The Innate Immune Sensor NLRC3 Acts as a Rheostat that Fine-Tunes T Cell Responses in Infection and Autoimmunity.

Appropriate immune responses require a fine balance between immune activation and attenuation. NLRC3, a non-inflammasome-forming member of the NLR innate immune receptor family, attenuates inflammation in myeloid cells and proliferation in epithelial cells. T lymphocytes express the highest amounts of Nlrc3 transcript where its physiologic relevance is unknown. We show that NLRC3 attenuated interferon-γ and TNF expression by CD4+ T cells and reduced T helper 1 (Th1) and Th17 cell proliferation. Nlrc3-/- mice exhibited increased and prolonged CD4+ T cell responses to lymphocytic choriomeningitis virus infection and worsened experimental autoimmune encephalomyelitis (EAE). These functions of NLRC3 were executed in a T-cell-intrinsic fashion: NLRC3 reduced K63-linked ubiquitination of TNF-receptor-associated factor 6 (TRAF6) to limit NF-κB activation, lowered phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and diminished glycolysis and oxidative phosphorylation. This study reveals an unappreciated role for NLRC3 in attenuating CD4+ T cell signaling and metabolism.

Central Autonomic Network and heart rate variability in premature neonates.

INTRODUCTION: The Central Autonomic Network (CAN) is a hierarchy of brain structures that collectively influence cardiac autonomic input, mediating the majority of brain-heart interactions, but has never been studied in premature neonates. In this study, we use heart rate variability (HRV), which has been described as the "primary output" of the CAN, and resting state functional MRI to characterize brain-heart relationships in premature neonates. METHODS: We studied premature neonates who underwent resting state functional MRI (rsfMRI) at term, (37-weeks postmenstrual age [PMA] or above) and had HRV data recorded during the same week of their MRI. HRV was derived from continuous electrocardiogram data during the week of the rsfMRI scan. For rsfMRI, a seed-based approach was used to define regions of interest (ROI) pertinent to the CAN, and blood oxygen level-dependent signal was correlated between each ROI as a measure of functional connectivity. HRV was correlated with CAN connectivity (CANconn) for each region, and sub-group analysis was performed based on sex and clinical comorbidities. RESULTS: Forty-seven premature neonates were included in this study, with a mean gestational age at birth of 28.1 +/- 2.6 weeks. Term CANconn was found to be significantly correlated with HRV in approximately one-fifth of CAN connections. Two distinct patterns emerged among these HRV-CANconn relationships. In the first, increased HRV was associated with stronger CANconn of limbic regions. In the second pattern, stronger CANconn at the precuneus was associated with impaired HRV maturation. These patterns were especially pronounced in male premature neonates. CONCLUSION: We report for the first time evidence of brain-heart relationships in premature neonates and an emerging CAN, most striking in male neonates, suggesting that the brain-heart axis may be more vulnerable in male premature neonates. Signatures in the heart rate may eventually become an important non-invasive tool to identify premature males at highest risk for neurodevelopmental impairment.

Correlation of In Vitro Kinetic Stability to Preclinical In Vivo Pharmacokinetics for a Panel of Anti-PD-1 Monoclonal Antibody Interleukin 21 Mutein Immunocytokines.

The development of therapeutic fusion protein drugs is often impeded by the unintended consequences that occur from fusing together domains from independent naturally occurring proteins, consequences such as altered biodistribution, tissue uptake, or rapid clearance and potential immunogenicity. For therapeutic fusion proteins containing globular domains, we hypothesized that aberrant in vivo behavior could be related to low kinetic stability of these domains leading to local unfolding and susceptibility to partial proteolysis and/or salvage and uptake. Herein we describe an assay to measure kinetic stability of therapeutic fusion proteins by way of their sensitivity to the protease thermolysin. The results indicate that in vivo pharmacokinetics of a panel of anti-programmed cell death protein 1 monocolonal antibody:interleukin 21 immunocytokines in both mice and nonhuman primates are highly correlated with their in vitro susceptibility to thermolysin-mediated proteolysis. This assay can be used as a tool to quickly identify in vivo liabilities of globular domains of therapeutic proteins, thus aiding in the optimization and development of new multispecific drug candidates. SIGNIFICANCE STATEMENT: This work describes a novel assay utilizing protein kinetic stability to identify preclinical in vivo pharmacokinetic liabilities of multispecific therapeutic fusion proteins. This provides an efficient, inexpensive method to ascertain inherent protein stability in vitro before conducting in vivo studies, which can rapidly increase the speed of preclinical drug development.

Inflammasomes Coordinate Pyroptosis and Natural Killer Cell Cytotoxicity to Clear Infection by a Ubiquitous Environmental Bacterium.

Defective neutrophils in patients with chronic granulomatous disease (CGD) cause susceptibility to extracellular and intracellular infections. Microbes must first be ejected from intracellular niches to expose them to neutrophil attack, so we hypothesized that inflammasomes detect certain CGD pathogens upstream of neutrophil killing. Here, we identified one such ubiquitous environmental bacterium, Chromobacterium violaceum, whose extreme virulence was fully counteracted by the NLRC4 inflammasome. Caspase-1 protected via two parallel pathways that eliminated intracellular replication niches. Pyroptosis was the primary bacterial clearance mechanism in the spleen, but both pyroptosis and interleukin-18 (IL-18)-driven natural killer (NK) cell responses were required for liver defense. NK cells cleared hepatocyte replication niches via perforin-dependent cytotoxicity, whereas interferon-γ was not required. These insights suggested a therapeutic approach: exogenous IL-18 restored perforin-dependent cytotoxicity during infection by the inflammasome-evasive bacterium Listeria monocytogenes. Therefore, inflammasomes can trigger complementary programmed cell death mechanisms, directing sterilizing immunity against intracellular bacterial pathogens.

T Follicular Helper Cell-Dependent Clearance of a Persistent Virus Infection Requires T Cell Expression of the Histone Demethylase UTX.

Epigenetic changes, including histone methylation, control T cell differentiation and memory formation, though the enzymes that mediate these processes are not clear. We show that UTX, a histone H3 lysine 27 (H3K27) demethylase, supports T follicular helper (Tfh) cell responses that are essential for B cell antibody generation and the resolution of chronic viral infections. Mice with a T cell-specific UTX deletion had fewer Tfh cells, reduced germinal center responses, lacked virus-specific immunoglobulin G (IgG), and were unable to resolve chronic lymphocytic choriomeningitis virus infections. UTX-deficient T cells showed decreased expression of interleukin-6 receptor-α and other Tfh cell-related genes that were associated with increased H3K27 methylation. Additionally, Turner Syndrome subjects, who are predisposed to chronic ear infections, had reduced UTX expression in immune cells and decreased circulating CD4(+) CXCR5(+) T cell frequency. Thus, we identify a critical link between UTX in T cells and immunity to infection.

Gestational age-related changes in the fetal functional connectome: in utero evidence for the global signal.

The human brain begins to develop in the third gestational week and rapidly grows and matures over the course of pregnancy. Compared to fetal structural neurodevelopment, less is known about emerging functional connectivity in utero. Here, we investigated gestational age (GA)-associated in vivo changes in functional brain connectivity during the second and third trimesters in a large dataset of 110 resting-state functional magnetic resonance imaging scans from a cohort of 95 healthy fetuses. Using representational similarity analysis, a multivariate analytical technique that reveals pair-wise similarity in high-order space, we showed that intersubject similarity of fetal functional connectome patterns was strongly related to between-subject GA differences (r = 0.28, P < 0.01) and that GA sensitivity of functional connectome was lateralized, especially at the frontal area. Our analysis also revealed a subnetwork of connections that were critical for predicting age (mean absolute error = 2.72 weeks); functional connectome patterns of individual fetuses reliably predicted their GA (r = 0.51, P < 0.001). Lastly, we identified the primary principal brain network that tracked fetal brain maturity. The main network showed a global synchronization pattern resembling global signal in the adult brain.

Robust sex differences in functional brain connectivity are present in utero.

Sex-based differences in brain structure and function are observable throughout development and are thought to contribute to differences in behavior, cognition, and the presentation of neurodevelopmental disorders. Using multiple support vector machine (SVM) models as a data-driven approach to assess sex differences, we sought to identify regions exhibiting sex-dependent differences in functional connectivity and determine whether they were robust and sufficiently reliable to classify sex even prior to birth. To accomplish this, we used a sample of 110 human fetal resting state fMRI scans from 95 fetuses, performed between 19 and 40 gestational weeks. Functional brain connectivity patterns classified fetal sex with 73% accuracy. Across SVM models, we identified features (functional connections) that reliably differentiated fetal sex. Highly consistent predictors included connections in the somatomotor and frontal areas alongside the hippocampus, cerebellum, and basal ganglia. Moreover, high consistency features also implicated a greater magnitude of cross-region connections in females, while male weighted features were predominately within anatomically bounded regions. Our findings indicate that these differences, which have been observed later in childhood, are present and reliably detectable even before birth. These results show that sex differences arise before birth in a manner that is consistent and reliable enough to be highly identifiable.

Experience of early-life pain in premature infants is associated with atypical cerebellar development and later neurodevelopmental deficits.

BACKGROUND: Infants born very and extremely premature (V/EPT) are at a significantly elevated risk for neurodevelopmental disorders and delays even in the absence of structural brain injuries. These risks may be due to earlier-than-typical exposure to the extrauterine environment, and its bright lights, loud noises, and exposures to painful procedures. Given the relative underdeveloped pain modulatory responses in these infants, frequent pain exposures may confer risk for later deficits. METHODS: Resting-state fMRI scans were collected at term equivalent age from 148 (45% male) infants born V/EPT and 99 infants (56% male) born at term age. Functional connectivity analyses were performed between functional regions correlating connectivity to the number of painful skin break procedures in the NICU, including heel lances, venipunctures, and IV placements. Subsequently, preterm infants returned at 18 months, for neurodevelopmental follow-up and completed assessments for autism risk and general neurodevelopment. RESULTS: We observed that V/EPT infants exhibit pronounced hyperconnectivity within the cerebellum and between the cerebellum and both limbic and paralimbic regions correlating with the number of skin break procedures. Moreover, skin breaks were strongly associated with autism risk, motor, and language scores at 18 months. Subsample analyses revealed that the same cerebellar connections strongly correlating with breaks at term age were associated with language dysfunction at 18 months. CONCLUSIONS: These results have significant implications for the clinical care of preterm infants undergoing painful exposures during routine NICU care, which typically occurs without anesthesia. Repeated pain exposures appear to have an increasingly detrimental effect on brain development during a critical period, and effects continue to be seen even 18 months later.

Modulation of Ligand-Gated Glycine Receptors Via Functional Monoclonal Antibodies.

Ion channels are targets of considerable therapeutic interest to address a wide variety of neurologic indications, including pain perception. Current pharmacological strategies have focused mostly on small molecule approaches that can be limited by selectivity requirements within members of a channel family or superfamily. Therapeutic antibodies have been proposed, designed, and characterized to alleviate this selectivity limitation; however, there are no Food and Drug Administration-approved therapeutic antibody-based drugs targeting ion channels on the market to date. Here, in an effort to identify novel classes of engineered ion channel modulators for potential neurologic therapeutic applications, we report the generation and characterization of six (EC50 < 25nM) Cys-loop receptor family monoclonal antibodies with modulatory function against rat and human glycine receptor alpha 1 (GlyRα1) and/or GlyRα3. These antibodies have activating (i.e., positive modulator) or inhibiting (i.e., negative modulator) profiles. Moreover, GlyRα3 selectivity was successfully achieved for two of the three positive modulators identified. When dosed intravenously, the antibodies achieved sufficient brain exposure to cover their calculated in vitro EC50 values. When compared head-to-head at identical exposures, the GlyRα3-selective antibody showed a more desirable safety profile over the nonselective antibody, thus demonstrating, for the first time, an advantage for GlyRα3-selectivity. Our data show that ligand-gated ion channels of the glycine receptor family within the central nervous system can be functionally modulated by engineered biologics in a dose-dependent manner and that, despite high protein homology between the alpha subunits, selectivity can be achieved within this receptor family, resulting in future therapeutic candidates with more desirable drug safety profiles. SIGNIFICANCE STATEMENT: This study presents immunization and multiplatform screening approaches to generate a diverse library of functional antibodies (agonist, potentiator, or inhibitory) raised against human glycine receptors (GlyRs). This study also demonstrates the feasibility of acquiring alpha subunit selectivity, a desirable therapeutic profile. When tested in vivo, these tool molecules demonstrated an increased safety profile in favor of GlyRα3-selectivity. These are the first reported functional GlyR antibodies that may open new avenues to treating central nervous system diseases with subunit selective biologics.

The joy of balancers.

Balancer chromosomes are multiply inverted and rearranged chromosomes that are widely used in Drosophila genetics. First described nearly 100 years ago, balancers are used extensively in stock maintenance and complex crosses. Recently, the complete molecular structures of several commonly used balancers were determined by whole-genome sequencing. This revealed a surprising amount of variation among balancers derived from a common progenitor, identified genes directly affected by inversion breakpoints, and cataloged mutations shared by balancers. These studies emphasized that it is important to choose the optimal balancer, because different inversions suppress meiotic recombination in different chromosomal regions. In this review, we provide a brief history of balancers in Drosophila, discuss how they are used today, and provide examples of unexpected recombination events involving balancers that can lead to stock breakdown.

Universal Automated Immunoaffinity Purification-CE-MS Platform for Accelerating Next Generation Biologic Design.

As the pharmaceutical industry places greater emphasis on pairing biological pathways with appropriate therapeutic intervention, an increase in the use of biologic drugs has emerged. With increasing complexity of biotherapeutics, absorption, distribution, metabolism, and excretion (ADME) studies have also become increasingly complex. The characterization of ADME properties is critical to tuning the pharmacokinetic profiles of next generation biologics (NGBs). The knowledge of the fate of a drug is essential for the enhancement of the design processes, elongation of exposure at the desired site of action, and achieving efficacy with minimum toxicity. In vivo proteolytic cleavage of biotherapeutics may lead to undesirable in vivo properties, such as rapid clearance, low bioavailability, and loss of pharmacodynamic effect. All of these may affect drug efficacy and/or generate safety concerns through increases in immunogenicity or off-target toxicity. The work herein describes the development of a robust, fully automated immunoaffinity purification (IA)-capillary electrophoresis-mass spectrometry (CE-MS) workflow. The reagents were carefully optimized to maximize isolation yields while minimizing the number of experimental steps to analytical results. The result is the development of a comprehensive integrated platform for the characterization of a wide range of biotherapeutics, including peptibodies, monoclonal antibodies, and bispecific antibodies. Empowered by this automated IA-CE-MS approach, implementing biotransformation studies at an early drug discovery stage can speed up the drug development process.

Resonant-antiresonant coupled cavity VCSELs.

The wavelength tuning range of a tunable vertical-cavity surface-emitting laser (VCSEL) is strongly influenced by the design of the interface between the semiconductor cavity and the air cavity. A simplified model is used to investigate the origin of the dramatic differences in free spectral range (FSR) and tuning slope observed in semiconductor cavity dominant, extended cavity, and air cavity dominant VCSELs. The differences arise from the positioning of the resonant and antiresonant wavelengths of the semiconductor cavity with respect to the center wavelength. The air cavity dominant design is realized by designing an antiresonant semiconductor cavity, resulting in a larger tuning slope near the center of the tuning range and a wider FSR toward the edges of the tuning range. The findings from the simplified model are confirmed with the simulation of a full VCSEL structure. Using an air cavity dominant design, an electrically pumped laser with a tuning range of 68.38 nm centered at 1056.7 nm at a 550 kHz sweep rate is demonstrated with continuous wave emission at room temperature. This epitaxial design rule can be used to increase the tuning range of tunable VCSELs, making them more applicable in swept-source optical coherence tomography and frequency-modulated continuous-wave LIDAR systems.

Silica optical fiber drawn from 3D printed preforms.

Silica optical fiber was drawn from a three-dimensional printed preform. Both single mode and multimode fibers are reported. The results demonstrate additive manufacturing of glass optical fibers and its potential to disrupt traditional optical fiber fabrication. It opens up fiber designs for novel applications hitherto not possible.

Targeting S-adenosylmethionine biosynthesis with a novel allosteric inhibitor of Mat2A.

S-Adenosyl-L-methionine (SAM) is an enzyme cofactor used in methyl transfer reactions and polyamine biosynthesis. The biosynthesis of SAM from ATP and L-methionine is performed by the methionine adenosyltransferase enzyme family (Mat; EC 2.5.1.6). Human methionine adenosyltransferase 2A (Mat2A), the extrahepatic isoform, is often deregulated in cancer. We identified a Mat2A inhibitor, PF-9366, that binds an allosteric site on Mat2A that overlaps with the binding site for the Mat2A regulator, Mat2B. Studies exploiting PF-9366 suggested a general mode of Mat2A allosteric regulation. Allosteric binding of PF-9366 or Mat2B altered the Mat2A active site, resulting in increased substrate affinity and decreased enzyme turnover. These data support a model whereby Mat2B functions as an inhibitor of Mat2A activity when methionine or SAM levels are high, yet functions as an activator of Mat2A when methionine or SAM levels are low. The ramification of Mat2A activity modulation in cancer cells is also described.

Rare recombination events generate sequence diversity among balancer chromosomes in Drosophila melanogaster.

Multiply inverted balancer chromosomes that suppress exchange with their homologs are an essential part of the Drosophila melanogaster genetic toolkit. Despite their widespread use, the organization of balancer chromosomes has not been characterized at the molecular level, and the degree of sequence variation among copies of balancer chromosomes is unknown. To map inversion breakpoints and study potential diversity in descendants of a structurally identical balancer chromosome, we sequenced a panel of laboratory stocks containing the most widely used X chromosome balancer, First Multiple 7 (FM7). We mapped the locations of FM7 breakpoints to precise euchromatic coordinates and identified the flanking sequence of breakpoints in heterochromatic regions. Analysis of SNP variation revealed megabase-scale blocks of sequence divergence among currently used FM7 stocks. We present evidence that this divergence arose through rare double-crossover events that replaced a female-sterile allele of the singed gene (sn(X2)) on FM7c with a sequence from balanced chromosomes. We propose that although double-crossover events are rare in individual crosses, many FM7c chromosomes in the Bloomington Drosophila Stock Center have lost sn(X2) by this mechanism on a historical timescale. Finally, we characterize the original allele of the Bar gene (B(1)) that is carried on FM7, and validate the hypothesis that the origin and subsequent reversion of the B(1) duplication are mediated by unequal exchange. Our results reject a simple nonrecombining, clonal mode for the laboratory evolution of balancer chromosomes and have implications for how balancer chromosomes should be used in the design and interpretation of genetic experiments in Drosophila.

Effects of Gene Dose, Chromatin, and Network Topology on Expression in Drosophila melanogaster.

Deletions, commonly referred to as deficiencies by Drosophila geneticists, are valuable tools for mapping genes and for genetic pathway discovery via dose-dependent suppressor and enhancer screens. More recently, it has become clear that deviations from normal gene dosage are associated with multiple disorders in a range of species including humans. While we are beginning to understand some of the transcriptional effects brought about by gene dosage changes and the chromosome rearrangement breakpoints associated with them, much of this work relies on isolated examples. We have systematically examined deficiencies of the left arm of chromosome 2 and characterize gene-by-gene dosage responses that vary from collapsed expression through modest partial dosage compensation to full or even over compensation. We found negligible long-range effects of creating novel chromosome domains at deletion breakpoints, suggesting that cases of gene regulation due to altered nuclear architecture are rare. These rare cases include trans de-repression when deficiencies delete chromatin characterized as repressive in other studies. Generally, effects of breakpoints on expression are promoter proximal (~100bp) or in the gene body. Effects of deficiencies genome-wide are in genes with regulatory relationships to genes within the deleted segments, highlighting the subtle expression network defects in these sensitized genetic backgrounds.

LAG-3 Confers a Competitive Disadvantage upon Antiviral CD8+ T Cell Responses.

Ongoing clinical trials are evaluating the benefits of systemic blockade of lymphocyte activation gene-3 (LAG-3) signals to improve immunity to tumors. Those studies are founded on the well-established inhibitory role of LAG-3 in regulating CD8(+) T cells during chronic virus infection and antitumor responses. However, the T cell response in LAG-3-deficient mice is similar in size and function to that in wild type animals, suggesting LAG-3 has nuanced immune-regulatory functions. We performed a series of adoptive transfer experiments in mice to better understand the T cell-intrinsic functions of LAG-3 in the regulation of CD8(+) T cell responses. Our results indicate that LAG-3 expression by CD8(+) T cells inhibits their competitive fitness and results in a slightly reduced rate of cell division in comparison with LAG-3-deficient cells. This cell-intrinsic effect of LAG-3 was consistent across both acute and chronic virus infections. These data show that LAG-3 directly modulates the size of the T cell response and support the use of LAG-3 blockade regimens to enhance CD8(+) T cell responses.

MEMS-tunable VCSELs using 2D high-contrast gratings.

We demonstrate the room-temperature operation of a two-dimensional (2D) high-contrast grating (HCG) vertical-cavity surface-emitting laser (VCSEL) at 1080 nm. To the best of our knowledge, this is the first tunable electrically pumped surface-emitting laser using a 2D HCG. Our theory successfully explains the mechanism of broadband ultrahigh reflection of 2D HCGs. Our monolithic integrated laser exhibits single-mode output power above 0.68 mW under continuous-wave operation. Wavelength tunability is demonstrated via microelectromechanical system-controlled voltage.

Use of a composite symptom score during challenge in patients with suspected aspirin-exacerbated respiratory disease.

BACKGROUND: Aspirin-exacerbated respiratory disease is characterized by asthma, chronic rhinosinusitis, nasal polyposis, and sensitivity to aspirin and other nonsteroidal anti-inflammatory drugs. Confirmation of the diagnosis requires provocation challenge with resulting upper and/or lower airways reactivity. Currently, determination of a positive challenge result is based solely on clinical judgment that synthesizes subjective symptoms and objective measures, as a concomitant increase in nasal or bronchial airways resistance is measured in only half of patients. OBJECTIVE: To describe a quantitative scoring system, based on symptoms typically reported during provocation challenge, used to identify a positive challenge result. METHODS: A total of 115 patients were asked to record 10 symptoms, rated on a scale from 1 (mild) to 10 (most severe), at regular intervals during intranasal ketorolac with modified oral aspirin challenge performed in our office. Composite scores, a simple sum of all individual scores, were calculated at each time point and compared with baseline, prechallenge values. RESULTS: One hundred of the 115 patients were determined to have a positive challenge result. A statistically significant difference in composite scores was observed in reactors vs nonreactors. All nonreactors recorded an increase in composite score of less than 5, whereas 69% of reactors recorded an increase of 5 or more. CONCLUSION: Our 10-symptom composite score provides a quantitative and comparable measure of symptoms that typically present during a challenge with a positive result. Although an external validation is needed to confirm its diagnostic performance characteristics, a change in composite score of 5 or more appears to be specific to reactors.

A 30-year-old woman with chronic hives, intermittent fevers, and joint pain.

Chronic urticaria with concomitant systemic symptoms may be seen in several rheumatologic and autoinflammatory conditions. Although most of these conditions tend to improve with corticosteroids, symptoms often recur with dose tapering. The appearance of the rash in addition to the symptom pattern and laboratory data must be considered to differentiate potential causes. We presented a unique case of chronic urticaria with fevers and arthralgias. A diagnosis was made, and the patient had rapid improvement with targeted therapy.