Background: The hairy ear (Eh) mutation in heterozygous mice (Eh/+) results in elongated and additional ear hairs, along with altered pinna morphology compared to wild-type (+/+) mice. Previous studies suggest that disruption of the Hoxc gene cluster caused by the Eh inversion influences the hair growth cycle. Methods: To elucidate the molecular basis of this phenotype, we performed RNA-seq analysis on ear tissues from four-week-old Eh/+ and +/+ mice and compared their transcriptomic profiles. Results: Differential expression analysis identified 2092 genes, and subsequent Gene Ontology (GO) and overrepresentation analysis revealed significant alterations in hair growth-related processes, including the hair cycle and canonical keratinization in Eh/+ ears. Notably, numerous hair keratin and keratin-associated protein (Krtap) genes were markedly upregulated in Eh/+ mice. Validation by quantitative real-time PCR confirmed increased expression of randomly selected keratin genes (Krt34, Krt39, Krt71, Krt81, Krt84) and keratin-associated proteins (Krtap4-16 and Krtap22-2). In contrast, epithelial keratin genes such as Krt2 and Krt14 were downregulated in Eh/+ ears. In addition, genes associated with hair follicle growth, Car6 and Gprc5d, showed elevated expression, while Dab2, a telogen-anagen transition marker linked to hair follicle stem cell activation, was slightly increased at the telogen stage in Eh/+ compared with +/+ mice. Conclusions: These findings provide new insights into the role of Hoxc cluster genes in orchestrating the expression of hair keratin and Krtap genes and highlight potential regulatory mechanisms underlying the hairy ear phenotype.

Excessive activation of NF-κB is implicated in the pathogenesis of numerous inflammatory and autoimmune diseases; however, conventional NF-κB inhibitors often cause widespread immunosuppression. In contrast, extracellular vesicles (EVs) are promising vehicles for therapeutic cargo delivery with advantages including reduced risk of replication. In this single-centre, randomized, double-blind, placebo-controlled phase 1 trial, we evaluated ILB-202, an engineered, allogeneic EV derived from HEK293 cells and loaded with a super-repressor IκBα. A single ascending intravenous dose of ILB-202 was administered to 18 healthy volunteers, and the short-term safety, tolerability, and preliminary pharmacodynamic effects were assessed. ILB-202 was well tolerated at all dose levels with no serious or dose-limiting toxicities; only minor adverse events, including a mild decrease in NK cell counts and one case of grade 1 neutropenia, were observed. The laboratory parameters, vital signs and cytokine profiles remained stable, indicating no systemic immunogenicity. Single-cell RNA sequencing revealed subtle, time-dependent modulation of NF-κB-associated pathways, enhanced TGF-β and visfatin signalling and reduced TNF signalling-suggesting a shift towards an anti-inflammatory state. These findings support the safety and immunomodulatory activity of ILB-202 and pave the way for future trials in diseases characterized by dysregulated NF-κB activation. Trial Registration: ClinicalTrials.gov identifier: NCT05843799.

The effectiveness of exosomes engineered to carry a dominantly active variant of inhibitor α of nuclear factor κB (NF-κB) (IκBα), super-repressor IκB (srIκB), that inhibits the expression of NF-κB in various animal models of inflammatory diseases has been demonstrated. In this study, we used a lipopolysaccharide (LPS)–induced chorioamnionitis model in pregnant nonhuman primates to explore the therapeutic potential and mode of action of srIκB-loaded exosomes (Exo-srIκBs). Intraamniotic injection of LPS induced infiltration of BCL2A1-positive neutrophils and CD68-positive macrophages in the extraplacental membranes, causing fetal lung injury. Conversely, administration of Exo-srIκB via intraamniotic and intravenous routes (6.9 × 10 10 and 4 × 10 11 particle numbers, respectively) ameliorated these effects. Single-cell RNA sequencing of the decidua and bulk RNA sequencing of the choriodecidua highlighted that Exo-srIκB treatment mitigated LPS-induced inflammatory pathways, particularly in macrophages, leading to a cascade effect on neutrophils through NF-κB signaling inhibition. These findings underscore the potential of Exo-srIκB as a therapeutic strategy for chorioamnionitis.

Extracellular vesicles, particularly exosomes, have emerged as promising drug delivery systems owing to their unique advantages, such as biocompatibility, immune tolerability, and target specificity. Various engineering strategies have been implemented to harness these innate qualities, with a focus on enhancing the pharmacokinetic and pharmacodynamic properties of exosomes via payload loading and surface engineering for active targeting. This concise review outlines the challenges in the development of exosomes as drug carriers and offers insights into strategies for their effective clinical translation. We also highlight preclinical studies that have successfully employed anti-inflammatory exosomes and suggest future directions for exosome therapeutics. These advancements underscore the potential for integrating exosome-based therapies into clinical practice, heralding promise for future medical interventions.

Given a pretrained encoder-based language model, how can we accurately compress it without retraining? Retraining-free structured pruning algorithms are crucial in pretrained language model compression due to their significantly reduced pruning cost and capability to prune large language models. However, existing retraining-free algorithms encounter severe accuracy degradation, as they fail to handle pruning errors, especially at high compression rates. In this paper, we propose KPrune (Knowledge-preserving pruning), an accurate retraining-free structured pruning algorithm for pretrained encoder-based language models. KPrune focuses on preserving the useful knowledge of the pretrained model to minimize pruning errors through a carefully designed iterative pruning process composed of knowledge measurement, knowledge-preserving mask search, and knowledge-preserving weight-tuning. As a result, KPrune shows significant accuracy improvements up to 58.02%p higher F1 score compared to existing retraining-free pruning algorithms under a high compression rate of 80% on the SQuAD benchmark without any retraining process.

Given a pretrained encoder-based language model, how can we accurately compress it without retraining? Retraining-free structured pruning algorithms are crucial in pretrained language model compression due to their significantly reduced pruning cost and capability to prune large language models. However, existing retraining-free algorithms encounter severe accuracy degradation, as they fail to handle pruning errors, especially at high compression rates. In this paper, we propose K-prune (Knowledge-preserving pruning), an accurate retraining-free structured pruning algorithm for pretrained encoder-based language models. K-prune focuses on preserving the useful knowledge of the pretrained model to minimize pruning errors through a carefully designed iterative pruning process composed of knowledge measurement, knowledge-preserving mask search, and knowledge-preserving weight-tuning. As a result, K-prune shows significant accuracy improvements up to 58.02%p higher F1 score compared to existing retraining-free pruning algorithms under a high compression rate of 80% on the SQuAD benchmark without any retraining process.

For the successful clinical advancement of exosome therapeutics, the biodistribution and pharmacokinetic profile of exogenous exosomes in various animal models must be determined. Compared with fluorescence or bioluminescence imaging, radionuclide imaging confers multiple advantages for the in vivo tracking of biomolecular therapeutics because of its excellent sensitivity for deep tissue imaging and potential for quantitative measurement. Herein, we assessed the quantitative biodistribution and pharmacokinetics of good manufacturing practice-grade therapeutic exosomes labeled with zirconium-89 (89Zr) after systemic intravenous administration in mice and rats. Quantitative biodistribution analysis by positron emission tomography/computed tomography and gamma counting in mice and rats revealed that the total 89Zr signals in the organs were lower in rats than in mice, suggesting a higher excretion rate of exosomes in rats. A prolonged 89Zr signal for up to 7 days in most organs indicated that substantial amounts of exosomes were taken up by the parenchymal cells in those organs, highlighting the therapeutic potential of exosomes for the intracellular delivery of therapeutics. Exosomes were mainly distributed in the liver and to a lesser extent in the spleen, while a moderately distributed in the kidney, lung, stomach, intestine, urinary bladder, brain, and heart. Exosomes were rapidly cleared from the blood circulation, with a rate greater than that of free 89Zr, indicating that exosomes might be rapidly taken up by cells and tissues.

Delivering therapeutics to the central nervous system (CNS) is difficult because of the blood–brain barrier (BBB). Therapeutic delivery across the tight junctions of the BBB can be achieved through various endogenous transportation mechanisms. Receptor-mediated transcytosis (RMT) is one of the most widely investigated and used methods. Drugs can hijack RMT by expressing specific ligands that bind to receptors mediating transcytosis, such as the transferrin receptor (TfR), low-density lipoprotein receptor (LDLR), and insulin receptor (INSR). Cell-penetrating peptides and viral components originating from neurotropic viruses can also be utilized for the efficient BBB crossing of therapeutics. Exosomes, or small extracellular vesicles, have gained attention as natural nanoparticles for treating CNS diseases, owing to their potential for natural BBB crossing and broad surface engineering capability. RMT-mediated transport of exosomes expressing ligands such as LDLR-targeting apolipoprotein B has shown promising results. Although surface-modified exosomes possessing brain targetability have shown enhanced CNS delivery in preclinical studies, the successful development of clinically approved exosome therapeutics for CNS diseases requires the establishment of quantitative and qualitative methods for monitoring exosomal delivery to the brain parenchyma in vivo as well as elucidation of the mechanisms underlying the BBB crossing of surface-modified exosomes.

Among extracellular vesicles, exosomes have gained great attention for their role as therapeutic vehicles for delivering various active pharmaceutical ingredients (APIs). Exosomes “armed” with anti-cancer therapeutics possess great potential for an efficient intracellular delivery of anti-cancer APIs and enhanced targetability to tumor cells. Various technologies are being developed to efficiently incorporate anti-cancer APIs such as genetic materials (miRNA, siRNA, mRNA), chemotherapeutics, and proteins into exosomes and to induce targeted delivery to tumor burden by exosomal surface modification. Exosomes can incorporate the desired therapeutic molecules via direct exogenous methods (e.g., electroporation and sonication) or indirect methods by modifying cells to produce “armed” exosomes. The targeted delivery of “armed” exosomes to tumor burden could be accomplished either by “passive” targeting using the natural tropism of exosomes or by “active” targeting via the surface engineering of exosomal membranes. Although anti-cancer exosome therapeutics demonstrated promising results in preclinical studies, success in clinical trials requires thorough validation in terms of chemistry, manufacturing, and control techniques. While exosomes possess multiple advantages over synthetic nanoparticles, challenges remain in increasing the loading efficiency of anti-cancer agents into exosomes, as well as establishing quantitative and qualitative analytical methods for monitoring the delivery of in vivo administered exosomes and exosome-incorporated anti-cancer agents to the tumor parenchyma.

The coronavirus disease 2019 (COVID-19) is an ongoing global pandemic caused by severe acute respiratory syndrome corona virus 2 (SARS-CoV-2). Patients with severe COVID-19 exhibit hyper-inflammatory responses characterized by excessive activation of myeloid cells, including monocytes, macrophages, and neutrophils, and a plethora of pro-inflammatory cytokines and chemokines. Accumulating evidence also indicates that hyper inflammation is a driving factor for severe progression of the disease, which has prompted the development of anti-inflammatory therapies for the treatment of patients with COVID-19. Corticosteroids, IL-6R inhibitors, and JAK inhibitors have demonstrated promising results in treating patients with severe disease. In addition, diverse forms of exosomes that exert anti-inflammatory functions have been tested experimentally for the treatment of COVID-19. Here, we briefly describe the immunological mechanisms of the hyper-inflammatory responses in patients with severe COVID-19. We also summarize current anti-inflammatory therapies for the treatment of severe COVID-19 and novel exosome-based therapeutics that are in experimental stages.

Process of manufacturing therapeutics exosome development for commercialization. The development of exosome treatment starts at the bench, and in order to be commercialized, it goes through the manufacturing, characterization, and formulation stages, production under Good Manufacturing Practice (GMP) conditions for clinical use, and close consultation with regulatory authorities. Exosome, a type of nanoparticles also known as small extracellular vesicles are gaining attention as novel therapeutics for various diseases because of their ability to deliver genetic or bioactive molecules to recipient cells. Although many pharmaceutical companies are gradually developing exosome therapeutics, numerous hurdles remain regarding manufacture of clinical-grade exosomes for therapeutic use. In this mini-review, we will discuss the manufacturing challenges of therapeutic exosomes, including cell line development, upstream cell culture, and downstream purification process. In addition, developing proper formulations for exosome storage and, establishing good manufacturing practice facility for producing therapeutic exosomes remains as challenges for developing clinicalgrade exosomes. However, owing to the lack of consensus regarding the guidelines for manufacturing therapeutic exosomes, close communication between regulators and companies is required for the successful development of exosome therapeutics. This review shares the challenges and perspectives regarding the manufacture and quality control of clinical grade exosomes.

Coronavirus disease 2019 (COVID-19) is an ongoing global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Type I and III interferon (IFN) responses act as the first line of defense against viral infection and are activated by the recognition of viruses by infected cells and innate immune cells. Dysregulation of host IFN responses has been known to be associated with severe disease progression in COVID-19 patients. However, the reported results are controversial and the roles of IFN responses in COVID-19 need to be investigated further. In the absence of a highly efficacious antiviral drug, clinical studies have evaluated recombinant type I and III IFNs, as they have been successfully used for the treatment of infections caused by two other epidemic coronaviruses, SARS-CoV-1 and Middle East respiratory syndrome (MERS)-CoV. In this review, we describe the strategies by which SARS-CoV-2 evades IFN responses and the dysregulation of host IFN responses in COVID-19 patients. In addition, we discuss the therapeutic potential of type I and III IFNs in COVID-19.

The objective of this study was to compare the effects of supplementation with two methionine isoforms, L-methionine (L-Met) or D-methionine (D-Met), on transcriptome expression in broiler chickens under acute heat stress. A total of 240 one-day-old chicks were randomly assigned to one of four treatments in a 2 × 2 factorial arrangement: thermo-neutral vs. acute heat-stress and L-Met vs. D-Met supplementation. On day 14, the heat-stressed group was exposed to 32°C for 5 h, while the others remained at 25°C. Six chicks were randomly selected per treatment and total RNA was isolated from whole blood, ileum, and liver tissues. Two RNA samples from each tissue of each treatment group were randomly selected and pooled in equal amounts. A total of 1.87 billion raw reads obtained from 36 samples (four treatments × three tissues × three composited replicates) were mapped to the reference genome build (Gallus_gallus-5.0) and used to identify differentially expressed genes (DEGs) using DESeq2. Functional enrichment of DEGs was tested using DAVID. Comparing the two isoforms of supplemented methionine, two, three, and ten genes were differentially expressed (> 1 or < -1 log2 fold change) in whole blood, ileum, and liver, respectively. A total of 38, 71, and 16 genes were differentially expressed in response to the interaction between heat stress and Met isoforms in the blood, ileum, and liver, respectively. Three-tissue-specific DEGs were functionally enriched for regulation of cholesterol homeostasis and metabolism, glucose metabolism, and vascular patterning. Chicks fed with L-Met had lower immune (e.g., IL4I1 and SERPINI1) and intestinal angiogenic responses (e.g., FLT1 and FGD5), and stable glucose and lipid metabolism (e.g., PCK1 and LDLR) under heat stress conditions. In conclusion, unlike D-Met, L-Met supplementation seems to help maintain physiological homeostasis and enhances cellular defense systems against external stresses like high environmental temperature.

Exosomes are cell-secreted nano-sized vesicles which deliver diverse biological molecules for intercellular communication. Due to their therapeutic potential, exosomes have been engineered in numerous ways for efficient delivery of active pharmaceutical ingredients to various target organs, tissues, and cells. In vivo administered exosomes are normally delivered to the liver, spleen, kidney, lung, and gastrointestinal tract and show rapid clearance from the blood circulation after systemic injection. The biodistribution and pharmacokinetics (PK) of exosomes can be modulated by engineering various factors such as cellular origin and membrane protein composition of exosomes. Recent advances accentuate the potential of targeted delivery of engineered exosomes even to the most challenging organs including the central nervous system. Major breakthroughs have been made related to various imaging techniques for monitoring in vivo biodistribution and PK of exosomes, as well as exosomal surface engineering technologies for inducing targetability. For inducing targeted delivery, therapeutic exosomes can be engineered to express various targeting moieties via direct modification methods such as chemically modifying exosomal surfaces with covalent/non-covalent bonds, or via indirect modification methods by genetically engineering exosome-producing cells. In this review, we describe the current knowledge of biodistribution and PK of exosomes, factors determining the targetability and organotropism of exosomes, and imaging technologies to monitor in vivo administered exosomes. In addition, we highlight recent advances in strategies for inducing targeted delivery of exosomes to specific organs and cells.

As extracellular vesicles that play an active role in intercellular communication by transferring cellular materials to recipient cells, exosomes offer great potential as a natural therapeutic drug delivery vehicle. The inflammatory responses in various disease models can be attenuated through introduction of super-repressor IκB (srIκB), which is the dominant active form of IκBα and can inhibit translocation of nuclear factor κB into the nucleus. An optogenetically engineered exosome system (EXPLOR) that we previously developed was implemented for loading a large amount of srIκB into exosomes. We showed that intraperitoneal injection of purified srIκB-loaded exosomes (Exo-srIκBs) attenuates mortality and systemic inflammation in septic mouse models. In a biodistribution study, Exo-srIκBs were observed mainly in the neutrophils, and in monocytes to a lesser extent, in the spleens and livers of mice. Moreover, we found that Exo-srIκB alleviates inflammatory responses in monocytic THP-1 cells and human umbilical vein endothelial cells.

Swine leukocyte antigens play indispensable roles in immune responses by recognizing a large number of foreign antigens and thus, their genetic diversity plays a critical role in their functions. In this study, we developed a new high-resolution typing method for pig SLA-1 and successfully typed 307 individuals from diverse genetic backgrounds including 11 pure breeds, 1 cross bred, and 12 cell lines. We identified a total of 52 alleles including 18 novel alleles and 9 SLA-1 duplication haplotypes, including 4 new haplotypes. We observed significant differences in the distribution of SLA-1 alleles among the different pig breeds, including the breed specific alleles. SLA-1 duplication was observed in 33% of the chromosomes and was especially high in the biomedical model breeds such as SNU (100%) and NIH (76%) miniature pigs. Our analysis showed that SLA-1 duplication is associated with the increased level of SLA-1 mRNA expression in porcine cells compared to that of the single copy haplotype. Therefore, we provide here the results of the most extensive genetic analysis on pig SLA-1.

Mesenchymal stem cells (MSCs) are one of the most easily accessible stem cells that can be obtained from various human tissues. They have raised considerable interests for their potential applications in tissue repair, anti-cancer therapy, and inflammation suppression. Stem cell-based therapy was first used to treat muscular dystrophies and has been studied intensively for its efficacy in various disease models, including myocardial infarction, kidney injuries, liver injuries, and cancers. In this review, we summarized the potential mechanisms underlying MSC-derived EVs therapy as a drug delivery platform. Additionally, based on currently published data, we predicted a potential therapeutic role of cargo proteins shuttled by EVs from MSCs. These data may support the therapeutic strategy of using the MSC-derived EVs to accelerate this strategy from bench to bedside. Stem Cells Translational Medicine 2019;8:880&886

Descriptive retrospective cohort of 52 pediatric patients with mucopolysaccharidosis (MPS) and spinal cord disease and surgical outcomes in a reference hospital.To describe clinical manifestations and surgical management and outcomes of spinal lesions.All medical records of 52 patients with mucopolysaccharidosis (I, II, III, IV, and VI) diagnosed between 1992 and 2011 were identified and followed at a single spine center of a pediatric hospital. Demographic, clinical manifestations, spinal cord lesions, and surgical management were the focus of the descriptive report.A total of 52 patients (32 males and 20 females), mean age at diagnosis of 8 ± 4 years (range 1–19), and with a mean follow-up of 11 ± 8 years were identified. Forty-three had cervical disease (the most frequent affection found was odontoid hypoplasia followed by atlantoaxial instability) and 14 patients had thoracolumbar kyphosis. Twenty-one patients presented neurologic compromise before surgery (quadriparesis as the most frequent manifestation), with the progression of neurologic impairment being the most common surgical indication. Surgery was performed in 38 patients (25 cervical and 13 thoracolumbar). Of the 21 patients with preoperative neurologic deficit, 6 patients showed neurologic improvement. The most common surgical complication found was proximal junctional kyphosis.This is the largest series published of mucopolysaccharidosis pediatric patients with a surgically treated spinal condition. We recommend early spinal cord decompression in mucopolysaccharidosis spine pathology to prevent or potentially reverse neurologic impairment.Level IV.

In this study, we investigated the effect of methionine isomers (D- and L-methionine) on growth performance, blood metabolite levels, nutrient digestibility, intestinal morphology, and foot pad dermatitis in broilers challenged with acute heat stress. In total, 240 broilers were randomly allocated in a 2×2 factorial arrangement consisting of two dietary treatments (D- vs. L-methionine) and two thermal environmental conditions (thermo-neutral vs. acute heat stress). Methionine isomers were added to the diet as an ingredient according to the diet formulation. The broilers were exposed to acute heat stress at 33°C for 5 h on day 14. The average daily gain and feed conversion ratio of birds fed L-methionine were higher than those fed D-methionine (P<0.05) from the time of hatching till 21 days. Induced acute heat stress impaired (P<0.05) the daily gain and feed intake of the broilers on day 21. Furthermore, the blood urea nitrogen levels of birds subjected to acute heat stress on days 14 and 21 were higher (P<0.05) than those of their counterparts. Longer villi (P<0.05) were observed in broilers fed L-methionine-supplemented diet than in those fed D-methionine-supplemented diet on day 14, irrespective of thermal environmental conditions. Heat stress reduced (P<0.01) nutrient digestibility of the broilers on days 14 and 21. Higher incidence and severity of foot pad dermatitis were observed (P<0.05) in broilers fed diet containing D-methionine than in those fed L-methionine-supplemented diet. In conclusion, L-methionine-supplemented diet improved growth performance, overcame growth depression, and reduced the incidence of foot pad dermatitis when broilers were exposed to acute heat stress in the starter period.

No conflict of interest was claimed. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.