Biology subjects

Lee, H.

Publications and source records attributed to Lee, H..

At least 199 records · Page 11Linked to original sources

bioRxiv2024

Lignocellulose Degradation in Bacteria and Fungi for Biomass Conversion

Lignocellulose biomass is one of the most abundant resources for sustainable biofuels. However, scaling up the biomass-to-biofuels conversion process for widespread usage is still pending. Bottlenecks during the process of enzymatic hydrolysis are the high cost of enzymes and the labor-intensive need for substrate-dependent enzyme mixtures. Current research efforts are therefore targeted at searching for or engineering lignocellulolytic enzymes of high efficiency. One way is to engineer multi-enzyme complexes that mimic the bacterial cellulosomal system, known to increase degradation efficiency up to 50-fold when compared to freely-secreted enzymes. However, these designer cellulosomes are instable and less efficient than wild type cellulosomes. Fungi cellulosomes discovered in recent years have significant differences from bacterial counterparts and hold great potential for industrial applications, both as designer cellulosomes and as additions to the enzymatic repertoire. Up to date, they are only found in a few anaerobic fungi. In this review, we extensively compared the degradation mechanisms in bacteria and fungi, and highlighted the essential gaps in applying these mechanisms in industrial applications. To better understand cellulosomes in microorganisms, we examined their sequences in 66,252 bacterial species and 823 fungal species and identified several bacterial species that are potentially cellulosome-producing. These findings act as a valuable resource in the biomass community for further proteomic and genetic sequence analysis. We also collated the current strategies of bioengineering lignocellulose degradation to suggest concepts that could be favorable for industrial usage.

microbiology↗

bioRxiv2024

Exploring N6-Methyladenosine and long transcripts in glioma cells and extracellular vesicles.

N6-methyladenosine (m6A) is the most prevalent internal mRNA modification, enriched in the CNS yet poorly characterized in glioma. Using long-read RNA sequencing, we mapped m6A in an in vitro glioma model following knockdown (KD) of the reader IGF2BP2, writer METTL3, and eraser ALKBH5, with naive glioma cells and astrocytes as controls. Glioma cells exhibited a two-fold reduction in global m6A, suggesting progressive loss from healthy to malignant states. Integrated analysis revealed that m6A mediated control of gene expression is influenced by modification topology (CDS:3'UTR), transcript biotype, and length. Regulator KD, particularly ALKBH5 induced redistribution of m6A toward 3'UTR with consequent gene upregulation. We also identified m6A-mediated isoform switching, with a higher usage of retained intron and nonsense-mediated decay isoforms. Structural and splicing alterations at the isoform level were identified unique to each KD condition indicating m6A driven aberrant alternative splicing. At the functional level, KD specific remodeling of oncogenic signaling was also observed. ALKBH5 KD suppressed MYC targets and pro-apoptotic signaling while METTL3 KD enhanced mTOR and PI3K-AKT signaling. Collectively, these results demonstrate that m6A mediated regulation in glioma is highly context-dependent, defining distinct clinically relevant phenotypes. This has implications for future biomarker discovery and development of targeted therapeutics.

cancer biology↗

bioRxiv2024

A high throughput single molecule platform to study DNA supercoiling effect on protein-DNA interactions

DNA supercoiling significantly influences DNA metabolic pathways. To examine its impact on DNA-protein interactions at the single-molecule level, we developed a highly efficient and reliable protocol to modify plasmid DNA at specific sites, allowing us to label plasmids with fluorophores and biotin. We then induced negative and positive supercoiling in these plasmids using gyrase and reverse gyrase, respectively. Comparing supercoiled DNA with relaxed circular DNA, we assessed the effects of supercoiling on CRISPR-Cas9 and mismatch repair protein MutS. We found that negative DNA supercoiling exacerbates off-target effects in DNA unwinding by Cas9. For MutS, we observed both negative and positive DNA supercoiling enhances the binding interaction between MutS and a mismatched base pair but does not affect the rate of ATP-induced sliding clamp formation. These findings not only underscore the versatility of our protocol but also opens new avenues for exploring the intricate dynamics of protein-DNA interactions under the influences of supercoiling.

biophysics↗

bioRxiv2024

A brain-shuttled antibody targeting alpha synuclein aggregates for the treatment of synucleinopathies

Parkinsons disease and multiple system atrophy are members of a class of devastating neurodegenerative diseases called synucleinopathies, which are characterized by the presence of alpha-synuclein (-Syn) rich aggregates in the brains of patients. Passive immunotherapy targeting these aggregates is an attractive disease-modifying strategy. Such an approach must not only demonstrate target selectivity towards -Syn aggregates, but also achieve appropriate brain exposure to have the desired therapeutic effect. Here we present preclinical data for a next-generation antibody for the treatment of synucleinopathies. SAR446159 (ABL301) is a bispecific antibody composed of an -Syn-binding immunoglobulin (IgG) and an engineered insulin-like growth factor receptor 1 (IGF1R) binding single-chain variable fragment (scFv), acting as a shuttle to transport an antibody across the blood-brain barrier (BBB). SAR446159 binds tightly and preferentially to -Syn aggregates and prevents their seeding capacity in vitro and in vivo. Incubation with SAR446159 reduced -Syn preformed fibrils (PFFs) uptake in neurons and facilitated uptake and clearance by microglia. In wild type mice injected in the striatum with -Syn PFFs, treatment with SAR446159 reduced the spread of aSyn pathology as measured by phosphorylated -Syn staining and lessened the severity of motor phenotypes. Additionally, in 9-month-old transgenic mice overexpressing -Syn (mThy1--Syn, Line 61), repeated treatment with SAR446159 reduced markers of -Syn aggregation in the brain. SAR446159 had significantly higher brain and CSF penetration over a sustained period than its monospecific counterpart (1E4) in rats and monkeys. The binding properties of SAR446159 combined with its brain-shuttle technology make it a potent, next-generation immunotherapeutic for treating synucleinopathies.

neuroscience↗

bioRxiv2024

Glucose-dependent glycosphingolipid biosynthesis fuels CD8+ T cell function and tumor control

Glucose is essential for T cell proliferation and function, yet its specific metabolic roles in vivo remain poorly defined. Here, we identify glycosphingolipid (GSL) biosynthesis as a key pathway fueled by glucose that enables CD8+ T cell expansion and cytotoxic function in vivo. Using 13C-based stable isotope tracing, we demonstrate that CD8+ effector T cells use glucose to synthesize uridine diphosphate-glucose (UDP-Glc), a precursor for glycogen, glycan, and GSL biosynthesis. Inhibiting GSL production by targeting the enzymes UGP2 or UGCG impairs CD8+ T cell expansion and cytolytic activity without affecting glucose-dependent energy production. Mechanistically, we show that glucose-dependent GSL biosynthesis is required for plasma membrane lipid raft integrity and aggregation following TCR stimulation. Moreover, UGCG-deficient CD8+ T cells display reduced granzyme expression and tumor control in vivo. Together, our data establish GSL biosynthesis as a critical metabolic fate of glucose--independent of energy production--required for CD8+ T cell responses in vivo.

immunology↗

bioRxiv2024

A Small-molecule Antagonist Radiotracer for Positron Emission Tomography Imaging of the Mu Opioid Receptor.

The opioid crisis is a catastrophic health emergency catalyzed by the misuse of opioids that target and activate the mu opioid receptor. Traditional radioligands used to study the mu opioid receptor are often tightly regulated owing to their abuse and respiratory depression potential. In the present study, we sought to design and characterize a library of 24 non-agonist ligands for the mu opioid receptor. Ligands were evaluated for the binding affinity, intrinsic activity, and predicted blood-brain barrier permeability. Several ligands demonstrated single-digit nM binding affinity for the mu opioid receptor while also demonstrating selectivity over the delta and kappa opioid receptors. The antagonist behavior of 1A and 3A at the mu opioid receptor indicate that these ligands would likely not induce opioid-dependent respiratory depression. Therefore, these ligands can enable a safer means to interrogate the endogenous opioid system. Based on binding affinity, selectivity, and potential off-target binding, [11C]1A was prepared via metallophotoredox of the aryl-bromide functional group to [11C]methyl iodide. The nascent radiotracer demonstrated brain uptake in a rhesus macaque model and accumulation in the caudate and putamen. Naloxone was able to reduce [11C]1A binding, though the interactions were not as pronounced as naloxones ability to displace [11C]carfentanil. These results suggest that GSK1521498 and related congeners are amenable to radioligand design and can offer a safer way to query opioid neurobiology.

neuroscience↗

bioRxiv2024

Inhibition of Androgen Receptor Exposes Replication Stress Vulnerability in Prostate Cancer

Standard initial systemic treatment for patients with metastatic prostate cancer includes agents that target androgen receptor (AR) signaling. Despite an initial positive response to these AR pathway inhibitors (ARPIs), acquired resistance remains a significant challenge. We show that treatment of AR-positive prostate cancer cells with the frontline ARPI enzalutamide induces DNA replication stress. Such stress is exacerbated by suppression of translesion DNA synthesis (TLS), leading to aberrant accumulation of single-stranded DNA (ssDNA) gaps and persistent DNA damage biomarkers. We further demonstrate that the TLS inhibitor, JH-RE-06, markedly sensitizes AR-positive prostate cancer cells, but not AR-negative benign cells, to enzalutamide in vitro. Combination therapy with enzalutamide and JH-RE-06 significantly suppresses cancer growth in a syngeneic murine tumor model over vehicle control or individual treatment groups. These findings suggest that AR inhibition broadly triggers DNA replication stress in hormone-sensitive prostate cancer, thereby exposing a unique vulnerability that can be exploited by a TLS-disrupting adjuvant for targeted therapy.

cancer biology↗

bioRxiv2024

Neural dynamics of spontaneous memory recall and future thinking in the continuous flow of thoughts

The human brain constantly recalls past experiences and anticipates future events, generating a continuous flow of thoughts. However, the neural mechanisms underlying the natural transitions and trajectories of thoughts during spontaneous memory recall and future thinking remain underexplored. To address this gap, we conducted a functional magnetic resonance imaging study using a think-aloud paradigm, where participants verbalize their uninterrupted stream of thoughts during rest. We found that transitions between thoughts, particularly those involving significant shifts in semantic content, activate the brains default and control networks. These neural responses to internally generated thought boundaries produce activation patterns resembling those triggered by external event boundaries. Moreover, interactions within and between these networks shape the overall semantic structure of thought trajectories: stronger functional connectivity within the medial temporal subsystem of the default network predicts greater variability in thoughts, while stronger connectivity between the control and core default networks is associated with reduced variability. Together, our findings highlight how the default and control networks guide the dynamic transitions and structure of naturally arising memory and future thinking.

neuroscience↗

bioRxiv2024

EXPERIMENTAL DESIGN ISSUES ASSOCIATED WITH CLASSIFICATIONS OF HYPERSPECTRAL IMAGING DATA

Hyperspectral imaging has emerged as a pivotal tool to classify plant materials (seeds, leaves, and whole plants), pharmaceutical products, food items, and many other objects. This communication addresses two issues, which appear to be over-looked or ignored in >99% of hyperspectral imaging studies: 1) the "small N, large P" problem, when number of spectral bands (explanatory variables, "P") surpasses number of observations, ("N") leading to potential model over-fitting, and 2) absence of independent validation data in performance assessments of classification models. Based on simulations of randomly generated data, we illustrate risks associated with these issues. We explore and discuss consequences of over-fitting and risks of misleadingly high accuracy that can result from having a large number of variables relative to observations. We highlight connections of these issues with radiometric repeatability (levels of stochastic noise). A method is proposed wherein a theoretical dataset is generated to mirror the structure of an actual dataset, with the classification of this theoretical dataset serving as a reference. By shedding light on important and common experimental design issues, we aim to enhance methodological rigor and transparency in classifications of hyperspectral imaging data and foster improved and effective applications across various science domains.

bioinformatics↗

bioRxiv2024

Evidence for belief updating in decision-variable space: past decisions with finer granularity attract future ones more strongly

When engaged in decision-making tasks, humans are known to create decision variables. Much effort has focused on the cognitive processes involved in forming decision variables. However, there is limited understanding of how decision variables, once formed, are utilized to adapt to the environment. We reason that decision-makers would benefit from updating the belief of decision-variable. As one such belief updating, we hypothesize that a decision commitment restricts the probabilistic belief distribution of decision variable to a range corresponding to that decision. This implies that past decisions not only attract future ones but also exert a greater pull when those decisions are made with finer granularity-- dubbed the granularity effect. Here, we present the findings of seven psychophysical experiments that confirm these implications. Further, as a unified account of the granularity effect, we offer a Bayesian model. Our work demonstrates how humans leverage the decision-variable to effectively adapt to their surroundings.

neuroscience↗

bioRxiv2024

Acute systemic macrophage depletion in osteoarthritic mice alleviates pain-related behaviors and does not affect joint damage

BackgroundOsteoarthritis (OA) is a painful degenerative joint disease and a leading source of years lived with disability globally due to inadequate treatment options. Neuroimmune interactions reportedly contribute to OA pain pathogenesis. Notably, in rodents, macrophages in the DRG are associated with onset of persistent OA pain. Our objective was to determine the effects of acute systemic macrophage depletion on pain-related behaviors and joint damage using surgical mouse models in both sexes. MethodsWe depleted CSF1R+ macrophages by treating male macrophage Fas-induced apoptosis (MaFIA) transgenic mice 8-or 16-weeks post destabilization of the medial meniscus (DMM) with AP20187 or vehicle control (10 mg/kg i.p., 1x/day for 5 days), or treating female MaFIA mice 12 weeks post partial meniscectomy (PMX) with AP20187 or vehicle control. We measured pain-related behaviors 1-3 days before and after depletion, and, 3-4 days after the last injection we examined joint histopathology and performed flow cytometry of the dorsal root ganglia (DRGs). In a separate cohort of male 8-week DMM mice or age-matched naive vehicle controls, we conducted DRG bulk RNA-sequencing analyses after the 5-day vehicle or AP20187 treatment. ResultsEight-and 16-weeks post DMM in male mice, AP20187-induced macrophage depletion resulted in attenuated mechanical allodynia and knee hyperalgesia. Female mice showed alleviation of mechanical allodynia, knee hyperalgesia, and weight bearing deficits after macrophage depletion at 12 weeks post PMX. Macrophage depletion did not affect the degree of cartilage degeneration, osteophyte width, or synovitis in either sex. Flow cytometry of the DRG revealed that macrophages and neutrophils were reduced after AP20187 treatment. In addition, in the DRG, only MHCII+ M1-like macrophages were significantly decreased, while CD163+MHCII-M2-like macrophages were not affected in both sexes. DRG bulk RNA-seq revealed that Cxcl10 and Il1b were upregulated with DMM surgery compared to naive mice, and downregulated in DMM after acute macrophage depletion. ConclusionsAcute systemic macrophage depletion reduced the levels of pro-inflammatory macrophages in the DRG and alleviated pain-related behaviors in established surgically induced OA in mice of both sexes, without affecting joint damage. Overall, these studies provide insight into immune cell regulation in the DRG during OA.

neuroscience↗

bioRxiv2024

Regulation of Ferroptosis in Obesity: Muscle Type-Specific Effects of Dietary Restriction and Exercise

BackgroundObesity is a significant global health issue and a risk factor for numerous diseases. Ferroptosis, an iron-dependent regulated cell death, is triggered by iron overload and the excessive accumulation of lipid peroxidation mediated by reactive oxygen species. Studies has identified a strong association between ferroptosis and obesity. Additionally, dietary restriction (DR) and DR combined with exercise (DR+Ex) are effective strategies for managing obesity and ferroptosis. However, the regulation of ferroptosis and its signaling pathways in skeletal muscle under conditions of obesity, DR, and DR+Ex remains poorly understood. MethodsMice were divided into four groups: normal diet, high-fat diet, high-fat DR, and high-fat DR+Ex. All mice were fed ad libitum with either a normal or high-fat diet for the first 14 weeks, followed by the respective interventions for the subsequent 8 weeks. Mice muscle ferroptosis were examined by immunohistochemistry, Hematoxylin & Eosin, Massons trichrome, Prussian blue staining, and Western-Immunoblot. ResultsThe high-fat diet resulted in increased inflammatory cell infiltration, fibrosis, and iron accumulation. Red and white muscle showed increased expression of 4-HNE, regulated by GPX4 and NCAO4, respectively. DR and DR+Ex reduced downstream 4-HNE expression by regulating GPX4 in red muscle. ConclusionDue to metabolic differences, obesity-induced ferroptosis in skeletal muscle and the regulation by DR+Ex exhibiting fiber type-specificity. Specifically, red and white muscle respond to obesity-induced ferroptosis through different pathways; red muscle can inhibit obesity-induced ferroptosis through DR+Ex by GPX4. This deepens the understanding of mechanisms related to skeletal muscle cell death and provides scientific data support for the personalized treatment of related diseases.

physiology↗

bioRxiv2024

Contributions of genetic variation in astrocytes to cell and molecular mechanisms of risk and resilience to late onset Alzheimer's disease

Reactive astrocytes are associated with Alzheimers disease (AD), and several AD genetic risk variants are associated with genes highly expressed in astrocytes. However, the contribution of genetic risk within astrocytes to cellular processes relevant to the pathogenesis of AD remains ill-defined. Here we present a resource for studying AD genetic risk in astrocytes using a large collection of induced pluripotent stem cell (iPSC) lines from deeply phenotyped individuals with a range of neuropathological and cognitive outcomes. IPSC lines from forty-four individuals were differentiated into astrocytes followed by unbiased molecular profiling using RNA sequencing and tandem mass tag-mass spectrometry. We demonstrate the utility of this resource in examining gene- and pathway-level associations with clinical and neuropathological traits, as well as in analyzing genetic risk and resilience factors through parallel analyses of iPSC-astrocytes and brain tissue from the same individuals. Our analyses reveal that genes and pathways altered in iPSC-derived astrocytes from AD individuals are concordantly dysregulated in AD brain tissue. This includes increased prefoldin proteins, extracellular matrix factors, COPI-mediated trafficking components and reduced proteins involved in cellular respiration and fatty acid oxidation. Additionally, iPSC-derived astrocytes from individuals resilient to high AD neuropathology show elevated basal levels of interferon response proteins and increased secretion of interferon gamma. Correspondingly, higher polygenic risk scores for AD are associated with lower levels of interferon response proteins. This study establishes an experimental system that integrates genetic information with a heterogeneous set of iPSCs to identify genetic contributions to molecular pathways affecting AD risk and resilience.

neuroscience↗

bioRxiv2024

The valine-arginine dipeptide repeat protein encoded by mammalian telomeric RNA appears highly expressed in mitosis and may repress global translation

Translation of mammalian telomeric G-rich RNA via the Repeat Associated non-AUG translation mechanism can produce two dipeptide repeat proteins: repeating valine-arginine (VR) and repeating glycine-leucine (GL). Their potentially toxic nature suggests that one or both must play a needed role in the cell. Using light microscopy combined with antibody staining we discovered that cultured human cells stain brightly for VR during mitosis with VR staining co-localizing with ribosomes. In vitro, VR protein represses translation in a firefly luciferase assay. Affinity purification combined with mass spectrometry identified ribosomal proteins as the major class of VR interacting proteins. Extension to mouse embryonic cerebral cortical development showed strong staining in the ventricular zone where high mitotic index neural progenitor cells proliferate and in the cortical plate where new neurons settle. These observations point to VR playing a key role in mitosis very possibly depressing global translation, a role mediated by the telomere. TeaserThe telomeric valine-arginine dipeptide repeat protein is highly expressed in mitotic cells in culture and in mouse embryonic neural tissue.

molecular biology↗

bioRxiv2024

Glutamate is a key nutrient for Porphyromonas gingivalis growth and survival during intracellular autophagic life under nutritionally limited conditions

Porphyromonas gingivalis survives in special autophagic vacuoles that serve as major replicative habitats in human primary gingival epithelial cells (GECs). As an asaccharolytic strict anaerobe, P. gingivalis is dependent on amino acids and peptides for nutrient sources. However, it is largely unknown as to P. gingivalis metabolic processing under the nutritionally limited intracellular environments such the vacuoles, especially the preferred amino acids and associated-metabolic machineries. Here we elucidate that a Glutamate (Glu) catabolic enzyme, glutamate dehydrogenase (GdhA) is highly enriched in the isolated P. gingivalis-containing vacuoles. Interestingly, we found that P. gingivalis induces conversion of intracellular glutamine pool to Glu determined by analyses of the P. gingivalis-containing vacuoles and the whole infected-GECs. Critically, exogenous Glu-Glu dipeptide, a simple precursor of Glu, significantly increases the size of isolated intact P. gingivalis containing-vacuoles and live wild-type P. gingivalis numbers in GECs. In contrast, the isogenic GdhA-deficient-strain, {Delta}gdhA displayed a significant growth defect with collapsed-vacuoles in GECs. Next, we confirmed that P. gingivalis uptakes 14C-Glu and it preferentially utilizes Glu-Glu-dipeptide using a nutritionally reduced Tryptic-Soy-Broth (TSB) media supplemented with Glu-Glu. Contrary, {Delta}gdhA-strain showed no detectable growth especially in nutritionally reduced TSB media with Glu-Glu. Using Atomic-Force-Microscopy, we observed that, wild-type P. gingivalis but not {Delta}gdhA strain notably increased the cell volume upon Glu-Glu supplementation, an indicator of higher metabolism and growth. Utilization of a human gingiva-mimicking organoid-system further validated the importance of Glu as an essential nutrient for the intramucosal colonization of P. gingivalis via the protected replicative vacuoles in GECs. ImportanceThis study reveals that P. gingivalis heavily depends on preferential utilization of Glutamate (Glu) for autophagic vacuolar growth and survival in human GECs. Several novel observations are made to support this: (i) GdhA of P. gingivalis is highly enriched in these vacuoles, (ii) P. gingivalis induces a large conversion of intracellular glutamine to Glu, (iii) size of vacuoles are significantly increased in the presence of Glu-Glu in P. gingivalis wild-type strain infection which is opposite in a {Delta}gdhA strain, (iv) P. gingivalis uptakes 14C-Glu and preferentially utilizes Glu-Glu dipeptide, (v) similarly, wild-type strain shows growth increase in a nutritionally reduced bacterial culture media, and (vi) finally, Glu-Glu supplementation increases bacterial cell-volume of P. gingivalis wild-type but not {Delta}gdhA strain, an indicator of higher metabolism and growth. Taken together, this study highlights the pathophysiological importance of Glu for P. gingivalis growth-rate, biomass induction and survival in nutritionally limited host subcellular environments.

microbiology↗

bioRxiv2024

NRF2-dependent regulation of the prostacyclin receptor PTGIR drives CD8 T cell exhaustion

The progressive decline of CD8 T cell effector function--also known as terminal exhaustion--is a major contributor to immune evasion in cancer. Yet, the molecular mechanisms that drive CD8 T cell dysfunction remain poorly understood. Here, we report that the Kelch-like ECH-associated protein 1 (KEAP1)-Nuclear factor erythroid 2-related factor 2 (NRF2) signaling axis, which mediates cellular adaptations to oxidative stress, directly regulates CD8 T cell exhaustion. Transcriptional profiling of dysfunctional CD8 T cells from chronic infection and cancer reveals enrichment of NRF2 activity in terminally exhausted (Texterm) CD8 T cells. Increasing NRF2 activity in CD8 T cells (via conditional deletion of KEAP1) promotes increased glutathione production and antioxidant defense yet accelerates the development of terminally exhausted (PD-1+TIM-3+) CD8 T cells in response to chronic infection or tumor challenge. Mechanistically, we identify PTGIR, a receptor for the circulating eicosanoid prostacyclin, as an NRF2-regulated protein that promotes CD8 T cell dysfunction. Silencing PTGIR expression restores the anti-tumor function of KEAP1-deficient T cells. Moreover, lowering PTGIR expression in CD8 T cells both reduces terminal exhaustion and enhances T cell effector responses (i.e. IFN-{gamma} and granzyme production) to chronic infection and cancer. Together, these results establish the KEAP1-NRF2 axis as a metabolic sensor linking oxidative stress to CD8 T cell dysfunction and identify the prostacyclin receptor PTGIR as an NRF2-regulated immune checkpoint that regulates CD8 T cell fate decisions between effector and exhausted states. One Sentence SummaryThe KEAP1-NRF2 pathway is hyperactivated in terminally exhausted CD8 T cells and drives T cell dysfunction via transcriptional regulation of the prostacyclin receptor, Ptgir.

immunology↗

bioRxiv2024

Clearing-enabled light sheet microscopy as a novel method for three-dimensional mapping of the sensory innervation of the mouse knee

A major barrier that hampers our understanding of the precise anatomic distribution of pain sensing nerves in and around the joint is the limited view obtained from traditional two dimensional (D) histological approaches. Therefore, our objective was to develop a workflow that allows examination of the innervation of the intact mouse knee joint in 3D by employing clearing-enabled light sheet microscopy. We first surveyed existing clearing protocols (SUMIC, PEGASOS, and DISCO) to determine their ability to clear the whole mouse knee joint, and discovered that a DISCO protocol provided the most optimal transparency for light sheet microscopy imaging. We then modified the DISCO protocol to enhance binding and penetration of antibodies used for labeling nerves. Using the pan-neuronal PGP9.5 antibody, our protocol allowed 3D visualization of innervation in and around the mouse knee joint. We then implemented the workflow in mice intra-articularly injected with nerve growth factor (NGF) to determine whether changes in the nerve density can be observed. Both 3D and 2D analytical approaches of the light sheet microscopy images demonstrated quantifiable changes in midjoint nerve density following 4 weeks of NGF injection in the medial but not in the lateral joint compartment. We provide, for the first time, a comprehensive workflow that allows detailed and quantifiable examination of mouse knee joint innervation in 3D.

neuroscience↗

bioRxiv2024

Timing antigenic escape in multiple myeloma treated with T-cell redirecting immunotherapies

Recent data highlight genomic events driving antigen escape as a recurring cause of chimeric antigen receptor T-cell (CAR-T) and bispecific T-cell engager (TCE) resistance in multiple myeloma (MM). Yet, it remains unclear if these events, leading to clonal dominance at progression, result from acquisition under treatment selection or selection of pre-existing undetectable clones. This differentiation gains importance as these immunotherapies progress to earlier lines of treatment, prompting the need for innovative diagnostic testing to detect these events early on. By reconstructing phylogenetic trees and exploring chemotherapy mutational signatures as temporal barcodes in 11 relapsed refractory MM patients with available whole genome sequencing data before and after CART/TCE treatment, we demonstrated that somatic antigen escape mechanisms for BCMA- and GPRC5D-targeting therapies are acquired post-diagnosis, likely during CART/TCE treatment. Longitudinal tracking of these mutations using digital PCR in 4 patients consistently showed that genomic events promoting antigen escape were not detectable during the initial months of therapy but began to emerge nearly 1 year post therapy initiation. This finding reduces the necessity for a diagnostic panel to identify these events before CART/TCE. Instead, it underscores the importance of surveillance and identifying patients at higher risk of acquiring these events. KEY POINTSO_LIGenomic events driving antigen escape are recurrent mechanisms of resistance to CART and T-cell engagers in multiple myeloma. C_LIO_LIUsing chemotherapy mutational signatures, we demonstrated that these events are most likely acquired during treatment. C_LI

genomics↗