Human Pancreatic α-Cell Heterogeneity and Trajectory Inference Analysis Using Integrated Single Cell- and Single Nucleus-RNA Sequencing Platforms.

Prior studies have shown that pancreatic α-cells can transdifferentiate into ß-cells, and that ß-cells de-differentiate and are prone to acquire an α-cell phenotype in type 2 diabetes (T2D). However, the specific human α-cell and ß-cell subtypes that are involved in α-to-ß-cell and ß-to-α-cell transitions are unknown. Here, we have integrated single cell RNA sequencing (scRNA-seq) and single nucleus RNA-seq (snRNA-seq) of isolated human islets and human islet grafts and provide additional insight into α-ß cell fate switching. Using this approach, we make seven novel observations. 1) There are five different GCG -expressing human α-cell subclusters [α1, α2, α-ß-transition 1 (AB-Tr1), α-ß-transition 2 (AB-Tr2), and α-ß (AB) cluster] with different transcriptome profiles in human islets from non-diabetic donors. 2) The AB subcluster displays multihormonal gene expression, inferred mostly from snRNA-seq data suggesting identification by pre-mRNA expression. 3) The α1, α2, AB-Tr1, and AB-Tr2 subclusters are enriched in genes specific for α-cell function while AB cells are enriched in genes related to pancreatic progenitor and ß-cell pathways; 4) Trajectory inference analysis of extracted α- and ß-cell clusters and RNA velocity/PAGA analysis suggests a bifurcate transition potential for AB towards both α- and ß-cells. 5) Gene commonality analysis identifies ZNF385D, TRPM3, CASR, MEG3 and HDAC9 as signature for trajectories moving towards ß-cells and SMOC1, PLCE1, PAPPA2, ZNF331, ALDH1A1, SLC30A8, BTG2, TM4SF4, NR4A1 and PSCK2 as signature for trajectories moving towards α-cells. 6) Remarkably, in contrast to the events in vitro , the AB subcluster is not identified in vivo in human islet grafts and trajectory inference analysis suggests only unidirectional transition from α-to-ß-cells in vivo . 7) Analysis of scRNA-seq datasets from adult human T2D donor islets reveals a clear unidirectional transition from ß-to-α-cells compatible with dedifferentiation or conversion into α-cells. Collectively, these studies show that snRNA-seq and scRNA-seq can be leveraged to identify transitions in the transcriptional status among human islet endocrine cell subpopulations in vitro , in vivo , in non-diabetes and in T2D. They reveal the potential gene signatures for common trajectories involved in interconversion between α- and ß-cells and highlight the utility and power of studying single nuclear transcriptomes of human islets in vivo . Most importantly, they illustrate the importance of studying human islets in their natural in vivo setting.

Peptide-conjugated antimiRs improve myotonic dystrophy type 1 phenotypes by promoting endogenous MBNL1 expression.

Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by a CTG repeat expansion in the DMPK gene that generates toxic RNA with a myriad of downstream alterations in RNA metabolism. A key consequence is the sequestration of alternative splicing regulatory proteins MBNL1/2 by expanded transcripts in the affected tissues. MBNL1/2 depletion interferes with a developmental alternative splicing switch that causes the expression of fetal isoforms in adults. Boosting the endogenous expression of MBNL proteins by inhibiting the natural translational repressors miR-23b and miR-218 has previously been shown to be a promising therapeutic approach. We designed antimiRs against both miRNAs with a phosphorodiamidate morpholino oligonucleotide (PMO) chemistry conjugated to cell-penetrating peptides (CPPs) to improve delivery to affected tissues. In DM1 cells, CPP-PMOs significantly increased MBNL1 levels. In some candidates, this was achieved using concentrations less than two orders of magnitude below the median toxic concentration, with up to 5.38-fold better therapeutic window than previous antagomiRs. In HSALR mice, intravenous injections of CPP-PMOs improve molecular, histopathological, and functional phenotypes, without signs of toxicity. Our findings place CPP-PMOs as promising antimiR candidates to overcome the treatment delivery challenge in DM1 therapy.

A modular RNA delivery system comprising spherical nucleic acids built on endosome-escaping polymeric nanoparticles.

Nucleic acid therapeutics require delivery systems to reach their targets. Key challenges to be overcome include avoidance of accumulation in cells of the mononuclear phagocyte system and escape from the endosomal pathway. Spherical nucleic acids (SNAs), in which a gold nanoparticle supports a corona of oligonucleotides, are promising carriers for nucleic acids with valuable properties including nuclease resistance, sequence-specific loading and control of receptor-mediated endocytosis. However, SNAs accumulate in the endosomal pathway and are thus vulnerable to lysosomal degradation or recycling exocytosis. Here, an alternative SNA core based on diblock copolymer PMPC25-PDPA72 is investigated. This pH-sensitive polymer self-assembles into vesicles with an intrinsic ability to escape endosomes via osmotic shock triggered by acidification-induced disassembly. DNA oligos conjugated to PMPC25-PDPA72 molecules form vesicles, or polymersomes, with DNA coronae on luminal and external surfaces. Nucleic acid cargoes or nucleic acid-tagged targeting moieties can be attached by hybridization to the coronal DNA. These polymeric SNAs are used to deliver siRNA duplexes against C9orf72, a genetic target with therapeutic potential for amyotrophic lateral sclerosis, to motor neuron-like cells. By attaching a neuron-specific targeting peptide to the PSNA corona, effective knock-down is achieved at doses of 2 particles per cell.

Patient Satisfaction with Healthcare Services and the Techniques Used for its Assessment: A Systematic Literature Review and a Bibliometric Analysis.

Patient satisfaction with healthcare provision services and the factors influencing it are be-coming the main focus of many scientific studies. Assuring the quality of the provided services is essential for the fulfillment of patients' expectations and needs. Thus, this systematic review seeks to find the determinants of patient satisfaction in a global setting. We perform an analysis to evaluate the collected literature and to fulfill the literature gap of bibliometric analysis within this theme. This review follows the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) approach. We conducted our database search in Scopus, Web of Science, and PubMed in June 2022. Studies from 2000-2021 that followed the inclusion and exclusion criteria and that were written in English were included in the sample. We ended up with 157 articles to review. A co-citation and bibliographic coupling analysis were employed to find the most relevant sources, authors, and documents. We divided the factors influencing patient satisfaction into criteria and explanatory variables. Medical care, communication with the patient, and patient's age are among the most critical factors for researchers. The bibliometric analysis revealed the countries, institutions, documents, authors, and sources most productive and significant in patient satisfaction.

Application of Antisense Conjugates for the Treatment of Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is one of the most common muscular dystrophies and can be potentially treated with antisense therapy decreasing mutant DMPK, targeting miRNAs or their binding sites or via a blocking mechanism for MBNL1 displacement from the repeats. Unconjugated antisense molecules are able to correct the disease phenotype in mouse models, but they show poor muscle penetration upon systemic delivery in DM1 patients. In order to overcome this challenge, research has focused on the improvement of the therapeutic window and biodistribution of antisense therapy using bioconjugation to lipids, cell penetrating peptides or antibodies. Antisense conjugates are able to induce the long-lasting correction of DM1 pathology at both molecular and functional levels and also efficiently penetrate hard-to-reach tissues such as cardiac muscle. Delivery to the CNS at clinically relevant levels remains challenging and the use of alternative administration routes may be necessary to ameliorate some of the symptoms experienced by DM1 patients. With several antisense therapies currently in clinical trials, the outlook for achieving a clinically approved treatment for patients has never looked more promising.

Peptide-Conjugated PMOs for the Treatment of Myotonic Dystrophy.

Antisense oligonucleotides (ASOs) have shown great therapeutic potential in the treatment of many neuromuscular diseases including myotonic dystrophy 1 (DM1). However, systemically delivered ASOs display poor biodistribution and display limited penetration into skeletal muscle. The conjugation of cell-penetrating peptides (CPPs) to phosphorodiamidate morpholino oligonucleotides (PMOs), a class of ASOs with a modified backbone, can be used to enhance ASO skeletal muscle penetration. Peptide-PMOs (P-PMOs) have been shown to be highly effective in correcting the DM1 skeletal muscle phenotype in both murine and cellular models of DM1 and at a molecular and functional level. Here we describe the synthesis and conjugation of P-PMOs and methods for analyzing their biodistribution and toxicity in the HSA-LR DM1 mouse model and their efficacy both in vitro and in vivo using FISH and RT-PCR splicing analysis.

Proof of concept of peptide-linked blockmiR-induced MBNL functional rescue in myotonic dystrophy type 1 mouse model.

Myotonic dystrophy type 1 is a debilitating neuromuscular disease causing muscle weakness, myotonia, and cardiac dysfunction. The phenotypes are caused by muscleblind-like (MBNL) protein sequestration by toxic RNA in the DM1 protein kinase (DMPK) gene. DM1 patients exhibit a pathogenic number of repetitions in DMPK, which leads to downstream symptoms. Another disease characteristic is altered microRNA (miRNA) expression. It was previously shown that miR-23b regulates the translation of MBNL1 into protein. Antisense oligonucleotide (AON) treatment targeting this miRNA can improve disease symptoms. Here, we present a refinement of this strategy targeting a miR-23b binding site on the MBNL1 3' UTR in DM1 model cells and mice by using AONs called blockmiRs. BlockmiRs linked to novel cell-penetrating peptide chemistry showed an increase in MBNL1 protein in DM1 model cells and HSALR mice. They also showed an increase in muscle strength and significant rescue of downstream splicing and histological phenotypes in mice without disturbing the endogenous levels of other miR-23b target transcripts.

Gastric Pneumatosis: An Atypical Presentation of Desmoid Tumor.

CASE PRESENTATION: A middle-aged woman presented to the emergency department with a chief complaint of abdominal pain, fever, vomiting, and diarrhea. Abdominal computed tomography revealed gastric pneumatosis and air in the portal system. The patient had an unfavorable clinical course with pneumoperitoneum and septic shock due to secondary peritonitis. She underwent emergency laparotomy where neoformation of the mesentery root was found with infiltration of the small intestine and jejunal perforation. The anatomopathological study of the tumor revealed that it was a desmoid tumor. DISCUSSION: To our knowledge this is the first report in the literature of gastric pneumatosis as the initial presentation of a mesenteric desmoid tumor. Although it usually has a benign clinical course, its locally invasive characteristics can lead to critical illness. Physicians shouldn't overlook these types of complications, as early identification and surgical intervention can modify the prognosis and shorten hospital stay.

Preclinical characterization of antagomiR-218 as a potential treatment for myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by expansion of unstable CTG repeats in a non-coding region of the DMPK gene. CUG expansions in mutant DMPK transcripts sequester MBNL1 proteins in ribonuclear foci. Depletion of this protein is a primary contributor to disease symptoms such as muscle weakness and atrophy and myotonia, yet upregulation of endogenous MBNL1 levels may compensate for this sequestration. Having previously demonstrated that antisense oligonucleotides against miR-218 boost MBNL1 expression and rescue phenotypes in disease models, here we provide preclinical characterization of an antagomiR-218 molecule using the HSALR mouse model and patient-derived myotubes. In HSALR, antagomiR-218 reached 40-60 pM 2 weeks after injection, rescued molecular and functional phenotypes in a dose- and time-dependent manner, and showed a good toxicity profile after a single subcutaneous administration. In muscle tissue, antagomiR rescued the normal subcellular distribution of Mbnl1 and did not alter the proportion of myonuclei containing CUG foci. In patient-derived cells, antagomiR-218 improved defective fusion and differentiation and rescued up to 34% of the gene expression alterations found in the transcriptome of patient cells. Importantly, miR-218 was found to be upregulated in DM1 muscle biopsies, pinpointing this microRNA (miRNA) as a relevant therapeutic target.

Effectiveness of Delayed Intrapleural Alteplase Instillation for Infected Residual Traumatic Hemothorax. Case Report.

In thoracic trauma, many cases may present with hemothorax, and, of those, a portion can complicate in empyema. These cases can reveal themselves to be of difficult management, particularly in peripheral hospitals with complicated access to thoracic surgery. Intrapleural fibrinolytic instillation can be of use and has been widely reported, mostly in the case of empyema. In the literature, the use of fibrinolytics in hemothorax mostly pertained to the older fibrinolytics, such as streptokinase and urokinase. Recent studies describe the use of alteplase in these patients but mostly in the first days after the trauma, when it becomes clear that the first chest tube is not being effective. We report a case of residual traumatic hemothorax that could not be evacuated after multiple chest tubes placements and was finally cleared after instillation of alteplase late in the course of the disease.

Cytotoxicity of Nucleotide-Stabilized Graphene Dispersions on Osteosarcoma and Healthy Cells: On the Way to Safe Theranostics Agents.

An appealing strategy that overcomes the hydrophobicity of pristine graphene and favors its interaction with biological media is colloidal stabilization in aqueous medium with the support of a biomolecule, such as flavin mononucleotide (FMN), as exfoliating/dispersing agent. However, to establish FMN-stabilized graphene (PG-FMN) as suitable for use in biomedicine, its biocompatibility must be proved by a complete assessment of cytotoxicity at the cellular level. Furthermore, if PG-FMN is to be proposed as a theranostic agent, such a study should include both healthy and tumoral cells and its outcome should reveal the nanomaterial as selectively toxic to the latter. Here, we provide an in-depth comparative in vitro analysis of the response of Saos-2 human sarcoma osteoblasts (model tumor cells) and MC3T3-E1 murine preosteoblasts (undifferentiated healthy cells) upon incubation with different concentrations (10-50 µg mL-1) of PG-FMN dispersions constituted by flakes with different average lateral size (90 and 270 nm). Specifically, the impact of PG-FMN on the viability and cell proliferation, reactive oxygen species (ROS) production, and the cellular incorporation process, cell-cycle progression, and apoptosis has been evaluated. PG-FMN was found to be toxic to both types of cells by increasing ROS production and triggering cell-cycle arrest. The present results constitute a cautionary tale on the need to establish the effect of a nanomaterial not only on tumor cells but also on healthy ones before proposing it as anticancer agent.

Application of CRISPR-Cas9-Mediated Genome Editing for the Treatment of Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is a debilitating multisystemic disorder, caused by expansion of a CTG microsatellite repeat in the 3' untranslated region of the DMPK (dystrophia myotonica protein kinase) gene. To date, novel therapeutic approaches have focused on transient suppression of the mutant, repeat-expanded RNA. However, recent developments in the field of genome editing have raised the exciting possibility of inducing permanent correction of the DM1 genetic defect. Specifically, repurposing of the prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) system has enabled programmable, site-specific, and multiplex genome editing. CRISPR-based strategies for the treatment of DM1 can be applied either directly to patients, or indirectly through the ex vivo modification of patient-derived cells, and they include excision of the repeat expansion, insertion of synthetic polyadenylation signals upstream of the repeat, steric interference with RNA polymerase II procession through the repeat leading to transcriptional downregulation of DMPK, and direct RNA targeting of the mutant RNA species. Potential obstacles to such therapies are discussed, including the major challenge of Cas9 and guide RNA transgene/ribonuclear protein delivery, off-target gene editing, vector genome insertion at cut sites, on-target unintended mutagenesis (e.g., repeat inversion), pre-existing immunity to Cas9 or AAV antigens, immunogenicity, and Cas9 persistence.

A Single Amino Acid Residue Regulates PTEN-Binding and Stability of the Spinal Muscular Atrophy Protein SMN.

Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by decreased levels of the survival of motoneuron (SMN) protein. Post-translational mechanisms for regulation of its stability are still elusive. Thus, we aimed to identify regulatory phosphorylation sites that modulate function and stability. Our results show that SMN residues S290 and S292 are phosphorylated, of which SMN pS290 has a detrimental effect on protein stability and nuclear localization. Furthermore, we propose that phosphatase and tensin homolog (PTEN), a novel phosphatase for SMN, counteracts this effect. In light of recent advancements in SMA therapies, a significant need for additional approaches has become apparent. Our study demonstrates S290 as a novel molecular target site to increase the stability of SMN. Characterization of relevant kinases and phosphatases provides not only a new understanding of SMN function, but also constitutes a novel strategy for combinatorial therapeutic approaches to increase the level of SMN in SMA.

Development of LNA Gapmer Oligonucleotide-Based Therapy for ALS/FTD Caused by the C9orf72 Repeat Expansion.

Several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), have a complex genetic background, in addition to cases where the disease appears to manifest sporadically. The recent discovery of the hexanucleotide repeat expansion in the C9orf72 gene as the causative agent of ALS (C9ALS) gives rise to the opportunity to develop new therapies directed at this mutation , which is responsible for a large proportion of ALS and/or frontotemporal dementia cases. Mammalian models conscientiously replicating the late-onset motor defects and cellular pathologies seen in human patients do not exist. In this context, patient-derived cells give us a platform to test potential antisense oligonucleotide therapies, which could be the key to treat this subtype of motor neuron disease. Recently, we described that locked nucleic acid gapmer oligonucleotide-based treatment targeting C9orf72 repeat expanded transcripts resulted in recovery from the disease-related phenotypes in patient-derived fibroblasts. Our findings highlight the therapeutic potential of C9ALS using this gapmer oligonucleotide-based approach.

Macrophage inflammatory and metabolic responses to graphene-based nanomaterials differing in size and functionalization.

The preparation of graphene-based nanomaterials (GBNs) with appropriate stability and biocompatibility is crucial for their use in biomedical applications. In this work, three GBNs differing in size and/or functionalization have been synthetized and characterized, and their in vitro biological effects were compared. Pegylated graphene oxide (GO-PEG, 200-500 nm) and flavin mononucleotide-stabilized pristine graphene with two different sizes (PG-FMN, 200-400 nm and 100-200 nm) were administered to macrophages, chosen as cellular model due to their key role in the processing of foreign materials and the regulation of inflammatory responses. The results showed that cellular uptake of GBNs was mainly influenced by their lateral size, while the inflammatory potential depended also on the type of functionalization. PG-FMN nanomaterials (both sizes) triggered significantly higher nitric oxide (NO) release, together with some intracellular metabolic changes, similar to those induced by the prototypical inflammatory stimulus LPS. NMR metabolomics revealed that macrophages incubated with smaller PG-FMN displayed increased levels of succinate, itaconate, phosphocholine and phosphocreatine, together with decreased creatine content. The latter two variations were also detected in cells incubated with larger PG-FMN nanosheets. On the other hand, GO-PEG induced a decrease in the inflammatory metabolite succinate and a few other changes distinct from those seen in LPS-stimulated macrophages. Assessment of TNF-α secretion and macrophage surface markers (CD80 and CD206) further corroborated the low inflammatory potential of GO-PEG. Overall, these findings revealed distinct phenotypic and metabolic responses of macrophages to different GBNs, which inform on their immunomodulatory activity and may contribute to guide their therapeutic applications.