CRISPR/Cas9 and genetic screens in malaria parasites : small genomes, big impact

The ∼30 Mb genomes of the Plasmodium parasites that cause malaria each encode ∼5000 genes, but the functions of the majority remain unknown. This is due to a paucity of functional annotation from sequence homology, which is compounded by low genetic tractability compared with many model organisms. In recent years technical breakthroughs have made forward and reverse genome-scale screens in Plasmodium possible. Furthermore, the adaptation of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-Associated protein 9 (CRISPR/Cas9) technology has dramatically improved gene editing efficiency at the single gene level. Here, we review the arrival of genetic screens in malaria parasites to analyse parasite gene function at a genome-scale and their impact on understanding parasite biology. CRISPR/Cas9 screens, which have revolutionised human and model organism research, have not yet been implemented in malaria parasites due to the need for more complex CRISPR/Cas9 gene targeting vector libraries. We therefore introduce the reader to CRISPR-based screens in the related apicomplexan Toxoplasma gondii and discuss how these approaches could be adapted to develop CRISPR/Cas9 based genome-scale genetic screens in malaria parasites. Moreover, since more than half of Plasmodium genes are required for normal asexual blood-stage reproduction, and cannot be targeted using knockout methods, we discuss how CRISPR/Cas9 could be used to scale up conditional gene knockdown approaches to systematically assign function to essential genes.Instituto de BiotecnologíaFil: Ishizaki, Takahiro. Umeå University. Department of Molecular Biology; SueciaFil: Ishizaki, Takahiro. The Laboratory for Molecular Infection Medicine Sweden (MIMS); SueciaFil: Hernandez, Sophia. Umeå University. Department of Molecular Biology; SueciaFil: Hernandez, Sophia. The Laboratory for Molecular Infection Medicine Sweden (MIMS); SueciaFil: Paoletta, Martina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Paoletta, Martina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Paoletta, Martina. Umeå University. Department of Molecular Biology; SueciaFil: Paoletta, Martina. The Laboratory for Molecular Infection Medicine Sweden (MIMS); SueciaFil: Sanderson, Theo. Francis Crick Institute; Reino UnidoFil: Bushell, Ellen S. C. Umeå University. Department of Molecular Biology; SueciaFil: Bushell, Ellen S. C. The Laboratory for Molecular Infection Medicine Sweden (MIMS); Sueci

Efficacy and Effectiveness of Extracorporeal Shockwave Therapy in Patients with Myofascial Pain or Fibromyalgia: A Scoping Review

Myofascial pain syndrome (MPS) and fibromyalgia (FM) are underestimated painful musculoskeletal conditions that could impact function and quality of life. A consensus about the most appropriate therapeutic approach is still not reached. Considering the long course of the diseases, prolonged assumption of drugs, such as NSAIDs and pain killers, could increase the risk of adverse events, often leading affected patients and physicians to prefer non-pharmacological approaches. Among these, radial and focused extracorporeal shock waves therapies (ESWT) are widely used in the management of painful musculoskeletal conditions, despite the fact that the mechanisms of action in the context of pain modulation should be further clarified. We performed a scoping review on PubMed using Mesh terms for analyzing the current evidence about the efficacy and effectiveness of ESWT for patients with MPS or FM. We included 19 clinical studies (randomized controlled trials and observational studies); 12 used radial ESWT, and 7 used focused ESWT for MPS. Qualitative analysis suggests a beneficial role of ESWT for improving clinical and functional outcomes in people with MPS, whereas no evidence was found for FM. Considering this research gap, we finally suggested a therapeutic protocol for this latter condition according to the most recent diagnostic criteria

Efficacy and Effectiveness of Physical Agent Modalities in Complex Regional Pain Syndrome Type I: A Scoping Review

Complex regional pain syndrome type I (CRPS-I) is a rare condition with huge variability in triggering factors and clinical scenarios. The complexity of the pathophysiology of this condition fosters the proposal of several therapeutic options with different mechanisms of action in both research and clinical practice. An interdisciplinary and multimodal approach, including pharmacological and non-pharmacological interventions, particularly physical therapy, is recommended by international guidelines, but the benefits and harms of available interventions are poorly known. In this scoping review, the clinical rationale for use of physical agent modalities for patients with CRPS-I will be presented. We found 10 studies addressing the role of electromagnetic field therapy, electrotherapy, and laser therapy. Our findings suggest that physical therapy modalities, in particular transcutaneous electrical nerve stimulation (TENS) and pulsed electromagnetic field therapy (PEMF), may contribute to reduce pain and improve function in patients with CRPS-1

The Rationale for the Intra-Articular Administration of Clodronate in Osteoarthritis

Background: Several pharmacological therapeutic approaches have been proposed to manage osteoarthritis (OA), including intra-articular (IA) injections. Although the discovery of clodronate, a bisphosphonate, dates back to the 1960s and the effects of its IA administration have been investigated for decades in animal models, mechanisms of action of this drug are not quite clear, particularly in OA. This scoping review is an overview of the biological as well as the clinical role of clodronic acid in OA. Method: A scoping review based on the PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews) model was performed to characterize the mechanisms of action of IA clodronate in OA and to evaluate its efficacy from a clinical point of view. Results: Several effects of clodronate have been observed in animal models of OA, including depletion of synovial lining cells that results in reduced production of chemokines (IL-1, TNF- α), growth factors (TGF-β, BMP 2/4), and metalloproteases (MMP 2/3/9); prevention of cartilage damage, synovial hyperplasia, and proteoglycans loss; reduction in joint inflammation, joint swelling, and osteophyte formation. From a clinical perspective, patients with knee OA treated with IA clodronate experienced improvements in pain and joint mobility. Conclusion: Clodronate appears to have different mechanisms of action interfering with the pathogenic processes contributing to OA development and progression. This intervention demonstrated positive effects for patients affected by knee OA

Physical Agent Modalities in Early Osteoarthritis: A Scoping Review

Early osteoarthritis (EOA) still represents a challenge for clinicians. Although there is no consensus on its definition and diagnosis, a prompt therapeutic intervention in the early stages can have a significant impact on function and quality of life. Exercise remains a core treatment for EOA; however, several physical modalities are commonly used in this population. The purpose of this paper is to investigate the role of physical agents in the treatment of EOA. A technical expert panel (TEP) of 8 medical specialists with expertise in physical agent modalities and musculoskeletal conditions performed the review following the PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews) model. The TEP searched for evidence of the following physical modalities in the management of EOA: “Electric Stimulation Therapy”, “Pulsed Electromagnetic field”, “Low-Level Light Therapy”, “Laser Therapy”, “Magnetic Field Therapy”, “Extracorporeal Shockwave Therapy”, “Hyperthermia, Induced”, “Cryotherapy”, “Vibration therapy”, “Whole Body Vibration”, “Physical Therapy Modalities”. We found preclinical and clinical data on transcutaneous electrical nerve stimulation (TENS), extracorporeal shockwave therapy (ESWT), low-intensity pulsed ultrasound (LIPUS), pulsed electromagnetic fields stimulation (PEMF), and whole-body vibration (WBV) for the treatment of knee EOA. We found two clinical studies about TENS and PEMF and six preclinical studies—three about ESWT, one about WBV, one about PEMF, and one about LIPUS. The preclinical studies demonstrated several biological effects on EOA of physical modalities, suggesting potential disease-modifying effects. However, this role should be better investigated in further clinical studies, considering the limited data on the use of these interventions for EOA patients

Role of the Endocannabinoid/Endovanilloid System in the Modulation of Osteoclast Activity in Paget’s Disease of Bone

The role of the endocannabinoid/endovanilloid (EC/EV) system in bone metabolism has recently received attention. Current literature evidences the modulation of osteoclasts and osteoblasts through the activation or inhibition of cannabinoid receptors in various pathological conditions with secondary involvement of bone tissue. However, this role is still unclear in primary bone diseases. Paget’s disease of the bone (PDB) could be considered a disease model for analyzing the role of the EC/EV system on osteoclasts (OCs), speculating the potential use of specific agents targeting this system for managing metabolic bone disorders. The aim of the study is to analyze OCs expression of EC/EV system in patients with PDB and to compare OCs activity between this population and healthy people. Finally, we investigate whether specific agents targeting EC/EV systems are able to modulate OCs activity in this metabolic bone disorder. We found a significant increase in cannabinoid receptor type 2 (CB2) protein expression in patients with PDB, compared to healthy controls. Moreover, we found a significant reduction in multi-nucleated tartrate-resistant acid phosphatase (TRAP)–positive OCs and resorption areas after treatment with JWH-133. CB2 could be a molecular target for reducing the activity of OCs in PDB, opening new therapeutic scenarios for the management of this condition

The role of bone anabolic drugs in the management of periodontitis: a scoping review

The aim of this scoping review was to summarise current knowledge about the effects of bone anabolic drugs on periodontitis, in order to identify new therapeutic strategies for preventing disease progression and reducing tooth loss. A technical expert panel (TEP) was established of 11 medical specialists, including periodontists and bone specialists that followed the PRISMA-ScR model to perform the scoping review and considered for eligibility both pre-clinical and clinical studies published in the English language up to September 2020. 716 items were initially found. After duplicate removal and screening of articles for eligibility criteria, 25 articles published between 2001 and 2019 were selected. Only studies concerning teriparatide, strontium ranelate, sclerostin antibodies and DKK1 antibodies met the eligibility criteria. In particular, only for teriparatide were there both clinical studies and experimental studies available, while for other bone anabolic drugs only animal studies were found. Available evidence about the use of bone anabolic drugs in periodontology demonstrates beneficial effects of these agents on biological pathways and histological parameters involved in periodontal tissue regeneration that suggest relevant clinical implications for the management of periodontitis

Inherited dysfunctional platelet P2Y12 receptor mutations associated with bleeding disorders

The platelet adenosine 5'-diphosphate (ADP) receptor P2Y12 (P2Y12R) plays a critical role in platelet aggregation. The present report illustrates an update of dysfunctional platelet P2Y12R mutations diagnosed with congenital lifelong bleeding problems. Described patients with heterozygous or homozygous substitution in the P2Y12R gene and qualitative abnormalities of the platelet P2Y12R are summarized. Recently, a further dysfunctional variant of P2Y12R has been identified in two brothers who presented with a lifelong severe bleeding disorder. During in vitro aggregation studies, the patient's platelets show a markedly reduced and rapid reversible ADP-promoted aggregation. A homozygous c.561T > A substitution that changes the codon for His187 to Gln (p.His187Gln) in the P2Y12R gene has been identified. This mutation causes no change in receptor expression but decreases the affinity of the ligand for the receptor, even at high concentrations. Structure modelling studies indicated that the p.His187Gln mutation, located in the fifth transmembrane spanning domain (TM5), impairs conformational changes of the receptor. Structural integrity of the TM5 region is necessary for agonist and antagonist binding and for correct receptor function

Structure-Activity Analysis of Biased Agonism at the Human Adenosine A 3 Receptor s

ABSTRACT Biased agonism at G protein-coupled receptors (GPCRs) has significant implications for current drug discovery, but molecular determinants that govern ligand bias remain largely unknown. The adenosine A 3 GPCR (A 3 AR) is a potential therapeutic target for various conditions, including cancer, inflammation, and ischemia, but for which biased agonism remains largely unexplored. We now report the generation of bias "fingerprints" for prototypical ribose containing A 3 AR agonists and rigidified (N)-methanocarba 59-N-methyluronamide nucleoside derivatives with regard to their ability to mediate different signaling pathways. Relative to the reference prototypical agonist IB-MECA, (N)-methanocarba 59-Nmethyluronamide nucleoside derivatives with significant N 6 or C2 modifications, including elongated aryl-ethynyl groups, exhibited biased agonism. Significant positive correlation was observed between the C2 substituent length (in Å) and bias toward cell survival. Molecular modeling suggests that extended C2 substituents on (N)-methanocarba 59-N-methyluronamide nucleosides promote a progressive outward shift of the A 3 AR transmembrane domain 2, which may contribute to the subset of A 3 AR conformations stabilized on biased agonist binding