Optical stimulation of cardiac cells with a polymer-supported silicon nanowire matrix.

Electronic pacemakers can treat electrical conduction disorders in hearts; however, they are invasive, bulky, and linked to increased incidence of infection at the tissue-device interface. Thus, researchers have looked to other more biocompatible methods for cardiac pacing or resynchronization, such as femtosecond infrared light pulsing, optogenetics, and polymer-based cardiac patches integrated with metal electrodes. Here we develop a biocompatible nongenetic approach for the optical modulation of cardiac cells and tissues. We demonstrate that a polymer-silicon nanowire composite mesh can be used to convert fast moving, low-radiance optical inputs into stimulatory signals in target cardiac cells. Our method allows for the stimulation of the cultured cardiomyocytes or ex vivo heart to beat at a higher target frequency.

Rational Design of Semiconductor Nanostructures for Functional Subcellular Interfaces.

One of the fundamental questions guiding research in the biological sciences is how cellular systems process complex physical and environmental cues and communicate with each other across multiple length scales. Importantly, aberrant signal processing in these systems can lead to diseases that can have devastating impacts on human lives. Biophysical studies in the past several decades have demonstrated that cells can respond to not only biochemical cues but also mechanical and electrical ones. Thus, the development of new materials that can both sense and modulate all of these pathways is necessary. Semiconducting nanostructures are an emerging class of discovery platforms and tools that can push the limits of our ability to modulate and sense biological behaviors for both fundamental research and clinical applications. These materials are of particular interest for interfacing with cellular systems due to their matched dimension with subcellular components (e.g., cytoskeletal filaments), and easily tunable properties in the electrical, optical and mechanical regimes. Rational design via traditional or new approaches, such as nanocasting and mesoscale chemical lithography, can allow us to control micro- and nanoscale features in nanowires to achieve new biointerfaces. Both processes endogenous to the target cell and properties of the material surface dictate the character of these interfaces. In this Account, we focus on (1) approaches for the rational design of semiconducting nanowires that exhibit unique structures for biointerfaces, (2) recent fundamental discoveries that yield robust biointerfaces at the subcellular level, (3) intracellular electrical and mechanical sensing, and (4) modulation of cellular behaviors through material topography and remote physical stimuli. In the first section, we discuss new approaches for the synthetic control of micro- and nanoscale features of these materials. In the second section, we focus on achieving biointerfaces with these rationally designed materials either intra- or extracellularly. We last delve into the use of these materials in sensing mechanical forces and electrical signals in various cellular systems as well as in instructing cellular behaviors. Future research in this area may shift the paradigm in fundamental biophysical research and biomedical applications through (1) the design and synthesis of new semiconductor-based materials and devices that interact specifically with targeted cells, (2) the clarification of many developmental, physiological, and anatomical aspects of cellular communications, (3) an understanding of how signaling between cells regulates synaptic development (e.g., information like this would offer new insight into how the nervous system works and provide new targets for the treatment of neurological diseases), (4) and the creation of new cellular materials that have the potential to open up completely new areas of application, such as in hybrid information processing systems.

Endogenous antigen tunes the responsiveness of naive B cells but not T cells.

In humans, up to 75% of newly generated B cells and about 30% of mature B cells show some degree of autoreactivity. Yet, how B cells establish and maintain tolerance in the face of autoantigen exposure during and after development is not certain. Studies of model B-cell antigen receptor (BCR) transgenic systems have highlighted the critical role of functional unresponsiveness or 'anergy'. Unlike T cells, evidence suggests that receptor editing and anergy, rather than deletion, account for much of B-cell tolerance. However, it remains unclear whether the mature diverse B-cell repertoire of mice contains anergic autoreactive B cells, and if so, whether antigen was encountered during or after their development. By taking advantage of a reporter mouse in which BCR signalling rapidly and robustly induces green fluorescent protein expression under the control of the Nur77 regulatory region, antigen-dependent and antigen-independent BCR signalling events in vivo during B-cell maturation were visualized. Here we show that B cells encounter antigen during development in the spleen, and that this antigen exposure, in turn, tunes the responsiveness of BCR signalling in B cells at least partly by downmodulating expression of surface IgM but not IgD BCRs, and by modifying basal calcium levels. By contrast, no analogous process occurs in naive mature T cells. Our data demonstrate not only that autoreactive B cells persist in the mature repertoire, but that functional unresponsiveness or anergy exists in the mature B-cell repertoire along a continuum, a fact that has long been suspected, but never yet shown. These results have important implications for understanding how tolerance in T and B cells is differently imposed, and how these processes might go awry in disease.

The structural wedge domain of the receptor-like tyrosine phosphatase CD45 enforces B cell tolerance by regulating substrate specificity.

CD45 is a receptor-like tyrosine phosphatase that positively regulates BCR signaling by dephosphorylating the inhibitory tyrosine of the Src family kinases. We showed previously that a single point mutation, E613R, introduced into the cytoplasmic membrane-proximal "wedge" domain of CD45 is sufficient to drive a lupus-like autoimmune disease on a susceptible genetic background. To clarify the molecular mechanism of this disease, we took advantage of a unique allelic series of mice in which the expression of CD45 is varied across a broad range. Although both E613R B cells and those with supraphysiologic CD45 expression exhibited hyperresponsive BCR signaling, they did so by opposite regulation of the Src family kinase Lyn. We demonstrated that the E613R allele of CD45 does not function as a hyper- or hypomorphic allele but rather alters the substrate specificity of CD45 for Lyn. Despite similarly enhancing BCR signaling, only B cells with supraphysiologic CD45 expression became anergic, whereas only mice harboring the E613R mutation developed frank autoimmunity on a susceptible genetic background. We showed that selective impairment of a Lyn-dependent negative-regulatory circuit in E613R B cells drove autoimmunity in E613R mice. This demonstrates that relaxing negative regulation of BCR signaling, rather than enhancing positive regulation, is critical for driving autoimmunity in this system.

Quantitative differences in CD45 expression unmask functions for CD45 in B-cell development, tolerance, and survival.

The receptor-like tyrosine phosphatase CD45 positively regulates antigen receptor signaling by dephosphorylating the inhibitory tyrosine of the src family kinases. CD45-deficient mice fail to fully unmask the role of CD45 in B cells because of the expression of a partially redundant tyrosine phosphatase, CD148. However, mice that are doubly deficient in CD45 and CD148 exhibit a very early block in B-cell development, thereby obscuring later roles for CD45. To overcome these limitations, here we take advantage of an allelic series of mice in which CD45 expression is titrated broadly (0-180%). Although high expression of CD45 inhibits T-cell receptor (TCR) signaling, we show that CD45 plays a purely positive regulatory role during B-cell receptor (BCR) signaling. In concert with exaggerated BCR signaling, increasing CD45 expression drives enhanced receptor editing in the bone marrow and profound loss of follicular and marginal zone B cells in the spleen. In the context of the IgHEL/sHEL model of B-cell tolerance, such high CD45 expression transforms anergy into deletion. Unexpectedly, elimination of the autoantigen sHEL in this model system in order to block clonal deletion fails to rescue survival of mature B cells. Rather, high CD45 expression reduces B-cell activating factor receptor (BAFFR) expression and inhibits B-cell activating factor (BAFF)-induced B-cell survival in a cell-intrinsic manner. Taken together, our findings reveal how CD45 function diverges in T cells and B cells, as well as how autoreactive B cells are censored as they transit development.

Impact of Sexual Assault Survivor Identity on Patient Care in the Emergency Department.

Interactions between emergency department (ED) staff and sexual assault (SA) survivors can be a source of retraumatization for survivors, increasing their risk of posttraumatic stress and decreasing utilization of longitudinal medical care. Little is known about nationwide trends in ED staff attitudes and behaviors toward survivors, including the impact of survivor identity. We conducted a survey to determine if survivor identity influenced ED staff behaviors. A nationwide survey of SA patient advocates was conducted between June and August 2021. Advocates are volunteers or staff dispatched from rape crisis centers to support survivors during ED care. Advocates participated in an online survey to determine the frequency of observing six potentially retraumatizing provider attitudes and behaviors. Adaptive questioning was used to explore the impact of survivor identity on each attitude or behavior and which identity groups were more likely to be affected. Three hundred fifteen advocates responded to the survey. Less than 10% indicated that ED staff often or always convey disbelief or blame to survivors. Almost 75% of advocates indicated that they often or always observe ED staff showing empathy to survivors. Disparities were found in provider attitudes. Over 75% of advocates observed that survivors' mental health status or substance use impacted conveyed belief from providers. Patients who were intoxicated when assaulted, had psychiatric disorders, were Black, Hispanic/LatinX, or indigenous, or were not cis-females were more likely to experience disbelief. Patients who were white and/or cis-gender females were more likely to be pressured by ED staff to complete the forensic exam and/or report to the police. Our study documents disparities in ED staff behavior towards SA survivors according to survivor identity. Given that post-assault ED interactions are critical turning points in survivors' future medical processes, disparities in ED care may be linked to larger disparities in healing from trauma.

Emergency Department Preparedness to Care for Sexual Assault Survivors: A Nationwide Study.

INTRODUCTION: Emergency departments (ED) provide trauma-informed care to sexual assault (SA) survivors and connect them with comprehensive services. Through surveying SA survivor advocates, we aimed to 1) document updated trends in the quality of care and resources offered to SA survivors and 2) identify potential disparities according to geographic regions in the US, urban vs rural clinic locations, and the availability of sexual assault nurse examiners (SANE). METHODS: We conducted a cross-sectional study between June-August 2021, surveying SA advocates who were dispatched from rape crisis centers to support survivors during ED care. Survey questions addressed two major themes in quality of care: staff preparedness to provide trauma-response care; and available resources. Staff preparedness to provide trauma-informed care was assessed through observations of staff behaviors. We used Wilcoxon rank-sum and Kruskal-Wallis tests to analyze differences in responses according to geographic regions and SANE presence. RESULTS: A total of 315 advocates from 99 crisis centers completed the survey. The survey had a participation rate of 88.7% and a completion rate of 87.9%. Advocates who indicated that a higher proportion of their cases were attended by SANEs were more likely to report higher rates of trauma-informed staff behaviors. For example, the recalled rate of staff asking patients for consent at every step of the exam was significantly associated with SANE presence (P < 0.001). With respect to access to resources, 66.7% of advocates reported that hospitals often or always have evidence collection kits available; 30.6% reported that resources such as transportation and housing are often or always available, and 55.3% reported that SANEs are often or always part of the care team. The SANEs were reported to be more frequently available in the Southwest than in other US regions (P < 0.001) and in urban as opposed to rural areas (P < 0.001). CONCLUSION: Our study indicates that support from sexual assault nurse examiners is highly associated with trauma-informed staff behaviors and comprehensive resources. Urban-rural and regional disparities exist regarding access to SANEs, suggesting that elevating nationwide quality and equity in care of survivors of sexual assault requires increased investments in SANE training and coverage.

Patient Advocates' Perspectives on the Care of Sexual Assault Survivors in Chicago-Area Emergency Departments.

Emergency departments (EDs) providing care and forensic examinations for sexual assault (SA) survivors are often supported by SA patient advocates. This study explored advocates' perspectives regarding problems and potential solutions in SA patient care through a focus group with 12 advocates. Thematic analysis identified two major themes: provider-patient interactions and ED-hospital systems. Challenging aspects of provider-patient interactions included (a) provider attitudes and (b) disempowering behaviors. Within ED-hospital systems, themes included time constraints, efficiencies, and hospital preparation. Advocates surveyed were optimistic about an increased presence of SA nurse examiners and enhanced protocols and provider training to improve survivors' experiences.

Gold-Decorated Silicon Nanowire Photocatalysts for Intracellular Production of Hydrogen Peroxide.

Hydrogen peroxide (H2O2) plays diverse biological roles, and its effects in part depend on its spatiotemporal presence, in both intra- and extracellular contexts. A full understanding of the physiological effects of H2O2 in both healthy and disease states is hampered by a lack of tools to controllably produce H2O2. Here, we address this issue by showing visible-light-induced production of exogenous H2O2 by free-standing, gold-decorated silicon nanowires internalized in human umbilical vein endothelial cells. We further show that the photocatalytic production of H2O2 is a general phenomenon of gold-silicon hybrid materials and is enhanced upon annealing.

A Needs Assessment and Educational Intervention Addressing the Care of Sexual Assault Patients in the Emergency Department.

Emergency department (ED) providers serve as the primary point-of-contact for many survivors of sexual assault but are often ill-prepared to address their unique treatment needs. Sexual assault nurse examiners (SANEs) are therefore an important resource for training other ED providers. The objective of this project was to create a SANE-led educational intervention addressing this training gap. We achieved this objective by (a) conducting a needs assessment of ED providers' self-reported knowledge of, and comfort with, sexual assault patient care at an urban academic adult ED and, (b) using these results to create and implement a SANE-led educational intervention to improve emergency medicine residents' ability to provide sexual assault patient care. From the needs assessment survey, ED providers reported confidence in medical management but not in providing trauma-informed care, conducting forensic examinations, or understanding hospital policies or state laws. Less than half of the respondents felt confident in their ability to avoid retraumatizing sexual assault patients, and only 29% felt comfortable conducting a forensic examination. On the basis of these results, a SANE-led educational intervention was developed for emergency medicine residents, consisting of a didactic lecture, two standardized patient cases, and a forensic pelvic examination simulation. Preintervention and postintervention surveys showed an increase in respondents' self-perceived ability to avoid retraumatizing patients, comfort with conducting forensic examinations, and understanding of laws and policies. These results show the value of an interprofessional collaboration between physicians and SANEs to train ED providers on sexual assault patient care.

Nongenetic optical neuromodulation with silicon-based materials.

Optically controlled nongenetic neuromodulation represents a promising approach for the fundamental study of neural circuits and the clinical treatment of neurological disorders. Among the existing material candidates that can transduce light energy into biologically relevant cues, silicon (Si) is particularly advantageous due to its highly tunable electrical and optical properties, ease of fabrication into multiple forms, ability to absorb a broad spectrum of light, and biocompatibility. This protocol describes a rational design principle for Si-based structures, general procedures for material synthesis and device fabrication, a universal method for evaluating material photoresponses, detailed illustrations of all instrumentation used, and demonstrations of optically controlled nongenetic modulation of cellular calcium dynamics, neuronal excitability, neurotransmitter release from mouse brain slices, and brain activity in the mouse brain in vivo using the aforementioned Si materials. The entire procedure takes ~4-8 d in the hands of an experienced graduate student, depending on the specific biological targets. We anticipate that our approach can also be adapted in the future to study other systems, such as cardiovascular tissues and microbial communities.

Photoelectrochemical modulation of neuronal activity with free-standing coaxial silicon nanowires.

Optical methods for modulating cellular behaviour are promising for both fundamental and clinical applications. However, most available methods are either mechanically invasive, require genetic manipulation of target cells or cannot provide subcellular specificity. Here, we address all these issues by showing optical neuromodulation with free-standing coaxial p-type/intrinsic/n-type silicon nanowires. We reveal the presence of atomic gold on the nanowire surfaces, likely due to gold diffusion during the material growth. To evaluate how surface gold impacts the photoelectrochemical properties of single nanowires, we used modified quartz pipettes from a patch clamp and recorded sustained cathodic photocurrents from single nanowires. We show that these currents can elicit action potentials in primary rat dorsal root ganglion neurons through a primarily atomic gold-enhanced photoelectrochemical process.

Rational design of silicon structures for optically controlled multiscale biointerfaces.

Silicon-based materials have been widely used. However, remotely controlled and interconnect-free silicon configurations have been rarely explored, because of limited fundamental understanding of the complex physicochemical processes that occur at interfaces between silicon and biological materials. Here, we describe rational design principles, guided by biology, for establishing intracellular, intercellular and extracellular silicon-based interfaces, where the silicon and the biological targets have matched properties. We focused on light-induced processes at these interfaces, and developed a set of matrices to quantify and differentiate the capacitive, Faradaic and thermal outputs from about 30 different silicon materials in saline. We show that these interfaces are useful for the light-controlled non-genetic modulation of intracellular calcium dynamics, of cytoskeletal structures and transport, of cellular excitability, of neurotransmitter release from brain slices, and of brain activity in vivo.

3D calcite heterostructures for dynamic and deformable mineralized matrices.

Scales are rooted in soft tissues, and are regenerated by specialized cells. The realization of dynamic synthetic analogues with inorganic materials has been a significant challenge, because the abiological regeneration sites that could yield deterministic growth behavior are hard to form. Here we overcome this fundamental hurdle by constructing a mutable and deformable array of three-dimensional calcite heterostructures that are partially locked in silicone. Individual calcite crystals exhibit asymmetrical dumbbell shapes and are prepared by a parallel tectonic approach under ambient conditions. The silicone matrix immobilizes the epitaxial nucleation sites through self-templated cavities, which enables symmetry breaking in reaction dynamics and scalable manipulation of the mineral ensembles. With this platform, we devise several mineral-enabled dynamic surfaces and interfaces. For example, we show that the induced growth of minerals yields localized inorganic adhesion for biological tissue and reversible focal encapsulation for sensitive components in flexible electronics.Minerals are rarely explored as building blocks for dynamic inorganic materials. Here, the authors derive inspiration from fish scales to create mutable surfaces based on arrays of calcite crystals, in which one end of each crystal is immobilized in and regenerated from silicone, and the other functional end is left exposed.

Cellular uptake and dynamics of unlabeled freestanding silicon nanowires.

The ability to seamlessly merge electronic devices with biological systems at the cellular length scale is an exciting prospect for exploring new fundamental cell biology and in designing next-generation therapeutic devices. Semiconductor nanowires are well suited for achieving this goal because of their intrinsic size and wide range of possible configurations. However, current studies have focused primarily on delivering substrate-bound nanowire devices through mechanical abrasion or electroporation, with these bulkier substrates negating many of the inherent benefits of using nanoscale materials. To improve on this, an important next step is learning how to distribute these devices in a drug-like fashion, where cells can naturally uptake and incorporate these electronic components, allowing for truly noninvasive device integration. We show that silicon nanowires (SiNWs) can potentially be used as such a system, demonstrating that label-free SiNWs can be internalized in multiple cell lines (96% uptake rate), undergoing an active "burst-like" transport process. Our results show that, rather than through exogenous manipulation, SiNWs are internalized primarily through an endogenous phagocytosis pathway, allowing cellular integration of these materials. To study this behavior, we have developed a robust set of methodologies for quantitatively examining high-aspect ratio nanowire-cell interactions in a time-dependent manner on both single-cell and ensemble levels. This approach represents one of the first dynamic studies of semiconductor nanowire internalization and offers valuable insight into designing devices for biomolecule delivery, intracellular sensing, and photoresponsive therapies.

p19ARF is a critical mediator of both cellular senescence and an innate immune response associated with MYC inactivation in mouse model of acute leukemia.

MYC-induced T-ALL exhibit oncogene addiction. Addiction to MYC is a consequence of both cell-autonomous mechanisms, such as proliferative arrest, cellular senescence, and apoptosis, as well as non-cell autonomous mechanisms, such as shutdown of angiogenesis, and recruitment of immune effectors. Here, we show, using transgenic mouse models of MYC-induced T-ALL, that the loss of either p19ARF or p53 abrogates the ability of MYC inactivation to induce sustained tumor regression. Loss of p53 or p19ARF, influenced the ability of MYC inactivation to elicit the shutdown of angiogenesis; however the loss of p19ARF, but not p53, impeded cellular senescence, as measured by SA-beta-galactosidase staining, increased expression of p16INK4A, and specific histone modifications. Moreover, comparative gene expression analysis suggested that a multitude of genes involved in the innate immune response were expressed in p19ARF wild-type, but not null, tumors upon MYC inactivation. Indeed, the loss of p19ARF, but not p53, impeded the in situ recruitment of macrophages to the tumor microenvironment. Finally, p19ARF null-associated gene signature prognosticated relapse-free survival in human patients with ALL. Therefore, p19ARF appears to be important to regulating cellular senescence and innate immune response that may contribute to the therapeutic response of ALL.

Nanoscale Semiconductor Devices as New Biomaterials.

Research on nanoscale semiconductor devices will elicit a novel understanding of biological systems. First, we discuss why it is necessary to build interfaces between cells and semiconductor nanoelectronics. Second, we describe some recent molecular biophysics studies with nanowire field effect transistor sensors. Third, we present the use of nanowire transistors as electrical recording devices that can be integrated into synthetic tissues and targeted intra- or extracellularly to study single cells. Lastly, we discuss future directions and challenges in further developing this area of research, which will advance biology and medicine.

Monovalent and multivalent ligation of the B cell receptor exhibit differential dependence upon Syk and Src family kinases.

The Src and Syk families of kinases are two distinct sets of kinases that play critical roles in initiating membrane-proximal B cell receptor (BCR) signaling. However, unlike in other lymphocytes, such as T cells, the "division of labor" between Src family kinases (SFKs) and Syk in B cells is not well separated because both Syk and SFKs can phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) present in proteins comprising the BCR. To understand why B cells require both SFKs and Syk for activation, we investigated the roles of both families of kinases in BCR signaling with computational modeling and in vitro experiments. Our computational model suggested that positive feedback enabled Syk to substantially compensate for the absence of SFKs when spatial clustering of BCRs was induced by multimeric ligands. We confirmed this prediction experimentally. In contrast, when B cells were stimulated by monomeric ligands that failed to produce BCR clustering, both Syk and SFKs were required for complete and rapid BCR activation. Our data suggest that SFKs could play a pivotal role in increasing BCR sensitivity to monomeric antigens of pathogens and in mediating a rapid response to soluble multimeric antigens of pathogens that can induce spatial BCR clustering.