Decreased bone speed of sound in children with growing pains measured by quantitative ultrasound

OBJECTIVE: The most common cause of recurring childhood musculoskeletal pain is termed growing pains (GP). We hypothesized that GP may represent a local overuse syndrome and therefore may be associated with decreased bone speed of sound (SOS) measured by quantitative ultrasound (US). METHODS: We studied 39 children with GP. Bone SOS was measured by US in both mid-tibial and radius bones according to a validated protocol. Unpaired Student t test was used to compare patients and norms of healthy controls. RESULTS: Tibial SOS was significantly reduced in children with GP compared to controls (Z score -0.546 for boys and -0.891 for girls; p = 0.004, p < 0.001, respectively). Radius SOS was significantly reduced only in girls with GP (Z score -0.692, p = 0.006). No correlation was found between bone SOS and various demographic and clinical factors besides the child's ethnicity and body mass index. CONCLUSION: Bone SOS was significantly reduced in children with GP, especially in painful tibial regions. GP may represent a local overuse syndrome.     

Microcephaly,intractable seizures and developmental delay caused by biallelic variants in TBCD: further delineation of a new chaperone‐mediated tubulinopathy

Microtubule dynamics play a crucial role in neuronal development and function, and several neurodevelopmental disorders have been linked to mutations in genes encoding tubulins and functionally related proteins. Most recently, variants in the tubulin cofactor D (TBCD) gene, which encodes one of the five co‐chaperones required for assembly and disassembly of α/β‐tubulin heterodimer, were reported to underlie a recessive neurodevelopmental/neurodegenerative disorder. We report on five patients from three unrelated families, who presented with microcephaly, intellectual disability, intractable seizures, optic nerve pallor/atrophy, and cortical atrophy with delayed myelination and thinned corpus callosum on brain imaging. Exome sequencing allowed the identification of biallelic variants in TBCD segregating with the disease in the three families. TBCD protein level was significantly reduced in cultured fibroblasts from one patient, supporting defective TBCD function as the event underlying the disorder. Such reduced expression was associated with accelerated microtubule re‐polymerization. Morpholino‐mediated TBCD knockdown in zebrafish recapitulated several key pathological features of the human disease, and TBCD overexpression in the same model confirmed previous studies documenting an obligate dependency on proper TBCD levels during development. Our findings confirm the link between inactivating TBCD variants and this newly described chaperone‐associated tubulinopathy, and provide insights into the phenotype of this disorder.     

Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria

The increasing threat of pathogen resistance to antibiotics requires the development of novel antimicrobial strategies. Here we present a proof of concept for a genetic strategy that aims to sensitize bacteria to antibiotics and selectively kill antibiotic-resistant bacteria. We use temperate phages to deliver a functional clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) system into the genome of antibiotic-resistant bacteria. The delivered CRISPR-Cas system destroys both antibiotic resistance-conferring plasmids and genetically modified lytic phages. This linkage between antibiotic sensitization and protection from lytic phages is a key feature of the strategy. It allows programming of lytic phages to kill only antibiotic-resistant bacteria while protecting antibiotic-sensitized bacteria. Phages designed according to this strategy may be used on hospital surfaces and hand sanitizers to facilitate replacement of antibiotic-resistant pathogens with sensitive ones.The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins have evolved in prokaryotes to protect against phage attack and undesired plasmid replication by targeting foreign DNA or RNA (1–3). These systems target nucleic acids, based on short DNA sequences, called spacers, that exist between repeats in the CRISPR array. Transcribed spacers guide Cas proteins to homologous sequences within the foreign nucleic acid, called protospacers, which are subsequently cleaved. The CRISPR-Cas systems have revolutionized molecular biology by providing efficient tools to precisely engineer genomes and manipulate gene expression in various organisms (4–10). CRISPR-Cas systems have also recently been used to phenotypically correct genetic diseases in live animals (11), and their utility is being explored for various therapeutic approaches in mammals. Nevertheless, only limited studies have shown the use of CRISPR-Cas systems to target antibiotic resistance genes or a specific population of virulent bacterial strains (12–17).Two recent elegant studies demonstrated that phage-transferable CRISPR-Cas systems are capable of specifically killing pathogens or resensitizing them to antibiotics (16, 17). These studies, and another study (13), also showed that the transferred CRISPR-Cas system is capable of eliminating specific bacterial populations. Furthermore, they demonstrated that the system might be used against pathogens to effectively treat infected animals. Consequently, it was suggested that the system could be used as a potent antimicrobial agent. Nevertheless, although the results of these studies highlight the potential of a phage-transferable CRISPR-Cas system, the concept of using the system as a direct antimicrobial is similar to conventional phage therapy, which currently faces various obstacles (18). One major obstacle is phage administration into infected tissues; this stems from the phages’ immunogenicity and relative large size compared with antibiotics. One may argue that it would be more efficient to directly kill a pathogen by a lytic phage if it were possible to deliver the CRISPR-Cas–encoding cassette into this pathogen by a phage. Moreover, using the proposed systems in infected patients to resensitize pathogens to antibiotics while antibiotics counterselect for these sensitized pathogens would most likely fail due to escape mutants that are selected by the antibiotics.Here we demonstrate a strategy to counteract the emerging threat of antibiotic-resistant bacteria that evades the above shortcomings. Instead of directly killing the pathogens, we propose to sensitize the pathogens on surfaces or in the human skin flora while concomitantly enriching for these sensitized populations. Patients infected by these antibiotic-sensitive bacteria would thus be treatable by traditional antibiotics. In this strategy, the CRISPR-Cas system is used to destroy specific DNAs that confer antibiotic resistance and to concurrently confer a selective advantage to antibiotic-sensitive bacteria by virtue of resistance to lytic phages. The selective advantage enables to efficiently displace populations of nonsensitized bacteria by killing them with lytic phages. In contrast to conventional phage therapy, this approach does not require administration of phages into the host’s tissues. In addition, it does not aim to directly kill treated bacteria but rather to sensitize them to antibiotics and to kill the nonsensitized bacteria. Therefore, there is no counterselection against the sensitization. The strategy relies on CRISPR spacers that can be rationally designed to target any DNA sequence, including those that encode resistance genes and lytic phages. It thus allows genetically linking a trait that is beneficial to the bacteria (i.e., spacers protecting from lytic phage) with a trait that reverses drug resistance (i.e., spacers targeting resistance genes). The genetic linkage enables selecting antibiotic-sensitized bacterial population by using lytic phages. The integrated construct is designed not only to actively eradicate existing resistance genes but also to eliminate horizontal transfer of these genes between bacteria. Extended use of this technology should thus reduce drug-resistant populations of pathogens on major sources of contamination. Consequently, well-established antibiotics for which resistance currently exists could once again be effective.     

Anti-tumor activity of selective inhibitors of XPO1/CRM1-mediated nuclear export in diffuse malignant peritoneal mesothelioma: the role of survivin

Survivin, which is highly expressed and promotes cell survival in diffuse malignant peritoneal mesothelioma (DMPM), exclusively relies on exportin 1 (XPO1/CRM1) to be shuttled into the cytoplasm and perform its anti-apoptotic function. Here, we explored the efficacy of Selective Inhibitors of Nuclear Export (SINE), KPT-251, KPT-276 and the orally available, clinical stage KPT-330 (selinexor), in DMPM preclinical models. Exposure to SINE induced dose-dependent inhibition of cell growth, cell cycle arrest at G1-phase and caspase-dependent apoptosis, which were consequent to a decrease of XPO1/CRM1 protein levels and the concomitant nuclear accumulation of its cargo proteins p53 and CDKN1a. Cell exposure to SINE led to a time-dependent reduction of cytoplasmic survivin levels. In addition, after an initial accumulation, the nuclear protein abundance progressively decreased, as a consequence of an enhanced ubiquitination and proteasome-dependent degradation. SINE and the survivin inhibitor YM155 synergistically cooperated in reducing DMPM cell proliferation. Most importantly, orally administered SINE caused a significant anti-tumor effect in subcutaneous and orthotopic DMPM xenografts without appreciable toxicity. Overall, we have demonstrated a marked efficacy of SINE in DMPM preclinical models that may relay on the interference with survivin intracellular distribution and function. Our study suggests SINE-mediated XPO1/CRM1 inhibition as a novel therapeutic option for DMPM.     

Methicillin-Resistant Staphylococcus aureus Sequence Type 239-III, Ohio, USA, 2007�C2009

Methicillin-resistant Staphylococcus aureus (MRSA) is a human pathogen that has diverse molecular heterogeneity. Most MRSA strains in the United States are pulsed-field gel electrophoresis USA100 sequence type (ST) 5 and USA300 ST8. Infections with MRSA ST239-III are common and found during health care–associated outbreaks. However, this strain has been rarely reported in the United States. As part of a study supported by the Prevention Epicenter Program of the Centers for Disease Control and Prevention (Atlanta, GA, USA), which evaluated transmission of MRSA among hospitals in Ohio, molecular typing identified 78 (6%) of 1,286 patients with MRSA ST239-III infections. Ninety-five percent (74/78) of these infections were health care associated, and 65% (51/78) of patients had histories of invasive device use. The crude case-fatality rate was 22% (17/78). Identification of these strains, which belong to a virulent clonal group, emphasizes the need for molecular surveillance.Staphylococcus aureus is a major human pathogen that possesses multiple toxins and virulence mechanisms (1). Antimicrobial drug resistance in S. aureus has added to the complexity of treating serious infections caused by this bacteria, and methicillin-resistant S. aureus (MRSA) appears to have greater virulence than methicillin-susceptible strains (2,3). Most MRSA strains in the United States are pulsed-field gel electrophoresis (PFGE) types USA100 and USA300, corresponding to multilocus sequence typing (MLST) ST5 and ST8, respectively (4). MRSA belonging to MLST ST239 and harboring staphylococcal cassette chromosome mec (SCCmec) type III (MRSA ST239-III) are associated with infections in health care settings, outbreaks, increased resistance to antimicrobial drugs, and capacity for invasive disease (5–7).MRSA ST239-III has a history of successful dissemination in many regions, leading to a diverse array of regionally prevalent clones. These clones include the Brazilian; British Epidemic 1, 4, 7, 9, and 11; Canadian Epidemic 3/Punjab; Czech; Eastern Australian 2 and 3; Georgian; Hungarian; Lublin; Nanjing/Taipei (ST241); Portuguese; and Vienna clones (8,9). Although it is common worldwide, MRSA ST239-III has not played any predominant role in the United States; infections with MRSA ST239-III have been rarely reported in the United States since the 1990s (9–13). Recently, only 2 reports of this strain in the United States involving sporadic nasal colonization and bloodstream infections have been published (13,14).In this study, we describe clinical epidemiologic characteristics and molecular analysis of clinical infections with MRSA ST239-III in the midwestern United States. Identification of a strain from such a virulent clonal group in the United States with wide dissemination in other parts of the world represents a potential public health concern.     

RNA interference--small RNAs effectively fight viral hepatitis.

RNA interference (RNAi) is the process of sequence-specific gene silencing, initiated by double-stranded RNA that is homologous in sequence to the target gene. This unique phenomenon has been extensively investigated during the last few years not only in the context of its mechanism and its possible role in the regulation of gene expression and cell function, but also as a potential powerful tool for gene therapy. Targeting essential viral genes or oncogenic alleles are only some of the possible applications of RNAi in the field of gene-directed therapy. This review covers the potential use of RNAi against hepatitis B and hepatitis C viruses, the main pathogens causing chronic liver disease. The major milestones along the discovery of RNAi will also be covered.     

Transcription and DNA Methylation Patterns of Blood-Derived CD8+ T Cells Are Associated With Age and Inflammatory Bowel Disease But Do Not Predict Prognosis

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Vascular inflammation and endothelial dysfunction in fracture healing

Angiogenesis is an important step in bone fracture healing. In this article, we report on the healing of long bone fractures, and the involvement of the vascular and the inflammatory systems in the process. We conducted a prospective study of 20 healthy adults with traumatic long bone fracture. One week after fracture, and then 1 month later, we evaluated markers of inflammation: vascular responsiveness (brachial endothelial function and ankle brachial index) and inflammatory and cytokine levels osteopontin [OPN], E-selectin, and vascular endothelial growth factor [VEGF]). Long bone fractures caused intense vascular and inflammatory responses, represented by high levels of OPN, Eselectin, and VEGF. In vivo measurements demonstrated severe endothelial dysfunction, which could support the idea that the vascular system is recruited to build new blood vessels that support bone regeneration.