HyperSCP: Combining Isotopic and Isobaric Labeling for Higher Throughput Single-Cell Proteomics.

Recent developments in mass spectrometry-based single-cell proteomics (SCP) have resulted in dramatically improved sensitivity, yet the relatively low measurement throughput remains a limitation. Isobaric and isotopic labeling methods have been separately applied to SCP to increase throughput through multiplexing. Here we combined both forms of labeling to achieve multiplicative scaling for higher throughput. Two-plex stable isotope labeling of amino acids in cell culture (SILAC) and isobaric tandem mass tag (TMT) labeling enabled up to 28 single cells to be analyzed in a single liquid chromatography-mass spectrometry (LC-MS) analysis, in addition to carrier, reference, and negative control channels. A custom nested nanowell chip was used for nanoliter sample processing to minimize sample losses. Using a 145-min total LC-MS cycle time, ∼280 single cells were analyzed per day. This measurement throughput could be increased to ∼700 samples per day with a high-duty-cycle multicolumn LC system producing the same active gradient. The labeling efficiency and achievable proteome coverage were characterized for multiple analysis conditions.

RAGE signaling during tobacco smoke-induced lung inflammation and potential therapeutic utility of SAGEs.

BACKGROUND: Smoke exposure culminates as a progressive lung complication involving airway inflammation and remodeling. While primary smoke poses the greatest risk, nearly half of the US population is also at risk due to exposure to secondhand smoke (SHS). METHODS: We used WT, RAGE-/- (KO), and Tet-inducible lung-specific RAGE overexpressing transgenic (TG) mice to study the role of RAGE during short-term responses to SHS. We evaluated SHS effects in mice with and without semi-synthetic glycosaminoglycan ethers (SAGEs), which are anionic, partially lipophilic sulfated polysaccharide derivatives known to inhibit RAGE signaling. TG Mice were weaned and fed doxycycline to induce RAGE at postnatal day (PN) 30. At PN40, mice from each line were exposed to room air (RA) or SHS from three Kentucky 3R4F research cigarettes via a nose-only delivery system (Scireq Scientific, Montreal, Canada) five days a week and i.p. injections of PBS or SAGE (30 mg/kg body weight) occurred three times per week from PN40-70 before mice were sacrificed on PN70. RESULTS: RAGE mRNA and protein expression was elevated following SHS exposure of control and TG mice and not detected in RAGE KO mice. Bronchoalveolar lavage fluid (BALF) analysis revealed RAGE-mediated influence on inflammatory cell diapedesis, total protein, and pro-inflammatory mediators following exposure. Lung histological assessment revealed indistinguishable morphology following exposure, yet parenchymal apoptosis was increased. Inflammatory signaling intermediates such as Ras and NF-κB, as well as downstream responses were influenced by the availability of RAGE, as evidenced by RAGE KO and SAGE treatment. CONCLUSIONS: These data provide fascinating insight suggesting therapeutic potential for the use of RAGE inhibitors in lungs exposed to SHS smoke.

The ECM: To Scaffold, or Not to Scaffold, That Is the Question.

The extracellular matrix (ECM) has pleiotropic effects, ranging from cell adhesion to cell survival. In tissue engineering, the use of ECM and ECM-like scaffolds has separated the field into two distinct areas-scaffold-based and scaffold-free. Scaffold-free techniques are used in creating reproducible cell aggregates which have massive potential for high-throughput, reproducible drug screening and disease modeling. Though, the lack of ECM prevents certain cells from surviving and proliferating. Thus, tissue engineers use scaffolds to mimic the native ECM and produce organotypic models which show more reliability in disease modeling. However, scaffold-based techniques come at a trade-off of reproducibility and throughput. To bridge the tissue engineering dichotomy, we posit that finding novel ways to incorporate the ECM in scaffold-free cultures can synergize these two disparate techniques.

PTRH2 gene mutation causes progressive congenital skeletal muscle pathology.

Peptidyl-tRNA hydrolase 2 (PTRH2) regulates integrin-mediated pro-survival and apoptotic signaling. PTRH2 is critical in muscle development and regulates myogenic differentiation. In humans a biallelic mutation in the PTRH2 gene causes infantile-onset multisystem disease with progressive muscle weakness. We report here that the Ptrh2 knockout mouse model recapitulates the progressive congenital muscle pathology observed in patients. Ptrh2 null mice demonstrate multiple degenerating and regenerating muscle fibers, increased central nuclei, elevated creatine kinase activity and endomysial fibrosis. This progressive muscle pathology resembles the muscular dystrophy phenotype in humans and mice lacking the α7 integrin. We demonstrate that in normal muscle Ptrh2 associates in a complex with the α7ß1 integrin at the sarcolemma and Ptrh2 expression is decreased in α7 integrin null muscle. Furthermore, Ptrh2 expression is altered in skeletal muscle of classical congenital muscular dystrophy mouse models. Ptrh2 levels were up-regulated in dystrophin deficient mdx muscle, which correlates with the elevated levels of the α7ß1 integrin observed in mdx muscle and Duchenne muscular dystrophy patients. Similar to the α7 integrin, Ptrh2 expression was decreased in laminin-α2 dyW null gastrocnemius muscle. Our data establishes a PTRH2 mutation as a novel driver of congenital muscle degeneration and identifies a potential novel target to treat muscle myopathies.

Bit-1 is an essential regulator of myogenic differentiation.

Muscle differentiation requires a complex signaling cascade that leads to the production of multinucleated myofibers. Genes regulating the intrinsic mitochondrial apoptotic pathway also function in controlling cell differentiation. How such signaling pathways are regulated during differentiation is not fully understood. Bit-1 (also known as PTRH2) mutations in humans cause infantile-onset multisystem disease with muscle weakness. We demonstrate here that Bit-1 controls skeletal myogenesis through a caspase-mediated signaling pathway. Bit-1-null mice exhibit a myopathy with hypotrophic myofibers. Bit-1-null myoblasts prematurely express muscle-specific proteins. Similarly, knockdown of Bit-1 expression in C2C12 myoblasts promotes early differentiation, whereas overexpression delays differentiation. In wild-type mice, Bit-1 levels increase during differentiation. Bit-1-null myoblasts exhibited increased levels of caspase 9 and caspase 3 without increased apoptosis. Bit-1 re-expression partially rescued differentiation. In Bit-1-null muscle, Bcl-2 levels are reduced, suggesting that Bcl-2-mediated inhibition of caspase 9 and caspase 3 is decreased. Bcl-2 re-expression rescued Bit-1-mediated early differentiation in Bit-1-null myoblasts and C2C12 cells with knockdown of Bit-1 expression. These results support an unanticipated yet essential role for Bit-1 in controlling myogenesis through regulation of Bcl-2.

Laminin-111 improves muscle repair in a mouse model of merosin-deficient congenital muscular dystrophy.

Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a severe and fatal muscle-wasting disease with no cure. MDC1A patients and the dy(W-/-) mouse model exhibit severe muscle weakness, demyelinating neuropathy, failed muscle regeneration and premature death. We have recently shown that laminin-111, a form of laminin found in embryonic skeletal muscle, can substitute for the loss of laminin-211/221 and prevent muscle disease progression in the dy(W-/-) mouse model. What is unclear from these studies is whether laminin-111 can restore failed regeneration to laminin-α2-deficient muscle. To investigate the potential of laminin-111 protein therapy to improve muscle regeneration, laminin-111 or phosphate-buffered saline-treated laminin-α2-deficient muscle was damaged with cardiotoxin and muscle regeneration quantified. Our results show laminin-111 treatment promoted an increase in myofiber size and number, and an increased expression of α7ß1 integrin, Pax7, myogenin and embryonic myosin heavy chain, indicating a restoration of the muscle regenerative program. Together, our results show laminin-111 restores muscle regeneration to laminin-α2-deficient muscle and further supports laminin-111 protein as a therapy for the treatment of MDC1A.

Galectin-1 Protein Therapy Prevents Pathology and Improves Muscle Function in the mdx Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease caused by mutations in the dystrophin gene, leading to the loss of a critical component of the sarcolemmal dystrophin glycoprotein complex. Galectin-1 is a small 14 kDa protein normally found in skeletal muscle and has been shown to be a modifier of immune response, muscle repair, and apoptosis. Galectin-1 levels are elevated in the muscle of mouse and dog models of DMD. Together, these findings led us to hypothesize that Galectin-1 may serve as a modifier of disease progression in DMD. To test this hypothesis, recombinant mouse Galectin-1 was produced and used to treat myogenic cells and the mdx mouse model of DMD. Here we show that intramuscular and intraperitoneal injections of Galectin-1 into mdx mice prevented pathology and improved muscle function in skeletal muscle. These improvements were a result of enhanced sarcolemmal stability mediated by elevated utrophin and α7ß1 integrin protein levels. Together our results demonstrate for the first time that Galectin-1 may serve as an exciting new protein therapeutic for the treatment of DMD.

Integrated biocompatible 3D printed isoporous membranes with 7 µm pores.

In this work, we present a new 3D printing technique that enables the realization of native digital micro-mirror device (DMD) resolution in negative features of a 3D printed part without improving 3D printer hardware and demonstrate the fabrication of fully integrated, biocompatible isoporous membranes with pore sizes as small as 7 µm. We utilize this technique to construct a microfluidic device that mimics an established organ-on-a-chip configuration, including an integrated isoporous membrane. Two cell populations are seeded on either side of the membrane and imaged as a proof of concept for other organ-on-a-chip applications. These 3D printed isoporous membranes can be leveraged for a wide variety of other mechanical and biological applications, creating new possibilities for seamlessly integrated, 3D printed microfluidic devices.

CEBPA Overexpression Enhances ß-Cell Proliferation and Survival.

A commonality between type 1 and type 2 diabetes is the decline in functional ß-cell mass. The transcription factor Nkx6.1 regulates ß-cell development and is integral for proper ß-cell function. We have previously demonstrated that Nkx6.1 depends on c-Fos mediated upregulation and the nuclear hormone receptors Nr4a1 and Nr4a3 to increase ß-cell insulin secretion, survival, and replication. Here, we demonstrate that Nkx6.1 overexpression results in upregulation of the bZip transcription factor CEBPA and that CEBPA expression is independent of c-Fos regulation. In turn, CEBPA overexpression is sufficient to enhance INS-1 832/13 ß-cell and primary rat islet proliferation. CEBPA overexpression also increases the survival of ß-cells treated with thapsigargin. We demonstrate that increased survival in response to ER stress corresponds with changes in expression of various genes involved in the unfolded protein response, including decreased Ire1a expression. These data show that CEBPA is sufficient to enhance functional ß-cell mass by increasing ß-cell proliferation and modulating the unfolded protein response.

De novo design of highly selective miniprotein inhibitors of integrins αvß6 and αvß8.

The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between the two closely related integrin proteins and other RGD integrins, stabilize specific conformational states, and have sufficient stability enabling tissue restricted administration could have considerable therapeutic utility. Existing small molecules and antibody inhibitors do not have all of these properties, and hence there is a need for new approaches. Here we describe a method for computationally designing hyperstable RGD-containing miniproteins that are highly selective for a single RGD integrin heterodimer and conformational state, and use this strategy to design inhibitors of αvß6 and αvß8 with high selectivity. The αvß6 and αvß8 inhibitors have picomolar affinities for their targets, and >1000-fold selectivity over other RGD integrins. CryoEM structures are within 0.6-0.7Å root-mean-square deviation (RMSD) to the computational design models; the designed αvß6 inhibitor and native ligand stabilize the open conformation in contrast to the therapeutic anti-αvß6 antibody BG00011 that stabilizes the bent-closed conformation and caused on-target toxicity in patients with lung fibrosis, and the αvß8 inhibitor maintains the constitutively fixed extended-closed αvß8 conformation. In a mouse model of bleomycin-induced lung fibrosis, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics when delivered via oropharyngeal administration mimicking inhalation, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

De novo design of highly selective miniprotein inhibitors of integrins αvß6 and αvß8.

The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous αvß6 and αvß8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective αvß6 and αvß8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very close to the computational design models, and show that the inhibitors stabilize specific conformational states of the αvß6 and the αvß8 integrins. In a lung fibrosis mouse model, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

Evaluating Therapeutic Activity of Galectin-1 in Sarcolemma Repair of Skeletal Muscle.

Galectin-1 is a small (14.5 kDa) multifunctional protein with cell-cell and cell-ECM adhesion due to interactions with the carbohydrate recognition domain (CRD). In two types of muscular dystrophies, this lectin protein has shown therapeutic properties, including positive regulation of skeletal muscle differentiation and regeneration. Both Duchenne and limb-girdle muscular dystrophy 2B (LGMD2B) are subtypes of muscular dystrophies characterized by deficient membrane repair, muscle weakness, and eventual loss of ambulation. This chapter explains confocal techniques such as laser injury, calcium imaging, and galectin-1 localization to examine the effects of galectin-1 on membrane repair in injured LGMD2B models.

Soluble ECM promotes organotypic formation in lung alveolar model.

Micropatterned suspension culture creates consistently sized and shaped cell aggregates but has not produced organotypic structures from stable cells, thus restricting its use in accurate disease modeling. Here, we show that organotypic structure is achieved in hybrid suspension culture via supplementation of soluble extracellular matrix (ECM). We created a viable lung organoid from epithelial, endothelial, and fibroblast human stable cell lines in suspension culture. We demonstrate the importance of soluble ECM in organotypic patterning with the emergence of lumen-like structures with airspace showing feasible gas exchange units, formation of branching, perfusable vasculature, and long-term 70-day maintenance of lumen structure. Our results show a dependent relationship between enhanced fibronectin fibril assembly and the incorporation of ECM in the organoid. We successfully applied this technology in modeling lung fibrosis via bleomycin induction and test a potential antifibrotic drug in vitro while maintaining fundamental cell-cell interactions in lung tissue. Our human fluorescent lung organoid (hFLO) model represents features of pulmonary fibrosis which were ameliorated by fasudil treatment. We also demonstrate a 3D culture method with potential of creating organoids from mature cells, thus opening avenues for disease modeling and regenerative medicine, enhancing understanding of lung cell biology in health and lung disease.

Spatially and optically tailored 3D printing for highly miniaturized and integrated microfluidics.

Traditional 3D printing based on Digital Light Processing Stereolithography (DLP-SL) is unnecessarily limiting as applied to microfluidic device fabrication, especially for high-resolution features. This limitation is due primarily to inherent tradeoffs between layer thickness, exposure time, material strength, and optical penetration that can be impossible to satisfy for microfluidic features. We introduce a generalized 3D printing process that significantly expands the accessible spatially distributed optical dose parameter space to enable the fabrication of much higher resolution 3D components without increasing the resolution of the 3D printer. Here we demonstrate component miniaturization in conjunction with a high degree of integration, including 15 µm × 15 µm valves and a 2.2 mm × 1.1 mm 10-stage 2-fold serial diluter. These results illustrate our approach's promise to enable highly functional and compact microfluidic devices for a wide variety of biomolecular applications.

An Improved Scalable Hydrogel Dish for Spheroid Culture.

Research in fields studying cellular response to surface tension and mechanical forces necessitate cell culture tools with tunability of substrate stiffness. We created a scalable hydrogel dish design to facilitate scaffold-free formation of multiple spheroids in a single dish. Our novel design features inner and outer walls, allowing efficient media changes and downstream experiments. The design is easily scalable, accommodating varying numbers of microwells per plate. We report that non-adherent hydrogel stiffness affects spheroid morphology and compaction. We found that spheroid morphology and viability in our hydrogel dishes were comparable to commercially available Aggrewell™800 plates, with improved tunability of surface stiffness and imaging area. Device function was demonstrated with a migration assay using two investigational inhibitors against EMT. We successfully maintained primary-derived spheroids from murine and porcine lungs in the hydrogel dish. These features increase the ability to produce highly consistent cell aggregates for biological research.

Therapeutic Benefit of Galectin-1: Beyond Membrane Repair, a Multifaceted Approach to LGMD2B.

Two of the main pathologies characterizing dysferlinopathies are disrupted muscle membrane repair and chronic inflammation, which lead to symptoms of muscle weakness and wasting. Here, we used recombinant human Galectin-1 (rHsGal-1) as a therapeutic for LGMD2B mouse and human models. Various redox and multimerization states of Gal-1 show that rHsGal-1 is the most effective form in both increasing muscle repair and decreasing inflammation, due to its monomer-dimer equilibrium. Dose-response testing shows an effective 25-fold safety profile between 0.54 and 13.5 mg/kg rHsGal-1 in Bla/J mice. Mice treated weekly with rHsGal-1 showed downregulation of canonical NF-κB inflammation markers, decreased muscle fat deposition, upregulated anti-inflammatory cytokines, increased membrane repair, and increased functional movement compared to non-treated mice. Gal-1 treatment also resulted in a positive self-upregulation loop of increased endogenous Gal-1 expression independent of NF-κB activation. A similar reduction in disease pathologies in patient-derived human cells demonstrates the therapeutic potential of Gal-1 in LGMD2B patients.

Biocompatible PEGDA Resin for 3D Printing.

We report a non-cytotoxic resin compatible with and designed for use in custom high-resolution 3D printers that follow the design approach described in Gong et al., Lab Chip 17, 2899 (2017). The non-cytotoxic resin is based on a poly(ethylene glycol) diacrylate (PEGDA) monomer with avobenzone as the UV absorber instead of 2-nitrophenyl phenyl sulfide (NPS). Both NPS-PEGDA and avobenzone-PEGDA (A-PEGDA) resins were evaluated for cytotoxicity and cell adhesion. We show that NPS-PEGDA can be made effectively non-cytotoxic with a post-print 12-hour ethanol wash, and that A-PEGDA, as-printed, is effectively non-cytotoxic. 3D prints made with either resin do not support strong cell adhesion in their as-printed state; however, cell adhesion increases dramatically with a short plasma treatment. Using A-PEGDA, we demonstrate spheroid formation in ultra-low adhesion 3D printed wells, and cell migration from spheroids on plasma-treated adherent surfaces. Given that A-PEGDA can be 3D printed with high resolution, it has significant promise for a wide variety of cell-based applications using 3D printed microfluidic structures.

Treatment with galectin-1 improves myogenic potential and membrane repair in dysferlin-deficient models.

Limb-girdle muscular dystrophy type 2B (LGMD2B) is caused by mutations in the dysferlin gene, resulting in non-functional dysferlin, a key protein found in muscle membrane. Treatment options available for patients are chiefly palliative in nature and focus on maintaining ambulation. Our hypothesis is that galectin-1 (Gal-1), a soluble carbohydrate binding protein, increases membrane repair capacity and myogenic potential of dysferlin-deficient muscle cells and muscle fibers. To test this hypothesis, we used recombinant human galectin-1 (rHsGal-1) to treat dysferlin-deficient models. We show that rHsGal-1 treatments of 48 h-72 h promotes myogenic maturation as indicated through improvements in size, myotube alignment, myoblast migration, and membrane repair capacity in dysferlin-deficient myotubes and myofibers. Furthermore, increased membrane repair capacity of dysferlin-deficient myotubes, independent of increased myogenic maturation is apparent and co-localizes on the membrane of myotubes after a brief 10min treatment with labeled rHsGal-1. We show the carbohydrate recognition domain of Gal-1 is necessary for observed membrane repair. Improvements in membrane repair after only a 10 min rHsGal-1treatment suggest mechanical stabilization of the membrane due to interaction with glycosylated membrane bound, ECM or yet to be identified ligands through the CDR domain of Gal-1. rHsGal-1 shows calcium-independent membrane repair in dysferlin-deficient and wild-type myotubes and myofibers. Together our novel results reveal Gal-1 mediates disease pathologies through both changes in integral myogenic protein expression and mechanical membrane stabilization.

Human Galectin-1 Improves Sarcolemma Stability and Muscle Vascularization in the mdx Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a devastating disease caused by mutations in the dystrophin gene that result in the complete absence of dystrophin protein. We have shown previously that recombinant mouse Galectin-1 treatment improves physiological and histological outcome measures in the mdx mouse model of DMD. Because recombinant human Galectin-1 (rHsGal1) will be used to treat DMD patients, we performed a dose-ranging study and intraperitoneal or intravenous delivery to determine the efficacy of rHsGal1 to improve preclinical outcome measures in mdx mice. Our studies showed that the optimal dose of rHsGal1 delivered intraperitoneally was 20 mg/kg and that this treatment improved muscle strength, sarcolemma stability, and capillary density in skeletal muscle. We next examined the efficacy of intravenous delivery and found that a dose of 2.5 mg/kg rHsGal1 was well tolerated and improved outcome measures in the mdx mouse model. Our studies identified that intravenous doses of rHsGal1 exceeding 2.5 mg/kg resulted in toxicity, indicating that dosing using this delivery mechanism will need to be carefully monitored. Our results support the idea that rHsGal1 treatment can improve outcome measures in the mdx mouse model and support further development as a potential therapeutic agent for DMD.

The Sweet-Side of Leukocytes: Galectins as Master Regulators of Neutrophil Function.

Among responders to microbial invasion, neutrophils represent one of the earliest and perhaps most important factors that contribute to initial host defense. Effective neutrophil immunity requires their rapid mobilization to the site of infection, which requires efficient extravasation, activation, chemotaxis, phagocytosis, and eventual killing of potential microbial pathogens. Following pathogen elimination, neutrophils must be eliminated to prevent additional host injury and subsequent exacerbation of the inflammatory response. Galectins, expressed in nearly every tissue and regulated by unique sensitivity to oxidative and proteolytic inactivation, appear to influence nearly every aspect of neutrophil function. In this review, we will examine the impact of galectins on neutrophils, with a particular focus on the unique biochemical traits that allow galectin family members to spatially and temporally regulate neutrophil function.