We evaluated the efficacy of intravenous PTR-01, a recombinant type VII collagen (C7) expressed in Chinese Hamster Ovary (CHO) cells, in a Phase 2 trial involving six patients with recessive dystrophic epidermolysis bullosa (RDEB). Results showed that PTR-01 was well-tolerated and improved wound healing; however, no anchoring fibrils (AFs) were detected at the basement membrane zone despite C7 deposition at the dermal-epidermal junction. To investigate this, we compared PTR-01 to fibroblast-derived wild-type C7. Non-reducing western blots showed additional low-molecular-weight bands in PTR-01, indicating reduced stability. Under reducing conditions, PTR-01 appeared as a doublet, with domain-specific antibodies revealing proteolytic maturation at the C-terminus in the NC2 domain. Proteomic analysis demonstrated altered glycosylation and a mixture of 40% wild-type C7 and 60% C7 containing a D1033Y substitution in the FN type III domain of NC1. This variant exhibited dominant-negative effects, reducing thermal stability and impairing binding to laminin-332 and collagen IV. These findings indicate that systemic C7 delivery to the skin is feasible and clinically beneficial in RDEB but suggest that impaired ligand binding, lower stability, and premature NC2 maturation hinder AF assembly. We have identified CHO clones that express only wild-type C7, which we plan to advance as an improved alternative to PTR-01.

The role of N6-methyladenosine (m6A) in shaping the tumor microenvironment remains incompletely understood. Here, we investigated the function of the m6A writer RBM15 in bladder cancer (BC). Single-cell sequencing and spatial transcriptomics demonstrated that RBM15 is predominantly expressed in malignant epithelial cells and exerts oncogenic effects. Integrated m6A-seq and lactylation proteomics analyses indicated that RBM15 could regulate both glycolysis and immunity through m6A modification and lactylation. Mechanistically, RIP-qPCR, MeRIP-qPCR, proteomic profiling, luciferase reporter assays, RNA stability tests, and rescue experiments revealed that RBM15 increased m6A modification and stability of PFKFB4 mRNA. We also revealed that RBM15-mediated PFKFB4 mRNA activation relied on the IGF2BP3-dependent pathway. Downregulation of RBM15 and IGF2BP3 suppressed glycolysis while enhancing the anti-tumor potential of CD8+ T cells, whereas PFKFB4 overexpression reversed these effects, and vice versa. In vivo, silencing RBM15 with lipid nanoparticle (LNP)-delivered siRNA enhanced the efficacy of anti-PD1 therapy and increased CD8+ T cell infiltration. Collectively, our findings demonstrate that RBM15 stabilizes PFKFB4 expression in BC through an m6A-IGF2BP3-dependent mechanism and thus promotes the glycolysis and inhibits CD8+ T cell function. Targeting RBM15 sensitizes tumors to PD1 blockade and provides a promising therapeutic strategy for BC.

Radiotherapy-induced salivary gland (SG) hypofunction substantially impairs the quality of life for patients with head and neck cancer, while effective preventive treatments remain limited. This study investigated the protective effects of laminarin against SG injury in mice and its association with Nrf2-related antioxidant responses. Mice received a single 15-Gy head and neck irradiation and were assessed 28 days later. Body weight, food and water intake, stimulated salivary flow rate, submandibular gland weight, histopathological changes, AQP5 expression, oxidative stress indicators, and Nrf2-associated proteins were evaluated. Irradiation reduced body weight, food intake, SG weight, salivary secretion, and AQP5 expression, while increasing water intake, histopathological injury, ROS, and MDA levels. Laminarin ameliorated these changes, preserved SG function, increased MnSOD expression, and reduced oxidative stress. Laminarin treatment was also accompanied by higher nuclear Nrf2 protein levels and increased HO-1 and NQO1 expression. These findings indicate that laminarin attenuates irradiation-induced SG hypofunction and oxidative injury in mice and suggest a possible association with enhanced Nrf2-associated antioxidant responses. The present study provides preclinical evidence supporting further evaluation of laminarin as a potential radioprotective strategy for preserving SG function during head and neck radiotherapy.

Recessive dystrophic epidermolysis bullosa (RDEB) and junctional epidermolysis bullosa (JEB) are severe blistering skin disorders caused by mutations in genes encoding type VII collagen (COL7A1) and laminin 332 (LAMA3, LAMB3, or LAMC2), respectively. In RDEB, 25% of patients carry nonsense mutations that result in premature termination codons (PTCs), while in JEB, the majority of mutations in LAMB3 are nonsense mutations (80%). CC-90009, an eRF3a degrader, is effective in inducing PTC readthrough in various in vitro models of diseases caused by nonsense mutations. This study evaluated CC-90009's ability, in combination with gentamicin, to suppress PTCs and promote the expression of type VII collagen (C7) in primary RDEB keratinocytes and fibroblasts, as well as laminin 332 in primary JEB keratinocytes with nonsense mutations. While CC-90009 alone demonstrated limited efficacy, its combination with low-dose gentamicin led to a dose-dependent increase in C7 and laminin β3 production, surpassing the effects of high-dose gentamicin alone. Furthermore, CC-90009/gentamicin reversed the hypermotility and poor substratum attachment characteristic of EB cells. Finally, C7 and laminin 332 induced by CC-90009/gentamicin localized to the dermal-epidermal junction in RDEB and JEB skin equivalents. Therefore, CC-90009/gentamicin may present a novel and safe treatment option for RDEB, JEB, and other inherited skin diseases arising from nonsense mutations.

Epidermolysis Bullosa (EB) comprises a group of inherited blistering disorders caused by pathogenic variants in genes essential for skin and mucosal integrity. Nonsense mutations, which generate premature termination codons (PTCs), result in reduced or absent protein expression and contribute to severe disease phenotypes in EB. Readthrough therapies, which may continue translation past PTCs to restore full-length functional proteins, have emerged as promising approaches. This review summarizes findings from preclinical studies investigating readthrough therapies in EB models, clinical studies demonstrating efficacy in EB patients, and emerging readthrough agents with potential application to EB. Preclinical and clinical studies with gentamicin have demonstrated restored type VII collagen and laminin-332 expression, leading to measurable clinical improvements. Parallel development of novel compounds—including aminoglycoside analogs (e.g., ELX-02), translation termination factor degraders (e.g., CC-90009, SRI-41315, SJ6986), tRNA post-transcriptional inhibitors (e.g., 2,6-diaminopurine, NV848), and nucleoside analogs (e.g., clitocine)—has expanded the therapeutic pipeline. Although challenges remain regarding toxicity, codon specificity, and variable protein restoration thresholds, continued advances in molecular targeting and combination therapies offer the potential to establish readthrough therapies as localized or systemic treatments addressing both cutaneous and extracutaneous disease manifestations in EB.

The role of adipose-derived stromal stem cells (ADSCs) in BLCA progression is unclear. We investigated the effects of invasion, stemness, Epithelial-mesenchymal transition (EMT), and drug resistance of BLCA cells co-cultured with ADSCs for a long period of time. Cells were divided into six groups: ADSCs group, ADSCs: T24 group (10:1, 3:1 and 1:1 groups), ADSCs-derived conditioned medium group (CM) and T24 cell group (T24), and cells in each group were cultured to 14 days, and puromycin (puro) was added to the co-cultured cell to remove ADSCs cells without puro resistance, and then the function of T24 cells (10:1-COC, 3:1-COC and 1:1-COC) after co-culture was studied; CCK-8 assay, Transwell, Wound healing, Flow cytometry, RNA-sequencing, qRT-PCR and Western Bloting assay were used to detect cell proliferation, invasion, migration, apoptosis and cellular mRNA and protein expression levels, respectively. We unexpectedly found enhanced stemness and drug resistance of BLCA cells after prolonged contact culture with ADSCs. T24 cells after co-culture mediated cell proliferation, invasion, EMT, stemness, drug resistance and immune escape by up-regulating MDM2, mt-P53 and PD-L1, compared to CM and T24 groups. The inhibitor Atezo and CP-31,398 eliminated mt-P53 and PD-L1-mediated T24 cell drug resistance and stemness, respectively. This study demonstrated that after prolonged co-culture of BLCA cells with ADSCs, the stemness, drug resistance, and immune evasion of T24 cells were dramatically enhanced, suggesting that long-term resident ADSCs in the bladder cancer tumor microenvironment play a procarcinogenic role.

All previous IND (investigational new drug) applications to US FDA for launching clinical trials with Hsp90 ATP-binding inhibitors only provided a partial, if not misleading, account of the inhibitors’ actual MOA (mechanism of action). Since 2004, studies have repeatedly shown a previously unanticipated “extra effect” of these inhibitors, but it has been incomprehensively ignored by the Hsp90 community. Membrane-impermeable, otherwise structurally identical, ATP-binding Hsp90 inhibitors show robust inhibition of tumor cell invasion in vitro and metastasis in vivo. Based on this new finding, the reported outcomes of around 90 monotherapy clinical trials with Hsp90 ATP-binding inhibitors since 1999 were actually a combined effect of targeting both intracellular Hsp90 chaperone and extracellular Hsp90 (eHsp90) non-chaperone functions by the inhibitors. A critical unanswered question remains: which form of the dual inhibitions caused the observed toxicity in humans that led to the spectacular failure of the trials and which underlies the limited efficacy that might be the real reason for the only approval of the orally administered ATP-binding inhibitor, Pimitespib (TAS-116), in 2022 by Japan? We suggest that addressing this question could prompt a paradigm shift in the design of next-generation anti-Hsp90 cancer therapeutics.

The treatment of diabetic wounds remains a significant challenge in the medical field. In this study, we present a novel approach using photothermally responsive graphene hybrid dry powders for the treatment of diabetic wounds. These powders, derived from polyacrylic acid (PAA) and polyethyleneimine (PEI), exhibit rapid water absorption at the interface, leading to thein situformation of physically crosslinked hydrogels due to interactions between polymers. Furthermore, by incorporating graphene into the PAA/PEI powder mixture, we establish a multifunctional platform with capabilities such as photothermal antibacterial effects and drug release. Given the outstanding performance of this hybrid material, we demonstrate its potential in wound healing by incorporating the tumor necrosis factor-alpha (TNF-α) inhibitor Etanercept into the PAA/PEI powder. This intervention resulted in a significant improvement in the wound healing process in diabetic rats, as evidenced by the downregulation of inflammatory factors, promotion of collagen deposition, and enhanced vascularization. These remarkable attributes underscore the enormous potential value of the presented hydrogel patches in the field of biomedicine.

Recessive dystrophic epidermolysis bullosa (RDEB) and junctional epidermolysis bullosa (JEB) are lethal blistering skin disorders resulting from mutations in genes coding for type VII collagen ( COL7A1 ) and laminin 332 ( LAMA3 , LAMB3, or LAMC2), respectively. In RDEB, 25% of patients harbor nonsense mutations causing premature termination codons (PTCs). In JEB, a majority of mutations in LAMB3 are nonsense mutations (80%). ELX-02, an aminoglycoside analog, has demonstrated superior PTC readthrough activity and lower toxicity compared to gentamicin in various genetic disorders. This study investigated the ability of ELX-02 to suppress PTCs and promote the expression of C7 and laminin 332 in primary RDEB keratinocytes/fibroblasts and primary JEB keratinocytes harboring nonsense mutations. ELX-02 induced a dose-dependent production of C7 or laminin b 3 that surpassed the results achieved with gentamicin. ELX-02 reversed RDEB and JEB cellular hypermotility and improved poor cell-substratum adhesion in JEB cells. Importantly, ELX-02-induced C7 and laminin 332 localized to the dermal-epidermal junction. This is the fi rst study demonstrating that ELX-02 can induce PTC readthrough and restore functional C7 and laminin 332 in RDEB and JEB caused by nonsense mutations. Therefore, ELX-02 may offer a novel and safe therapy for RDEB, JEB, and other inherited skin diseases caused by nonsense mutations.

Targeting the heat shock protein-90 (Hsp90) chaperone machinery in various cancers with 200 monotherapy or combined-therapy clinical trials since 1999 has not yielded any success of food and drug administration approval. Blames for the failures were unanimously directed at the Hsp90 inhibitors or tumors or both. However, analyses of recent cellular and genetic studies together with the Hsp90 data from the Human Protein Atlas database suggest that the vast variations in Hsp90 expression among different organs in patients might have been the actual cause. It is evident now that Hsp90β is the root of dose-limiting toxicity (DLT), whereas Hsp90α is a buffer of penetrated Hsp90 inhibitors. The more Hsp90α, the safer Hsp90β, and the lower DLT are for the host. Unfortunately, the dramatic variations of Hsp90, from total absence in the eye, muscle, pancreas, and heart to abundance in reproduction organs, lung, liver, and gastrointestinal track, would cause the selection of any fair toxicity biomarker and an effective maximum tolerable dose (MTD) of Hsp90 inhibitor extremely challenging. In theory, a safe MTD for the organs with high Hsp90 could harm the organs with low Hsp90. In reverse, a safe MTD for organs with low or undetectable Hsp90 would have little impact on the tumors, whose cells exhibit average 3-7% Hsp90 over the average 2-3% Hsp90 in normal cells. Moreover, not all tumor cell lines tested follow the "inhibitor binding-client protein degradation" paradigm. It is likely why the oral Hsp90 inhibitor TAS-116 (Pimitespib), which bypasses blood circulation and other organs, showed some beneficiary efficacy by conveniently hitting tumors along the gastrointestinal track. The critical question is what the next step will be for the Hsp90 chaperone as a cancer therapeutic target.

Agricultural waste has great potential to be used as biomaterial raw materials that can be used in medical applications, especially for bone tissue regeneration. Nanocellulose, which is produced from natural cellulose, offers good mechanical properties and high biocompatibility. This research aims to develop nanocellulose-based biomaterials from agricultural waste for bone regeneration applications. The purpose of this study is to explore the potential of agricultural waste, such as rice straw, peanut husks, and corn leaves, in producing high-quality nanocellulose that can be used for applications in the field of bone tissue regeneration. This study uses an experimental design with a laboratory approach. Agricultural waste is treated through nanocellulose extraction using certain chemical techniques. Material characterization was carried out using scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR), as well as biocompatibility tests using osteoblast cell cultures. The results show that rice straw produces nanocellulose with the highest cellulose content (65%) and has optimal tensile strength and degradation time for bone tissue applications. Peanut husks and corn leaves also show good results, although not as good as rice straw. Agricultural waste, especially rice straw, has great potential to be used as a raw material for nanocellulose that can be used in bone tissue regeneration applications. This research opens up opportunities to develop more sustainable and affordable biomaterials for medical applications.

Delayed wound healing is one vital complication of diabetes mellitus (DM) that adversely impacts patient quality of life. Infliximab (INF), a monoclonal tumor necrosis factor cti (TNF-cti) antibody, has been investigated for its therapeutic potential across various diseases through displaying anti-inflammation ability. However, the regulatory mechanisms by which INF influences and correlates with delayed wound healing in DM remain unclear. In this study, we first induced diabetes in mice using streptozotocin (STZ) to establish a DM model, and then excision wounds were generated. We observed that wound closure was significantly retarded in DM mice, but this effect was reversed following INF treatment (5 mg/kg). Moreover, INF treatment attenuated the heightened inflammation observed in DM mice. Furthermore, we found that INF expedited the M2 phenotype polarization in DM mice. Mechanistically, INF was shown to delay activation of the nuclear factor kappa-B (NF-icB) pathway. To sum up, our findings demonstrate that in diabetic mice, INF facilitates wound healing by modulating macrophage polarization, and refrained the NF-icB pathway, supporting INF as a promising treatment approach for improving wound healing in diabetics.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and most often severe genetic disease characterized by recurrent blistering and erosions of the skin and mucous membranes after minor trauma, leading to major local and systemic complications. The disease is caused by loss-of-function variants in COL7A1 encoding type VII collagen (C7), the main component of anchoring fibrils, which form attachment structures stabilizing the cutaneous basement membrane zone. Alterations in C7 protein structure and/or expression lead to abnormal, rare or absent anchoring fibrils resulting in loss of dermal-epidermal adherence and skin blistering. To date, more than 1,200 distinct COL7A1 deleterious variants have been reported and 19% are splice variants. Here, we describe two RDEB patients for whom we identified two pathogenic deep intronic pathogenic variants in COL7A1 . One of these variants (c.7795-97C > G) promotes the inclusion of a pseudoexon between exons 104 and 105 in the COL7A1 transcript, while the other causes partial or complete retention of intron 51. We used antisense oligonucleotide (ASO) mediated exon skipping to correct these aberrant splicing events in vitro. This led to increased normal mRNA splicing above 94% and restoration of C7 protein expression at a level (up to 56%) that should be sufficient to reverse the phenotype. This first report of exon skipping applied to counteract deep intronic variants in COL7A1 represents a promising therapeutic strategy for personalized medicine directed at patients with intronic variants at a distance of consensus splice sites.

Cell secretion repairs tissue damage and restores homeostasis throughout adult life. The extracellular heat shock protein-90alpha (eHsp90α) has been reported as an exosome cargo and a potential driver of wound healing. However, neither the mechanism of secretion nor the genetic evidence for eHsp90α in wound healing has been substantiated. Herein, we show that tissue injury causes massive deposition of eHsp90α in tissues and secretion of eHsp90α by cells. Sequential centrifugations of conditioned medium from relevant cell lines revealed the relative distributions of eHsp90α in microvesicle, exosome and trypsin-sensitive supernatant fractions to be approximately <2%, <4% and >95%, respectively. Establishing the cell-number-to-interstitial-fluid-volume (CIF) ratio for the microenvironment of human tissues as 1 × 109 cells: 1 mL interstitial fluid enabled us to predict the corresponding tissue concentrations of eHsp90α in these fractions as 3.74 μg/mL, 5.61 μg/mL and 178 μg/mL. Remarkably, the 178 μg/mL eHsp90α matches the previously reported 100–300 μg/mL of recombinant eHsp90α whose topical application promotes maximum wound healing in animal models. More importantly, we demonstrate that two parallel secretory autophagy-regulating gene families, the autophagy-regulating (AR) genes and the Golgi reassembly-stacking protein (GRASP) genes work together to mediate the secretion of the physiological concentration of eHsp90α to promote wound healing. Thus, utilization of the CIF ratio-based extrapolation method may enable investigators to rapidly predict biomarker targets from cell-conditioned-medium data.

Background Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable widespread blistering skin disorder caused by mutations in the gene encoding for type VII collagen (C7), the major component of anchoring fibrils. Objectives To evaluate the efficacy and safety of intravenous (IV) gentamicin readthrough therapy in patients with RDEB harbouring nonsense mutations. The primary outcomes were increased expression of C7 in patients' skin and safety assessments (ototoxicity, nephrotoxicity, autoimmune response); secondary outcomes included measuring wound healing in target wounds and assessment by a validated Epidermolysis Bullosa Disease Activity and Scarring Index (EBDASI) scoring system. Methods An open-label pilot trial to assess two different IV gentamicin regimens between August 2018 and March 2020 with follow-up through to 180 days post-treatment was carried out. Three patients with RDEB with confirmed nonsense mutations in COL7A1 in either one or two alleles and decreased baseline expression of C7 at the dermal-epidermal junction (DEJ) of their skin participated in the study. Three patients received gentamicin 7.5 mg kg-1 daily for 14 days and two of the three patients further received 7.5 mg kg-1 IV gentamicin twice weekly for 12 weeks. Patients who had pre-existing auditory or renal impairment, were currently using ototoxic or nephrotoxic medications, or had allergies to aminoglycosides or sulfate compounds were excluded. Results After gentamicin treatment, skin biopsies from all three patients (age range 18-28 years) exhibited increased C7 in their DEJ. With both regimens, the new C7 persisted for at least 6 months post-treatment. At 1 and 3 months post-treatment, 100% of the monitored wounds exhibited > 85% closure. Both IV gentamicin infusion regimens decreased EBDASI total activity scores. Of the patients assessed with the EBDASI, all exhibited decreased total activity scores 3 months post-treatment. All three patients completed the study; no adverse effects or anti-C7 antibodies were detected. Conclusions IV gentamicin induced the readthrough of nonsense mutations in patients with RDEB and restored functional C7 in their skin, enhanced wound healing and improved clinical parameters. IV gentamicin may be a safe, efficacious, low-cost and readily available treatment for this population of patients with RDEB.

XLS file - 8K, describing KEGG pathway analysis results

legend to supplementary figure 1

Since the only and the milestone FDA approval of becaplermin gel (RegranexTM, 0.01% human recombinant PDGF-BB) as a (diabetic) wound healing therapeutic more than 25 years ago, no new therapeutic (excluding physical therapies, devices, dressings, anti-microbial agents, or other preventive treatments) for any type of wound healing has advanced to clinical applications. During the same period of time, the FDA has approved additional 250 new drugs for various human tumors, which were famously described as "wounds that do not heal". Two similar pathological conditions have experienced such a dramatic difference in therapeutics. More surprisingly, few in the wound healing community seem to be alarmed by this mysterious deficit. As it is often said, "damaging is far easier than re-building". In contrast to the primary duty of a cancer drug to damage a single molecule of the signaling network, a wound healing drug must be able to re-build the multi-level damages in the wound. No known single molecule alone is capable of repairing multi-cell-type and multi-pathway damages all at once. We argue that the previous single molecule-based strategy for developing wound healing therapeutics is profoundly flawed in theory. The future success of effective wound healing therapeutics requires a fundamental change in the paradigm.