Simultaneous Positron Emission Tomography and Molecular Magnetic Resonance Imaging of Cardiopulmonary Fibrosis in a Mouse Model of Left Ventricular Dysfunction.

BACKGROUND: Aging-associated left ventricular dysfunction promotes cardiopulmonary fibrogenic remodeling, Group 2 pulmonary hypertension (PH), and right ventricular failure. At the time of diagnosis, cardiac function has declined, and cardiopulmonary fibrosis has often developed. Here, we sought to develop a molecular positron emission tomography (PET)-magnetic resonance imaging (MRI) protocol to detect both cardiopulmonary fibrosis and fibrotic disease activity in a left ventricular dysfunction model. METHODS AND RESULTS: Left ventricular dysfunction was induced by transverse aortic constriction (TAC) in 6-month-old senescence-accelerated prone mice, a subset of mice that received sham surgery. Three weeks after surgery, mice underwent simultaneous PET-MRI at 4.7 T. Collagen-targeted PET and fibrogenesis magnetic resonance (MR) probes were intravenously administered. PET signal was computed as myocardium- or lung-to-muscle ratio. Percent signal intensity increase and Δ lung-to-muscle ratio were computed from the pre-/postinjection magnetic resonance images. Elevated allysine in the heart (P=0.02) and lungs (P=0.17) of TAC mice corresponded to an increase in myocardial magnetic resonance imaging percent signal intensity increase (P<0.0001) and Δlung-to-muscle ratio (P<0.0001). Hydroxyproline in the heart (P<0.0001) and lungs (P<0.01) were elevated in TAC mice, which corresponded to an increase in heart (myocardium-to-muscle ratio, P=0.02) and lung (lung-to-muscle ratio, P<0.001) PET measurements. Pressure-volume loop and echocardiography demonstrated adverse left ventricular remodeling, function, and increased right ventricular systolic pressure in TAC mice. CONCLUSIONS: Administration of collagen-targeted PET and allysine-targeted MR probes led to elevated PET-magnetic resonance imaging signals in the myocardium and lungs of TAC mice. The study demonstrates the potential to detect fibrosis and fibrogenesis in cardiopulmonary disease through a dual molecular PET-magnetic resonance imaging protocol.

Lung-specific interleukin 6 mediated transglutaminase 2 activation and cardiopulmonary fibrogenesis.

Pulmonary hypertension (PH) pathogenesis is driven by inflammatory and metabolic derangements as well as glycolytic reprogramming. Induction of both interleukin 6 (IL6) and transglutaminase 2 (TG2) expression participates in human and experimental cardiovascular diseases. However, little is known about the role of TG2 in these pathologic processes. The current study aimed to investigate the molecular interactions between TG2 and IL6 in mediation of tissue remodeling in PH. A lung-specific IL6 over-expressing transgenic mouse strain showed elevated right ventricular (RV) systolic pressure as well as increased wet and dry tissue weights and tissue fibrosis in both lungs and RVs compared to age-matched wild-type littermates. In addition, IL6 over-expression induced the glycolytic and fibrogenic markers, hypoxia-inducible factor 1α, pyruvate kinase M2 (PKM2), and TG2. Consistent with these findings, IL6 induced the expression of both glycolytic and pro-fibrogenic markers in cultured lung fibroblasts. IL6 also induced TG2 activation and the accumulation of TG2 in the extracellular matrix. Pharmacologic inhibition of the glycolytic enzyme, PKM2 significantly attenuated IL6-induced TG2 activity and fibrogenesis. Thus, we conclude that IL6-induced TG2 activity and cardiopulmonary remodeling associated with tissue fibrosis are under regulatory control of the glycolytic enzyme, PKM2.

Results of a national survey of substance use treatment services for youth under community supervision.

BACKGROUND: Despite the heightened risk for substance use (SU) among youth in the juvenile justice system, many do not receive the treatment that they need. OBJECTIVES: The purpose of this study is to examine the extent to which youth under community supervision by juvenile justice agencies receive community-based SU services and the factors associated with access to such services. METHODS: Data are from a nationally representative sample of Community Supervision (CS) agencies and their primary behavioral health (BH) partners. Surveys were completed by 192 CS and 271 BH agencies. RESULTS: SU services are more often available through BH than CS for all treatment modalities. EBPs are more likely to be used by BH than by CS. Co-location of services occurs most often in communities with fewer treatment options and is associated with higher interagency collaboration. Youth are more likely to receive services in communities with higher EBP use, which mediates the relationship between the availability of SU treatment modalities and the proportion of youth served. CONCLUSION: Findings identify opportunities to strengthen community systems and improve linkage to care.

Vascular smooth muscle ROCK1 contributes to hypoxia-induced pulmonary hypertension development in mice.

Rho kinase (ROCK) is implicated in the development of pulmonary arterial hypertension (PAH) in which abnormal pulmonary vascular smooth muscle (VSM) contractility and remodeling lead to right heart failure. Pharmacologic ROCK inhibitors block experimental pulmonary hypertension (PH) development in rodents but can have off-target effects and do not distinguish between the two ROCK forms, ROCK1 and ROCK2, encoded by separate genes. An earlier study using gene knock out (KO) in mice indicated that VSM ROCK2 is required for experimental PH development, but the role of ROCK1 is not well understood. Here we investigated the in vivo role of ROCK1 in PH development by generating a VSM-targeted homozygous ROCK1 gene KO mouse strain. Adult control mice exposed to Sugen5416 (Su)/hypoxia treatment to induce PH had significantly increased right ventricular systolic pressures (RVSP) and RV hypertrophy versus normoxic controls. In contrast, Su/hypoxia-exposed VSM ROCK1 KO mice did not exhibit significant RVSP elevation, and RV hypertrophy was blunted. Su/hypoxia-induced pulmonary small vessel muscularization was similarly elevated in both control and VSM ROCK1 KO animals. siRNA-mediated ROCK1 knock-down (KD) in human PAH pulmonary arterial SM cells (PASMC) did not affect cell growth. However, ROCK1 KD led to reduced AKT and MYPT1 signaling in serotonin-treated PAH PASMC. The findings suggest that like VSM ROCK2, VSM ROCK1 actively contributes to PH development, but in distinction acts via nonproliferative pathways to promote hypoxemia, and thus may be a distinct therapeutic target in PH.

Vascular cell-specific roles of mineralocorticoid receptors in pulmonary hypertension.

Abnormalities that characterize pulmonary arterial hypertension include impairment in the structure and function of pulmonary vascular endothelial and smooth muscle cells. Aldosterone levels are elevated in human pulmonary arterial hypertension and in experimental pulmonary hypertension, while inhibition of the aldosterone-binding mineralocorticoid receptor attenuates pulmonary hypertension in multiple animal models. We explored the role of mineralocorticoid receptor in endothelial and smooth muscle cells in using cell-specific mineralocorticoid receptor knockout mice exposed to sugen/hypoxia-induced pulmonary hypertension. Treatment with the mineralocorticoid receptor inhibitor spironolactone significantly reduced right ventricular systolic pressure. However, this is not reproduced by selective mineralocorticoid receptor deletion in smooth muscle cells or endothelial cells. Similarly, spironolactone attenuated the increase in right ventricular cardiomyocyte area independent of vascular mineralocorticoid receptor with no effect on right ventricular weight or interstitial fibrosis. Right ventricular perivascular fibrosis was significantly decreased by spironolactone and this was reproduced by specific deletion of mineralocorticoid receptor from endothelial cells. Endothelial cell-mineralocorticoid receptor deletion attenuated the sugen/hypoxia-induced increase in the leukocyte-adhesion molecule, E-selectin, and collagen IIIA1 in the right ventricle. Spironolactone also significantly reduced pulmonary arteriolar muscularization, independent of endothelial cell-mineralocorticoid receptor or smooth muscle cell-mineralocorticoid receptor. Finally, the degree of pulmonary perivascular inflammation was attenuated by mineralocorticoid receptor antagonism and was fully reproduced by smooth muscle cell-specific mineralocorticoid receptor deletion. These studies demonstrate that in the sugen/hypoxia pulmonary hypertension model, systemic-mineralocorticoid receptor blockade significantly attenuates the disease and that mineralocorticoid receptor has cell-specific effects, with endothelial cell-mineralocorticoid receptor contributing to right ventricular perivascular fibrosis and smooth muscle cell-mineralocorticoid receptor participating in pulmonary vascular inflammation. As mineralocorticoid receptor antagonists are being investigated to treat pulmonary arterial hypertension, these findings support novel mechanisms and potential mineralocorticoid receptor targets that mediate therapeutic benefits in patients.

HIV/STI Service Delivery Within Juvenile Community Supervision Agencies: A National Survey of Practices and Approaches to Moving High-Risk Youth Through the HIV Care Cascade.

Justice-involved youth (JIY) are at considerable risk for human immunodeficiency virus (HIV), but are disconnected from treatment and prevention. Juvenile justice agencies providing community supervision (CS) are well positioned to provide HIV prevention, testing, and prompt referral to treatment for JIY. However, we lack an understanding of juvenile CS agency responses to HIV/sexually transmitted infection (STI) needs among JIY. We conducted a nationwide systematic assessment of how juvenile CS agencies identify, refer, and move youth through the HIV care cascade using a nationally representative sample of 195 juvenile CS agencies across 20 states. Two-thirds of CS agencies did not offer any HIV-/STI-related services, and 82% reported no collaboration with health agencies. Screening or referral for HIV risk behaviors was reported by 32% of the CS agencies and 12% for any intervention or prevention for HIV/STI risk behaviors. Between 21% and 30% of agencies were unaware of the location of local HIV/STI services. HIV/STI prevention training was not a priority for directors and was ranked second to last out of 16 training topics. Agencies where staff expressed need for HIV risk training and where specific court programming was available were more likely to provide or refer for HIV/STI screening and/or testing. Agencies were more likely to provide or refer for services if they provided pre-trial/pre-adjudication supervision, parole, or court programming. Considering the low provision of HIV/STI-related services and limited collaboration between health and justice agencies, interventions that promote cross-system collaboration designed to minimize barriers and facilitate identification, referral, and linkage to HIV services for JIY are necessary.

Glycolysis regulated transglutaminase 2 activation in cardiopulmonary fibrogenic remodeling.

The pathophysiology of pulmonary hypertension (PH) and heart failure (HF) includes fibrogenic remodeling associated with the loss of pulmonary arterial (PA) and cardiac compliance. We and others have previously identified transglutaminase 2 (TG2) as a participant in adverse fibrogenic remodeling. However, little is known about the biologic mechanisms that regulate TG2 function. We examined physiological mouse models of experimental PH, HF, and type 1 diabetes that are associated with altered glucose metabolism/glycolysis and report here that TG2 expression and activity are elevated in pulmonary and cardiac tissues under all these conditions. We additionally used PA adventitial fibroblasts to test the hypothesis that TG2 is an intermediary between enhanced tissue glycolysis and fibrogenesis. Our in vitro results show that glycolytic enzymes and TG2 are upregulated in fibroblasts exposed to high glucose, which stimulates cellular glycolysis as measured by Seahorse analysis. We examined the relationship of TG2 to a terminal glycolytic enzyme, pyruvate kinase M2 (PKM2), and found that PKM2 regulates glucose-induced TG2 expression and activity as well as fibrogenesis. Our studies further show that TG2 inhibition blocks glucose-induced fibrogenesis and cell proliferation. Our findings support a novel role for glycolysis-mediated TG2 induction and tissue fibrosis associated with experimental PH, HF, and hyperglycemia.

Bioinspired Three-Dimensional Human Neuromuscular Junction Development in Suspended Hydrogel Arrays.

The physical connection between motoneurons and skeletal muscle targets is responsible for the creation of neuromuscular junctions (NMJs), which allow electrical signals to be translated to mechanical work. NMJ pathology contributes to the spectrum of neuromuscular, motoneuron, and dystrophic disease. Improving in vitro tools that allow for recapitulation of the physiology of the neuromuscular connection will enable researchers to better understand the development and maturation of NMJs, and will help to decipher mechanisms leading to NMJ degeneration. In this work, we first describe robust differentiation of bungarotoxin-positive human myotubes, as well as a reproducible method for encapsulating and aligning human myoblasts in three-dimensional (3D) suspended culture using bioprinted silk fibroin cantilevers as cell culture supports. Further analysis with coculture of motoneuron-like cells demonstrates feasibility of fully human coculture using two-dimensional and 2.5-dimensional culture methods, with appropriate differentiation of both cell types. Using these coculture differentiation conditions with motoneuron-like cells added to monocultures of 3D suspended human myotubes, we then demonstrate synaptic colocalization in coculture as well as acetylcholine and glutamic acid stimulation of human myocytes. This method represents a unique platform to coculture suspended human myoblast-seeded 3D hydrogels with integrated motoneuron-like cells derived from human induced neural stem cells. The platform described is fully customizable using 3D freeform printing into standard laboratory tissue culture materials, and allows for human myoblast alignment in 3D with precise motoneuron integration into preformed myotubes. The coculture method will ideally be useful in observation and analysis of neurite outgrowth and myogenic differentiation in 3D with quantification of several parameters of muscle innervation and function.

Developing and applying control charts to detect changes in water chemistry parameters measured in the Athabasca River near the oil sands: A tool for surveillance monitoring.

Control charting is a simple technique to identify change and is well suited for use in water quality programs. Control charts accounting for covariation associated with discharge and in some cases time were used to explore example and representative variables routinely measured in the Athabasca River near the oil sands area for indications of change. The explored variables include 5 major ions (chloride, sodium, sulfate, calcium, magnesium), 5 total metals (aluminum, iron, thallium, molybdenum, vanadium), and total suspended solids at two sites straddling the developments north of Fort McMurray. Regression equations developed from reference data (1988-2009) were used to predict observations and calculate residuals from later test data (2010-2016). Evidence of change was sought in the deviation of residual errors from the test period compared with the patterns expected and defined from probability distributions of the reference residuals using the odds ratio. In most cases, the patterns in test residuals were not statistically different from those expected from the reference period at either site, especially when data were examined annually. However, differences were found at both locations, more were found at the downstream site, and more differences emerged as data accumulated and were analyzed over time. In sum, the analyses at the downstream site suggest higher concentrations than predicted in most major ions, but the source of the changes is uncertain. In contrast, the concentrations of most metals at during the test period were lower than expected, which may be related to deposition patterns of materials or weathering of minerals during construction activities of the 2000s which influence the reference data used. The analyses also suggest alternative approaches may be necessary to understand change in some variables. Despite this, the results support the use of control charts to detect changes in water chemistry parameters and the value of the tool in surveillance phases of long-term and adaptive monitoring programs. Environ Toxicol Chem 2018;37:2296-2311. © 2018 SETAC.

Calpain-1 regulates platelet function in a humanized mouse model of sickle cell disease.

One of the major contributors to sickle cell disease (SCD) pathobiology is the hemolysis of sickle red blood cells (RBCs), which release free hemoglobin and platelet agonists including adenosine 5'-diphosphate (ADP) into the plasma. While platelet activation/aggregation may promote tissue ischemia and pulmonary hypertension in SCD, modulation of sickle platelet dysfunction remains poorly understood. Calpain-1, a ubiquitous calcium-activated cysteine protease expressed in hematopoietic cells, mediates aggregation of platelets in healthy mice. We generated calpain-1 knockout Townes sickle (SSCKO) mice to investigate the role of calpain-1 in steady state and hypoxia/reoxygenation (H/R)-induced sickle platelet activation and aggregation, clot retraction, and pulmonary arterial hypertension. Using multi-electrode aggregometry, which measures platelet adhesion and aggregation in whole blood, we determined that steady state SSCKO mice exhibit significantly impaired PAR4-TRAP-stimulated platelet aggregation as compared to Townes sickle (SS) and humanized control (AA) mice. Interestingly, the H/R injury induced platelet hyperactivity in SS and SSCKO, but not AA mice, and partially rescued the aggregation defect in SSCKO mice. The PAR4-TRAP-stimulated GPIIb-IIIa (αIIbß3) integrin activation was normal in SSCKO platelets suggesting that an alternate mechanism mediates the impaired platelet aggregation in steady state SSCKO mice. Taken together, we provide the first evidence that calpain-1 regulates platelet hyperactivity in sickle mice, and may offer a viable pharmacological target to reduce platelet hyperactivity in SCD.

Transglutaminase 2 in pulmonary and cardiac tissue remodeling in experimental pulmonary hypertension.

Tissue matrix remodeling and fibrosis leading to loss of pulmonary arterial and right ventricular compliance are important features of both experimental and clinical pulmonary hypertension (PH). We have previously reported that transglutaminase 2 (TG2) is involved in PH development while others have shown it to be a cross-linking enzyme that participates in remodeling of extracellular matrix in fibrotic diseases in general. In the present studies, we used a mouse model of experimental PH (Sugen 5416 and hypoxia; SuHypoxia) and cultured primary human cardiac and pulmonary artery adventitial fibroblasts to evaluate the relationship of TG2 to the processes of fibrosis, protein cross-linking, extracellular matrix collagen accumulation, and fibroblast-to-myofibroblast transformation. We report here that TG2 expression and activity as measured by serotonylated fibronectin and protein cross-linking activity along with fibrogenic markers are significantly elevated in lungs and right ventricles of SuHypoxic mice with PH. Similarly, TG2 expression and activity, protein cross-linking activity, and fibrogenic markers are significantly increased in cultured cardiac and pulmonary artery adventitial fibroblasts in response to hypoxia exposure. Pharmacological inhibition of TG2 activity with ERW1041E significantly reduced hypoxia-induced cross-linking activity and synthesis of collagen 1 and α-smooth muscle actin in both the in vivo and in vitro studies. TG2 short interfering RNA had a similar effect in vitro. Our results suggest that TG2 plays an important role in hypoxia-induced pulmonary and right ventricular tissue matrix remodeling in the development of PH.

Using adaptive processes and adverse outcome pathways to develop meaningful, robust, and actionable environmental monitoring programs.

The primary goals of environmental monitoring are to indicate whether unexpected changes related to development are occurring in the physical, chemical, and biological attributes of ecosystems and to inform meaningful management intervention. Although achieving these objectives is conceptually simple, varying scientific and social challenges often result in their breakdown. Conceptualizing, designing, and operating programs that better delineate monitoring, management, and risk assessment processes supported by hypothesis-driven approaches, strong inference, and adverse outcome pathways can overcome many of the challenges. Generally, a robust monitoring program is characterized by hypothesis-driven questions associated with potential adverse outcomes and feedback loops informed by data. Specifically, key and basic features are predictions of future observations (triggers) and mechanisms to respond to success or failure of those predictions (tiers). The adaptive processes accelerate or decelerate the effort to highlight and overcome ignorance while preventing the potentially unnecessary escalation of unguided monitoring and management. The deployment of the mutually reinforcing components can allow for more meaningful and actionable monitoring programs that better associate activities with consequences. Integr Environ Assess Manag 2017;13:877-891. © 2017 The Authors. Integrated Environmental Assessment and Management Published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Plasma 12- and 15-hydroxyeicosanoids are predictors of survival in pulmonary arterial hypertension.

This study aimed to characterize alterations in select eicosanoids in experimental and human pulmonary arterial hypertension (PAH) and to assess their potential utility as predictors of outcome. Using liquid chromatography-mass spectrometry, we performed targeted lipidomic analyses of the lungs and right ventricles (RVs) of chronically hypoxic rats and plasma of consecutive PAH patients and healthy controls. In rat lungs, chronic hypoxia was associated with significantly decreased lung prostacyclin (PGI2)/thromboxane B2 (TXB2) ratio and elevated lung 8-hydroxyeicosanoid (HETE) acid concentrations. RV eicosanoids did not exhibit any changes with chronic hypoxia. PAH treatment-naïve patients had significantly increased plasma concentrations of TXB2 and 5-, 8-, 12-, and 15-HETE. The PGI2/TXB2 ratio was lower in PAH patients than in controls, especially in the treatment-naïve cohort (median: 2.1, 0.3, and 1.3 in controls, treatment-naïve, and treated patients, respectively, P = 0.001). Survival was significantly worse in PAH patients with 12-HETEhigh (≥57 pg/mL) and 15-HETEhigh (≥256 pg/mL) in unadjusted and adjusted analyses (hazard ratio [HR]: 2.8 [95% confidence interval (CI): 1.1-7.3], P = 0.04 and HR: 4.3 [95% CI: 1.6-11.8], P = 0.004, respectively; adjustment was performed with the REVEAL [Registry to Evaluate Early and Long-Term PAH Disease Management] risk score). We demonstrate significant alterations in eicosanoid pathways in experimental and human PAH. We found that 12- and 15-HETE were independent predictors of survival in human PAH, even after adjusting for the REVEAL score, suggesting their potential role as novel biomarkers.

Evolution of Bioinks and Additive Manufacturing Technologies for 3D Bioprinting.

3D printing is an additive manufacturing (AM) technique that has quickly disrupted traditional design and manufacturing strategies. New structures can be manufactured that could not be fabricated using other methods. These new capabilities are considered by many to hallmark a historic shift representative of a new industrial revolution. Exciting utilities of this evolving technology are the fields of biomedical engineering and translational medicine, particularly in applying three-dimensional (3D) printing toward enabling on-demand fabrication of customized tissue scaffolds and medical device geometries. AM techniques are promising a future where on-demand production of patient-specific living tissues is a reality. In this review, we cover the rapid evolution and widespread concepts of a bio-"ink" and bioprinted devices and tissues from the past two decades as well as review the various additive manufacturing methods that have been used toward 3D bioprinting of cells and scaffolds with a special look at the benefits and practical considerations for each method. Despite being a young technology, the evolution and impact of AM in the fields of tissue engineering and regenerative medicine has progressed rapidly. We finish the review by looking toward the future of bioprinting and identify some of the current bottlenecks facing the blossoming industry.

Trauma and Morbidities Among Female Detainees in a Large Urban Jail.

Using data from 810 women entering the Department of Women's Justice Services in the Cook County Jail (Chicago) from 2010 to 2013, this study examines patterns of trauma exposure and the relationship between trauma exposure and mental disorders. Female detainees averaged 6.1 (SD = 4.90) types of trauma in their lifetimes, with greater trauma exposure associated with earlier age of trauma onset, more recent trauma exposure, and higher rates of fear for life or injury. Higher rates of trauma exposure were also correlated with higher rates of past-year symptoms of posttraumatic stress disorder as well as other internalizing, externalizing, and substance use disorders. Behavioral health programming for female detainees in jail settings should include more trauma-sensitive mental health and substance use disorder treatments.

Three-dimensional Printing in Developing Countries.

The advent of 3-dimensional (3D) printing technology has facilitated the creation of customized objects. The lack of regulation in developing countries renders conventional means of addressing various healthcare issues challenging. 3D printing may provide a venue for addressing many of these concerns in an inexpensive and easily accessible fashion. These may potentially include the production of basic medical supplies, vaccination beads, laboratory equipment, and prosthetic limbs. As this technology continues to improve and prices are reduced, 3D printing has the potential ability to promote initiatives across the entire developing world, resulting in improved surgical care and providing a higher quality of healthcare to its residents.

Anthrax lethal toxin-induced lung injury and treatment by activating MK2.

Anthrax is associated with severe vascular leak, which is caused by the bacterial lethal toxin (LeTx). Pleural effusions and pulmonary edema that occur in anthrax are believed to reflect endothelial injury caused by the anthrax toxin. Since vascular leak can also be observed consistently in rats injected intravenously with LeTx, the latter might present a simple physiologically relevant animal model of acute lung injury (ALI). Such a model could be utilized in evaluating and developing better treatment for ALI or acute respiratory distress syndrome (ARDS), as other available rodent models do not consistently produce the endothelial permeability that is a major component of ARDS. The biological activity of LeTx resides in the lethal factor metalloprotease that specifically degrades MAP kinase kinases (MKKs). Recently, we showed that LeTx inactivation of p38 MAP kinase signaling via degradation of MKK3 in pulmonary vascular endothelial cells can be linked to compromise of the endothelial permeability barrier. LeTx effects were linked specifically to blocking activation of p38 substrate and MAP kinase-activated protein kinase 2 (MAPKAPK2 or MK2) and phosphorylation of the latter's substrate, heat shock protein 27 (HSP27). We have now designed a peptide that directly and specifically activates MK2, causing HSP27 phosphorylation in cells and in vivo. The MK2-activating peptide (MK2-AP) also blocks the effects of LeTx on endothelial barriers in cultured cells and reduces LeTx-induced pulmonary vascular leak in rats. Hence, MK2-AP has the therapeutic potential to counteract anthrax or pulmonary edema and vascular leak due to other causes.

Polyol-Silk Bioink Formulations as Two-Part Room-Temperature Curable Materials for 3D Printing.

Silk-based bioinks were developed for 2D and 3D printing. By incorporating nontoxic polyols into silk solutions, two-part formulations with self-curing features at room temperature were generated. By varying the formulations the crystallinity of the silk polymer matrix could be controlled to support printing in 2D and 3D formats interfaced with CAD geometry and with good feature resolution. The self-curing phenomenon was tuned and exploited in order to demonstrate the formation of both structural and support materials. Biocompatible aqueous protein inks for printing that avoid the need for chemical or photo initiators and that form aqueous-stable structures with good resolution at ambient temperatures provide useful options for biofunctionalization and a broad range of applications.