Noninvasive In Vivo Imaging of T-Cells during Cancer Immunotherapy Using Rare-Earth Nanoparticles.

Only a minority of patients respond positively to cancer immunotherapy, and addressing this variability is an active area of immunotherapy research. Infiltration of tumors by immune cells is one of the most significant prognostic indicators of response and disease-free survival. However, the ability to noninvasively sample the tumor microenvironment for immune cells remains limited. Imaging in the near-infrared-II region using rare-earth nanocrystals is emerging as a powerful imaging tool for high-resolution deep-tissue imaging. In this paper, we demonstrate that these nanoparticles can be used for noninvasive in vivo imaging of tumor-infiltrating T-cells in a highly aggressive melanoma tumor model. We present nanoparticle synthesis and surface modification strategies for the generation of small, ultrabright, and biocompatible rare-earth nanocrystals necessary for deep tissue imaging of rare cell types. The ability to noninvasively monitor the immune contexture of a tumor during immunotherapy could lead to early identification of nonresponding patients in real time, leading to earlier interventions and better outcomes.

TET2 guards against unchecked BATF3-induced CAR T cell expansion.

Further advances in cell engineering are needed to increase the efficacy of chimeric antigen receptor (CAR) and other T cell-based therapies1-5. As T cell differentiation and functional states are associated with distinct epigenetic profiles6,7, we hypothesized that epigenetic programming may provide a means to improve CAR T cell performance. Targeting the gene that encodes the epigenetic regulator ten-eleven translocation 2 (TET2)8 presents an interesting opportunity as its loss may enhance T cell memory9,10, albeit not cause malignancy9,11,12. Here we show that disruption of TET2 enhances T cell-mediated tumour rejection in leukaemia and prostate cancer models. However, loss of TET2 also enables antigen-independent CAR T cell clonal expansions that may eventually result in prominent systemic tissue infiltration. These clonal proliferations require biallelic TET2 disruption and sustained expression of the AP-1 factor BATF3 to drive a MYC-dependent proliferative program. This proliferative state is associated with reduced effector function that differs from both canonical T cell memory13,14 and exhaustion15,16 states, and is prone to the acquisition of secondary somatic mutations, establishing TET2 as a guardian against BATF3-induced CAR T cell proliferation and ensuing genomic instability. Our findings illustrate the potential of epigenetic programming to enhance T cell immunity but highlight the risk of unleashing unchecked proliferative responses.

HLA-independent T cell receptors for targeting tumors with low antigen density.

Chimeric antigen receptors (CARs) are receptors for antigen that direct potent immune responses. Tumor escape associated with low target antigen expression is emerging as one potential limitation of their efficacy. Here we edit the TRAC locus in human peripheral blood T cells to engage cell-surface targets through their T cell receptor-CD3 complex reconfigured to utilize the same immunoglobulin heavy and light chains as a matched CAR. We demonstrate that these HLA-independent T cell receptors (HIT receptors) consistently afford high antigen sensitivity and mediate tumor recognition beyond what CD28-based CARs, the most sensitive design to date, can provide. We demonstrate that the functional persistence of HIT T cells can be augmented by constitutive coexpression of CD80 and 4-1BBL. Finally, we validate the increased antigen sensitivity afforded by HIT receptors in xenograft mouse models of B cell leukemia and acute myeloid leukemia, targeting CD19 and CD70, respectively. Overall, HIT receptors are well suited for targeting cell surface antigens of low abundance.

Discovery of a new flavin N5-adduct in a tyrosine to phenylalanine variant of d-Arginine dehydrogenase.

d-Arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) catalyzes the flavin-dependent oxidation of d-arginine and other d-amino acids. Here, we report the crystal structure at 1.29 Å resolution for PaDADH-Y249F expressed and co-crystallized with d-arginine. The overall structure of PaDADH-Y249F resembled PaDADH-WT, but the electron density for the flavin cofactor was ambiguous, suggesting the presence of modified flavins. Electron density maps and mass spectrometric analysis confirmed the presence of both N5-(4-guanidino-oxobutyl)-FAD and 6-OH-FAD in a single crystal of PaDADH-Y249F and helped with the further refinement of the X-ray crystal structure. The versatility of the reduced flavin is apparent in the PaDADH-Y249F structure and is evidenced by the multiple functions it can perform in the same active site.

Clinical profile and outcomes of neovascular glaucoma in the era of anti-vascular endothelial growth factor.

PURPOSE: To report the clinical profile and visual impairment in various stages of neovascular glaucoma (NVG) at a tertiary eye center in East India. METHODS: The electronic medical records of the hospital database of patients with neovascular glaucoma seen between 2013 and 2020 were reviewed. Gonioscopic details were used to stratify patients into nonspecified NVG (Group 1), open-angle NVG (Group 2), and closed-angle NVG (Group 3). The clinical profile, angle features, cause of NVG, systemic associations, visual impairment, and blindness (defined as logarithm of the minimum angle of resolution, LogMar >1.3 at baseline and at final follow-up), and outcomes of medical/surgical interventions were compared between the three groups. RESULTS: Of 846 eyes of 810 patients with NVG (Group 1, n = 564 eyes, Group 2, n = 61 eyes, and Group 3, n = 220 eyes), at baseline, the blindness rates in Groups 3 and 2 were 90 and 75%, respectively. The time from a previous intervention to the onset of NVG ranged from 3 to 5 months, while the median duration of NVG was about 4-4.5 months (0.03-120 months). Multivariate regression identified a longer duration of NVG as the only variable associated with poor final visual acuity. CONCLUSION: Visual morbidity by NVG remains as high as 75-90% in developing countries, even with the availability of anti-VEGFs and after improved management/investigative at all stages.

Knowledge-based classification of fine-grained immune cell types in single-cell RNA-Seq data.

Single-cell RNA sequencing (scRNA-Seq) is an emerging strategy for characterizing immune cell populations. Compared to flow or mass cytometry, scRNA-Seq could potentially identify cell types and activation states that lack precise cell surface markers. However, scRNA-Seq is currently limited due to the need to manually classify each immune cell from its transcriptional profile. While recently developed algorithms accurately annotate coarse cell types (e.g. T cells versus macrophages), making fine distinctions (e.g. CD8+ effector memory T cells) remains a difficult challenge. To address this, we developed a machine learning classifier called ImmClassifier that leverages a hierarchical ontology of cell type. We demonstrate that its predictions are highly concordant with flow-based markers from CITE-seq and outperforms other tools (+15% recall, +14% precision) in distinguishing fine-grained cell types with comparable performance on coarse ones. Thus, ImmClassifier can be used to explore more deeply the heterogeneity of the immune system in scRNA-Seq experiments.

A Single-Point Mutation in d-Arginine Dehydrogenase Unlocks a Transient Conformational State Resulting in Altered Cofactor Reactivity.

Proteins are inherently dynamic, and proper enzyme function relies on conformational flexibility. In this study, we demonstrated how an active site residue changes an enzyme's reactivity by modulating fluctuations between conformational states. Replacement of tyrosine 249 (Y249) with phenylalanine in the active site of the flavin-dependent d-arginine dehydrogenase yielded an enzyme with both an active yellow FAD (Y249F-y) and an inactive chemically modified green FAD, identified as 6-OH-FAD (Y249F-g) through various spectroscopic techniques. Structural investigation of Y249F-g and Y249F-y variants by comparison to the wild-type enzyme showed no differences in the overall protein structure and fold. A closer observation of the active site of the Y249F-y enzyme revealed an alternative conformation for some active site residues and the flavin cofactor. Molecular dynamics simulations probed the alternate conformations observed in the Y249F-y enzyme structure and showed that the enzyme variant with FAD samples a metastable conformational state, not available to the wild-type enzyme. Hybrid quantum/molecular mechanical calculations identified differences in flavin electronics between the wild type and the alternate conformation of the Y249F-y enzyme. The computational studies further indicated that the alternate conformation in the Y249F-y enzyme is responsible for the higher spin density at the C6 atom of flavin, which is consistent with the formation of 6-OH-FAD in the variant enzyme. The observations in this study are consistent with an alternate conformational space that results in fine-tuning the microenvironment around a versatile cofactor playing a critical role in enzyme function.

Surface Plasmon-Enhanced Short-Wave Infrared Fluorescence for Detecting Sub-Millimeter-Sized Tumors.

Short-wave infrared (SWIR, 900-1700 nm) enables in vivo imaging with high spatiotemporal resolution and penetration depth due to the reduced tissue autofluorescence and decreased photon scattering at long wavelengths. Although small organic SWIR dye molecules have excellent biocompatibility, they have been rarely exploited as compared to their inorganic counterparts, mainly due to their low quantum yield. To increase their brightness, in this work, the SWIR dye molecules are placed in close proximity to gold nanorods (AuNRs) for surface plasmon-enhanced emission. The fluorescence enhancement is optimized by controlling the dye-to-AuNR number ratio and up to ≈45-fold enhancement factor is achieved. In addition, the results indicate that the highest dye-to-AuNR number ratio gives the highest emission intensity per weight and this is used for synthesizing SWIR imaging probes using layer-by-layer (LbL) technique with polymer coating protection. Then, the SWIR imaging probes are applied for in vivo imaging of ovarian cancer and the surface coating effect on intratumor distribution of the imaging probes is investigated in two orthotopic ovarian cancer models. Lastly, it is demonstrated that the plasmon-enhanced SWIR imaging probe has great potential for fluorescence imaging-guided surgery by showing its capability to detect sub-millimeter-sized tumors.

An evolutionarily conserved Lhx2-Ldb1 interaction regulates the acquisition of hippocampal cell fate and regional identity.

The protein co-factor Ldb1 regulates cell fate specification by interacting with LIM-homeodomain (LIM-HD) proteins in a tetrameric complex consisting of an LDB:LDB dimer that bridges two LIM-HD molecules, a mechanism first demonstrated in the Drosophila wing disc. Here, we demonstrate conservation of this interaction in the regulation of mammalian hippocampal development, which is profoundly defective upon loss of either Lhx2 or Ldb1 Electroporation of a chimeric construct that encodes the Lhx2-HD and Ldb1-DD (dimerization domain) in a single transcript cell-autonomously rescues a comprehensive range of hippocampal deficits in the mouse Ldb1 mutant, including the acquisition of field-specific molecular identity and the regulation of the neuron-glia cell fate switch. This demonstrates that the LHX:LDB complex is an evolutionarily conserved molecular regulatory device that controls complex aspects of regional cell identity in the developing brain.

Axonal precursor miRNAs hitchhike on endosomes and locally regulate the development of neural circuits.

Various species of non-coding RNAs (ncRNAs) are enriched in specific subcellular compartments, but the mechanisms orchestrating their localization and their local functions remain largely unknown. We investigated both aspects using the elongating retinal ganglion cell axon and its tip, the growth cone, as models. We reveal that specific endogenous precursor microRNAs (pre-miRNAs) are actively trafficked to distal axons by hitchhiking primarily on late endosomes/lysosomes. Upon exposure to the axon guidance cue semaphorin 3A (Sema3A), pre-miRNAs are processed specifically within axons into newly generated miRNAs, one of which, in turn, silences the basal translation of tubulin beta 3 class III (TUBB3), but not amyloid beta precursor protein (APP). At the organismal level, these mature miRNAs are required for growth cone steering and a fully functional visual system. Overall, our results uncover a novel mode of ncRNA transport from one cytosolic compartment to another within polarized cells. They also reveal that newly generated miRNAs are critical components of a ncRNA-based signaling pathway that transduces environmental signals into the structural remodeling of subcellular compartments.

Neuronal diversity and reciprocal connectivity between the vertebrate hippocampus and septum.

A hallmark of the nervous system is the precision with which myriad cell types are integrated into functional networks that control complex behaviors. The limbic system governs evolutionarily conserved processes essential for survival. The septum and the hippocampus are central to the limbic system, and control not only emotion-related behaviors but also learning and memory. Here, we provide a developmental and evolutionary perspective of the hippocampus and septum and highlight the neuronal diversity and circuitry that connects these two central components of the limbic system. This article is categorized under: Nervous System Development > Vertebrates: Regional Development Nervous System Development > Vertebrates: General Principles Comparative Development and Evolution > Regulation of Organ Diversity.

Elucidating synergistic dependencies in lung adenocarcinoma by proteome-wide signaling-network analysis.

To understand drug combination effect, it is necessary to decipher the interactions between drug targets-many of which are signaling molecules. Previously, such signaling pathway models are largely based on the compilation of literature data from heterogeneous cellular contexts. Indeed, de novo reconstruction of signaling interactions from large-scale molecular profiling is still lagging, compared to similar efforts in transcriptional and protein-protein interaction networks. To address this challenge, we introduce a novel algorithm for the systematic inference of protein kinase pathways, and applied it to published mass spectrometry-based phosphotyrosine profile data from 250 lung adenocarcinoma (LUAD) samples. The resulting network includes 43 TKs and 415 inferred, LUAD-specific substrates, which were validated at >60% accuracy by SILAC assays, including "novel' substrates of the EGFR and c-MET TKs, which play a critical oncogenic role in lung cancer. This systematic, data-driven model supported drug response prediction on an individual sample basis, including accurate prediction and validation of synergistic EGFR and c-MET inhibitor activity in cells lacking mutations in either gene, thus contributing to current precision oncology efforts.

Organotypic Explants of the Embryonic Rodent Hippocampus: An Accessible System for Transgenesis.

This protocol describes the technique of ex-vivo electroporation to target embryonic hippocampal progenitors in an organotypic slice preparation. This technique allows gene perturbation for examining developmental processes in the embryonic hippocampus while retaining the environment and connectivity of the cells. Gene perturbation can include Cre-mediated recombination, RNAi-mediated knockdown, gene overexpression, or a combination of any of these. Ex-vivo electroporation can be performed at a wide range of embryonic stages, giving temporal control to the experimenter. Spatial control can be achieved more easily by preparing the brain in a Petri dish to target particular regions of the hippocampus. The electroporated explant cultures provide a highly tractable system for the study of developmental processes that include progenitor proliferation, migration and cell fate acquisition.

Cross-Cohort Analysis Identifies a TEAD4-MYCN Positive Feedback Loop as the Core Regulatory Element of High-Risk Neuroblastoma.

High-risk neuroblastomas show a paucity of recurrent somatic mutations at diagnosis. As a result, the molecular basis for this aggressive phenotype remains elusive. Recent progress in regulatory network analysis helped us elucidate disease-driving mechanisms downstream of genomic alterations, including recurrent chromosomal alterations. Our analysis identified three molecular subtypes of high-risk neuroblastomas, consistent with chromosomal alterations, and identified subtype-specific master regulator proteins that were conserved across independent cohorts. A 10-protein transcriptional module-centered around a TEAD4-MYCN positive feedback loop-emerged as the regulatory driver of the high-risk subtype associated with MYCN amplification. Silencing of either gene collapsed MYCN-amplified (MYCNAmp) neuroblastoma transcriptional hallmarks and abrogated viability in vitro and in vivo Consistently, TEAD4 emerged as a robust prognostic marker of poor survival, with activity independent of the canonical Hippo pathway transcriptional coactivators YAP and TAZ. These results suggest novel therapeutic strategies for the large subset of MYCN-deregulated neuroblastomas.Significance: Despite progress in understanding of neuroblastoma genetics, little progress has been made toward personalized treatment. Here, we present a framework to determine the downstream effectors of the genetic alterations sustaining neuroblastoma subtypes, which can be easily extended to other tumor types. We show the critical effect of disrupting a 10-protein module centered around a YAP/TAZ-independent TEAD4-MYCN positive feedback loop in MYCNAmp neuroblastomas, nominating TEAD4 as a novel candidate for therapeutic intervention. Cancer Discov; 8(5); 582-99. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.

Associations of Vitamin D Levels and Vitamin D Receptor Genotypes with Patient-Reported Outcome/Disease Activity in Patients with Rheumatoid Arthritis.

BACKGROUND: The aim of this study is to evaluate the prevalence of vitamin-D insufficiency and vitamin-D receptor (VDR) polymorphisms in rheumatoid arthritis (RA) patients and its association with disease activity and patient reported outcomes (PROs). METHODS: Eighty-two individuals were included in a cross-sectional study (41 RA patients, 41 controls). Prior to assessment, each patient completed a PRO questionnaire. Serum vitamin-D levels and genotyping for VDR were assessed. Vitamin-D deficient patients received vitamin-D supplementation. Re-assessment of disease activity (DAS28) was performed after 9-months. RESULTS: Low vitamin-D levels were more frequent in RA patients (p < 0.01). A negative, but insignificant, association with DAS-28 score was identified; whereas, there was a significant negative association with the PROs (p < 0.01). Vitamin-D supplementation was associated with significant improvement in the patients' scores for pain, fatigue, global assessment, physical disability, and quality of life. In contrast to the control group, the frequency of the recessive TaqI and FokI genotypes was higher in RA patients. CONCLUSIONS: In RA patients, serum vitamin-D level was significantly and inversely associated with both PROs and disease activity. The TaqI and FokI fragment length polymorphisms of VDR significantly contributed to the risk of RA. Having a significant positive impact on patient reported outcomes, vitamin-D supplementation may have a role in RA management.

Urinary levels of triclosan and triclocarban in several Asian countries, Greece and the USA: Association with oxidative stress.

Triclosan (TCS) and Triclocarban (TCC) are widely used as antimicrobial preservatives in personal care products (PCPs). Because of their potential for endocrine disrupting effects, human exposure to these chemicals is a concern. Biomonitoring studies of human exposure to TCS and TCC have shown widespread exposure of populations in western European countries and the USA. However, exposure to TCC and TCS by populations in Asian countries is less well known. In this study, concentrations of TCS and TCC were determined in human urine collected from seven Asian countries (China, India, Korea, Kuwait, Japan, Saudi Arabia, and Vietnam), and Greece and the USA. A total of 430 urine samples were analyzed for TCS and TCC, of which 355 (83%) and 82 (19%), respectively, contained measurable levels of these chemicals. The overall geometric mean [GM] concentrations of TCS and TCC, were 1.36 and 0.03ng/mL, respectively. The highest mean concentration of TCS was found in urine from China (100ng/mL) and the lowest concentration was found in urine from Vietnam (2.34ng/mL). We also analyzed urinary 8-OHdG, a marker of oxidative stress, to elucidate the association with TCS and TCC levels for samples from Saudi Arabia (n=130) and a positive correlation between Ln-transformed TCC levels and 8-OHdG was found, although this was not statistically significant. This is the first study to report urinary levels of TCS and TCC in several Asian countries, especially for Vietnam, Kuwait, and Japan.

Association of Paraoxonase 1 Polymorphism and Serum 25-Hydroxyvitamin D with the Risk of Cardiovascular Disease in Patients with Rheumatoid Arthritis.

BACKGROUND: Patients with rheumatoid arthritis (RA) have significantly increased cardiovascular (CV) morbidity and mortality that are not accounted for by traditional risk factors alone. Paraoxonase 1 (PON1) and 25-hydroxyvitamin D have been shown to be involved in the pathogenesis of CV diseases. Objective: This study aimed to investigate PON1 gene polymorphism and serum 25-hydroxyvitamin D concentrations in RA patients, and to determine their association with CV risk in RA. METHODS: Serum samples from 46 RA patients and 45 healthy controls were tested for PON1 R192Q genotypes and serum vitamin D concentrations. The cardiovascular risks were assessed by Q-risk. Lipoprotein cholesterol levels, traditional CV risk factors, medication use, and RA disease activity status were also assessed. RESULTS: PON1 polymorphism and low serum 25-hydroxyvitamin D were significantly associated with increased CV risk (p < 0.05). Compared to patients with either the PON1 QQ genotype or the QR genotype, patients with the RR genotype demonstrated decreased CV risk on multivariate analysis, controlling for traditional CV risk factors, C-reactive protein levels, prednisone use, and cholesterol-lowering medication use (p < 0.05). CONCLUSIONS: There was a relationship of the genetic determinants of paraoxonase 1 (PON1 192) and serum 25-hydroxyvitamin D to CV risk in RA patients. Paired measurement of paraoxonase 1 genotype and serum 25-hydroxyvitamin D can be used as biomarkers of CV risk in RA patients.

Amine oxidation by d-arginine dehydrogenase in Pseudomonas aeruginosa.

d-Arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) is a flavin-dependent oxidoreductase, which is part of a novel two-enzyme racemization system that functions to convert d-arginine to l-arginine. PaDADH contains a noncovalently linked FAD that shows the highest activity with d-arginine. The enzyme exhibits broad substrate specificity towards d-amino acids, particularly with cationic and hydrophobic d-amino acids. Biochemical studies have established the structure and the mechanistic properties of the enzyme. The enzyme is a true dehydrogenase because it displays no reactivity towards molecular oxygen. As established through solvent and multiple kinetic isotope studies, PaDADH catalyzes an asynchronous CH and NH bond cleavage via a hydride transfer mechanism. Steady-state kinetic studies with d-arginine and d-histidine are consistent with the enzyme following a ping-pong bi-bi mechanism. As shown by a combination of crystallography, kinetic and computational data, the shape and flexibility of loop L1 in the active site of PaDADH are important for substrate capture and broad substrate specificity.

miR-182 Regulates Slit2-Mediated Axon Guidance by Modulating the Local Translation of a Specific mRNA.

During brain wiring, cue-induced axon behaviors such as directional steering and branching are aided by localized mRNA translation. Different guidance cues elicit translation of subsets of mRNAs that differentially regulate the cytoskeleton, yet little is understood about how specific mRNAs are selected for translation. MicroRNAs (miRNAs) are critical translational regulators that act through a sequence-specific mechanism. Here, we investigate the local role of miRNAs in mRNA-specific translation during pathfinding of Xenopus laevis retinal ganglion cell (RGC) axons. Among a rich repertoire of axonal miRNAs, miR-182 is identified as the most abundant. Loss of miR-182 causes RGC axon targeting defects in vivo and impairs Slit2-induced growth cone (GC) repulsion. We find that miR-182 targets cofilin-1 mRNA, silencing its translation, and Slit2 rapidly relieves the repression without causing miR-182 degradation. Our data support a model whereby miR-182 reversibly gates the selection of transcripts for fast translation depending on the extrinsic cue.

Analysis of SNPs of MC4R, GNB3 and FTO gene polymorphism in obese Saudi subjects.

BACKGROUND: The goal of this study was to analyze the association between the FTO rs17817449 (G>T), G protein beta3 subunit (GNB3) C825T and Melanocortin 4 receptor (MC4R) A822G single nucleotide polymorphism (SNP) with obesity in Saudi subjects. METHODS: The subjects were divided into 2 groups according to BMI: Obese (BMI> 29.9) and non- obese control (BMI<24.9). Genotyping of the target genes were determined by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis (RFLP). RESULTS: We demonstrated the association of the FTO genotype TT with increased weight, BMI and leptin levels in both males and females. However, there was no association of genotype TT with fasting blood glucose, triglycerides and cholesterol levels. Regarding GNB3 rs5443 polymorphism, the likelihood of obesity was linked to the TT genotype which was also associated with increased leptin levels. On the other hand, the SNP of MC4R A822G did not exhibit any significant association with obesity among studied subjects and showed only the presence of homozygous AA genotype. CONCLUSION: The polymorphism of FTO gene rs17817449 and GNB3 gene rs5443 (C825T) may be a genetic determinant of obesity in Saudi population whereas impact of MC4R Asn274Ser change could not be detected.