Carlos A. Aguilar

Integration of solid-state nanopores in microfluidic networks via transfer printing of suspended membranes

Solid-state nanopores have emerged as versatile single-molecule sensors for applications including DNA sequencing, protein unfolding, micro-RNA detection, label-free detection of single nucleotide polymorphisms, and mapping of DNA-binding proteins involved in homologous recombination. While machining nanopores in dielectric membranes provides nanometer-scale precision, the rigid silicon support for the membrane contributes capacitive noise and limits integration with microfluidic networks for sample preprocessing. Herein, we demonstrate a technique to directly transfer solid-state nanopores machined in dielectric membranes from a silicon support into a microfluidic network. The resulting microfluidic-addressable nanopores can sense single DNA molecules at high bandwidths and with low noise, owing to significant reductions in membrane capacitance. This strategy will enable large-scale integration of solid-state nanopores with microfluidic upstream and downstream processing and permit new functions with nanopores such as complex manipulations for multidimensional analysis and parallel sensing in two and three-dimensional architectures.

Probing electronic properties of molecular engineered zinc oxide nanowires with photoelectron spectroscopy.

ZnO nanowires (NWs) are emerging as key elements for new lasing, photovoltaic and sensing applications but elucidation of their fundamental electronic properties has been hampered by a dearth of characterization tools capable of probing single nanowires. Herein, ZnO NWs were synthesized in solution and integrated into a low energy photoelectron spectroscopy system, where quantitative optical measurements of the NW work function and Fermi level location within the band gap were collected. Next, the NWs were decorated with several dipolar self-assembled monolayers (SAMs) and control over the electronic properties is demonstrated, yielding a completely tunable hybrid electronic material. Using this new metrology approach, a host of other extraordinary interfacial phenomena could be explored on nanowires such as spatial dopant profiling or heterostructures.

Shaping biodegradable polymers as nanostructures: Fabrication and applications

The interest in micro- and nano-devices based on biodegradable polymers for in vivo applications is growing rapidly and the key to these applications lies in the fashioning of features analogous to the size of cells. This paper presents fabrication techniques for leveraging micro- and nanostructures on biodegradable polymers. Innovative approaches such as replication molding, laser interference lithography, nanosphere lithography, and block copolymer lithography are discussed. These techniques demonstrate excellent potential for fabricating biodegradable polymeric devices in both a laboratory and industry scale.:

In vivo Monitoring of Transcriptional Dynamics After Lower-Limb Muscle Injury Enables Quantitative Classification of Healing

Traumatic lower-limb musculoskeletal injuries are pervasive amongst athletes and the military and typically an individual returns to activity prior to fully healing, increasing a predisposition for additional injuries and chronic pain. Monitoring healing progression after a musculoskeletal injury typically involves different types of imaging but these approaches suffer from several disadvantages. Isolating and profiling transcripts from the injured site would abrogate these shortcomings and provide enumerative insights into the regenerative potential of an individual’s muscle after injury. In this study, a traumatic injury was administered to a mouse model and healing progression was examined from 3 hours to 1 month using high-throughput RNA-Sequencing (RNA-Seq). Comprehensive dissection of the genome-wide datasets revealed the injured site to be a dynamic, heterogeneous environment composed of multiple cell types and thousands of genes undergoing significant expression changes in highly regulated networks. Four independent approaches were used to determine the set of genes, isoforms, and genetic pathways most characteristic of different time points post-injury and two novel approaches were developed to classify injured tissues at different time points. These results highlight the possibility to quantitatively track healing progression in situ via transcript profiling using high- throughput sequencing.

Unwavering Pathobiology of Volumetric Muscle Loss Injury.

Volumetric muscle loss (VML) resulting from extremity trauma presents chronic and persistent functional deficits which ultimately manifest disability. Acellular biological scaffolds, or decellularized extracellular matrices (ECMs), embody an ideal treatment platform due to their current clinical use for soft tissue repair, off-the-shelf availability, and zero autogenous donor tissue burden. ECMs have been reported to promote functional skeletal muscle tissue remodeling in small and large animal models of VML injury, and this conclusion was reached in a recent clinical trial that enrolled 13 patients. However, numerous other pre-clinical reports have not observed ECM-mediated skeletal muscle regeneration. The current study was designed to reconcile these discrepancies. The capacity of ECMs to orchestrate functional muscle tissue remodeling was interrogated in a porcine VML injury model using unbiased assessments of muscle tissue regeneration and functional recovery. Here, we show that VML injury incites an overwhelming inflammatory and fibrotic response that leads to expansive fibrous tissue deposition and chronic functional deficits, which ECM repair does not augment.

Transcriptional and Chromatin Dynamics of Muscle Regeneration after Severe Trauma

Summary Following injury, adult skeletal muscle undergoes a well-coordinated sequence of molecular and physiological events to promote repair and regeneration. However, a thorough understanding of the in vivo epigenomic and transcriptional mechanisms that control these reparative events is lacking. To address this, we monitored the in vivo dynamics of three histone modifications and coding and noncoding RNA expression throughout the regenerative process in a mouse model of traumatic muscle injury. We first illustrate how both coding and noncoding RNAs in tissues and sorted satellite cells are modified and regulated during various stages after trauma. Next, we use chromatin immunoprecipitation followed by sequencing to evaluate the chromatin state of cis-regulatory elements (promoters and enhancers) and view how these elements evolve and influence various muscle repair and regeneration transcriptional programs. These results provide a comprehensive view of the central factors that regulate muscle regeneration and underscore the multiple levels through which both transcriptional and epigenetic patterns are regulated to enact appropriate repair and regeneration.

RNA transcript expression of IGF-I/PI3K pathway components in regenerating skeletal muscle is sensitive to initial injury intensity.

OBJECTIVE Skeletal muscle regeneration is a complex process involving the coordinated input from multiple stimuli. Of these processes, actions of the insulin-like growth factor-I (IGF-I) and phosphoinositide 3-kinase (PI3K) pathways are vital; however, whether IGF-I or PI3K expression is modified during regeneration relative to initial damage intensity is unknown. The objective of this study was to determine whether mRNA expression of IGF-I/PI3K pathway components was differentially regulated during muscle regeneration in mice in response to traumatic injury induced by freezing of two different durations. DESIGN Traumatic injury was imposed by applying a 6-mm diameter cylindrical steel probe, cooled to the temperature of dry ice (-79°C), to the belly of the left tibialis anterior muscle of 12-week-old C57BL/6J mice for either 5s (5s) or 10s (10s). The right leg served as the uninjured control. RNA was obtained from injured and control muscles following 3, 7, and 21days recovery and examined by real-time PCR. Expression of transcripts within the IGF, PI3K, and Akt families, as well as for myogenic regulatory factors and micro-RNAs were studied. RESULTS Three days following injury, there was significantly increased expression of Igf1, Igf2, Igf1r, Igf2r, Pik3cb, Pik3cd, Pik3cg, Pik3r1, Pik3r5, Akt1, and Akt3 in response to either 5s or 10s injury compared to uninjured control muscle. There was a significantly greater expression of Pik3cb, Pik3cd, Pik3cg, Pik3r5, Akt1, and Akt3 in 10s injured muscle compared to 5s injured muscle. Seven days following injury, we observed significantly increased expression of Igf1, Igf2, Pik3cd, and Pik3cg in injured muscle compared to control muscle in response to 10s freeze injury. We also observed significantly reduced expression of Igf1r and miR-133a in response to 5s freeze injury compared to control muscle, and significantly reduced expression of Ckm, miR-1 and miR-133a in response to 10s freeze injury as compared to control. Twenty-one days following injury, 5s freeze-injured muscle exhibited significantly increased expression of Igf2, Igf2r, Pik3cg, Akt3, Myod1, Myog, Myf5, and miR-206 compared to control muscle, while 10s freeze-injured muscles showed significantly increased expression of Igf2, Igf2r, Pik3cb, Pik3cd, Pik3r5, Akt1, Akt3, and Myog compared to control. Expression of miR-1 was significantly reduced in 10s freeze-injured muscle compared to control muscle at this time. There were no significant differences in RNA expression between 5s and 10s injury at either 7d or 21d recovery in any transcript examined. CONCLUSIONS During early skeletal muscle regeneration in mice, transcript expressions for some components of the IGF-I/PI3K pathway are sensitive to initial injury intensity induced by freeze damage.

Direct Analysis of Gene Synthesis Reactions Using Solid-State Nanopores

Synthetic nucleic acids offer rich potential to understand and engineer new cellular functions, yet an unresolved limitation in their production and usage is deleterious products, which restrict design complexity and add cost. Herein, we employ a solid-state nanopore to differentiate molecules of a gene synthesis reaction into categories of correct and incorrect assemblies. This new method offers a solution that provides information on gene synthesis reactions in near-real time with higher complexity and lower costs. This advance can permit insights into gene synthesis reactions such as kinetics monitoring, real-time tuning, and optimization of factors that drive reaction-to-reaction variations as well as open venues between nanopore-sensing, synthetic biology, and DNA nanotechnology.

Multiscale analysis of a regenerative therapy for treatment of volumetric muscle loss injury

Skeletal muscle possesses a remarkable capacity to regenerate when injured, but when confronted with major traumatic injury resulting in volumetric muscle loss (VML), the regenerative process consistently fails. The loss of muscle tissue and function from VML injury has prompted development of a suite of therapeutic approaches but these strategies have proceeded without a comprehensive understanding of the molecular landscape that drives the injury response. Herein, we administered a VML injury in an established rodent model and monitored the evolution of the healing phenomenology over multiple time points using muscle function testing, histology, and expression profiling by RNA sequencing. The injury response was then compared to a regenerative medicine treatment using orthotopic transplantation of autologous minced muscle grafts (~1 mm3 tissue fragments). A chronic inflammatory and fibrotic response was observed at all time points following VML. These results suggest that the pathological response to VML injury during the acute stage of the healing response overwhelms endogenous and therapeutic regenerative processes. Overall, the data presented delineate key molecular characteristics of the pathobiological response to VML injury that are critical effectors of effective regenerative treatment paradigms.Correction to:Cell Death and Discovery (2018) 4, 33; https://doi.org/10.1038/s41420-018-0027-8; published online 20 February 2018.

Robust inflammatory and fibrotic signaling following volumetric muscle loss: a barrier to muscle regeneration.

Skeletal muscle has a remarkable regenerative capacity, which is conferred by a pool of resident stem cells, known as satellite cells. After damage, satellite cells proliferate, differentiate, and fuse to form new or repair existing multinucleated myofibers. However, after surgical or traumatic loss of a critical mass of muscle, also known as volumetric muscle loss (VML), this endogenous regenerative competence is overwhelmed. Rather VML has been shown to induce robust scar deposition, fibrotic supplantation, loss of function, and serious morbidity. These outcomes have been postulated to result from the ablation of resident regenerative progenitors in addition to connective tissue and basement membrane, which provide structural, biochemical, and mechanical cues to guide regeneration. Regenerative therapies that aim to restore these elements, such as autologous tissue or stem cell transfer from an uninjured site, or implantation of an instructive scaffold that recruits and guides reparative cells, have yielded incomplete recovery of muscle volume, strength, and function. The development of successful regenerative therapies for VML has been hindered by an incomplete understanding of the molecular phenomena driving and mediating injury repair. In this issue of Cell Death and Discovery, Aguilar et al. addresses this issue by characterizing the pathophysiologic response to VML using a multi-scale approach, and contrasting those results to surgical implantation of a regenerative therapy (minced muscle grafts-MMGs). The investigators tracked the molecular phenomenology after VML over 56 days using muscle function testing, histology, and gene expression profiling using high-throughput sequencing (RNAsequencing). Consistent with previous reports, histological analysis showed progressive fibrosis, macrophage infiltration, and minimal muscle fiber regeneration throughout the period observed . Using RNA-seq on the injured tissues and several types of bioinformatics analyses, the investigators found a series of enriched gene sets associated with chemotaxis and inflammation that were followed by pathways associated with excessive extracellular matrix (ECM) deposition and remodeling. These results were in contrast to many muscle regenerative studies, where inflammatory pathways subsided after several days. Instead, VML injury appears to stimulate complement, Wnt and TGF-β signaling in a sustained fashion, which in turn activates fibrosis development. These pathways, coupled with inefficient debris clearance, have been shown to influence the actions of multipotent mesenchymal progenitors, called fibro-adipogenic progenitors (FAPs), triggering their proliferation and differentiation into fibroblasts or adipocytes and their production of excess matrix. Uniquely, when VML was treated with MMGs, the transcriptional landscape of the tissue did not vary considerably and the deleterious pathways described above were marginally affected. The authors described multiple programs that could be contributing to this effect, including a sustained inflammatory response, dysregulated and stiff ECM (which would confer alterations in integrin signaling), as well as increases in expression of transcription factors (Smad2/3, Snai1, Id2, Id3, Bmp1) that block differentiation-promoting myogenic transcription factors (MyoD, MyoG, Mef2). Aguilar et al. then stipulate that VML injury drives muscle into a myogenesis-inhibitive feedback loop, where perturbations, such as those delivered from MMGs, do not impact