Selective Expression Research Articles

Effect of optogenetic excitation of non-orexinergic neurons in the hypothalamic perifornical area on motor behaviors and cardiovascular parameters in rats

Central command, a motor volition originating in the rostral part of the brain, plays a pivotal role in the precise regulation of autonomic nervous and cardiovascular systems. Central neuronal substrates responsible for transmitting central command signals remain incompletely understood. This study aimed to investigate the effect of optogenetic excitation of non-orexinergic (NOrx) neurons in the hypothalamic perifornical area (PeFA), where orexinergic neurons are densely distributed, on motor behaviors and cardiovascular parameters in rats. An adeno-associated viral serotype 2 vector carrying the human synapsin promoter encoding channelrhodopsin 2 (ChR2) fused to EYFP was injected into the PeFA of Sprague-Dawley rats, resulting in selective expression of ChR2-EYFP in NOrx PeFA neurons. In conscious rats, optogenetic excitation of NOrx PeFA neurons rapidly elicited walking or biting behavior, simultaneously causing pressor and tachycardiac responses regardless of the observed behavioral patterns. Under anesthesia, this excitation rapidly increased renal sympathetic nerve activity, immediately followed by sympathoinhibition. These findings suggest that NOrx PeFA neurons transmit central command signals, concurrently regulating somatomotor and autonomic nervous systems for locomotor exercise or biting behavior.

Reference Gene Selection and Gene Expression Analysis during Gall Development of Zizania latifolia

Reference Gene Selection and Gene Expression Analysis during Gall Development of Zizania latifolia

ISLET-1 expression in soft tissue neoplasms reveals high sensitivity but moderate specificity for desmoplastic small round cell tumors and potential utility as a diagnostic biomarker

ISLET-1 (ISL1) is a LIM-homeodomain transcription factor. Selective ISL1 expression is shown in neuroendocrine, non-neuroendocrine, and some soft tissue tumors including desmoplastic small round cell tumor (DSRCT). We assessed the specificity of ISL1 (clone EP283, 1:500, Cell Marque) in 288 soft tissue tumors, which included 17 DSRCTs and other histologic mimics. Positive staining threshold for ISL1 was set to >10 % of neoplastic cell nuclei at moderate intensity. ISL1 IHC was positive in 15/16 (94 %) DSRCTs with 75 % showing diffuse (>50 %) expression. ISL1 was positive in 1/10 (10 %) Ewing sarcomas (EWS), 7/13 (54 %) alveolar rhabdomyosarcoma (RMS), 14/22 (63 %) embryonal RMS, 7/14 (50 %) synovial sarcomas, 15/16 (93 %) neuroblastoma, 1/5 (20 %) Wilms tumor, 2/4 (50 %) olfactory neuroblastoma, and all 9 Merkel cell carcinomas. Other tumors, including all CIC::DUX4 sarcomas, were negative except 3/27 leiomyosarcomas, and 1 each of angiosarcoma, myxoid liposarcomas, inflammatory myofibroblastic tumor, malignant peripheral nerve sheath tumor, tenosynovial giant cell tumor, dedifferentiated LPS, and 1 ectomesenchymoma. In summary, among the soft tissue tumors tested, ISL1 is a highly sensitive but moderately specific marker for DSRCT and may be useful to distinguish from round cell mimics including EWS and CIC::DUX4 sarcomas. The oncogenic role of ISL1 in these tumors warrants further investigation.

Discovery of optimal cell type classification marker genes from single cell RNA sequencing data.

The use of single cell/nucleus RNA sequencing (scRNA-seq) technologies that quantitively describe cell transcriptional phenotypes is revolutionizing our understanding of cell biology, leading to new insights in cell type identification, disease mechanisms, and drug development. The tremendous growth in scRNA-seq data has posed new challenges in efficiently characterizing data-driven cell types and identifying quantifiable marker genes for cell type classification. The use of machine learning and explainable artificial intelligence has emerged as an effective approach to study large-scale scRNA-seq data. NS-Forest is a random forest machine learning-based algorithm that aims to provide a scalable data-driven solution to identify minimum combinations of necessary and sufficient marker genes that capture cell type identity with maximum classification accuracy. Here, we describe the latest version, NS-Forest version 4.0 and its companion Python package (https://github.com/JCVenterInstitute/NSForest), with several enhancements to select marker gene combinations that exhibit highly selective expression patterns among closely related cell types and more efficiently perform marker gene selection for large-scale scRNA-seq data atlases with millions of cells. By modularizing the final decision tree step, NS-Forest v4.0 can be used to compare the performance of user-defined marker genes with the NS-Forest computationally-derived marker genes based on the decision tree classifiers. To quantify how well the identified markers exhibit the desired pattern of being exclusively expressed at high levels within their target cell types, we introduce the On-Target Fraction metric that ranges from 0 to 1, with a metric of 1 assigned to markers that are only expressed within their target cell types and not in cells of any other cell types. NS-Forest v4.0 outperforms previous versions on its ability to identify markers with higher On-Target Fraction values for closely related cell types and outperforms other marker gene selection approaches at classification with significantly higher F-beta scores when applied to datasets from three human organs - brain, kidney, and lung.

5′ Transgenes drive leaky expression of 3′ transgenes in Cre-inducible bi-cistronic vectors

Molecular cloning techniques enabling contemporaneous expression of two or more protein-coding sequences provide an invaluable tool for understanding the molecular regulation of cellular functions. The Cre-lox system is used for inducing the expression of recombinant proteins encoded within a bi/poly-cistronic cassette. However, leak expression of transgenes is often observed in the absence of Cre recombinase activity, compromising the utility of this approach. To investigate the mechanism of leak expression, we generated Cre-inducible bicistronic vectors to monitor the expression of transgenes positioned either 5′ or 3′ of a 2A peptide or IRES sequence. Cells transfected with these bicistronic vectors exhibited Cre-independent leak expression specifically of transgenes positioned 3′ of the 2A peptide or IRES sequence. Similarly, AAV-FLEX vectors encoding bicistronic cassettes or fusion proteins revealed the selective Cre-independent leak expression of transgenes positioned at the 3′ end of the open-reading frame. Our data demonstrate that 5′ transgenes confer promoter-like activity that drives the expression of 3′ transgenes. An additional lox-STOP-lox cassette between the 2A sequence and 3′ transgene dramatically reduced Cre-independent transgene expression. Our findings highlight the need for appropriate experimental controls when using Cre-inducible bi/poly-cistronic constructs and inform improved design of vectors for more tightly regulated inducible transgene expression.

Identifying Novel Proteins for Chronic Pain: Integration of Human Brain Proteomes and Genome-wide Association Data

Numerous genome-wide association studies have identified risk genes for chronic pain, yet the mechanisms by which genetic variants modify susceptibility have remained elusive. We sought to identify key genes modulating chronic pain risk by regulating brain protein expression. We integrated brain proteomic data with the largest genome-wide dataset for multisite chronic pain (N = 387,649) in a proteome-wide association study (PWAS) using discovery and confirmatory proteomic datasets (N = 376 and 152) from the dorsolateral prefrontal cortex. Leveraging summary data-based Mendelian randomization and Bayesian colocalization analysis, we pinpointed potential causal genes, while a transcriptome-wide association study integrating 452 human brain transcriptomes investigated whether cis-effects on protein abundance extended to the transcriptome. Single-cell RNA-sequencing data and single-nucleus transcriptomic data revealed cell-type-specific expression patterns for identified causal genes in the dorsolateral prefrontal cortex and dorsal root ganglia (DRG), complemented by RNA microarray analysis of expression profiles in other pain-related brain regions. Of the 22 genes cis-regulating protein abundance identified by the discovery PWAS, 18 (82%) were deemed causal by summary data-based Mendelian randomization or Bayesian colocalization analysis analyses, with 7 of these 18 genes (39%) replicating in the confirmatory PWAS, including guanosine diphosphate-mannose pyrophosphorylase B, which also associated at the transcriptome level. Several causal genes exhibited selective expression in excitatory and inhibitory neurons, oligodendrocytes, and astrocytes, while most identified genes were expressed across additional pain-related brain regions. This integrative proteogenomic approach identified 18 high-confidence causal genes for chronic pain, regulated by cis-effects on brain protein levels, suggesting promising avenues for treatment research and indicating a contributory role for the DRG. PerspectiveThe current post genome-wide association study analyses identified 18 high-confidence causal genes regulating chronic pain risk via cis-modulation of brain protein abundance, suggesting promising avenues for future chronic pain therapies. Additionally, the significant expression of these genes in the DRG indicated a potential contributory role, warranting further investigation.

Reelin marks cocaine-activated striatal ensembles, promotes neuronal excitability, and regulates cocaine reward.

Drugs of abuse activate defined neuronal ensembles in brain reward structures such as the nucleus accumbens (NAc), which are thought to promote the enduring synaptic, circuit, and behavioral consequences of drug exposure. While the molecular and cellular effects arising from experience with drugs like cocaine are increasingly well understood, the mechanisms that sculpt NAc ensemble participation are largely unknown. Here, we leveraged unbiased single-nucleus transcriptional profiling to identify expression of the secreted glycoprotein Reelin (encoded by the Reln gene) as a marker of cocaine-activated neuronal ensembles within the rat NAc. Multiplexed in situ detection confirmed selective expression of the immediate early gene Fos in Reln+ neurons after cocaine experience, and also revealed enrichment of Reln mRNA in Drd1 + medium spiny neurons (MSNs) in both the rat and human brain. Using a novel CRISPR interference strategy enabling selective Reln knockdown in the adult NAc, we observed altered expression of genes linked to calcium signaling, emergence of a transcriptional trajectory consistent with loss of cocaine sensitivity, and a striking decrease in MSN intrinsic excitability. At the behavioral level, loss of Reln prevented cocaine locomotor sensitization, abolished cocaine place preference memory, and decreased cocaine self-administration behavior. Together, these results identify Reelin as a critical mechanistic link between ensemble participation and cocaine-induced behavioral adaptations.

Super-Transparent Soil for In Situ Observation of Root Phenotypes.

Transparent soil (TS) presents immense potential for root phenotyping due to its ability to facilitate high-resolution imaging. However, challenges related to transparency, mechanical properties, and cost hinder its development. Herein, we introduce super-transparent soil (s-TS) prepared via the droplet method using low acyl gellan gum and hydroxyethyl cellulose crosslinked with magnesium ions. The refractive index of the hydroxyethyl cellulose solution (1.345) closely aligns with that of water (1.333) and the low acyl gellan gum solution (1.340), thereby significantly enhancing the transmittance of hydrogel-based transparent soil. Optimal transmittance (98.45%) is achieved with polymer concentrations ranging from 0.8 to 1.6 wt.% and ion concentrations between 0.01 and 0.09 mol·L-1. After 60 days of plant cultivation, s-TS maintains a transmittance exceeding 89.5%, enabling the detailed visualization of root growth dynamics. Furthermore, s-TS exhibits remarkable mechanical properties, withstanding a maximum compressive stress of 477 kPa and supporting a maximum load-bearing depth of 186 cm. This innovative approach holds promising implications for advanced root phenotyping studies, fostering the investigation of root heterogeneity and the development of selective expression under controlled conditions.

CRPA1A2: A novel TCR-based T-cell engager targeting MAGEA1-positive solid tumors.

e14589 Background: MAGEA1, identified as a cancer/testis antigen, is highly expression in various solid tumors, including hepatocellular carcinoma, non-small cell lung cancer, but absent in normal tissues except male germ cells and placenta, both of which lacking HLA class molecules. This selective expression of HLA-MAGEA1 epitope makes it a promising target for cancer therapy. However, its intracellular localization poses a challenge for conventional antibodies designed to target surface-expressed antigens. TCR-based T-cell engagers, such as Tebentafusp, overcome this hurdle by targeting intracellular protein-derived peptides presented on cancer cell surfaces via HLA, enabling precise cytotoxic T cell attacks. Notably, Tebentafusp has shown significant clinical benefits by improving survival rates in metastatic uveal melanoma, underscoring the efficacy of TCR-based T-cell engagers in treating MAGEA1-positive tumors. Methods: We have developed an advanced structural format, CR62, consisting of a anti-CD3 single chain fragment, a soluble modified TCR, and an Fc region. Additionally, the use of CorreGene's SMART-TCR system has facilitated the acquisition of TCRs with high affinities in the pico-molar range. By leveraging CR62 and a high-affinity TCR, we have conceptualized the TCR-based T-cell engager, CRPA1A2. Comprehensive preclinical assessments have been conducted to evaluate CRPA1A2's safety and efficacy. Results: CRPA1A2 is designed to specifically target the HLA-A*02:01-restricted MAGEA1 epitope, effectively inhibiting tumor growth. CRPA1A2 is characterized by its exceptional soluble expression in CHO cells, minimal homodimer miss-assembly and simplifying purification. It is strategically engineered with a high-affinity TCR and a low-affinity anti-CD3 scFv to optimize the efficacy and safety. In vitro studies, CRPA1A2 showed strong ability to eliminate tumor cells and induce IFN-γ release, effective even at low MAGEA1 epitope densities, with a therapeutic window that spans over three orders of magnitude, suggesting a 1000-fold greater activity against antigen-positive cancer cells compared to antigen-negative cells. In vivo, using a tumor-bearing mouse model, CRPA1A2 has been shown to recruit T cells into subcutaneous solid tumors, expand CD8+ T cell populations, and significantly reduce tumor mass. Notably, CRPA1A2 has an extended half-life of 120 hours and demonstrates preferential accumulation in tumor tissues, thereby enhancing its therapeutic potential. Moreover, the safety assessments for CRPA1A2 highlight its exceptional specificity, with in vitro studies showing no evidence of off-target toxicity, on-target off-tumor toxicity, or alloreactivity and favorable tolerance in mice during in vivo studies. Conclusions: CRPA1A2 has emerged as a superior TCR-based T-cell engager with exceptional antitumor efficacy and promising safety profile.

TROCEPT: An oncolytic virus platform engineered for αvß6 integrin targeted tumor-localised expression of an immune checkpoint inhibitor following intravenous delivery.

e14579 Background: TROCEPT is a novel tumor-selective replicating, oncolytic adenovirus type 5 (Ad5) rationally engineered to remove all natural tissue tropisms. This engineering addresses the main limitation of other viral therapies which infect normal tissues and are rapidly removed by the liver and are therefore limited to local delivery. TROCEPT has been further engineered to specifically bind to αvβ6 integrin which is expressed at high frequency in the majority of epithelial cancers, so that following intravenous delivery the virus targets tumor tissue where it replicates and amplifies. In addition, the TROCEPT platform encodes transgenes which enables in-tumor production of powerful therapeutic drugs. The lead programme, TROCEPT-01, encodes a fully human, full length immune checkpoint inhibitor (ICI) antibody. TROCEPT-01 is currently undergoing IND-enabling studies and is expected to enter First in Human studies in 2024 in multiple solid tumor indications. Methods: Proof of concept studies, both in vitro and in vivo, to demonstrate TROCEPT-01’s tumor selectivity and toxicity profile have been performed. Results: In vitro experiments demonstrate that viral replication, transgene expression and oncolytic cell death following infection with TROCEPT-01 is highly selective for αvβ6 integrin positive tumor cells compared to a panel of normal human primary cells. Intravenous delivery in in vivo SKOV3 ovarian human tumor xenograft mouse models demonstrate virus delivery, replication, and transgene expression in the tumor. In comparison to the parental Ad5 virus, the engineering of TROCEPT-01 leads to more viral delivery to the tumour, less delivery to normal tissue including liver, reduced markers of liver toxicity and reduced inflammatory cytokine release, all of which validate the tumour selectivity and safety profile of the virus. Conclusions: We have demonstrated that TROCEPT engineering enables highly selective tumor targeting and significant tumor exposure following intravenous delivery. TROCEPT-01’s in-tumor selective expression of ICIs enables high local drug concentration in the tumor, potentially reducing systemic toxicity and increasing efficacy. TROCEPT-01 is currently undergoing IND-enabling studies and is expected to enter First in Human studies in 2024 in multiple solid tumor indications.

CFLIP in the molecular regulation of astroglia-driven neuroinflammation in experimental glaucoma

BackgroundRecent experimental studies of neuroinflammation in glaucoma pointed to cFLIP as a molecular switch for cell fate decisions, mainly regulating cell type-specific caspase-8 functions in cell death and inflammation. This study aimed to determine the importance of cFLIP for regulating astroglia-driven neuroinflammation in experimental glaucoma by analyzing the outcomes of astroglia-targeted transgenic deletion of cFLIP or cFLIPL.MethodsGlaucoma was modeled by anterior chamber microbead injections to induce ocular hypertension in mouse lines with or without conditional deletion of cFLIP or cFLIPL in astroglia. Morphological analysis of astroglia responses assessed quantitative parameters in retinal whole mounts immunolabeled for GFAP and inflammatory molecules or assayed for TUNEL. The molecular analysis included 36-plexed immunoassays of the retina and optic nerve cytokines and chemokines, NanoString-based profiling of inflammation-related gene expression, and Western blot analysis of selected proteins in freshly isolated samples of astroglia.ResultsImmunoassays and immunolabeling of retina and optic nerve tissues presented reduced production of various proinflammatory cytokines, including TNFα, in GFAP/cFLIP and GFAP/cFLIPL relative to controls at 12 weeks of ocular hypertension with no detectable alteration in TUNEL. Besides presenting a similar trend of the proinflammatory versus anti-inflammatory molecules displayed by immunoassays, NanoString-based molecular profiling detected downregulated NF-κB/RelA and upregulated RelB expression of astroglia in ocular hypertensive samples of GFAP/cFLIP compared to ocular hypertensive controls. Analysis of protein expression also revealed decreased phospho-RelA and increased phospho-RelB in parallel with an increase in caspase-8 cleavage products.ConclusionsA prominent response limiting neuroinflammation in ocular hypertensive eyes with cFLIP-deletion in astroglia values the role of cFLIP in the molecular regulation of glia-driven neuroinflammation during glaucomatous neurodegeneration. The molecular responses accompanying the lessening of neurodegenerative inflammation also seem to maintain astroglia survival despite increased caspase-8 cleavage with cFLIP deletion. A transcriptional autoregulatory response, dampening RelA but boosting RelB for selective expression of NF-κB target genes, might reinforce cell survival in cFLIP-deleted astroglia.

Application Analysis of Graphic Design Elements in the Teaching of Environmental Art Specialty

This paper aims to analyze the application of graphic design elements in the teaching of environmental art major. Firstly, by reviewing the definition and development of graphic design, the basic concept and classification of graphic design elements are introduced. Then, combined with the characteristics of environmental art, the application of graphic design elements in different environments and scenes is discussed. It includes color application, composition principle, font selection and graphic expression. Finally, the importance of graphic design elements in the teaching of environmental art is summarized, and the prospect of further research is put forward.

GHSR in a Subset of GABA Neurons Controls Food Deprivation-Induced Hyperphagia in Male Mice.

The growth hormone secretagogue receptor (GHSR), primarily known as the receptor for the hunger hormone ghrelin, potently controls food intake, yet the specific Ghsr-expressing cells mediating the orexigenic effects of this receptor remain incompletely characterized. Since Ghsr is expressed in gamma-aminobutyric acid (GABA)-producing neurons, we sought to investigate whether the selective expression of Ghsr in a subset of GABA neurons is sufficient to mediate GHSR's effects on feeding. First, we crossed mice that express a tamoxifen-dependent Cre recombinase in the subset of GABA neurons that express glutamic acid decarboxylase 2 (Gad2) enzyme (Gad2-CreER mice) with reporter mice, and found that ghrelin mainly targets a subset of Gad2-expressing neurons located in the hypothalamic arcuate nucleus (ARH) and that is predominantly segregated from Agouti-related protein (AgRP)-expressing neurons. Analysis of various single-cell RNA-sequencing datasets further corroborated that the primary subset of cells coexpressing Gad2 and Ghsr in the mouse brain are non-AgRP ARH neurons. Next, we crossed Gad2-CreER mice with reactivable GHSR-deficient mice to generate mice expressing Ghsr only in Gad2-expressing neurons (Gad2-GHSR mice). We found that ghrelin treatment induced the expression of the marker of transcriptional activation c-Fos in the ARH of Gad2-GHSR mice, yet failed to induce food intake. In contrast, food deprivation-induced refeeding was higher in Gad2-GHSR mice than in GHSR-deficient mice and similar to wild-type mice, suggesting that ghrelin-independent roles of GHSR in a subset of GABA neurons is sufficient for eliciting full compensatory hyperphagia in mice.

Identification and Evolutionary Analysis of the Widely Distributed CAP Superfamily in Spider Venom.

Venom plays a crucial role in the defense and predation of venomous animals. Spiders (Araneae) are among the most successful predators and have a fascinating venom composition. Their venom mainly contains disulfide-rich peptides and large proteins. Here, we analyzed spider venom protein families, utilizing transcriptomic and genomic data, and highlighted their similarities and differences. We show that spiders have specific combinations of toxins for better predation and defense, typically comprising a core toxin expressed alongside several auxiliary toxins. Among them, the CAP superfamily is widely distributed and highly expressed in web-building Araneoidea spiders. Our analysis of evolutionary relationships revealed four subfamilies (subA-subD) of the CAP superfamily that differ in structure and potential functions. CAP proteins are composed of a conserved CAP domain and diverse C-terminal domains. CAP subC shares similar domains with the snake ion channel regulator svCRISP proteins, while CAP subD possesses a sequence similar to that of insect venom allergen 5 (Ag5). Furthermore, we show that gene duplication and selective expression lead to increased expression of CAP subD, making it a core member of the CAP superfamily. This study sheds light on the functional diversity of CAP subfamilies and their evolutionary history, which has important implications for fully understanding the composition of spider venom proteins and the core toxin components of web-building spiders.

Key Roles of CACNA1C/Cav1.2 and CALB1/Calbindin in Prefrontal Neurons Altered in Cognitive Disorders.

The risk of mental disorders is consistently associated with variants in CACNA1C (L-type calcium channel Cav1.2) but it is not known why these channels are critical to cognition, and whether they affect the layer III pyramidal cells in the dorsolateral prefrontal cortex that are especially vulnerable in cognitive disorders. To examine the molecular mechanisms expressed in layer III pyramidal cells in primate dorsolateral prefrontal cortices. The design included transcriptomic analyses from human and macaque dorsolateral prefrontal cortex, and connectivity, protein expression, physiology, and cognitive behavior in macaques. The research was performed in academic laboratories at Yale, Harvard, Princeton, and the University of Pittsburgh. As dorsolateral prefrontal cortex only exists in primates, the work evaluated humans and macaques. Outcome measures included transcriptomic signatures of human and macaque pyramidal cells, protein expression and interactions in layer III macaque pyramidal cells using light and electron microscopy, changes in neuronal firing during spatial working memory, and working memory performance following pharmacological treatments. Layer III pyramidal cells in dorsolateral prefrontal cortex coexpress a constellation of calcium-related proteins, delineated by CALB1 (calbindin), and high levels of CACNA1C (Cav1.2), GRIN2B (NMDA receptor GluN2B), and KCNN3 (SK3 potassium channel), concentrated in dendritic spines near the calcium-storing smooth endoplasmic reticulum. L-type calcium channels influenced neuronal firing needed for working memory, where either blockade or increased drive by β1-adrenoceptors, reduced neuronal firing by a mean (SD) 37.3% (5.5%) or 40% (6.3%), respectively, the latter via SK potassium channel opening. An L-type calcium channel blocker or β1-adrenoceptor antagonist protected working memory from stress. The layer III pyramidal cells in the dorsolateral prefrontal cortex especially vulnerable in cognitive disorders differentially express calbindin and a constellation of calcium-related proteins including L-type calcium channels Cav1.2 (CACNA1C), GluN2B-NMDA receptors (GRIN2B), and SK3 potassium channels (KCNN3), which influence memory-related neuronal firing. The finding that either inadequate or excessive L-type calcium channel activation reduced neuronal firing explains why either loss- or gain-of-function variants in CACNA1C were associated with increased risk of cognitive disorders. The selective expression of calbindin in these pyramidal cells highlights the importance of regulatory mechanisms in neurons with high calcium signaling, consistent with Alzheimer tau pathology emerging when calbindin is lost with age and/or inflammation.

Differential distribution of PINK1 and Parkin in the primate brain implies distinct roles.

JOURNAL/nrgr/04.03/01300535-202504000-00028/figure1/v/2024-07-06T104127Z/r/image-tiff The vast majority of in vitro studies have demonstrated that PINK1 phosphorylates Parkin to work together in mitophagy to protect against neuronal degeneration. However, it remains largely unclear how PINK1 and Parkin are expressed in mammalian brains. This has been difficult to address because of the intrinsically low levels of PINK1 and undetectable levels of phosphorylated Parkin in small animals. Understanding this issue is critical for elucidating the in vivo roles of PINK1 and Parkin. Recently, we showed that the PINK1 kinase is selectively expressed as a truncated form (PINK1-55) in the primate brain. In the present study, we used multiple antibodies, including our recently developed monoclonal anti-PINK1, to validate the selective expression of PINK1 in the primate brain. We found that PINK1 was stably expressed in the monkey brain at postnatal and adulthood stages, which is consistent with the findings that depleting PINK1 can cause neuronal loss in developing and adult monkey brains. PINK1 was enriched in the membrane-bound fractionations, whereas Parkin was soluble with a distinguishable distribution. Immunofluorescent double staining experiments showed that PINK1 and Parkin did not colocalize under physiological conditions in cultured monkey astrocytes, though they did colocalize on mitochondria when the cells were exposed to mitochondrial stress. These findings suggest that PINK1 and Parkin may have distinct roles beyond their well-known function in mitophagy during mitochondrial damage.

Desensitizing the autonomic nervous system to mitigate anti-GD2 monoclonal antibody side effects.

Anti-GD2 monoclonal antibodies (mAbs) have shown to improve the overall survival of patients with high-risk neuroblastoma (HR-NB). Serious adverse events (AEs), including pain, within hours of antibody infusion, have limited the development of these therapies. In this study, we provide evidence of Autonomic Nervous System (ANS) activation as the mechanism to explain the main side effects of anti-GD2 mAbs. Through confocal microscopy and computational super-resolution microscopy experiments we explored GD2 expression in postnatal nerves of infants. In patients we assessed the ANS using the Sympathetic Skin Response (SSR) test. To exploit tachyphylaxis, a novel infusion protocol (the Step-Up) was mathematically modelled and tested. Through confocal microscopy, GD2 expression is clearly visible in the perineurium surrounding the nuclei of nerve cells. By computational super-resolution microscopy experiments we showed the selective expression of GD2 on the cell membranes of human Schwann cells in peripheral nerves (PNs) significantly lower than on NB. In patients, changes in the SSR were observed 4 minutes into the anti-GD2 mAb naxitamab infusion. SSR latency quickly shortened followed by gradual decrease in the amplitude before disappearance. SSR response did not recover for 24 hours consistent with tachyphylaxis and absence of side effects in the clinic. The Step-Up protocol dissociated on-target off-tumor side effects while maintaining serum drug exposure. We provide first evidence of the ANS as the principal non-tumor target of anti-GD2 mAbs in humans. We describe the development and modeling of the Step-Up protocol exploiting the tachyphylaxis phenomenon we demonstrate in patients using the SSR test.

Estimation of mixed-gas selectivity of membranes using the linear friction-based model

The development of analytical models for predicting the transport properties of membranes in the separation of multicomponent mixtures is highly relevant because (1) measurements of permeability and sorption of gas mixtures in membrane materials are laborious and time-consuming, and (2) estimation of transport properties based on pure gas measurements is desirable, however, rather unreliable and can lead to serious errors. In this paper, within the framework of the well-known frictional-coefficient formalism, we present nonlinear equations for the fluxes of gas mixture components through the membrane, which take into account the diffusional coupling of the fluxes compared to independent gas transport. As a result, an analytical equation for the mixed-gas selectivity was obtained. This equation has an implicit form, which is not quite convenient for practical application, since it has to be solved numerically. A simple explicit expression for mixed gas selectivity was derived using the so-called linear transport model, which is a linearized version of the original nonlinear flux equations. It is important to emphasize that a comparison of the linear model with the formally more rigorous nonlinear model showed only minor quantitative differences. The input parameters for the model are the single gas transport parameters. Testing the linear model using experimental data for the separation of hydrocarbons (n-butane/methane, n-butane/i-butane, propylene/propane) revealed an acceptable fit. A comparison of the model with recent experimental data on the separation of CO2-containing mixtures using microporous polymers is also presented. It is found that the calculated CO2/N2 mixed gas selectivity is in good agreement with the experimental data, while only a semi-quantitative agreement is observed for the CO2/CH4 separation. Besides, it is shown that the model developed for binary gas separation can be extended to ternary gas mixtures.

Induction of FoxP3 Pre-mRNA Alternative Splicing to Enhance the Suppressive Activity of Regulatory T Cells from Amyotrophic Lateral Sclerosis Patients.

Forkhead box protein 3 (FoxP3) is a key transcription factor responsible for the development, maturation, and function of regulatory T cells (Tregs). The FoxP3 pre-mRNA is subject to alternative splicing, resulting in the translation of multiple splice variants. We have shown that Tregs from patients with amyotrophic lateral sclerosis (ALS) have reduced expression of full-length (FL) FoxP3, while other truncated splice variants are expressed predominantly. A correlation was observed between the reduced number of Tregs in the peripheral blood of ALS patients, reduced total FoxP3 mRNA, and reduced mRNA of its FL splice variant. Induction of FL FoxP3 was achieved using splice-switching oligonucleotides capable of base pairing with FoxP3 pre-mRNA and selectively modulating the inclusion of exons 2 and 7 in the mature mRNA. Selective expression of FL FoxP3 resulted in the induction of CD127low, CD152, and Helios-positive cells, while the cell markers CD4 and CD25 were not altered. Such Tregs had an increased proliferative activity and a higher frequency of cell divisions per day. The increased suppressive activity of Tregs with the induced FL FoxP3 splice variant was associated with the increased synthesis of the pro-apoptotic granzymes A and B, and perforin, IL-10, and IL-35, which are responsible for contact-independent suppression, and with the increased ability to suppress telomerase in target cells. The upregulation of Treg suppressive and proliferative activity using splice-switching oligonucleotides to induce the predominant expression of the FoxP3 FL variant is a promising approach for regenerative cell therapy in Treg-associated diseases.

Abstract PO3-18-04: MBRC-101: a novel antibody-drug conjugate (ADC) targeting the membrane-associated tyrosine kinase receptor EphA5 in breast cancer

Abstract Objectives: ADCs have emerged as a major advance in contemporary medical oncology. The receptor tyrosine kinase (RTK) EphA5 possesses the classical receptor kinase topology with an extracellular-binding domain, a single-pass transmembrane domain, and a cytoplasmic kinase domain. We have previously shown that EphA5 is highly expressed in non-small cell lung cancer and contributes to DNA-damage repair and radiation therapy resistance. Others have reported EphA5 expression in gastric, ovarian, and pancreatic cancers. Here, we investigated the expression of EphA5 in breast cancer (BC) and assessed EphA5 as a potential cancer-specific target using a novel ADC designated MBRC-101. Methods: MBRC-101 is composed of a humanized anti-EphA5 IgG1 kappa monoclonal antibody conjugated to the small molecule microtubule-disrupting agent monomethyl auristatin E (MMAE) via a protease cleavable valine-citrulline (vc) ThioBridge linker (ThioBridge™-Glu-[Val-Cit-PAB-MMAE] PEG [24u]). MBRC-101 averages a drug-to-antibody ratio (DAR)=4. The specificity profile of the anti-EphA5 monoclonal antibody was characterized using a Membrane Proteome Array (MPA). Receptor-mediated antibody internalization was determined by real-time live-cell analysis; cell killing assays used both live-cell analysis of cell confluence and the CellTiter-Glo® luminescent cell viability assay. Expression of EphA5 in archival human breast tumor tissue sections was evaluated by immunohistochemistry (IHC) using a commercial polyclonal antibody against human EphA5. In vivo efficacy studies used patient-derived xenograft (PDX) models of BC. Results: IHC demonstrated robust and selective EphA5 expression in 87% (20 of 23) of triple negative breast cancer (TNBC) and 88% (23 of 26) of hormone receptor-positive (HR+) patient tumors tested. EphA5 expression was not detected in adjacent, non-malignant breast tissue. Antibody-specificity profile testing showed that the anti-EphA5 antibody bound exclusively to EphA5 and did not cross-react with other members of the Eph receptor/ephrin ligand family. Binding of the anti-EphA5 antibody to EphA5 on the cell surface triggered rapid internalization and processing of the EphA5/antibody complex through the endosomal-lysosomal system. Consistently, MBRC-101 was cytotoxic to EphA5-expressing cells and its activity was concentration-dependent and commensurate to the levels of EphA5 on the cell surface. In PDX murine models of TNBC, once weekly administrations of intravenous (IV) MBRC-101 showed dose-dependent, robust, and reproducible anti-tumor activity in vivo. Partial tumor responses were observed at doses of 2.5 mg/Kg IV and complete and nearly complete tumor responses starting at 5 mg/Kg IV. Doses up to 10 mg/Kg IV were well-tolerated in tumor-bearing mice with no observable weight loss. Pre-clinical safety confirmed MBRC-101 is also well tolerated in rats and in cynomolgus monkeys. Conclusions: These results support EphA5 as a new and promising membrane-associated molecular target for the design and development of ADC-based therapies against hormone-positive breast cancer and TNBC and support the conduct of a Phase 1/1b study of MBRC-101 in patients with breast cancer. Citation Format: Fernanda Staquicini, Fenny Tang, Vanessa de Oliveira, Daniela Staquicini, Yongjian Wu, Kirstin Barnhart, J Kellogg Parsons, Wadih Arap, Isan Chen, Renata Pasqualini. MBRC-101: a novel antibody-drug conjugate (ADC) targeting the membrane-associated tyrosine kinase receptor EphA5 in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-18-04.