An overview of the current diagnostic approach to Periprosthetic Joint Infections.

The diagnosis of periprosthetic joint infections (PJI) presents a formidable challenge to orthopaedic surgeons due to its complex and diverse manifestations. Accurate diagnosis is of utmost importance, as even mild pain following joint replacement surgery may indicate PJI in the absence of a definitive gold standard diagnostic test. Numerous diagnostic modalities have been suggested in the literature, and international societies have continually updated diagnostic criteria for this debilitating complication. This review article aims to comprehensively examine the latest evidence-based approaches for diagnosing PJI. Through a thorough analysis of current literature, we explore promising diagnostic strategies that have demonstrated effectiveness in identifying PJI. These strategies encompass the utilization of laboratory markers, such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), alongside imaging techniques such as magnetic resonance imaging (MRI) and leukocyte scintigraphy. Additionally, we highlight the importance of synovial fluid analysis, including the potential role of alpha-defensin as a biomarker, and examine evolving international diagnostic criteria to standardize and improve diagnostic accuracy.

Periprosthetic tibial fracture as a complication of unicompartmental knee arthroplasty: Current insights.

Periprosthetic fracture following knee arthroplasty is a rare but devastating complication associated with significant morbidity. With unicompartmental knee arthroplasty being performed far less frequently than total knee arthroplasty, periprosthetic fracture following unicompartmental knee arthroplasty presents a particular challenge to orthopaedic surgeons, due to clinical unfamiliarity and sparsity of literature. An up-to-date review of the epidemiology, risk factors, and management strategies for PPF after UKA is presented.

Reduced capsaicin-induced mechanical allodynia and neuronal responses in the dorsal root ganglion in the presence of protein tyrosine phosphatase non-receptor type 6 overexpression.

Transient Receptor Potential Vanilloid 1 (TRPV1) is a nonselective cation channel expressed by pain-sensing neurons and has been an attractive target for the development of drugs to treat pain. Recently, Src homology region two domain-containing phosphatase-1 (SHP-1, encoded by Ptpn6) was shown to dephosphorylate TRPV1 in dorsal root ganglia (DRG) neurons, which was linked with alleviating different pain phenotypes. These previous studies were performed in male rodents only and did not directly investigate the role of SHP-1 in TRPV-1 mediated sensitization. Therefore, our goal was to determine the impact of Ptpn6 overexpression on TRPV1-mediated neuronal responses and capsaicin-induced pain behavior in mice of both sexes. Twelve-week-old male and female mice overexpressing Ptpn6 (Shp1-Tg) and their wild type (WT) littermates were used. Ptpn6 overexpression was confirmed in the DRG of Shp1-Tg mice by RNA in situ hybridization and RT-qPCR. Trpv1 and Ptpn6 were found to be co-expressed in DRG sensory neurons in both genotypes. Functionally, this overexpression resulted in lower magnitude intracellular calcium responses to 200 nM capsaicin stimulation in DRG cultures from Shp1-Tg mice compared to WTs. In vivo, we tested the effects of Ptpn6 overexpression on capsaicin-induced pain through a model of capsaicin footpad injection. While capsaicin injection evoked nocifensive behavior (paw licking) and paw swelling in both genotypes and sexes, only WT mice developed mechanical allodynia after capsaicin injection. We observed similar level of TRPV1 protein expression in the DRG of both genotypes, however, a higher amount of tyrosine phosphorylated TRPV1 was detected in WT DRG. These experiments suggest that, while SHP-1 does not mediate the acute swelling and nocifensive behavior induced by capsaicin, it does mediate a protective effect against capsaicin-induced mechanical allodynia in both sexes. The protective effect of SHP-1 might be mediated by TRPV1 dephosphorylation in capsaicin-sensitive sensory neurons of the DRG.

Multiple surgical resections for progressive IDH wildtype glioblastoma-is it beneficial?

PURPOSE: The role of repeat resection for recurrent glioblastoma (rGB) remains equivocal. This study aims to assess the overall survival and complications rates of single or repeat resection for rGB. METHODS: A single-centre retrospective review of all patients with IDH-wildtype glioblastoma managed surgically, between January 2014 and January 2022, was carried out. Patient survival and factors influencing prognosis were analysed, using Kaplan-Meier and Cox regression methods. RESULTS: Four hundred thirty-two patients were included, of whom 329 underwent single resection, 83 had two resections and 20 patients underwent three resections. Median OS (mOS) in the cohort who underwent a single operation was 13.7 months (95% CI: 12.7-14.7 months). The mOS was observed to be extended in patients who underwent second or third-time resection, at 22.9 months and 44.7 months respectively (p < 0.001). On second operation achieving > 95% resection or residual tumour volume of < 2.25 cc was significantly associated with prolonged survival. There was no significant difference in overall complication rates between primary versus second (p = 0.973) or third-time resections (p = 0.312). The use of diffusion tensor imaging (DTI) guided resection was associated with reduced post-operative neurological deficit (RR 0.37, p = 0.002), as was use of intraoperative ultrasound (iUSS) (RR 0.45, p = 0.04). CONCLUSIONS: This study demonstrates potential prolongation of survival for rGB patients undergoing repeat resection, without significant increase in complication rates with repeat resections. Achieving a more complete repeat resection improved survival. Moreover, the use of intraoperative imaging adjuncts can maximise tumour resection, whilst minimising the risk of neurological deficit.

An Induced Pluripotent Stem Cell-Derived Human Blood-Brain Barrier (BBB) Model to Test the Crossing by Adeno-Associated Virus (AAV) Vectors and Antisense Oligonucleotides.

The blood-brain barrier (BBB) is the specialised microvasculature system that shields the central nervous system (CNS) from potentially toxic agents. Attempts to develop therapeutic agents targeting the CNS have been hindered by the lack of predictive models of BBB crossing. In vitro models mimicking the human BBB are of great interest, and advances in induced pluripotent stem cell (iPSC) technologies and the availability of reproducible differentiation protocols have facilitated progress. In this study, we present the efficient differentiation of three different wild-type iPSC lines into brain microvascular endothelial cells (BMECs). Once differentiated, cells displayed several features of BMECs and exhibited significant barrier tightness as measured by trans-endothelial electrical resistance (TEER), ranging from 1500 to >6000 Ωcm2. To assess the functionality of our BBB models, we analysed the crossing efficiency of adeno-associated virus (AAV) vectors and peptide-conjugated antisense oligonucleotides, both currently used in genetic approaches for the treatment of rare diseases. We demonstrated superior barrier crossing by AAV serotype 9 compared to serotype 8, and no crossing by a cell-penetrating peptide-conjugated antisense oligonucleotide. In conclusion, our study shows that iPSC-based models of the human BBB display robust phenotypes and could be used to screen drugs for CNS penetration in culture.

Periprosthetic acetabular fractures as a complication of total hip arthroplasty.

Periprosthetic acetabular fractures are rare but potentially devastating complications of total hip arthroplasty. As the number of total hip arthroplasties performed annually increases, so has the incidence of periprosthetic fractures, with the topic being spotlighted more frequently in the orthopaedic community. There is a particular sparsity of literature regarding periprosthetic acetabular fractures, with periprosthetic femoral fractures after total hip arthroplasty being traditionally far more commonly reported. This article aims to provide an up-to-date review of the epidemiology, risk factors, diagnostic challenges, classifications, and management strategies for periprosthetic acetabular fractures after total hip arthroplasty.

Modulation of Pro-Inflammatory IL-6 Trans-Signaling Axis by Splice Switching Oligonucleotides as a Therapeutic Modality in Inflammation.

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a crucial role in maintaining normal homeostatic processes under the pathogenesis of various inflammatory and autoimmune diseases. This context-dependent effect from a cytokine is due to two distinctive forms of signaling: cis-signaling and trans-signaling. IL-6 cis-signaling involves binding IL-6 to the membrane-bound IL-6 receptor and Glycoprotein 130 (GP130) signal-transducing subunit. By contrast, in IL-6 trans-signaling, complexes of IL-6 and the soluble form of the IL-6 receptor (sIL-6R) signal via membrane-bound GP130. Various strategies have been employed in the past decade to target the pro-inflammatory effect of IL-6 in numerous inflammatory disorders. However, their development has been hindered since these approaches generally target global IL-6 signaling, also affecting the anti-inflammatory effects of IL-6 signaling too. Therefore, novel strategies explicitly targeting the pro-inflammatory IL-6 trans-signaling without affecting the IL-6 cis-signaling are required and carry immense therapeutic potential. Here, we have developed a novel approach to specifically decoy IL-6-mediated trans-signaling by modulating alternative splicing in GP130, an IL-6 signal transducer, by employing splice switching oligonucleotides (SSO), to induce the expression of truncated soluble isoforms of the protein GP130. This isoform is devoid of signaling domains but allows for specifically sequestering the IL-6/sIL-6R receptor complex with high affinity in serum and thereby suppressing inflammation. Using the state-of-the-art Pip6a cell-penetrating peptide conjugated to PMO-based SSO targeting GP130 for efficient in vivo delivery, reduced disease phenotypes in two different inflammatory mouse models of systemic and intestinal inflammation were observed. Overall, this novel gene therapy platform holds great potential as a refined therapeutic intervention for chronic inflammatory diseases.

A Purposefully Designed pH/GSH-Responsive MnFe-Based Metal-Organic Frameworks as Cascade Nanoreactor for Enhanced Chemo-Chemodynamic-Starvation Synergistic Therapy.

Metal-organic frameworks (MOFs) have emerged as promising novel therapeutics for treating malignancies due to their tunable porosity, biocompatibility, and modularity to functionalize with various chemotherapeutics drugs. However, the design and synthesis of dual-stimuli responsive MOFs for controlled drug release in tumor microenvironments are vitally essential but still challenging. Meanwhile, the catalytic effect of metal ions selection and ratio optimization in MOFs for enhanced chemodynamic therapy (CDT) is relatively unexplored. Herein, a series of MnFe-based MOFs with pH/glutathione (GSH)-sensitivity are synthesized and then combined with gold nanoparticles (Au NPs) and cisplatin prodrugs (DSCP) as a cascade nanoreactor (SMnFeCGH) for chemo-chemodynamic-starvation synergistic therapy. H+ and GSH can specifically activate the optimal SMnFeCGH nanoparticles in cancer cells to release Mn2+/4+ /Fe2+/3+ , Au NPs, and DSCP rapidly. The optimal ratio of Mn/Fe shows excellent H2 O2 decomposition efficiency for accelerating CDT. Au NPs can cut off the energy supply to cancer cells for starvation therapy and strengthen CDT by providing large amounts of H2 O2 . Then H2 O2 is catalyzed by Mn2+ /Fe2+ to generate highly toxic •OH with the depletion of GSH. Meanwhile, the reduced DSCP accelerates cancer cell regression for chemotherapy. The ultrasensitivity cascade nanoreactor can enhance the anticancer therapeutic effect by combining chemotherapy, CDT, and starvation therapy.

Tumor-targeted glycogen nanoparticles loaded with hemin and glucose oxidase to promote tumor synergistic therapy.

Strategies which are used to address the low levels of intracellular hydrogen peroxide and the development of biocompatible catalysts still need to be fulfilled in tumor chemodynamic therapy. Therefore, a novel tumor-targeted glycogen-based nanoparticle system (GN/He/GOx/HA) was developed to co-deliver hemin (He) and GOx, which can self-supply glucose formed upon degradation of glycogen by α-glycosidase in the lysosome environment, in order to achieve synergistic antitumor therapy. Hyaluronic acid (HA) was selected as the outer shell to protect the activity of GOx, and to increase the uptake by tumor cells via CD44 receptor-mediated endocytosis. GN/He/GOx/HA NPs had a good stability in the blood circulation, but fast release of the therapeutic cargos upon intracellular uptake. Hemin had a cascade catalytic reaction with GOx. Furthermore, GN/He/GOx/HA NPs had the strongest cytotoxicity in Hela cells in a glucose concentration dependent manner. The NPs could efficiently produce reactive oxygen species in tumor cells, resulting in a decrease in the mitochondrial membrane potential and apoptosis of tumor cells. The in vivo results showed that the drug-loaded nanoparticles had good safety, biocompatibility, and efficacious antitumor effect. Therefore, the glycogen-based nanoparticle delivery system provides potential application for self-enhancing CDT, which can be used for effective antitumor therapy.

Engineered bacteria combined with doxorubicin nanoparticles suppress angiogenesis and metastasis in murine melanoma models.

Methods capable of distributing antitumour therapeutics uniformly throughout an entire tumour and that can suppress metastasis at the same time, would be of great significance in improving cancer treatment. Bacteria-mediated synergistic therapies have been explored for better specificity, temporal and spatial controllability, as well for providing regulation of the immune microenvironment, in order to provide improved cancer treatment. To achieve this goal, here we developed an engineered bacteria delivery system (GDOX@HSEc) using synthetic biology and interfacial chemistry technologies. The engineered bacteria were concurrently modified to express heparin sulfatase 1 (HSulf-1) inside (HSEc), to attach doxorubicin-loaded glycogen nanoparticles (GDOX NPs) on their surface. Here we demonstrate that HSEc can actively target and colonise tumour sites resulting in HSulf-1 overexpression, thereby suppressing angiogenesis and metastasis. Simultaneously, the GDOX NPs were able to penetrate into tumour cells, leading to intracellular DNA damage. Our results confirmed that a combination of biotherapy and chemotherapy using GDOX@HSEc resulted in significant melanoma suppression in murine models, with reduced side effects. This study provides a powerful platform for the simultaneous delivery of biomacromolecules and chemotherapeutic drugs to tumours, representing an innovative strategy potentially more effective in treating solid tumours. STATEMENT OF SIGNIFICANCE: An original engineered bacteria-based system (GDOX@HSEc) was developed using synthetic biology and interfacial chemistry technologies to concurrently produce naturally occurring heparin sulfatase 1 (HSulf-1) inside and anchor doxorubicin-loaded glycogen nanoparticles on the surface. GDOX@HSEc allowed for combined local delivery of chemotherapeutic agents along with the enzymes and immunostimulatory bacterial adjuvants, which resulted in a synergistic action in the inhibition of tumour growth and metastasis. The study provides a potential therapeutic approach that allows therapeutic agents to be distributed in a spatiotemporally controllable manner in tumours for combinatorial enhanced therapy.

Non-uniform dystrophin re-expression after CRISPR-mediated exon excision in the dystrophin/utrophin double-knockout mouse model of DMD.

Duchenne muscular dystrophy (DMD) is the most prevalent inherited myopathy affecting children, caused by genetic loss of the gene encoding the dystrophin protein. Here we have investigated the use of the Staphylococcus aureus CRISPR-Cas9 system and a double-cut strategy, delivered using a pair of adeno-associated virus serotype 9 (AAV9) vectors, for dystrophin restoration in the severely affected dystrophin/utrophin double-knockout (dKO) mouse. Single guide RNAs were designed to excise Dmd exon 23, with flanking intronic regions repaired by non-homologous end joining. Exon 23 deletion was confirmed at the DNA level by PCR and Sanger sequencing, and at the RNA level by RT-qPCR. Restoration of dystrophin protein expression was demonstrated by western blot and immunofluorescence staining in mice treated via either intraperitoneal or intravenous routes of delivery. Dystrophin restoration was most effective in the diaphragm, where a maximum of 5.7% of wild-type dystrophin expression was observed. CRISPR treatment was insufficient to extend lifespan in the dKO mouse, and dystrophin was expressed in a within-fiber patchy manner in skeletal muscle tissues. Further analysis revealed a plethora of non-productive DNA repair events, including AAV genome integration at the CRISPR cut sites. This study highlights potential challenges for the successful development of CRISPR therapies in the context of DMD.

High Soluble Amyloid-ß42 Predicts Normal Cognition in Amyloid-Positive Individuals with Alzheimer's Disease-Causing Mutations.

BACKGROUND: In amyloid-positive individuals at risk for Alzheimer's disease (AD), high soluble 42-amino acid amyloid-ß (Aß42) levels are associated with normal cognition. It is unknown if this relationship applies longitudinally in a genetic cohort. OBJECTIVE: To test the hypothesis that high Aß42 preserves normal cognition in amyloid-positive individuals with Alzheimer's disease (AD)-causing mutations (APP, PSEN1, or PSEN2) to a greater extent than lower levels of brain amyloid, cerebrospinal fluid (CSF) phosphorylated tau (p-tau), or total tau (t-tau). METHODS: Cognitive progression was defined as any increase in Clinical Dementia Rating (CDR = 0, normal cognition; 0.5, very mild dementia; 1, mild dementia) over 3 years. Amyloid-positivity was defined as a standard uptake value ratio (SUVR) ≥1.42 by Pittsburgh compound-B positron emission tomography (PiB-PET). We used modified Poisson regression models to estimate relative risk (RR), adjusted for age at onset, sex, education, APOE4 status, and duration of follow-up. The results were confirmed with multiple sensitivity analyses, including Cox regression. RESULTS: Of 232 mutation carriers, 108 were PiB-PET-positive at baseline, with 43 (39.8%) meeting criteria for progression after 3.3±2.0 years. Soluble Aß42 levels were higher among CDR non-progressors than CDR progressors. Higher Aß42 predicted a lower risk of progression (adjusted RR, 0.36; 95% confidence interval [CI], 0.19-0.67; p = 0.002) better than lower SUVR (RR, 0.81; 95% CI, 0.68-0.96; p = 0.018). CSF Aß42 levels predicting lower risk of progression increased with higher SUVR levels. CONCLUSION: High CSF Aß42 levels predict normal cognition in amyloid-positive individuals with AD-causing genetic mutations.

Dysregulation of Tweak and Fn14 in skeletal muscle of spinal muscular atrophy mice.

BACKGROUND: Spinal muscular atrophy (SMA) is a childhood neuromuscular disorder caused by depletion of the survival motor neuron (SMN) protein. SMA is characterized by the selective death of spinal cord motor neurons, leading to progressive muscle wasting. Loss of skeletal muscle in SMA is a combination of denervation-induced muscle atrophy and intrinsic muscle pathologies. Elucidation of the pathways involved is essential to identify the key molecules that contribute to and sustain muscle pathology. The tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/TNF receptor superfamily member fibroblast growth factor-inducible 14 (Fn14) pathway has been shown to play a critical role in the regulation of denervation-induced muscle atrophy as well as muscle proliferation, differentiation, and metabolism in adults. However, it is not clear whether this pathway would be important in highly dynamic and developing muscle. METHODS: We thus investigated the potential role of the TWEAK/Fn14 pathway in SMA muscle pathology, using the severe Taiwanese Smn-/-; SMN2 and the less severe Smn2B/- SMA mice, which undergo a progressive neuromuscular decline in the first three post-natal weeks. We also used experimental models of denervation and muscle injury in pre-weaned wild-type (WT) animals and siRNA-mediated knockdown in C2C12 muscle cells to conduct additional mechanistic investigations. RESULTS: Here, we report significantly dysregulated expression of Tweak, Fn14, and previously proposed downstream effectors during disease progression in skeletal muscle of the two SMA mouse models. In addition, siRNA-mediated Smn knockdown in C2C12 myoblasts suggests a genetic interaction between Smn and the TWEAK/Fn14 pathway. Further analyses of SMA, Tweak-/-, and Fn14-/- mice revealed dysregulated myopathy, myogenesis, and glucose metabolism pathways as a common skeletal muscle feature, providing further evidence in support of a relationship between the TWEAK/Fn14 pathway and Smn. Finally, administration of the TWEAK/Fn14 agonist Fc-TWEAK improved disease phenotypes in the two SMA mouse models. CONCLUSIONS: Our study provides mechanistic insights into potential molecular players that contribute to muscle pathology in SMA and into likely differential responses of the TWEAK/Fn14 pathway in developing muscle.

AR cooperates with SMAD4 to maintain skeletal muscle homeostasis.

Androgens and androgen-related molecules exert a plethora of functions across different tissues, mainly through binding to the transcription factor androgen receptor (AR). Despite widespread therapeutic use and misuse of androgens as potent anabolic agents, the molecular mechanisms of this effect on skeletal muscle are currently unknown. Muscle mass in adulthood is mainly regulated by the bone morphogenetic protein (BMP) axis of the transforming growth factor (TGF)-ß pathway via recruitment of mothers against decapentaplegic homolog 4 (SMAD4) protein. Here we show that, upon activation, AR forms a transcriptional complex with SMAD4 to orchestrate a muscle hypertrophy programme by modulating SMAD4 chromatin binding dynamics and enhancing its transactivation activity. We challenged this mechanism of action using spinal and bulbar muscular atrophy (SBMA) as a model of study. This adult-onset neuromuscular disease is caused by a polyglutamine expansion (polyQ) in AR and is characterized by progressive muscle weakness and atrophy secondary to a combination of lower motor neuron degeneration and primary muscle atrophy. Here we found that the presence of an elongated polyQ tract impairs AR cooperativity with SMAD4, leading to an inability to mount an effective anti-atrophy gene expression programme in skeletal muscle in response to denervation. Furthermore, adeno-associated virus, serotype 9 (AAV9)-mediated muscle-restricted delivery of BMP7 is able to rescue the muscle atrophy in SBMA mice, supporting the development of treatments able to fine-tune AR-SMAD4 transcriptional cooperativity as a promising target for SBMA and other conditions associated with muscle loss.

Enhancing the Therapeutic Potential of Extracellular Vesicles Using Peptide Technology.

Extracellular vesicles are lipid-bilayer-enclosed nanoparticles present in the majority of biological fluids that mediate intercellular communication. EVs are able to transfer their contents (including nucleic acids, proteins, lipids, and small molecules) to recipient cells, and thus hold great promise as drug delivery vehicles. However, their therapeutic application is limited by lack of efficient cargo loading strategies, a need to improve EV tissue-targeting capabilities and a requirement to improve escape from the endolysosomal system. These challenges can be effectively addressed by modifying EVs with peptides which confer specific advantageous properties, thus enhancing their therapeutic potential. Here we provide an overview of the applications of peptide technology with respect to EV therapeutics. We focus on the utility of EV-modifying peptides for the purposes of promoting cargo loading, tissue-targeting and endosomal escape, leading to enhanced delivery of the EV cargo to desired cells/tissues and subcellular target locations. Both endogenous and exogenous methods for modifying EVs with peptides are considered.

GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain.

Extracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication, and potential as drug delivery vehicles. Here we demonstrate a role for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EV assembly and secretion. We observe high levels of GAPDH binding to the outer surface of EVs via a phosphatidylserine binding motif (G58), which promotes extensive EV clustering. Further studies in a Drosophila EV biogenesis model reveal that GAPDH is required for the normal generation of intraluminal vesicles in endosomal compartments, and promotes vesicle clustering. Fusion of the GAPDH-derived G58 peptide to dsRNA-binding motifs enables highly efficient loading of small interfering RNA (siRNA) onto the EV surface. Such vesicles efficiently deliver siRNA to multiple anatomical regions of the brain in a Huntington's disease mouse model after systemic injection, resulting in silencing of the huntingtin gene in different regions of the brain.

Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles.

Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.

RASSF1C oncogene elicits amoeboid invasion, cancer stemness, and extracellular vesicle release via a SRC/Rho axis.

Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces, and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here, we show that the novel oncogene RASSF1C drives mesenchymal-to-amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC-mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that RASSF1C-induced amoeboid cells display increased expression of cancer stem-like markers such as CD133, ALDH1, and Nanog, and are accompanied by higher invasive potential in vitro and in vivo. Further, RASSF1C-induced amoeboid cells employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C-driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early-stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification.

Gene therapy with AR isoform 2 rescues spinal and bulbar muscular atrophy phenotype by modulating AR transcriptional activity.

Spinal and bulbar muscular atrophy (SBMA) is an X-linked, adult-onset neuromuscular condition caused by an abnormal polyglutamine (polyQ) tract expansion in androgen receptor (AR) protein. SBMA is a disease with high unmet clinical need. Recent studies have shown that mutant AR-altered transcriptional activity is key to disease pathogenesis. Restoring the transcriptional dysregulation without affecting other AR critical functions holds great promise for the treatment of SBMA and other AR-related conditions; however, how this targeted approach can be achieved and translated into a clinical application remains to be understood. Here, we characterized the role of AR isoform 2, a naturally occurring variant encoding a truncated AR lacking the polyQ-harboring domain, as a regulatory switch of AR genomic functions in androgen-responsive tissues. Delivery of this isoform using a recombinant adeno-associated virus vector type 9 resulted in amelioration of the disease phenotype in SBMA mice by restoring polyQ AR-dysregulated transcriptional activity.

Immortalized Canine Dystrophic Myoblast Cell Lines for Development of Peptide-Conjugated Splice-Switching Oligonucleotides.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frameshift or nonsense mutations in the DMD gene, resulting in the loss of dystrophin from muscle membranes. Exon skipping using splice-switching oligonucleotides (SSOs) restores the reading frame of DMD pre-mRNA by generating internally truncated but functional dystrophin protein. To potentiate effective tissue-specific targeting by functional SSOs, it is essential to perform accelerated and reliable in vitro screening-based assessment of novel oligonucleotides and drug delivery technologies, such as cell-penetrating peptides, before their in vivo pharmacokinetic and toxicity evaluation. We have established novel canine immortalized myoblast lines by transducing murine cyclin-dependent kinase-4 and human telomerase reverse transcriptase genes into myoblasts isolated from beagle-based wild-type or canine X-linked muscular dystrophy in Japan (CXMDJ) dogs. These myoblast lines exhibited improved myogenic differentiation and increased proliferation rates compared with passage-15 primary parental myoblasts, and their potential to differentiate into myotubes was maintained in later passages. Using these dystrophin-deficient immortalized myoblast lines, we demonstrate that a novel cell-penetrating peptide (Pip8b2)-conjugated SSO markedly improved multiexon skipping activity compared with the respective naked phosphorodiamidate morpholino oligomers. In vitro screening using immortalized canine cell lines will provide a basis for further pharmacological studies on drug delivery tools.