Cavity-enhanced microwave readout of a solid-state spin sensor.

Overcoming poor readout is an increasingly urgent challenge for devices based on solid-state spin defects, particularly given their rapid adoption in quantum sensing, quantum information, and tests of fundamental physics. However, in spite of experimental progress in specific systems, solid-state spin sensors still lack a universal, high-fidelity readout technique. Here we demonstrate high-fidelity, room-temperature readout of an ensemble of nitrogen-vacancy centers via strong coupling to a dielectric microwave cavity, building on similar techniques commonly applied in cryogenic circuit cavity quantum electrodynamics. This strong collective interaction allows the spin ensemble's microwave transition to be probed directly, thereby overcoming the optical photon shot noise limitations of conventional fluorescence readout. Applying this technique to magnetometry, we show magnetic sensitivity approaching the Johnson-Nyquist noise limit of the system. Our results pave a clear path to achieve unity readout fidelity of solid-state spin sensors through increased ensemble size, reduced spin-resonance linewidth, or improved cavity quality factor.

Development and Characterization of a Preclinical Model for the Evaluation of CD205-Mediated Antigen Delivery Therapeutics in Type 1 Diabetes.

Dendritic cells (DCs) are crucial for the production of adaptive immune responses to disease-causing microbes. However, in the steady state (i.e., in the absence of an infection or when Ags are experimentally delivered without a DC-activating adjuvant), DCs present Ags to T cells in a tolerogenic manner and are important for the establishment of peripheral tolerance. Delivery of islet Ags to DCs using Ag-linked Abs to the DC endocytic receptor CD205 has shown promise in the NOD mouse model of type 1 diabetes (T1D). It is important to note, however, that all myeloid DCs express CD205 in humans, whereas in mice, only one of the classical DC subsets does (classical DC1; CD8α+ in spleen). Thus, the evaluation of CD205-targeted treatments in mice will likely not accurately predict the results observed in humans. To overcome this challenge, we have developed and characterized a novel NOD mouse model in which all myeloid DCs transgenically express human CD205 (hCD205). This NOD.hCD205 strain displays a similar T1D incidence profile to standard NOD mice. The presence of the transgene does not alter DC development, phenotype, or function. Importantly, the DCs are able to process and present Ags delivered via hCD205. Because Ags taken up via hCD205 can be presented on both class I and class II MHC, both CD4+ and CD8+ T cells can be modulated. As both T cell subsets are important for T1D pathogenesis, NOD.hCD205 mice represent a unique, patient-relevant tool for the development and optimization of DC-directed T1D therapies.

HLA-B*39:06 Efficiently Mediates Type 1 Diabetes in a Mouse Model Incorporating Reduced Thymic Insulin Expression.

Type 1 diabetes (T1D) is characterized by T cell-mediated destruction of the insulin-producing ß cells of the pancreatic islets. Among the loci associated with T1D risk, those most predisposing are found in the MHC region. HLA-B*39:06 is the most predisposing class I MHC allele and is associated with an early age of onset. To establish an NOD mouse model for the study of HLA-B*39:06, we expressed it in the absence of murine class I MHC. HLA-B*39:06 was able to mediate the development of CD8 T cells, support lymphocytic infiltration of the islets, and confer T1D susceptibility. Because reduced thymic insulin expression is associated with impaired immunological tolerance to insulin and increased T1D risk in patients, we incorporated this in our model as well, finding that HLA-B*39:06-transgenic NOD mice with reduced thymic insulin expression have an earlier age of disease onset and a higher overall prevalence as compared with littermates with typical thymic insulin expression. This was despite virtually indistinguishable blood insulin levels, T cell subset percentages, and TCR Vß family usage, confirming that reduced thymic insulin expression does not impact T cell development on a global scale. Rather, it will facilitate the thymic escape of insulin-reactive HLA-B*39:06-restricted T cells, which participate in ß cell destruction. We also found that in mice expressing either HLA-B*39:06 or HLA-A*02:01 in the absence of murine class I MHC, HLA transgene identity alters TCR Vß usage by CD8 T cells, demonstrating that some TCR Vß families have a preference for particular class I MHC alleles.

Improved Murine MHC-Deficient HLA Transgenic NOD Mouse Models for Type 1 Diabetes Therapy Development.

Improved mouse models for type 1 diabetes (T1D) therapy development are needed. T1D susceptibility is restored to normally resistant NOD.ß2m-/- mice transgenically expressing human disease-associated HLA-A*02:01 or HLA-B*39:06 class I molecules in place of their murine counterparts. T1D is dependent on pathogenic CD8+ T-cell responses mediated by these human class I variants. NOD.ß2m-/--A2.1 mice were previously used to identify ß-cell autoantigens presented by this human class I variant to pathogenic CD8+ T cells and for testing therapies to attenuate such effectors. However, NOD.ß2m-/- mice also lack nonclassical MHC I family members, including FcRn, required for antigen presentation, and maintenance of serum IgG and albumin, precluding therapies dependent on these molecules. Hence, we used CRISPR/Cas9 to directly ablate the NOD H2-Kd and H2-Db classical class I variants either individually or in tandem (cMHCI-/-). Ablation of the H2-Ag7 class II variant in the latter stock created NOD mice totally lacking in classical murine MHC expression (cMHCI/II-/-). NOD-cMHCI-/- mice retained nonclassical MHC I molecule expression and FcRn activity. Transgenic expression of HLA-A2 or -B39 restored pathogenic CD8+ T-cell development and T1D susceptibility to NOD-cMHCI-/- mice. These next-generation HLA-humanized NOD models may provide improved platforms for T1D therapy development.

Optical magnetic detection of single-neuron action potentials using quantum defects in diamond.

Magnetic fields from neuronal action potentials (APs) pass largely unperturbed through biological tissue, allowing magnetic measurements of AP dynamics to be performed extracellularly or even outside intact organisms. To date, however, magnetic techniques for sensing neuronal activity have either operated at the macroscale with coarse spatial and/or temporal resolution-e.g., magnetic resonance imaging methods and magnetoencephalography-or been restricted to biophysics studies of excised neurons probed with cryogenic or bulky detectors that do not provide single-neuron spatial resolution and are not scalable to functional networks or intact organisms. Here, we show that AP magnetic sensing can be realized with both single-neuron sensitivity and intact organism applicability using optically probed nitrogen-vacancy (NV) quantum defects in diamond, operated under ambient conditions and with the NV diamond sensor in close proximity (∼10 µm) to the biological sample. We demonstrate this method for excised single neurons from marine worm and squid, and then exterior to intact, optically opaque marine worms for extended periods and with no observed adverse effect on the animal. NV diamond magnetometry is noninvasive and label-free and does not cause photodamage. The method provides precise measurement of AP waveforms from individual neurons, as well as magnetic field correlates of the AP conduction velocity, and directly determines the AP propagation direction through the inherent sensitivity of NVs to the associated AP magnetic field vector.

An HLA-Transgenic Mouse Model of Type 1 Diabetes That Incorporates the Reduced but Not Abolished Thymic Insulin Expression Seen in Patients.

Type 1 diabetes (T1D) is an autoimmune disease characterized by T cell-mediated destruction of the pancreatic islet beta cells. Multiple genetic loci contribute to disease susceptibility in humans, with the most responsible locus being the major histocompatibility complex (MHC). Certain MHC alleles are predisposing, including the common HLA-A(∗)02:01. After the MHC, the locus conferring the strongest susceptibility to T1D is the regulatory region of the insulin gene, and alleles associated with reduced thymic insulin expression are predisposing. Mice express two insulin genes, Ins1 and Ins2. While both are expressed in beta cells, only Ins2 is expressed in the thymus. We have developed an HLA-A(∗)02:01-transgenic NOD-based T1D model that is heterozygous for a functional Ins2 gene. These mice exhibit reduced thymic insulin expression and accelerated disease in both genders. Immune cell populations are not grossly altered, and the mice exhibit typical signs of islet autoimmunity, including CD8 T cell responses to beta cell peptides also targeted in HLA-A(∗)02:01-positive type 1 diabetes patients. This model should find utility as a tool to uncover the mechanisms underlying the association between reduced thymic insulin expression and T1D in humans and aid in preclinical studies to evaluate insulin-targeted immunotherapies for the disease.

Characterization of the peptide binding specificity of the HLA class I alleles B*38:01 and B*39:06.

B*38:01 and B*39:06 are present with phenotypic frequencies <2% in the general population, but are of interest as B*39:06 is the B allele most associated with type 1 diabetes susceptibility and 38:01 is most protective. A previous study derived putative main anchor motifs for both alleles based on peptide elution data. The present study has utilized panels of single amino acid substitution peptide libraries to derive detailed quantitative motifs accounting for both primary and secondary influences on peptide binding. From these analyses, both alleles were confirmed to utilize the canonical position 2/C-terminus main anchor spacing. B*38:01 preferentially bound peptides with the positively charged or polar residues H, R, and Q in position 2 and the large hydrophobic residues I, F, L, W, and M at the C-terminus. B*39:06 had a similar preference for R in position 2, but also well-tolerated M, Q, and K. A more dramatic contrast between the two alleles was noted at the C-terminus, where the specificity of B*39:06 was clearly for small residues, with A as most preferred, followed by G, V, S, T, and I. Detailed position-by-position and residue-by-residue coefficient values were generated from the panels to provide detailed quantitative B*38:01 and B*39:06 motifs. It is hoped that these detailed motifs will facilitate the identification of T cell epitopes recognized in the context of two class I alleles associated with dramatically different dispositions towards type 1 diabetes, offering potential avenues for the investigation of the role of CD8 T cells in this disease.

Glucagon-reactive islet-infiltrating CD8 T cells in NOD mice.

Type 1 diabetes is characterized by T-cell-mediated destruction of the insulin-producing ß cells in pancreatic islets. A number of islet antigens recognized by CD8 T cells that contribute to disease pathogenesis in non-obese diabetic (NOD) mice have been identified; however, the antigenic specificities of the majority of the islet-infiltrating cells have yet to be determined. The primary goal of the current study was to identify candidate antigens based on the level and specificity of expression of their genes in mouse islets and in the mouse ß cell line MIN6. Peptides derived from the candidates were selected based on their predicted ability to bind H-2K(d) and were examined for recognition by islet-infiltrating T cells from NOD mice. Several proteins, including those encoded by Abcc8, Atp2a2, Pcsk2, Peg3 and Scg2, were validated as antigens in this way. Interestingly, islet-infiltrating T cells were also found to recognize peptides derived from proglucagon, whose expression in pancreatic islets is associated with α cells, which are not usually implicated in type 1 diabetes pathogenesis. However, type 1 diabetes patients have been reported to have serum autoantibodies to glucagon, and NOD mouse studies have shown a decrease in α cell mass during disease pathogenesis. Our finding of islet-infiltrating glucagon-specific T cells is consistent with these reports and suggests the possibility of α cell involvement in development and progression of disease.

Metal-specific interactions of H2 adsorbed within isostructural metal-organic frameworks.

Diffuse reflectance infrared (IR) spectroscopy performed over a wide temperature range (35-298 K) is used to study the dynamics of H(2) adsorbed within the isostructural metal-organic frameworks M(2)L (M = Mg, Mn, Co, Ni and Zn; L = 2,5-dioxidobenzene-1,4-dicarboxylate) referred to as MOF-74 and CPO-27. Spectra collected at H(2) concentrations ranging from 0.1 to 3.0 H(2) per metal cation reveal that strongly red-shifted vibrational modes arise from isolated H(2) bound to the available metal coordination site. The red shift of the bands associated with this site correlate with reported isosteric enthalpies of adsorption (at small surface coverage), which in turn depend on the identity of M. In contrast, the bands assigned to H(2) adsorbed at positions >3 Å from the metal site exhibit only minor differences among the five materials. Our results are consistent with previous models based on neutron diffraction data and independent IR studies, but they do not support a recently proposed adsorption mechanism that invokes strong H(2)···H(2) interactions (Nijem et al. J. Am. Chem. Soc.2010, 132, 14834-14848). Room temperature IR spectra comparable to those on which the recently proposed adsorption mechanism was based were only reproduced after contaminating the adsorbent with ambient air. Our interpretation that the uncontaminated spectral features result from stepwise adsorption at discrete framework sites is reinforced by systematic red shifts of adsorbed H(2) isotopologues and consistencies among overtone bands that are well-described by the Buckingham model of molecular interactions in vibrational spectroscopy.

Neural lineage differentiation of embryonic stem cells within alginate microbeads.

Cell replacement therapies, using renewable stem cell sources, hold tremendous potential to treat a wide range of degenerative diseases. Although many studies have established techniques to successfully differentiate stem cells into different mature cell lineages using growth factors or extracellular matrix protein supplementation in both two and three-dimensional configurations, they are often limited by lack of control and low yields of differentiated cells. Previously, we developed a scalable murine embryonic stem cell differentiation environment which maintained cell viability and supported ES cell differentiation to hepatocyte lineage cells. Differentiated hepatocyte function was contingent upon aggregate formation within the alginate microbeads. The present studies were designed to determine the feasibility of adapting the alginate encapsulation technique to neural lineage differentiation. The results of our studies indicate that by incorporating the soluble inducer, retinoic acid (RA), into the permeable microcapsule system, cell aggregation was decreased and neural lineage differentiation enhanced. In addition, we demonstrated that even in the absence of RA, differentiation could be directed away from the hepatocyte and toward the neural lineage by physical cell-cell aggregation blocking. In conjunction with the mechanical and physical characterization of the alginate crosslinking network, we determined that 2.2% alginate microencapsulation can be optimally adapted to ES neural differentiation. This study offers insights into targeting cellular differentiation toward both endodermal and ectodermal cell lineages, and could potentially be adaptable to differentiation of other stem cell types given the correct inducible factors and material properties.