Human Patients Research Articles

Overcoming Challenges in Interdisciplinary Collaboration Between Human and Veterinary Medicine

Companion animals, such as dogs and cats, have gained considerable attention in translational medicine due to their potential as models for human diseases. The use of these animals in research has opened new avenues for developing treatments that can benefit both human and veterinary patients, aligning with the One Health approach. Unlike traditional laboratory models like mice, rats, and rabbits, companion animals naturally develop diseases that closely mirror those in humans, including but not limited to diabetes, aging, cancer, and neurological disorders, making them particularly valuable in translational research. Recent advances have highlighted the role of companion animals in enhancing the effectiveness of novel therapies during clinical trials, as they are exposed to diverse environmental and lifestyle factors similar to those experienced by humans. However, the integration of companion animals into translational medicine presents challenges, particularly in terms of collaboration between veterinary and human medicine, where terminology differences in anatomy, clinical terminology, and animal classification can lead to miscommunication. In conclusion, these findings underscore the need for better implementation of the One Health approach by uniting the fragmented collaboration between veterinarians and doctors through interdisciplinary training and fostering unified efforts across both fields, with experts from various disciplines contributing their specialized knowledge in clinical practice and research.

Fecal Bile Acids in Canine Chronic Liver Disease: Results from 46 Dogs.

The concentrations of fecal and serum bile acids (BAs) are known to be altered in human patients with chronic liver diseases (CLDs), especially those with biliary tract involvement (BTD). Scarce literature is available regarding fecal BA modifications during canine CLDs. This study aimed to evaluate fecal BAs in canine CLDs according to different clinical and clinicopathological variables. Forty-six dogs were enrolled. Canine feces were analyzed by HPLC. Cholic Acid (CA), Chenodeoxycholic Acid (CDCA), Ursodeoxycholic Acid (UDCA), Deoxycholic Acid (DCA), and Lithocholic Acid (LCA) were measured, and primary BAs (CA + CDCA), secondary BAs (UDCA + DCA + LCA), and the primary/secondary (P/S) ratio were calculated. Primary BAs (p < 0.0001), CA (p = 0.0003), CDCA (p = 0.003), the P/S ratio (p = 0.002), and total BAs (p = 0.005) were significatively higher in BTD dogs (n = 18) compared to in non-BTD dogs (n = 28). Fecal secondary BAs did not statistically differ between BTD and non-BTD dogs. Gastrointestinal clinical signs (p = 0.028) and diarrhea (p = 0.03) were significantly more prevalent in BTD dogs compared to in non-BTD dogs, supporting the hypothesis of some pathological mechanisms assimilable to bile acid diarrhea (BAD). Our results could reflect imbalances of the fecal BA metabolism in dogs with CLDs. Further studies involving gut microbiome and metabolomic assessment are needed to better understand the possible clinical implications of BA metabolism disruption and their potential role in canine CLDs.

Intragenic duplication disrupting the reading frame of MFSD8 in Small Swiss Hounds with neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinosis (NCL) represents a heterogenous group of lysosomal storage diseases resulting in progressive neurodegeneration. We investigated two Small Swiss Hound littermates that showed progressive ataxia and loss of cognitive functions and vision starting around the age of 12 months. Both dogs had to be euthanized a few months after the onset of disease owing to the severity of their clinical signs. Pathological investigation of one affected dog revealed cerebral and cerebellar atrophy with cytoplasmic accumulation of autofluorescent material in degenerating neurons. The clinical signs in combination with the characteristic histopathology led to a tentative diagnosis of NCL. In the subsequent genetic investigation, the genome of one affected dog was sequenced. This revealed a duplication of 18 819 bp within the MFSD8 gene. The duplication breakpoints were located in intron 3 and exon 12 of the gene and were predicted to disrupt the reading frame. Both affected dogs carried the duplication in a homozygous state and there was perfect cosegregation of the genotypes with the phenotype in a large pedigree, consistent with autosomal recessive inheritance. MFSD8 loss-of-function variants are a known cause of NCL7 in human patients, dogs and other mammalian species. The existing knowledge on MFSD8 together with the experimental data strongly suggests that the identified intragenic MFSD8 duplication caused the disease in the Small Swiss Hounds. These results allow their diagnosis to be refined to NCL7 and enable genetic testing in the breed to avoid further unintentional carrier × carrier matings.

METTL16 inhibits pancreatic cancer proliferation and metastasis by promoting MROH8 RNA stability and inhibiting CAPN2 expression.

N6-methyladenosine (m6A) modification plays a crucial role in the progression of various cancers, including pancreatic cancer, by regulating gene expression. However, the specific mechanisms by which m6A affects pancreatic cancer metastasis remain unclear. This study aims to elucidate the role of METTL16, an m6A writer gene, in regulating core genes such as CAPN2 and MROH8, influencing tumor growth and metastasis. Transcriptomic data from pancreatic cancer patients in The Cancer Genome Atlas (TCGA) were analyzed to identify m6A-related genes. We performed correlation and survival analyses to uncover core genes influenced by m6A expression. Functional assays, including METTL16 knockdown and overexpression experiments, were conducted in pancreatic cancer cell lines, patient-derived organoids, and animal models. Immunofluorescence, co-immunoprecipitation (Co-IP), and m6A-specific quantitative PCR were used to validate protein interactions and m6A modifications. Chromatin immunoprecipitation (ChIP) analysis was utilized to investigate transcription factor binding at gene promoter regions. METTL16 and METTL3 were identified as key m6A regulators associated with improved prognosis in pancreatic cancer patients (P<0.05). CAPN2, CHMP2B, ITGA3, ITGA6, ITPR1, and RAC1 were identified as core genes linked to m6A expression, all significantly correlated with patient prognosis (P<0.05). METTL16 overexpression significantly inhibited tumor growth and metastasis (P<0.001) by downregulating CAPN2 through an indirect mechanism involving the transcription factor TBP and the gene MROH8. MROH8 negatively regulated CAPN2 by promoting TBP degradation, with METTL16 enhancing MROH8 mRNA stability through m6A modifications (P<0.01). Functional assays demonstrated that METTL16 and YTHDC2 (an m6A reader) collaboratively enhanced MROH8 mRNA stability, thereby inhibiting CAPN2 expression and reducing tumor proliferation and metastasis (P<0.001). This study reveals a novel regulatory axis involving METTL16, MROH8, and TBP that modulates CAPN2 expression, contributing to the suppression of pancreatic cancer progression. The METTL16-MROH8-TBP-CAPN2 pathway offers potential therapeutic targets for pancreatic cancer treatment, highlighting the significance of m6A modifications in tumor regulation. Further clinical validation is needed to confirm these findings in human patients.

Radiation dermatitis in the hairless mouse model mimics human radiation dermatitis

Over half of all people diagnosed with cancer receive radiation therapy. Moderate to severe radiation dermatitis occurs in most human radiation patients, causing pain, aesthetic distress, and a negative impact on tumor control. No effective prevention or treatment for radiation dermatitis exists. The lack of well-characterized, clinically relevant animal models of human radiation dermatitis contributes to the absence of strategies to mitigate radiation dermatitis. Here, we establish and characterize a hairless SKH-1 mouse model of human radiation dermatitis by correlating temporal stages of clinical and pathological skin injury. We demonstrate that a single ionizing radiation treatment of 30 Gy using 6 MeV electrons induces severe clinical grade 3 peak toxicity at 12 days, defined by marked erythema, desquamation and partial ulceration, with resolution occurring by 25 days. Histopathology reveals that radiation-induced skin injury features temporally unique inflammatory changes. Upregulation of epidermal and dermal TGF-ß1 and COX-2 protein expression occurs at peak dermatitis, with sustained epidermal TGF-ß1 expression beyond resolution. Specific histopathological variables that remain substantially high at peak toxicity and early clinical resolution, including epidermal thickening, hyperkeratosis and dermal fibroplasia/fibrosis, serve as specific measurable parameters for in vivo interventional preclinical studies that seek to mitigate radiation-induced skin injury.

NUMB dysfunction defines a novel mechanism underlying hyperuricemia and gout

Defective renal excretion and increased production of uric acid engender hyperuricemia that predisposes to gout. However, molecular mechanisms underlying defective uric acid excretion remain largely unknown. Here, we report a rare genetic variant of gout-unprecedented NUMB gene within a hereditary human gout family, which was identified by an unbiased genome-wide sequencing approach. This dysfunctional missense variant within the conserved region of the NUMB gene (NUMBR630H) underwent intracellular redistribution and degradation through an autophagy-dependent mechanism. Mechanistically, we identified the uric acid transporter, ATP Binding Cassette Subfamily G Member 2 (ABCG2), as a novel NUMB-binding protein through its intracellular YxNxxF motif. In polarized renal tubular epithelial cells (RTECs), NUMB promoted ABCG2 trafficking towards the apical plasma membrane. Genetic loss-of-function of NUMB resulted in redistribution of ABCG2 in the basolateral domain and ultimately defective excretion of uric acid. To recapitulate the clinical situation in human gout patients, we generated a NUMBR630H knock-in mouse strain, which showed marked increases of serum urate and decreased uric acid excretion. The NUMBR630H knock-in mice exhibited clinically relevant hyperuricemia. In summary, we have uncovered a novel NUMB-mediated mechanism of uric acid excretion and a functional missense variant of NUMB in humans, which causes hyperuricemia and gout.

Exploring the contribution of Zfp521/ZNF521 on primary hematopoietic stem/progenitor cells and leukemia progression.

Hematopoietic stem cells (HSCs) drive cellular turnover in the hematopoietic system by balancing self-renewal and differentiation. In the adult bone marrow (BM), these cells are regulated by a complex cellular microenvironment known as "niche," which involves dynamic interactions between diverse cellular and non-cellular elements. During blood cell maturation, lineage branching is guided by clusters of genes that interact or counteract each other, forming complex networks of lineage-specific transcription factors. Disruptions in these networks can lead to obstacles in differentiation, lineage reprogramming, and ultimately malignant transformation, including acute myeloid leukemia (AML). Zinc Finger Protein 521 (Znf521/Zfp521), a conserved transcription factor enriched in HSCs in both human and murine hematopoiesis, plays a pivotal role in regulating HSC self-renewal and differentiation. Its enforced expression preserves progenitor cell activity, while inhibition promotes differentiation toward the lymphoid and myeloid lineages. Transcriptomic analysis of human AML patient samples has revealed upregulation of ZNF521 in AMLs with the t(9;11) fusion gene MLL-AF9. In vitro studies have shown that ZNF521 collaborates with MLL-AF9 to enhance the growth of transformed leukemic cells, increase colony formation, and activate MLL target genes. Conversely, inhibition of ZNF521 using short-hairpin RNA (shRNA) results in decreased leukemia proliferation, reduced colony formation, and induction of cell cycle arrest in MLL-rearranged AML cell lines. In vivo experiments have demonstrated that mZFP521-deficient mice transduced with MLL-AF9 experience a delay in leukemia development. This review provides an overview of the regulatory network involving ZNF521, which plays a crucial role in controlling both HSC self-renewal and differentiation pathways. Furthermore, we examine the impact of ZNF521 on the leukemic phenotype and consider it a potential marker for MLL-AF9+ AML.

Blockade of brain alkaline phosphatase efficiently reduces amyloid-β plaque burden and associated cognitive impairment

BackgroundAlzheimer’s disease (AD) is the most prevalent neurodegenerative disease. Three new drugs for AD based on monoclonal antibodies against the amyloid-β peptide (Aβ) have recently been approved because they favor the reduction of the burden of senile plaque in the AD patient’s brain. Nonetheless, both drugs have very limited applicability and benefits and show several side effects. These limitations invite us to find alternative strategies for treating patients with AD. Here, we explored whether tissue-nonspecific alkaline phosphatase (TNAP), an ectoenzyme upregulated in the brain of AD patients and whose inhibition has beneficial effects on tau-induced pathology, is also efficient in reducing senile plaque burden.MethodsTo evaluate whether TNAP may reduce cerebral senile plaque loading and Aβ-related toxicity, we use both pharmacological and genetic approaches. We analyze postmortem samples from human AD patients, APP/PS1 mice (a mouse model that mimics amyloid pathology observed in AD patients) treated or not with TNAP inhibitors, and the newly generated transgenic mouse line, TNAP-deficient APP/PS1 mice.ResultsFor the first time, we describe that genetic or pharmacological blockade of TNAP effectively reduces senile plaque burden by promoting its clearance, which leads to amelioration of cognitive impairment caused by Aβ-induced toxicity. These beneficial effects of TNAP inhibition occur concomitantly with higher microglial recruitment toward the senile plaque and increased microglial phagocytic capacity of Aβ by a mechanism involving metalloprotease-depending osteopontin processing. In addition, we also found that TNAP blockade favors LRP1-mediated transport of Aβ through the BBB.ConclusionsHere, we have shown that TNAP inhibition effectively reduces brain senile plaque burden and associated behavioral defects. Furthermore, given that we had previously reported that TNAP blockade also ameliorates Tau-induced neurotoxicity and increases lifespan of P301S tauopathy mouse model, we can state that TNAP blockade may be a novel and efficient therapy for treating patients with AD.

A qualitative study on the dyadic coping experience of human papillomavirus-infected patients of childbearing age and their spouses.

China's recent policy initiatives and dedication of resources to heighten the public's awareness of cancer risks have led to increased cervical cancer screening and testing for human papillomavirus (HPV) which has resulted in a greater number of people diagnosed with HPV. The psychological stress experienced by women of childbearing age who are infected with HPV is also felt by their spouses, as the close relationship between spouses results in intertwined psychological distress and health statuses. Therefore, this study aimed to explore the dyadic coping experience of HPV-infected patients of childbearing age and their spouses in China and to provide a research basis for marital interventions for the disease. From July 2022 to January 2023, we used a purposive sampling method to select 11 pairs of HPV-infected patients of childbearing age and their spouses from a tertiary hospital. We conducted in-depth interviews with the patients and their spouses and analyzed the data using Colaizzi's seven-step method. We identified three main themes and eight subthemes: (a) stress perception (including negative psychological reactions, emotional relationship deterioration, and family social role imbalance), (b) stress communication (including enhancing communication awareness and changing communication methods), and (c) stress adjustment (including supporting each other emotionally, facing the disease together, and seeking social support). Health care professionals should assess the stress experienced by patients and their spouses. Moreover, they should encourage them to better cope with the disease as a team. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Fecal bile acid dysmetabolism and reduced ursodeoxycholic acid correlate with novel microbial signatures in feline chronic kidney disease.

Microbial-derived secondary bile acids (SBAs) are reabsorbed and sensed via host receptors modulating cellular inflammation and fibrosis. Feline chronic kidney disease (CKD) occurs with progressive renal inflammation and fibrosis, mirroring the disease pathophysiology of human CKD patients. Prospective cross-sectional study compared healthy cats (n = 6) with CKD (IRIS Stage 2 n = 17, Stage 3 or 4 n = 11). Single timepoint fecal samples from all cats underwent targeted bile acid metabolomics. 16S rRNA gene amplicon sequencing using DADA2 with SILVA taxonomy characterized the fecal microbiota. CKD cats had significantly reduced fecal concentrations (median 12.8 ng/mg, Mann-Whitney p = 0.0127) of the SBA ursodeoxycholic acid (UDCA) compared to healthy cats (median 39.4 ng/mg). Bile acid dysmetabolism characterized by <50% SBAs was present in 8/28 CKD and 0/6 healthy cats. Beta diversity significantly differed between cats with <50% SBAs and > 50% SBAs (PERMANOVA p < 0.0001). Twenty-six amplicon sequence variants (ASVs) with >97% nucleotide identity to Peptacetobacter hiranonis were identified. P. hiranonis combined relative abundance was significantly reduced (median 2.1%) in CKD cats with <50% SBAs compared to CKD cats with >50% SBAs (median 13.9%, adjusted p = 0.0002) and healthy cats with >50% SBAs (median 15.5%, adjusted p = 0.0112). P. hiranonis combined relative abundance was significantly positively correlated with the SBAs deoxycholic acid (Spearman r = 0.5218, adjusted p = 0.0407) and lithocholic acid (Spearman r = 0.5615, adjusted p = 0.0156). Three Oscillospirales ASVs and a Roseburia ASV were also identified as significantly correlated with fecal SBAs. The gut-kidney axis mediated through microbial-derived SBAs appears relevant to the spontaneous animal CKD model of domestic cats. This includes reduced fecal concentrations of the microbial-derived SBA UDCA, known to regulate inflammation and fibrosis and be reno-protective. Microbes correlated with fecal SBAs include bai operon containing P. hiranonis, as well as members of Oscillospirales, which also harbor a functional bai operon. Ultimately, CKD cats represent a translational opportunity to study the role of SBAs in the gut-kidney axis, including the potential to identify novel microbial-directed therapeutics to mitigate CKD pathogenesis in veterinary patients and humans alike.

A Novel PTPRQ c.3697del Variant Causes Autosomal Dominant Progressive Hearing Loss in Both Humans and Mice.

PTPRQ plays an important role in the development of inner ear hair cell stereocilia. While many autosomal recessive variants in PTPRQ have been identified as the pathogenic cause for nonsyndromic hearing loss (DFNB84A), so far only one autosomal dominant PTPRQ variant, c.6881G>A (p.Trp2294*), has been reported for late-onset, mild-to-severe hearing loss (DFNA73). By using targeted next-generation sequencing, this study identified a novel PTPRQ truncating pathogenic variant, c.3697del (p.Leu1233Phefs*11), from a Chinese Han family that co-segregated with autosomal dominant, postlingual, progressive hearing loss. A Ptprq-3700del knock-in mouse model was generated by CRISPR-Cas9 and characterized for its hearing function and inner ear morphology. While the homozygous knock-in mice exhibit profound hearing loss at all frequencies at the age of 3 weeks, the heterozygous mutant mice resemble the human patients in mild, progressive hearing loss from age 3 to 12 weeks, primarily affecting high frequencies. At this stage, the homozygous knock-in mice have a normal hair cell count but disorganized stereocilia. Cochlear proteosome analysis of the homozygous mutant mice revealed differentially expressed genes and pathways involved in oxidative phosphorylation, regulation of angiogenesis and synaptic vesicle cycling. Our study provides a valuable animal model for further functional studies of the pathogenic mechanisms underlying DFNA73.

Divergent and Convergent TMEM106B Pathology in Murine Models of Neurodegeneration and Human Disease.

TMEM106B is a lysosomal/late endosome protein that is a potent genetic modifier of multiple neurodegenerative diseases as well as general aging. Recently, TMEM106B was shown to form insoluble aggregates in postmortem human brain tissue, drawing attention to TMEM106B pathology and the potential role of TMEM106B aggregation in disease. In the context of neurodegenerative diseases, TMEM106B has been studied in vivo using animal models of neurodegeneration, but these studies rely on overexpression or knockdown approaches. To date, endogenous TMEM106B pathology and its relationship to known canonical pathology in animal models has not been reported. Here, we analyze histological patterns of TMEM106B in murine models of C9ORF72 -related amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD), SOD1-related ALS, and tauopathy and compare these to postmortem human tissue from patients with C9-ALS/FTD, Alzheimer's disease (AD), and AD with limbic-predominant age-related TDP-43 encephalopathy (AD/LATE). We show that there are significant differences between TMEM106B pathology in mouse models and human patient tissue. Importantly, we also identified convergent evidence from both murine models and human patients that links TMEM106B pathology to TDP-43 nuclear clearance specifically in C9-ALS. Similarly, we find a relationship at the cellular level between TMEM106B pathology and phosphorylated Tau burden in Alzheimer's disease. By characterizing endogenous TMEM106B pathology in both mice and human postmortem tissue, our work reveals considerations that must be taken into account when analyzing data from in vivo mouse studies and elucidates new insights supporting the involvement of TMEM106B in the pathogenesis and progression of multiple neurodegenerative diseases.

Klebsiella pneumoniae co-infection leads to fatal pneumonia in SARS-CoV-2-infected mice

SARS-CoV-2 patients have been reported to have high rates of secondary Klebsiella pneumoniae infections. K. pneumoniae is a commensal that is typically found in the respiratory and gastrointestinal tracts. However, it can cause severe disease when a person’s immune system is compromised. Despite a high number of K. pneumoniae cases reported in SARS-CoV-2 patients, a co-infection animal model evaluating the pathogenesis is not available. In our cohort of COVID-19-positive human patients, 38% exhibited the presence of K. pneumoniae. Therefore we developed a mouse model to study the disease pathogenesis of SARS-CoV-2 and K. pneumoniae co-infection. BALB/cJ mice were inoculated with mouse-adapted SARS-CoV-2 followed by a challenge with K. pneumoniae. Mice were monitored for body weight change, clinical signs, and survival during infection. The bacterial load, viral titers, immune cell accumulation and phenotype, and histopathology were evaluated in the lungs. The co-infected mice showed severe clinical disease and a higher mortality rate within 48 h of K. pneumoniae administration. The co-infected mice had significantly elevated bacterial load in the lungs, however, viral loads were similar between co-infected and single-infected mice. Histopathology of co-infected mice showed severe bronchointerstitial pneumonia with copious intralesional bacteria. Flow cytometry analysis showed significantly higher numbers of neutrophils and macrophages in the lungs. Collectively, our results demonstrated that co-infection of SARS-CoV-2 with K. pneumoniae causes severe disease with increased mortality in mice.

The mitochondrial long non-coding RNA lncMtloop regulates mitochondrial transcription and suppresses Alzheimer's disease.

Maintaining mitochondrial homeostasis is crucial for cell survival and organismal health, as evidenced by the links between mitochondrial dysfunction and various diseases, including Alzheimer's disease (AD). Here, we report that lncMtDloop, a non-coding RNA of unknown function encoded within the D-loop region of the mitochondrial genome, maintains mitochondrial RNA levels and function with age. lncMtDloop expression is decreased in the brains of both human AD patients and 3xTg AD mouse models. Furthermore, lncMtDloop binds to mitochondrial transcription factor A (TFAM), facilitates TFAM recruitment to mtDNA promoters, and increases mitochondrial transcription. To allow lncMtDloop transport into mitochondria via the PNPASE-dependent trafficking pathway, we fused the 3'UTR localization sequence of mitochondrial ribosomal protein S12 (MRPS12) to its terminal end, generating a specified stem-loop structure. Introducing this allotropic lncMtDloop into AD model mice significantly improved mitochondrial function and morphology, and ameliorated AD-like pathology and behavioral deficits of AD model mice. Taken together, these data provide insights into lncMtDloop as a regulator of mitochondrial transcription and its contribution to Alzheimer's pathogenesis.

Sensory-Motor Neuropathy in Mfn2 T105M Knock-in Mice and Its Reversal by a Novel Piperine-Derived Mitofusin Activator.

Mitochondrial dysfunction is a hallmark of many genetic neurodegenerative diseases, but therapeutic options to reverse mitochondrial dysfunction are limited. While recent studies support the possibility of improving mitochondrial fusion/fission dynamics and motility to correct mitochondrial dysfunction and resulting neurodegeneration in Charcot-Marie-Tooth disease (CMT) and other neuropathies, the clinical utility of reported compounds and relevance of preclinical models are uncertain. Here, we describe motor and sensory neuron dysfunction characteristic of clinical CMT type 2 A in a CRISPR/Casp-engineered Mfn2 Thr105Met (T105M) mutant knock-in mouse. We further demonstrate that daily oral treatment with a novel mitofusin activator derived from the natural product piperine can reverse these neurologic phenotypes. Piperine derivative 8015 promoted mitochondrial fusion and motility in Mfn2-deficient cells in a mitofusin-dependent manner and reversed mitochondrial dysfunction in cultured fibroblasts and reprogrammed motor neurons from a human CMT2A patient carrying the MFN2 T105M mutation. Like previous mitofusin activators, 8015 exhibited stereospecific functionality, but the more active stereoisomer, 8015-P2, is unique in that it has subnanomolar potency and undergoes entero-hepatic recirculation which extends its in vivo half-life. Daily administration of 8015-P2 to Mfn2 T105M knock-in mice for 6 weeks normalized neuromuscular and sensory dysfunction and corrected histological/ultrastructural neurodegeneration and neurogenic myoatrophy. These studies describe a more clinically relevant mouse model of CMT2A and an improved mitofusin activator derived from piperine. We posit that 8015-P2 and other piperine derivatives may benefit CMT2A or other neurodegenerative conditions wherein mitochondrial dysdynamism plays a contributory role. SIGNIFICANCE STATEMENT: Mitochondrial dysfunction is widespread and broadly contributory in neurodegeneration, but difficult to target therapeutically. Here, we describe 8015-P2, a new small molecule mitofusin activator with ∼10-fold greater potency and improved in vivo pharmacokinetics versus comparators, and demonstrate its rapid reversal of sensory and motor neuron dysfunction in an Mfn2 T105M knock-in mouse model of Charcot-Marie-Tooth disease type 2 A. These findings further support the therapeutic approach of targeting mitochondrial dysdynamism in neurodegeneration.

A dual-STING-activating nanosystem expands cancer immunotherapeutic temporal window.

Stimulator of interferon genes (STING) is a promising antitumor target via bridging innate and adaptive immunity, yet the transient nature of immune signal transduction renders small-molecule agonists susceptible to short time effectiveness. Here, we report a dual-STING-activating micelle system (D-SAM) to dynamically program STING kinetics. Mechanistically, the natural ligand cGAMP encapsulated in D-SAM initiates STING signaling, while the pH-sensitive polymeric agonist PC7A disassembled from micelle shell buffers lysosomal protons and retards STING degradation. This prolonged STING activity facilitates dendritic cell (DC) antigen presentation and extends cytotoxic T lymphocyte priming. D-SAM improves efficacy over single soluble or delivered agonists against established, metastatic, and recurring murine tumors. Specific depletion of STING in DCs or blockade of CD8+ Tcell infiltration abrogates therapeutic effects. The feasibility of immune modulation is further validated in resected human patient tissues. This work underscores the temporal rhythm of STING as crucial for mounting a potent and enduring antitumor immune response.

Large Scale Comparative Analysis of Canine and Human Osteosarcomas Uncovers Conserved Clinically Relevant Tumor Microenvironment Subtypes.

Osteosarcoma is an aggressive bone cancer lacking robust biomarkers for personalized treatment. Despite its scarcity in humans, it is relatively common in adult pet dogs. This study aimed to analyze clinically annotated bulk tumor transcriptomic datasets of canine and human osteosarcoma patients to identify potentially conserved patterns of disease progression. Bulk transcriptomic data from 245 pet dogs with treatment-naïve appendicular osteosarcoma were analyzed using deconvolution to characterize the tumor microenvironment (TME). The TME of both primary and metastatic tumors derived from the same dog was compared, and its impact on canine survival was assessed. A machine learning model was developed to classify the TME based on its inferred composition using canine tumor data. This model was applied to 8 independent human osteosarcoma datasets to assess its generalizability and prognostic value. This study found three distinct TME subtypes of canine osteosarcoma based on cell type composition of bulk tumor samples: Immune Enriched (IE), Immune Enriched Dense Extra-Cellular Matrix-like (IE-ECM), and Immune Desert (ID). These three TME-based subtypes of canine osteosarcomas were conserved in humans and could predict progression-free survival outcomes of human patients, independent of conventional prognostic factors such as percent tumor necrosis post standard of care chemotherapy treatment and disease stage at diagnosis. These findings demonstrate the potential of leveraging data from naturally occurring cancers in canines to model the complexity of the human osteosarcoma TME, offering a promising avenue for the discovery of novel biomarkers and developing more effective precision oncology treatments.

Spatial transcriptomics of human and murine Neurofibromatosis Type I fracture pseudarthroses reveal impaired BMP signaling

Abstract Introduction/Objective In Neurofibromatosis Type 1 (NF1), caused by mutations in the NF1 tumor-suppressor gene, children form fibrotic nonunions known as pseudarthroses following long bone fractures. Here, we integrate spatial transcriptomics of human and murine samples to investigate the pathogenesis underlying pseudoarthrosis development. Methods/Case Report We performed spatial transcriptomics on 10-day post-fracture calluses from control and periosteum-specific Nf1-deficient (Nf1Postn) mice as well as a human NF1 patient pseudarthrosis specimen. Differential gene expression and SpatialTime analyses were performed. BMP signaling was validated by immunohistochemical staining of tissues for phospho-SMAD1/5/8. Results (if a Case Study enter NA) Spatial transcriptomics detected eight cell clusters, including bone, cartilage, and muscle within mouse fracture calluses. In the control, clusters involved in fracture repair were enriched for genes associated with extracellular matrix and collagen formation (progenitor and cartilage clusters), cartilage and endochondral bone processes (ossifying perichondrium cluster), and bone remodeling (woven bone cluster). Expression of TGF-beta pathway genes was highest near the fracture plane, while BMP and Wnt pathways were activated further from the fracture. In the Nf1Postn fracture, healing was significantly delayed, lacking a robust callus and with a lower abundance of reparative skeletal cell clusters. While expression of TGF-beta pathway genes was similar to control, BMP pathway expression was minimal within the Nf1Postn fracture, which was further demonstrated by a lack of phospho-SMAD1/5/8 staining. In the human pseudarthrosis, while TGF-beta pathway genes were expressed near the fracture plane as expected, there was no enrichment of BMP pathway expression in the pseudarthrosis tissue and no significant phospho-SMAD1/5/8 staining. Conclusion Spatial transcriptomics allows for the simultaneous unbiased analysis of multiple signal transduction pathways within their native tissue environment. Our analyses 1) revealed the cellular diversity required for fracture healing, 2) demonstrated the spatially-restricted expression of key morphogenetic pathways within a fracture callus, and 3) provides in situ evidence for impaired BMP pathway activation associated with NF1 fracture pseudarthroses.

Axon guidance genes are regulated by TDP-43 and RGNEF through long-intron removal.

Rho guanine nucleotide exchange factor (RGNEF) is a guanine nucleotide exchange factor (GEF) mainly involved in regulating the activity of Rho-family GTPases. It is a bi-functional protein, acting both as a guanine exchange factor and as an RNA-binding protein. RGNEF is known to act as a destabilizing factor of neurofilament light chain RNA (NEFL) and it could potentially contribute to their sequestration in nuclear cytoplasmic inclusions. Most importantly, RGNEF inclusions in the spinal motor neurons of ALS patients have been shown to co-localize with inclusions of TDP-43, the major well-known RNA-binding protein aggregating in the brain and spinal cord of human patients. Therefore, it can be hypothesized that loss-of-function of both proteins following aggregation may contribute to motor neuron death/survival in ALS patients. To further characterize their relationship, we have compared the transcriptomic profiles of neuronal cells depleted of TDP-43 and RGNEF and show that these two factors predominantly act in an antagonistic manner when regulating the expression of axon guidance genes. From a mechanistic point of view, our experiments show that the effect of these genes on the processivity of long introns can explain their mode of action. Taken together, our results show that loss-of-function of factors co-aggregating with TDP-43 can potentially affect the expression of commonly regulated neuronal genes in a very significant manner, potentially acting as disease modifiers. This finding further highlights that neurodegenerative processes at the RNA level are the result of combinatorial interactions between different RNA-binding factors that can be co-aggregated in neuronal cells. A deeper understanding of these complex scenarios may lead to a better understanding of pathogenic mechanisms occurring in patients, where more than one specific protein may be aggregating in their neurons.

NEU4-mediated desialylation enhances the activation of the oncogenic receptors for the dissemination of ovarian carcinoma.

Glycosylation profoundly influences the interactions between cancer cells and microenvironmental stromal cells during the peritoneal disseminated metastasis of ovarian carcinoma (OC), which is the major cause of cancer-related death. Although the characteristic cancer glycoconjugates are widely used as biomarkers for cancer diagnosis, our knowledge about cancer glycome remains quite fragmented due to the technique limitations in analyzing glycan chains with tremendous structural and functional heterogeneity. Given the dysregulated cancer glycome is defined by the altered glycosylation machinery, here we performed a systematic loss-of-function screen on 498 genes involved in glycosylation for key regulators of OC dissemination. We identified neuraminidase 4 (NEU4), an enzyme capable of hydrolyzing terminal sialic acid from glycoconjugates, as a vital peritoneal dissemination-promoting modifier of OC glycome. In human patients with high-grade serous OC (HGSOC), increased NEU4 was detected in the disseminated OC cells when compared with that in the primary tumor cells, which significantly correlated with the worse survival. Among three alternative splice-generated isoforms of human NEU4, we revealed that only the plasma membrane-localized NEU4 isoform 2 (NEU4-iso2) and intracellular isoform 3 promoted the peritoneal dissemination of OC by enhancing the cell motility and epithelial-mesenchymal transition. We also identified NEU4-iso2-regulated cell surface glycoproteome and found that NEU4-iso2 desialylated the epithelial growth factor receptor (EGFR), in particular at N196 residue, for the hyperactivation of EGFR and its downstream tumor-promoting signaling cascades. Our results provide new insights into how the OC glycome is dysregulated during OC progression and reveal a functionally important glycosite on EGFR for its abnormal activation in cancer.