3′-Sialyllactose alleviates bone loss by regulating bone homeostasis

AUTHORS

Ahreum Baek, Dawoon Baek, Yoonhee Cho, Seongmoon Jo, Jinyoung Kim, Yoontaik Hong, Seunghee Cho, Sung Hoon Kim & Sung-Rae Cho

ABSTRACT

Osteoporosis is a common skeletal disease that results in an increased risk of fractures. However, there is no definitive cure, warranting the development of potential therapeutic agents. 3′-Sialyllactose (3′-SL) in human milk regulates many biological functions. However, its effect on bone metabolism remains unknown. This study aimed to investigate the molecular mechanisms underlying the effect of 3′-SL on bone homeostasis. Treatment of human bone marrow stromal cells (hBMSCs) with 3′-SL enhanced osteogenic differentiation and inhibited adipogenic differentiation of hBMSCs. RNA sequencing showed that 3′-SL enhanced laminin subunit gamma-2 expression and promoted osteogenic differentiation via the phosphatidylinositol 3‑kinase/protein kinase B signaling pathway. Furthermore, 3′-SL inhibited the receptor activator of nuclear factor κB ligand-induced osteoclast differentiation of bone marrow-derived macrophages through the nuclear factor κB and mitogen‑activated protein kinase signaling pathway, ameliorated osteoporosis in ovariectomized mice, and positively regulated bone remodeling. Our findings suggest 3′-SL as a potential drug for osteoporosis.

Inhibition of RANKL improves the skeletal phenotype of adenine-induced chronic kidney disease in mice

AUTHORS

Corinne E Metzger, Mizuho Kittaka, Alec N LaPlant, Yasuyoshi Ueki, Matthew R Allen

ABSTRACT

Skeletal fragility and high fracture rates are common in CKD. A key component of bone loss in CKD with secondary hyperparathyroidism is high bone turnover and cortical bone deterioration through both cortical porosity and cortical thinning. We hypothesized that RANKL drives high bone resorption within cortical bone leading to the development of cortical porosity in CKD (study 1) and that systemic inhibition of RANKL would mitigate the skeletal phenotype of CKD (study 2). In study 1, we assessed the skeletal properties of male and female Dmp1-cre RANKLfl/fl (cKO) and control genotype (Ranklfl/fl; Con) mice after 10 wk of adenine-induced CKD (AD; 0.2% dietary adenine). All AD mice regardless of sex or genotype had elevated blood urea nitrogen and high PTH. Con AD mice in both sexes had cortical porosity and lower cortical thickness as well as high osteoclast-covered trabecular surfaces and higher bone formation rate. cKO mice had preserved cortical bone microarchitecture despite high circulating PTH as well as no CKD-induced increases in osteoclasts. In study 2, male mice with established AD CKD were either given a single injection of an anti-RANKL antibody (5 mg/kg) 8 wk post-induction of CKD or subjected to 3×/wk dosing with risedronate (1.2 μg/kg) for 4 wk. Anti-RANKL treatment significantly reduced bone formation rate as well as osteoclast surfaces at both trabecular and cortical pore surfaces; risedronate treatment had little effect on these bone parameters. In conclusion, these studies demonstrate that bone-specific RANKL is critical for the development of high bone formation/high osteoclasts and cortical bone loss in CKD with high PTH. Additionally, systemic anti-RANKL ligand therapy in established CKD may help prevent the propagation of cortical bone loss via suppression of bone turnover.

Inhibition of PPP1R15A alleviates osteoporosis via suppressing RANKL-induced osteoclastogenesis

AUTHORS

Zong-bao Ding, Yan Chen, Yu-rong Zheng, Yi-yuan Wang, Wen-de Deng, Jie-huang Zheng, Qin Yang, Zi-ye Chen, Li-hong Li, Hui Jiang & Xiao-juan Li

ABSTRACT

Osteoporosis results from overactivation of osteoclasts. There are currently few drug options for treatment of this disease. Since the successful development of allosteric inhibitors, phosphatases have become attractive therapeutic targets. Protein phosphatase 1, regulatory subunit 15 A (PPP1R15A), is a stress-responsive protein, which promotes the UPR (unfolded protein response) and restores protein homeostasis. In this study we investigated the role of PPP1R15A in osteoporosis and osteoclastogenesis. Ovariectomy (OVX)-induced osteoporosis mouse model was established, osteoporosis was evaluated in the left femurs using micro-CT. RANKL-stimulated osteoclastogenesis was used as in vitro models. We showed that PPP1R15A expression was markedly increased in BMMs derived from OVX mice and during RANKL-induced osteoclastogenesis in vitro. Knockdown of PPP1R15A or application of Sephin1 (a PPP1R15A allosteric inhibitor in a phase II clinical trial) significantly inhibited osteoclastogenesis in vitro. Sephin1 (0.78, 3.125 and 12.5 μM) dose-dependently mitigated the changes in NF-κB, MAPK, and c-FOS and the subsequent nuclear factor of activated T cells 1 (NFATc1) translocation in RANKL-stimulated BMMs. Both Sephin1 and PPP1R15A knockdown increased the phosphorylated form of eukaryotic initiation factor 2α (eIF2α); knockdown of eIF2α reduced the inhibitory effects of Sephin1 on NFATc1-luc transcription and osteoclast formation. Furthermore, Sephin1 or PPP1R15A knockdown suppressed osteoclastogenesis in CD14+ monocytes from osteoporosis patients. In OVX mice, injection of Sephin1 (4, 8 mg/kg, i.p.) every two days for 6 weeks significantly inhibited bone loss, and restored bone destruction and decreased TRAP-positive cells. This study has identified PPP1R15A as a novel target for osteoclast differentiation, and genetic inhibition or allosteric inhibitors of PPP1R15A, such as Sephin1, can be used to treat osteoporosis.

Gene therapy with bidridistrogene xeboparvovec for limb-girdle muscular dystrophy type 2E/R4: phase 1/2 trial results

AUTHORS

Jerry R. Mendell, Eric R. Pozsgai, Sarah Lewis, Danielle A. Griffin, Linda P. Lowes, Lindsay N. Alfano, Kelly J. Lehman, Kathleen Church, Natalie F. Reash, Megan A. Iammarino, Brenna Sabo, Rachael Potter, Sarah Neuhaus, Xiaoxi Li, Herb Stevenson & Louise R. Rodino-Klapac

ABSTRACT

Limb-girdle muscular dystrophy 2E/R4 is caused by mutations in the β-sarcoglycan (SGCB) gene, leading to SGCB deficiency and consequent muscle loss. We developed a gene therapy approach based on functional replacement of the deficient SCB protein. Here we report interim results from a first-in-human, open-label, nonrandomized, phase 1/2 trial evaluating the safety and efficacy of bidridistrogene xeboparvovec, an adeno-associated virus-based gene therapy containing a codon-optimized, full-length human SGCB transgene. Patients aged 4–15 years with confirmed SGCB mutations at both alleles received one intravenous infusion of either 1.85 × 1013 vector genome copies kg−1 (Cohort 1, n = 3) or 7.41 × 1013 vector gene copies kg−1 (Cohort 2, n = 3). Primary endpoint was safety, and secondary endpoint was change in SGCB expression in skeletal muscle from baseline to Day 60. We report interim Year 2 results (trial ongoing). The most frequent treatment-related adverse events were vomiting (four of six patients) and gamma-glutamyl transferase increase (three of six patients). Serious adverse events resolved with standard therapies. Robust SGCB expression was observed: Day 60 mean (s.d.) percentage of normal expression 36.2% (2.7%) in Cohort 1 and 62.1% (8.7%) in Cohort 2. Post hoc exploratory analysis showed preliminary motor improvements using the North Star Assessment for Limb-girdle Type Muscular Dystrophies maintained through Year 2. The 2-year safety and efficacy of bidridistrogene xeboparvovec support clinical development advancement. Further studies are necessary to confirm the long-term safety and efficacy of this gene therapy. ClinicalTrials.gov registration: NCT03652259.

Novel enzyme-linked aptamer-antibody sandwich assay and hybrid lateral flow strip for SARS-CoV-2 detection

AUTHORS

Shih-Wei Wu, Ying-Ju Chen, Yu-Wen Chang, Cheng-Yang Huang, Biing-Hui Liu & Feng-Yih Yu

ABSTRACT

We have successfully generated oligonucleotide aptamers (Apts) and monoclonal antibodies (mAbs) targeting the recombinant nucleocapsid (N) protein of SARS-CoV-2. Apts were obtained through seven rounds of systematic evolution of ligands by exponential enrichment (SELEX), while mAbs were derived from the 6F6E11 hybridoma cell line. Leveraging these Apts and mAbs, we have successfully devised two innovative and remarkably sensitive detection techniques for the rapid identification of SARS-CoV-2 N protein in nasopharyngeal samples: the enzyme-linked aptamer-antibody sandwich assay (ELAAA) and the hybrid lateral flow strip (hybrid-LFS). ELAAA exhibited an impressive detection limit of 0.1 ng/mL, while hybrid-LFS offered a detection range of 0.1 – 0.5 ng/mL. In the evaluation using ten nasopharyngeal samples spiked with known N protein concentrations, ELAAA demonstrated an average recovery rate of 92%. Additionally, during the assessment of five nasopharyngeal samples from infected individuals and ten samples from healthy volunteers, hybrid-LFS displayed excellent sensitivity and specificity. Our study introduces a novel and efficient on-site approach for SARS-CoV-2 detection in nasopharyngeal samples. The reliable hybrid Apt-mAb strategy not only advances virus diagnostic methods but also holds promise in combating the spread of related diseases.

The morphology of the interfacial tissue between bighorn sheep horn and bony horncore increases contact surface to enhance strength and facilitate load transfer from the horn to the horncore

AUTHORS

Luca H. Fuller, Evan C. Marcet, Laura L. Agarkov, Prisha Singh, Seth W. Donahue

ABSTRACT

The horns of bighorn sheep rams are permanent cranial appendages used for high energy head-to-head impacts during interspecific combat. The horns attach to the underlying bony horncore by a layer of interfacial tissue that facilitates load transfer between the impacted horn and underlying horncore, which has been shown to absorb substantial energy during head impact. However, the morphology and mechanical properties of the interfacial tissue were previously unknown. Histomorphometry was used to quantify the interfacial tissue composition and morphology and lap-shear testing was used to quantify its mechanical properties. Histological analyses revealed the interfacial tissue is a complex network of collagen and keratin fibers, with collagen being the most abundant protein. Sharpey's fibers provide strong attachment between the interfacial tissue and horncore bone. The inner horn surface displayed microscopic porosity and branching digitations which increased the contact surface with the interfacial tissue by approximately 3-fold. Horn-horncore samples tested by lap-shear loading failed primarily at the horn surface, and the interfacial tissue displayed non-linear strain hardening behavior similar to other soft tissues. The elastic properties of the interfacial tissue (i.e., low- and high-strain shear moduli) were comparable to previously measured values for the equine laminar junction. The interfacial tissue contact surface was positively correlated with the interfacial tissue shear strength (1.23 ± 0.21 MPa), high-strain shear modulus (4.5 ± 0.7 MPa), and strain energy density (0.38 ± 0.07 MJ/m3).