammatory activation in microglia and is associated with worse functional outcomes. Conversely, increasing autophagy with trehalose, decreased inflammation and improved outcomes. These findings highlight the importance of autophagy in spinal cord microglia and its role in secondary injury after SCI., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Elderly patients with traumatic brain injury (TBI) have greater mortality and poorer outcomes than younger individuals. The extent to which old age alters long-term recovery and chronic microglial activation after TBI is unknown, and evidence for therapeutic efficacy in aged mice is sorely lacking. The present study sought to identify potential inflammatory mechanisms underlying age-related outcomes late after TBI. Controlled cortical impact was used to induce moderate TBI in young and old male C57BL/6 mice. At 12 weeks post-injury, aged mice exhibited higher mortality, poorer functional outcomes, larger lesion volumes, and increased microglial activation. Transcriptomic analysis identified age- and TBI-specific gene changes consistent with a disease-associated microglial signature in the chronically injured brain, including those involved with complement, phagocytosis, and autophagy pathways. Dysregulation of phagocytic and autophagic function in microglia was accompanied by increased neuroinflammation in old mice. As proof-of-principle that these pathways have functional importance, we administered an autophagic enhancer, trehalose, in drinking water continuously for 8 weeks after TBI. Old mice treated with trehalose showed enhanced functional recovery and reduced microglial activation late after TBI compared to the sucrose control group. Our data indicate that microglia undergo chronic changes in autophagic regulation with both normal aging and TBI that are associated with poorer functional outcome. Enhancing autophagy may therefore be a promising clinical therapeutic strategy for TBI, especially in older patients., (© 2022. The Author(s).)

RNA in situ hybridization based on the mechanism of the hybridization chain reaction (HCR) enables multiplexed, quantitative, high-resolution RNA imaging in highly autofluorescent samples, including whole-mount vertebrate embryos, thick brain slices and formalin-fixed paraffin-embedded tissue sections. Here, we extend the benefits of one-step, multiplexed, quantitative, isothermal, enzyme-free HCR signal amplification to immunohistochemistry, enabling accurate and precise protein relative quantitation with subcellular resolution in an anatomical context. Moreover, we provide a unified framework for simultaneous quantitative protein and RNA imaging with one-step HCR signal amplification performed for all target proteins and RNAs simultaneously., Competing Interests: Competing interests The authors declare competing financial interests in the form of patents, pending patent applications and the startup company Molecular Instruments., (© 2021. Published by The Company of Biologists Ltd.)

HIV-associated cardiomyopathy is a well-established sequela in people infected with HIV (PHIV). Despite significant advances in HIV management through the use of highly active anti-retroviral therapy (HAART), PHIV on HAART continue to have elevated risk of cardiomyopathy and heart failure, even when accounting for known cardiovascular risk factors. This review article will explore the proposed mechanisms by which chronic HIV infection induces cardiomyopathy and heart failure in the setting of HAART. Evaluation, work-up, and management of cardiomyopathy in PHIV will also be briefly discussed. The advent of HAART has altered the pathophysiology HIV-associated cardiomyopathy from a rapidly progressive cardiomyopathy, often with pericardial involvement, into a chronic process involving inflammation and persistent immune dysregulation. With the significant decrease in AIDS-related deaths, the prevalence of cardiomyopathy and the mortality associated with heart failure in PHIV have increased. Multiple immune-related and inflammatory mechanisms have been proposed, which may provide insight into evaluation and management of cardiomyopathy in PHIV.

There is a lack of quantitative and non-invasive clinical biomechanical assessment tools for diabetic foot ulcers. Our previous study reported that the indentation stiffness measured by an optical coherence tomography-based air-jet indentation system in a non-contact and non-invasive manner may reflect the tensile properties of diabetic wounds. As the tensile properties are known to be contributed by type I collagen, this study was aimed to establish the correlations between the indentation stiffness, and type I collagen abundance and organisation, in order to further justify and characterise the in vivo indentation stiffness measurement in diabetic wounds. In a male streptozotocin-induced diabetic rat model, indentation stiffness, and type I collagen abundance and organisation of excisional wounds were quantified and examined using the optical coherence tomography-based air-jet indentation system and picrosirius red polarised light microscopy, respectively, on post-wounding days 3, 5, 7, 10, 14, and 21. The results showed significant negative correlations between indentation stiffness at the wound centre, and the collagen abundance and organisation. The correlations between the indentation stiffness, as well as collagen abundance and organisation of diabetic wounds suggest that the optical coherence tomography-based air-jet indentation system can potentially be used to quantitatively and non-invasively monitor diabetic wound healing in clinical settings, clinical research or preclinical research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Choi, Cheung, Ng, Zheng, Jan and Cheing.)

Background: Inflammation plays a major role in the development and progression of cardiovascular disease (CVD) morbidity and mortality. The well-established relationship between periodontal disease (PD) and CVD may be causal. Left untreated, PD can lead to high systemic inflammation, thus contributing to inflammatory CVD, such as atherosclerosis. Multiple mechanisms have been proposed to elucidate the causal relationship between PD and its contribution to CVD., Objective: This review article highlights the current evidence supporting the role of PD in the development and progression of atherosclerosis., Methods: After creating a list of relevant medical subject heading (MeSH) terms, a systematic search within PubMed in English for each MeSH term between 2000 and 2019 was used to generate evidence for this review article., Conclusion: There is overwhelming evidence in the current literature that supports an association between PD and CVD that is independent of known CVD risk factors. However, the supporting evidence that PD directly causes CVD in humans continues to remain elusive. Multiple biologically plausible mechanisms have been proposed and investigated, yet most studies are limited to mouse models and in vitro cell cultures. Additional studies testing the various proposed mechanisms in longitudinal human studies are required to provide deeper insight into the mechanistic link between these 2 related diseases., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Psoriasis is associated with elevated risk of heart attack and increased accumulation of subclinical noncalcified coronary burden by coronary computed tomography angiography (CCTA). Machine learning algorithms have been shown to effectively analyze well-characterized data sets., Objective: In this study, we used machine learning algorithms to determine the top predictors of noncalcified coronary burden by CCTA in psoriasis., Methods: The analysis included 263 consecutive patients with 63 available variables from the Psoriasis Atherosclerosis Cardiometabolic Initiative. The random forest algorithm was used to determine the top predictors of noncalcified coronary burden by CCTA. We evaluated our results using linear regression models., Results: Using the random forest algorithm, we found that the top 10 predictors of noncalcified coronary burden were body mass index, visceral adiposity, total adiposity, apolipoprotein A1, high-density lipoprotein, erythrocyte sedimentation rate, subcutaneous adiposity, small low-density lipoprotein particle, cholesterol efflux capacity and the absolute granulocyte count. Linear regression of noncalcified coronary burden yielded results consistent with our machine learning output., Limitation: We were unable to provide external validation and did not study cardiovascular events., Conclusion: Machine learning methods identified the top predictors of noncalcified coronary burden in psoriasis. These factors were related to obesity, dyslipidemia, and inflammation, showing that these are important targets when treating comorbidities in psoriasis., (Published by Elsevier Inc.)

Background: Lipid-rich necrotic core (LRNC), a high-risk coronary plaque feature assessed by coronary computed tomography angiography, is associated with increased risk of future cardiovascular events in patients with subclinical, nonobstructive coronary artery disease. Psoriasis is a chronic inflammatory condition that is associated with increased prevalence of high-risk coronary plaque and risk of cardiovascular events. This study characterized LRNC in psoriasis and how LRNC modulates in response to biologic therapy., Methods: Consecutive biologic naïve psoriasis patients (n=209) underwent coronary computed tomography angiography at baseline and 1-year to assess changes in LRNC using a novel histopathologically validated software (vascuCAP Elucid Bioimaging, Boston, MA) before and after biologic therapy over 1 year., Results: Study participants were middle-aged, predominantly male with similar cardiometabolic and psoriasis status between treatment groups. In all participants at baseline, LRNC was associated with Framingham risk score (β [standardized β]=0.12 [95% CI, 0.00-0.15]; P =0.045), and psoriasis severity (β=0.13 [95% CI, 0.01-0.26]; P =0.029). At 1-year, participants receiving biologic therapy had a reduction in LRNC (mm 2 ; 3.12 [1.99-4.66] versus 2.97 [1.84-4.35]; P =0.028), while those who did not receive biologic therapy over 1 year demonstrated no significant change with nominally higher LRNC (3.12 [1.82-4.60] versus 3.34 [2.04-4.74]; P =0.06). The change in LRNC was significant compared with that of the nonbiologic treated group (ΔLRNC, -0.22 mm 2 versus 0.14 mm 2 , P =0.004) and remained significant after adjusting for cardiovascular risk factors and psoriasis severity (β=-0.09 [95% CI, -0.01 to -0.18]; P =0.033)., Conclusions: LRNC was associated with psoriasis severity and cardiovascular risk factors in psoriasis. Additionally, there was favorable modification of LRNC in those on biologic therapy. This study provides evidence of potential reduction in LRNC with treatment of systemic inflammation. Larger, longer follow-up prospective studies should be conducted to understand how changes in LRNC may translate into a reduction in future cardiovascular events in psoriasis.

Importance: Psoriasis, a chronic inflammatory skin disease associated with accelerated noncalcified coronary burden (NCB) by coronary computed tomography angiography (CCTA), accelerates lipoprotein oxidation in the form of oxidized modified lipoproteins. A transmembrane scavenger receptor for these oxidized modified lipoproteins is lectinlike oxidized low-density lipoprotein receptor-1 (LOX-1), which has been reported to be associated with coronary artery disease. It is unknown whether this receptor is associated with coronary artery disease in psoriasis., Objective: To assess the association between soluble LOX-1 (sLOX-1) and NCB in psoriasis over time., Design, Setting, and Participants: In a cohort study at the National Institutes of Health, 175 consecutive patients with psoriasis were referred from outpatient dermatology practices between January 1, 2013, and October 1, 2017. A total of 138 consecutively recruited patients with psoriasis were followed up at 1 year., Exposures: Circulating soluble lectinlike oxidized low-density lipoprotein receptor-1 levels were measured blindly by field scientists running undiluted serum using an enzyme-linked immunosorbent assay., Main Outcomes and Measures: Coronary computed tomography angiography scans were performed to quantify NCB in all 3 major epicardial coronary arteries by a reader blinded to patient demographics, visit, and treatment status., Results: Among the 175 patients with psoriasis, the mean (SD) age was 49.7 (12.6) years and 91 were men (55%). The cohort had relatively low median cardiovascular risk by Framingham risk score (median, 2.0 [interquartile range (IQR), 1.0-6.0]) and had a mean (SD) body mass index (calculated as weight in kilograms divided by height in meters squared) suggestive of overweight profiles (29.6 [6.0]). Elevated sLOX-1 levels were found in patients with psoriasis compared with age- and sex-matched controls (median, 210.3 [IQR, 110.9-336.2] vs 83.7 [IQR, 40.1-151.0]; P < .001), and were associated with Psoriasis Area Severity Index (PASI) score (β = 0.23; 95% CI, 0.082-0.374; P = .003). Moreover, sLOX-1 was associated with NCB independent of hyperlipidemia status (β = 0.11; 95% CI, 0.016-0.200; P = .023), an association which persisted after adjusting for traditional cardiovascular risk factors, statin use, and biologic psoriasis treatment (β = 0.10; 95% CI, 0.014-0.193; P = .03). At 1 year, in those who had clinical improvement in PASI (eg, >50% improvement), a reduction in sLOX-1 (median, 311.1 [IQR, 160.0-648.8] vs median, 224.2 [IQR, 149.1 - 427.4]; P = .01) was associated with a reduction in NCB (β = 0.14; 95% CI, 0.028-0.246; P = .02)., Conclusions and Relevance: Soluble lectinlike oxidized low-density lipoprotein receptor-1 levels were elevated in patients with psoriasis and were associated with severity of skin disease. Moreover, sLOX-1 associated with NCB independent of hyperlipidemia status, suggesting that inflammatory sLOX-1 induction may modulate lipid-rich NCB in psoriasis. Improvement of skin disease was associated with a reduction of sLOX-1 at 1 year, demonstrating the potential role of sLOX-1 in inflammatory atherogenesis in psoriasis.

In situ hybridization based on the mechanism of hybridization chain reaction (HCR) enables multiplexed quantitative mRNA imaging in diverse sample types. Third-generation in situ HCR (v3.0) provides automatic background suppression throughout the protocol, dramatically enhancing performance and ease of use. In situ HCR v3.0 supports two quantitative imaging modes: (1) qHCR imaging for analog mRNA relative quantitation with subcellular resolution and (2) dHCR imaging for digital mRNA absolute quantitation with single-molecule resolution. Here, we provide protocols for qHCR and dHCR imaging in mammalian cells on a slide.

In situ hybridization based on the mechanism of hybridization chain reaction (HCR) enables high-throughput expression profiling of mammalian or bacterial cells via flow cytometry. Third-generation in situ HCR (v3.0) provides automatic background suppression throughout the protocol, dramatically enhancing performance and ease of use. In situ HCR v3.0 supports analog mRNA relative quantitation via qHCR flow cytometry. Here, we provide protocols for multiplexed qHCR flow cytometry for mammalian or bacterial cells in suspension.

In situ hybridization based on the mechanism of hybridization chain reaction (HCR) enables multiplexed quantitative mRNA imaging in the anatomical context of whole-mount vertebrate embryos. Third-generation in situ HCR (v3.0) provides automatic background suppression throughout the protocol, dramatically enhancing performance and ease of use. In situ HCR v3.0 supports two quantitative imaging modes: (1) qHCR imaging for analog mRNA relative quantitation with subcellular resolution in an anatomical context and (2) dHCR imaging for digital mRNA absolute quantitation with single-molecule resolution in an anatomical context. Here, we provide protocols for qHCR and dHCR imaging in whole-mount zebrafish, chicken, and mouse embryos.

Purpose of Review: Traditional risk models, such as the Framingham risk score, fail to capture the increased cardiovascular disease risk seen in patients with chronic inflammatory diseases. This review will cover imaging modalities and their emerging applications in assessing subclinical cardiovascular disease for both research and clinical care in patients with chronic inflammatory diseases., Recent Findings: Multiple imaging modalities have been studied to assess for subclinical cardiovascular disease via functional/physiologic, inflammatory, and anatomic assessment in patients with chronic inflammatory diseases. The use of imaging to evaluate subclinical cardiovascular disease in patients with chronic inflammatory diseases has the potential to capture early sub-clinical atherosclerosis, to improve risk stratification of future cardiovascular events, and to guide effective disease management.

Background: The contribution of inflammation to the incidence of cardiovascular disease (CVD) has been increasingly recognized in recent years. We investigated the relationship of aortic vascular uptake of 18F-FDG by PET/CT and aortic wall thickness (AWT) by MRI in psoriasis, a chronic inflammatory disease with increased incidence of CVD. One hundred sixty-five patients with plaque psoriasis participated in an ongoing longitudinal cohort study. Subclinical atherosclerosis was assessed as aortic uptake of 18F-FDG by PET/CT reported as target-to-background ratio (TBR) and AWT by MRI reported as maximal thickness., Results: Patients with psoriasis were middle aged, predominantly male, and had mild CV risk by traditional risk factors. Psoriasis severity as measured by PASI score was a notable determinant of AWT (ρ = 0.20, p = 0.01). Moreover, aortic vascular uptake of 18F-FDG associated with AWT by MRI at baseline in unadjusted analysis (β = 0.27 p = 0.001) and following adjustment for traditional cardiovascular risk factors, waist-to-hip ratio, and statin use (β = 0.21 p = 0.01). Finally, following 1 year of psoriasis treatment, a decrease in aortic vascular uptake of 18F-FDG was associated with a reduction in AWT in fully adjusted models (β = 0.33, p = 0.02)., Conclusion: In conclusion, we demonstrate that psoriasis severity and aortic vascular uptake of 18F-FDG in the aorta were associated with AWT. Following treatment of psoriasis, a decrease in aortic vascular uptake of 18F-FDG was associated with a reduction in AWT at 1 year. These findings suggest that aortic vascular uptake of 18F-FDG is associated with early evidence of vascular disease assessed by aortic wall thickness. Prospective studies in larger populations including other inflammatory diseases are warranted.

Importance: Psoriasis is a chronic inflammatory skin disease associated with increased coronary plaque burden and cardiovascular events. Biologic therapy for psoriasis has been found to be favorably associated with luminal coronary plaque, but it is unclear whether these associations are attributable to direct anti-inflammatory effects on the coronary arteries., Objective: To investigate the association of biologic therapy with coronary inflammation in patients with psoriasis using the perivascular fat attenuation index (FAI), a novel imaging biomarker that assesses coronary inflammation by mapping spatial changes of perivascular fat composition via coronary computed tomography angiography (CCTA)., Design, Setting, and Participants: This prospective cohort study performed from January 1, 2013, through March 31, 2019, analyzed changes in FAI in patients with moderate to severe psoriasis who underwent CCTA at baseline and at 1 year and were not receiving biologic psoriasis therapy at baseline., Exposures: Biologic therapy for psoriasis., Main Outcomes and Measures: Perivascular FAI mapping was performed based on an established method by a reader blinded to patient demographics, visit, and treatment status., Results: Of the 134 patients (mean [SD] age, 51.1 [12.1] years; 84 [62.5%] male), most had low cardiovascular risk by traditional risk scores (median 10-year Framingham Risk Score, 3% [interquartile range, 1%-7%]) and moderate to severe skin disease. Of these patients, 82 received biologic psoriasis therapy (anti-tumor necrosis factor α, anti-interleukin [IL] 12/23, or anti-IL-17) for 1 year, and 52 did not receive any biologic therapy and were given topical or light therapy (control group). At baseline, 46 patients (27 in the treated group and 19 in the untreated group) had a focal coronary atherosclerotic plaque. Biologic therapy was associated with a significant decrease in FAI at 1 year (median FAI -71.22 HU [interquartile range (IQR), -75.85 to -68.11 HU] at baseline vs -76.09 HU [IQR, -80.08 to -70.37 HU] at 1 year; P < .001) concurrent with skin disease improvement (median PASI, 7.7 [IQR, 3.2-12.5] at baseline vs 3.2 [IQR, 1.8-5.7] at 1 year; P < .001), whereas no change in FAI was noted in those not receiving biologic therapy (median FAI, -71.98 [IQR, -77.36 to -65.64] at baseline vs -72.66 [IQR, -78.21 to -67.44] at 1 year; P = .39). The associations with FAI were independent of the presence of coronary plaque and were consistent among patients receiving different biologic agents, including anti-tumor necrosis factor α (median FAI, -71.25 [IQR, -75.86 to -66.89] at baseline vs -75.49 [IQR, -79.12 to -68.58] at 1 year; P < .001) and anti-IL-12/23 or anti-IL-17 therapy (median FAI, -71.18 [IQR, -75.85 to -68.80] at baseline vs -76.92 [IQR, -81.16 to -71.67] at 1 year; P < .001)., Conclusions and Relevance: In this study, biologic therapy for moderate to severe psoriasis was associated with reduced coronary inflammation assessed by perivascular FAI. This finding suggests that perivascular FAI measured by CCTA may be used to track response to interventions for coronary artery disease.

The autophagy-lysosomal pathway plays an essential role in cellular homeostasis as well as a protective function against a variety of diseases including neurodegeneration. Conversely, inhibition of autophagy, for example due to lysosomal dysfunction, can lead to pathological accumulation of dysfunctional autophagosomes and consequent neuronal cell death. We previously reported that autophagy is inhibited and contributes to neuronal cell death following spinal cord injury (SCI). In this study, we examined lysosomal function and explored the mechanism of lysosomal defects following SCI. Our data demonstrated that expression levels and processing of the lysosomal enzyme cathepsin D (CTSD) are decreased by 2 h after SCI. Enzymatic activity levels of CTSD and another lysosomal enzyme, N-acetyl-alpha-glucosaminidase, are both decreased 24 h post injury, indicating general lysosomal dysfunction. Subcellular fractionation and immunohistochemistry analysis demonstrated that this dysfunction is due to lysosomal membrane permeabilization and leakage of lysosomal contents into the cytosol. To directly assess extent and mechanisms of damage to lysosomal membranes, we performed mass spectrometry-based lipidomic analysis of lysosomes purified from SCI and control spinal cord. At 2 h post injury our data demonstrated increase in several classes of lysosophospholipids, the products of phospholipases (PLAs), as well as accumulation of PLA activators, ceramides. Phospholipase cPLA2, the main PLA species expressed in the CNS, has been previously implicated in mediation of secondary injury after SCI, but the mechanisms of its involvement remain unclear. Our data demonstrate that cPLA2 is activated within 2 h after SCI preferentially in the lysosomal fraction, where it colocalizes with lysosomal-associated membrane protein 2 in neurons. Inhibition of cPLA2 in vivo decreased lysosomal damage, restored autophagy flux, and reduced neuronal cell damage. Taken together our data implicate lysosomal defects in pathophysiology of SCI and for the first time indicate that cPLA2 activation leads to lysosomal damage causing neuronal autophagosome accumulation associated with neuronal cell death.

Background: Understanding the differences in skin blood flow (SBF) on the plantar and dorsal foot in people with diabetes mellitus (DM) may help to assess the influence of diabetes and neuropathy on microvascular dysfunction and risks of diabetic foot ulcers in this population. However, there is no study comparing SBF oscillations between the plantar and dorsal foot in people with DM and peripheral neuropathy (PN)., Objective: The objective of this study was to compare SBF oscillations between the plantar and dorsal foot in people with DM and PN and investigate the underlying mechanisms responsible for the differences., Methods: 18 people with Type 2 DM and PN and 8 healthy controls were recruited. Laser Doppler flowmetry (LDF) was used to measure SBF on the plantar and dorsal foot for 10 min when the subject was in the supine position. Wavelet analysis was used to quantify the relative amplitude of the characteristic frequency components of SBF oscillations. Sample entropy analysis was used to quantify the regularity degree of SBF oscillations., Results: People with DM and PN had a higher SBF on the plantar foot compared to the dorsal foot. The relative wavelet amplitudes of metabolic and myogenic frequency components on the plantar foot were respectively higher and lower compared to the dorsal foot. Sample entropy analysis showed that SBF on the plantar foot had a higher degree of regularity compared to the dorsal foot., Conclusions: In people with DM and PN, higher SBF on the plantar foot is attributed to the metabolic and myogenic controls, and SBF on the plantar foot exhibits a higher degree of regularity compared to the dorsal foot. People with DM and PN also had higher plantar and dorsal SBF compared to the healthy controls. This study provides evidence to document differences in SBF of the plantar and dorsal foot in people with DM and PN., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We have systematically assessed published cell studies and animal experimental reports on the efficacy of selected biophysical energies (BPEs) in the treatment of diabetic foot ulcers. These BPEs include electrical stimulation (ES), pulsed electromagnetic field (PEMF), extracorporeal shockwave (ECSW), photo energies and ultrasound (US). Databases searched included CINAHL, MEDLINE and PubMed from 1966 to 2018. Studies reviewed include animal and cell studies on treatment with BPEs compared with sham, control or other BPEs. Information regarding the objective measures of tissue healing and data was extracted. Eighty-two studies were eventually selected for the critical appraisal: five on PEMF, four each on ES and ECSW, sixty-six for photo energies, and three about US. Based on the percentage of original wound size affected by the BPEs, both PEMF and low-level laser therapy (LLL) demonstrated a significant clinical benefit compared to the control or sham treatment, whereas the effect of US did not reveal a significance. Our results indicate potential benefits of selected BPEs in diabetic wound management. However, due to the heterogeneity of the current clinical trials, comprehensive studies using well-designed trials are warranted to confirm the results.

[This corrects the article DOI: 10.1371/journal.pone.0191074.].

In situ hybridization based on the mechanism of the hybridization chain reaction (HCR) has addressed multi-decade challenges that impeded imaging of mRNA expression in diverse organisms, offering a unique combination of multiplexing, quantitation, sensitivity, resolution and versatility. Here, with third-generation in situ HCR, we augment these capabilities using probes and amplifiers that combine to provide automatic background suppression throughout the protocol, ensuring that reagents will not generate amplified background even if they bind non-specifically within the sample. Automatic background suppression dramatically enhances performance and robustness, combining the benefits of a higher signal-to-background ratio with the convenience of using unoptimized probe sets for new targets and organisms. In situ HCR v3.0 enables three multiplexed quantitative analysis modes: (1) qHCR imaging - analog mRNA relative quantitation with subcellular resolution in the anatomical context of whole-mount vertebrate embryos; (2) qHCR flow cytometry - analog mRNA relative quantitation for high-throughput expression profiling of mammalian and bacterial cells; and (3) dHCR imaging - digital mRNA absolute quantitation via single-molecule imaging in thick autofluorescent samples., Competing Interests: Competing interestsThe authors declare competing financial interests in the form of patents, pending patent applications and a startup company (Molecular Instruments)., (© 2018. Published by The Company of Biologists Ltd.)

Necroptosis, a regulated necrosis pathway mediated by the receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3), is induced following spinal cord injury (SCI) and thought to contribute to neuronal and glial cell death. However, mechanisms leading to activation of necroptosis after SCI remain unclear. We have previously shown that autophagy, a catabolic pathway facilitating degradation of cytoplasmic proteins and organelles in a lysosome-dependent manner, is inhibited following SCI in rats. Our current data confirm that inhibition of autophagy also occurs after thoracic contusive SCI in the mouse model, as indicated by accumulation of both the autophagosome marker, LC3-II and autophagy cargo protein, p62/SQSTM1. This was most pronounced in the ventral horn neurons and was caused by rapid inhibition of lysosomal function after SCI. Interestingly, RIPK1, RIPK3, and the necroptosis effector protein MLKL also rapidly accumulated after SCI and localized to neurons with disrupted autophagy, suggesting that these events may be related. To determine if lysosomal dysfunction could contribute to induction of necroptosis, we treated PC12 cells and primary rat cortical neurons with lysosomal inhibitors. This led to rapid accumulation of RIPK1 and RIPK3, confirming that they are normally degraded by the lysosomal pathway. In PC12 cells lysosomal inhibition also sensitized cells to necroptosis induced by tumor necrosis factor α (TNFα) and caspase inhibitor. Imaging studies confirmed that RIPK1 partially localized to lysosomes in both untreated and lysosomal inhibitor treated cells. Similarly, we detected presence of RIPK1, RIPK3 and MLKL in both cytosol and at lysosomes after SCI in vivo. Furthermore, stimulation of autophagy and lysosomal function with rapamycin treatment led to decreased accumulation of RIPK1 and attenuated cell death after SCI. These data suggest that lysosomal dysfunction after SCI may contribute to both inhibition of autophagy and sensitize cells to necroptosis by promoting RIPK1 and RIPK3 accumulation.

The present study investigated the effects of pulsed electromagnetic field (PEMF) on the tensile biomechanical properties of diabetic wounds at different phases of healing. Two intensities of PEMF were adopted for comparison. We randomly assigned 111 10-week-old male streptozotocin-induced diabetic Sprague-Dawley rats to two PEMF groups and a sham control group. Six-millimetre biopsy punched full thickness wounds were made on the lateral side of their hindlimbs. The PEMF groups received active PEMF delivered at 25 Hz with intensity of either 2 mT or 10 mT daily, while the sham group was handled in a similar way except they were not exposed to PEMF. Wound tissues were harvested for tensile testing on post-wounding days 3, 5, 7, 10, 14 and 21. Maximum load, maximum stress, energy absorption capacity, Young's modulus and thickness of wound tissue were measured. On post-wounding day 5, the PEMF group that received 10-mT intensity had significantly increased energy absorption capacity and showed an apparent increase in the maximum load. However, the 10-mT PEMF group demonstrated a decrease in Young's modulus on day 14. The 10-mT PEMF groups showed a significant increase in the overall thickness of wound tissue whereas the 2-mT group showed a significant decrease in the overall maximum stress of the wounds tissue. The present findings demonstrated that the PEMF delivered at 10 mT can improve energy absorption capacity of diabetic wounds in the early healing phase. However, PEMF (both 2-mT and 10-mT) seemed to impair the material properties (maximum stress and Young's modulus) in the remodelling phase. PEMF may be a useful treatment for promoting the recovery of structural properties (maximum load and energy absorption capacity), but it might not be applied at the remodelling phase to avoid impairing the recovery of material properties.

For decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting and/or homogenization. Here, we eliminate the trade-off between quantitation and anatomical context, using quantitative in situ hybridization chain reaction (qHCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos. Gene expression can be queried in two directions: read-out from anatomical space to expression space reveals co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space reveals those anatomical locations in which selected gene co-expression relationships occur. As we demonstrate by examining gene circuits underlying somitogenesis, quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving subcellular anatomical context, they enable bi-directional queries that open a new era for in situ hybridization., Competing Interests: Competing interestsThe authors declare competing financial interests in the form of patents (N.A.P. and S.E.F.), pending patent applications (N.A.P. and H.M.T.C.) and a pending startup company (N.A.P. and H.M.T.C.)., (© 2018. Published by The Company of Biologists Ltd.)

In melanoma, therapies with inhibitors to oncogenic BRAF V600E are highly effective but responses are often short-lived due to the emergence of drug-resistant tumor subpopulations. We describe here a mechanism of acquired drug resistance through the tumor microenvironment, which is mediated by human tumor-associated B cells. Human melanoma cells constitutively produce the growth factor FGF-2, which activates tumor-infiltrating B cells to produce the growth factor IGF-1. B-cell-derived IGF-1 is critical for resistance of melanomas to BRAF and MEK inhibitors due to emergence of heterogeneous subpopulations and activation of FGFR-3. Consistently, resistance of melanomas to BRAF and/or MEK inhibitors is associated with increased CD20 and IGF-1 transcript levels in tumors and IGF-1 expression in tumor-associated B cells. Furthermore, first clinical data from a pilot trial in therapy-resistant metastatic melanoma patients show anti-tumor activity through B-cell depletion by anti-CD20 antibody. Our findings establish a mechanism of acquired therapy resistance through tumor-associated B cells with important clinical implications.Resistance to BRAFV600E inhibitors often occurs in melanoma patients. Here, the authors describe a potential mechanism of acquired drug resistance mediated by tumor-associated B cells-derived IGF-1.

Circadian rhythms optimize physiology and behavior to the varying demands of the 24 h day. The master circadian clock is located in the suprachiasmatic nuclei (SCN) of the hypothalamus and it regulates circadian oscillators in tissues throughout the body to prevent internal desynchrony. Here, we demonstrate for the first time that, under standard 12 h:12 h light/dark (LD) cycles, object, visuospatial, and olfactory recognition performance in C57BL/6J mice is consistently better at midday relative to midnight. However, under repeated exposure to constant light ( r LL), recognition performance becomes desynchronized, with object and visuospatial performance better at subjective midday and olfactory performance better at subjective midnight. This desynchrony in behavioral performance is mirrored by changes in expression of the canonical clock genes Period1 and Period2 ( Per1 and Per2 ), as well as the immediate-early gene Fos in the SCN, dorsal hippocampus, and olfactory bulb. Under r LL, rhythmic Per1 and Fos expression is attenuated in the SCN. In contrast, hippocampal gene expression remains rhythmic, mirroring object and visuospatial performance. Strikingly, Per1 and Fos expression in the olfactory bulb is reversed, mirroring the inverted olfactory performance. Temporal desynchrony among these regions does not result in arrhythmicity because core body temperature and exploratory activity rhythms persist under r LL. Our data provide the first demonstration that abnormal lighting conditions can give rise to temporal desynchrony between autonomous circadian oscillators in different regions, with different consequences for performance across different sensory domains. Such a dispersed network of dissociable circadian oscillators may provide greater flexibility when faced with conflicting environmental signals. SIGNIFICANCE STATEMENT A master circadian clock in the suprachiasmatic nuclei (SCN) of the hypothalamus regulates physiology and behavior across the 24 h day by synchronizing peripheral clocks throughout the brain and body. Without the SCN, these peripheral clocks rapidly become desynchronized. Here, we provide a unique demonstration that, under lighting conditions in which the central clock in the SCN is dampened, peripheral oscillators in the hippocampus and olfactory bulb become desynchronized, along with the behavioral processes mediated by these clocks. Multiple clocks that adopt different phase relationships may enable processes occurring in different brain regions to be optimized to specific phases of the 24 h day. Moreover, such a dispersed network of dissociable circadian clocks may provide greater flexibility when faced with conflicting environmental signals (e.g., seasonal changes in photoperiod)., (Copyright © 2017 Tam et al.)

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences., Competing Interests: The authors declare competing financial interests in the form of patents and pending patent applications., (© 2016. Published by The Company of Biologists Ltd.)

Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas - from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (passive CLARITY technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ∼20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH., Competing Interests: The authors declare competing financial interests in the form of patents and pending patents., (© 2016. Published by The Company of Biologists Ltd.)

Targeting multiple components of the MAPK pathway can prolong the survival of patients with BRAFV600E melanoma. This approach is not curative, as some BRAF-mutated melanoma cells are intrinsically resistant to MAPK inhibitors (MAPKi). At the systemic level, our knowledge of how signaling pathways underlie drug resistance needs to be further expanded. Here, we have shown that intrinsically resistant BRAF-mutated melanoma cells with a low basal level of mitochondrial biogenesis depend on this process to survive MAPKi. Intrinsically resistant cells exploited an integrated stress response, exhibited an increase in mitochondrial DNA content, and required oxidative phosphorylation to meet their bioenergetic needs. We determined that intrinsically resistant cells rely on the genes encoding TFAM, which controls mitochondrial genome replication and transcription, and TRAP1, which regulates mitochondrial protein folding. Therefore, we targeted mitochondrial biogenesis with a mitochondrium-targeted, small-molecule HSP90 inhibitor (Gamitrinib), which eradicated intrinsically resistant cells and augmented the efficacy of MAPKi by inducing mitochondrial dysfunction and inhibiting tumor bioenergetics. A subset of tumor biopsies from patients with disease progression despite MAPKi treatment showed increased mitochondrial biogenesis and tumor bioenergetics. A subset of acquired drug-resistant melanoma cell lines was sensitive to Gamitrinib. Our study establishes mitochondrial biogenesis, coupled with aberrant tumor bioenergetics, as a potential therapy escape mechanism and paves the way for a rationale-based combinatorial strategy to improve the efficacy of MAPKi.

Multiplexed fluorescent hybridization chain reaction (HCR) and advanced imaging techniques can be used to evaluate combinatorial gene expression patterns in whole mouse embryos with unprecedented spatial resolution. Using HCR, DNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled DNA HCR hairpins self-assemble into tethered fluorescent amplification polymers. Each target mRNA is detected by a probe set containing one or more DNA probes, with each probe carrying two HCR initiators. For multiplexed experiments, probe sets for different target mRNAs carry orthogonal initiators that trigger orthogonal DNA HCR amplification cascades labeled by spectrally distinct fluorophores. As a result, in situ amplification is performed for all targets simultaneously, and the duration of the experiment is independent of the number of target mRNAs. We have used multiplexed fluorescent in situ HCR and advanced imaging technologies to address questions of cell heterogeneity and tissue complexity in craniofacial patterning and anterior neural development. In the sample protocol presented here, we detect three different mRNA targets: Tg(egfp), encoding the enhanced green fluorescent protein (GFP) transgene (typically used as a control); Twist1, encoding a transcription factor involved in cell lineage determination and differentiation; and Pax2, encoding a transcription factor expressed in the mid-hindbrain region of the mouse embryo., (© 2015 Cold Spring Harbor Laboratory Press.)

Hybridization chain reaction (HCR) provides multiplexed, isothermal, enzyme-free, molecular signal amplification in diverse settings. Within intact vertebrate embryos, where signal-to-background is at a premium, HCR in situ amplification enables simultaneous mapping of multiple target mRNAs, addressing a longstanding challenge in the biological sciences. With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The properties of HCR lead to straightforward multiplexing, deep sample penetration, high signal-to-background, and sharp subcellular signal localization within fixed whole-mount zebrafish embryos, a standard model system for the study of vertebrate development. However, RNA reagents are expensive and vulnerable to enzymatic degradation. Moreover, the stringent hybridization conditions used to destabilize nonspecific hairpin binding also reduce the energetic driving force for HCR polymerization, creating a trade-off between minimization of background and maximization of signal. Here, we eliminate this trade-off by demonstrating that low background levels can be achieved using permissive in situ amplification conditions (0% formamide, room temperature) and engineer next-generation DNA HCR amplifiers that maximize the free energy benefit per polymerization step while preserving the kinetic trapping property that underlies conditional polymerization, dramatically increasing signal gain, reducing reagent cost, and improving reagent durability.

Identifying microbes responsible for particular environmental functions is challenging, given that most environments contain an uncultivated microbial diversity. Here we combined approaches to identify bacteria expressing genes relevant to catabolite flow and to locate these genes within their environment, in this case the gut of a "lower," wood-feeding termite. First, environmental transcriptomics revealed that 2 of the 23 formate dehydrogenase (FDH) genes known in the system accounted for slightly more than one-half of environmental transcripts. FDH is an essential enzyme of H2 metabolism that is ultimately important for the assimilation of lignocellulose-derived energy by the insect. Second, single-cell PCR analysis revealed that two different bacterial types expressed these two transcripts. The most commonly transcribed FDH in situ is encoded by a previously unappreciated deltaproteobacterium, whereas the other FDH is spirochetal. Third, PCR analysis of fractionated gut contents demonstrated that these bacteria reside in different spatial niches; the spirochete is free-swimming, whereas the deltaproteobacterium associates with particulates. Fourth, the deltaproteobacteria expressing FDH were localized to protozoa via hybridization chain reaction-FISH, an approach for multiplexed, spatial mapping of mRNA and rRNA targets. These results underscore the importance of making direct vs. inference-based gene-species associations, and have implications in higher termites, the most successful termite lineage, in which protozoa have been lost from the gut community. Contrary to expectations, in higher termites, FDH genes related to those from the protozoan symbiont dominate, whereas most others were absent, suggesting that a successful gene variant can persist and flourish after a gut perturbation alters a major environmental niche.

DNA nanotechnology has emerged as a reliable and programmable way of controlling matter at the nanoscale through the specificity of Watson-Crick base pairing, allowing both complex self-assembled structures with nanometer precision and complex reaction networks implementing digital and analog behaviors. Here we show how two well-developed frameworks, DNA tile self-assembly and DNA strand-displacement circuits, can be systematically integrated to provide programmable kinetic control of self-assembly. We demonstrate the triggered and catalytic isothermal self-assembly of DNA nanotubes over 10 μm long from precursor DNA double-crossover tiles activated by an upstream DNA catalyst network. Integrating more sophisticated control circuits and tile systems could enable precise spatial and temporal organization of dynamic molecular structures.

In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos, representing a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. Here, we report a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reactions (HCR). With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability and sequence specificity of these amplification cascades enable multiple HCR amplifiers to operate orthogonally at the same time in the same sample. Robust performance is achieved when imaging five target mRNAs simultaneously in fixed whole-mount and sectioned zebrafish embryos. HCR amplifiers exhibit deep sample penetration, high signal-to-background ratios and sharp signal localization.

Synthesizing molecular tubes with monodisperse, programmable circumferences is an important goal shared by nanotechnology, materials science, and supermolecular chemistry. We program molecular tube circumferences by specifying the complementarity relationships between modular domains in a 42-base single-stranded DNA motif. Single-step annealing results in the self-assembly of long tubes displaying monodisperse circumferences of 4, 5, 6, 7, 8, 10, or 20 DNA helices.

In nature, self-assembling and disassembling complexes of proteins and nucleic acids bound to a variety of ligands perform intricate and diverse dynamic functions. In contrast, attempts to rationally encode structure and function into synthetic amino acid and nucleic acid sequences have largely focused on engineering molecules that self-assemble into prescribed target structures, rather than on engineering transient system dynamics. To design systems that perform dynamic functions without human intervention, it is necessary to encode within the biopolymer sequences the reaction pathways by which self-assembly occurs. Nucleic acids show promise as a design medium for engineering dynamic functions, including catalytic hybridization, triggered self-assembly and molecular computation. Here, we program diverse molecular self-assembly and disassembly pathways using a 'reaction graph' abstraction to specify complementarity relationships between modular domains in a versatile DNA hairpin motif. Molecular programs are executed for a variety of dynamic functions: catalytic formation of branched junctions, autocatalytic duplex formation by a cross-catalytic circuit, nucleated dendritic growth of a binary molecular 'tree', and autonomous locomotion of a bipedal walker.

A theoretical examination of kinetic mechanisms for forming knots and links in nucleic acid structures suggests that molecules involving base pairs between loops are likely to become topologically trapped in persistent frustrated states through the mechanism of 'helix-driven wrapping'. Augmentation of the state space to include both secondary structure and topology in describing the free energy landscape illustrates the potential for topological effects to influence the kinetics and function of nucleic acid strands. An experimental study of metastable complementary 'kissing hairpins' demonstrates that the topological constraint of zero linking number between the loops effectively prevents conversion to the minimum free energy helical state. Introduction of short catalyst strands that break the topological constraint causes rapid conversion to full duplex.