Expression of Cre recombinase in chondrocytes causes abnormal craniofacial and skeletal development

The cranial base synchondroses are growth centers that drive cranial and upper facial growth. The intersphenoid synchondrosis (ISS) and the spheno-occipital synchondrosis (SOS) are two major synchondroses located in the middle of the cranial base and are maintained at early developmental stages to sustain cranial base elongation. In this study, we report unexpected premature ossification of ISS and SOS when Cre recombinase is activated in a chondrocyte-specific manner. We used a Cre transgenic line expressing Aggrecan enhancer-driven, Tetracycline-inducible Cre (ATC), of which expression is controlled by a Col2a1 promoter. Neonatal doxycycline injection or doxycycline diet fed to breeders was used to activate Cre recombinase. The premature ossification of ISS and/or SOS led to a reduction in cranial base length and subsequently a dome-shaped skull. Furthermore, the mice carrying either heterozygous or homozygous conditional deletion of Tsc1 or Fip200 using ATC mice developed similar craniofacial abnormalities, indicating that Cre activity itself but not conditional deletion of Tsc1 or Fip200 gene, is the major contributor of this phenotype. In contrast, the Col2a1-Cre mice carrying Cre expression in both perichondrium and chondrocytes and the mice carrying the conditional deletion of Tsc1 or Fip200 using Col2a1-Cre did not manifest the same skull abnormalities. In addition to the defective craniofacial bone development, our data also showed that the Cre activation in chondrocytes significantly compromised bone acquisition in femur. Our data calls for the consideration of the potential in vivo adverse effects caused by Cre expression in chondrocytes and reinforcement of the importance of including Cre-containing controls to facilitate accurate phenotype interpretation in transgenic research.

     

Synthesis, characterization, and biological properties of small branched RNA fragments containing chiral (Rp and Sp) 2',5'-phosphorothioate linkages

Synthetic branched RNA fragments were prepared to examine the stereochemical requirements for hydrolysis of RNA lariats by the yeast debranching enzyme (yDBR). Specifically, two branched trinucleoside diphosphates and a tetranucleoside triphosphate containing a 2',5'-linked phosphorothioate linkage of defined stereochemistry, namely Rp-A(2'ps5'G)pC, Sp-A(2'ps5'G)pC and Sp-ApA(2'ps5'G)pC, were prepared via solution-phase methods. Unlike the all-phosphodiester control, A(2'p5'G)pC, the Rp-thioated trimer was not cleaved by yDBR, demonstrating that changing the pro-Rp oxygen at the 2',5' phosphodiester bond averts hydrolysis by the enzyme. In contrast, the Sp branched compounds (trimer and tetramer) were cleaved yDBR, albeit with reduced efficiency relative to the corresponding all-phosphodiester branched compounds. Furthermore, the small branched RNAs (5 nt) were not cleaved as efficiently as a 18-nt bRNA, suggesting that the enzyme appears to have a stronger preference for larger bRNA substrates. The non-hydrolyzable branched RNA fragments prepared during these studies may be promising candidates for the future co-crystallization and X-ray analyses of DBR:bRNA complexes.     

Antimicrobial screening involving Helicobacter pylori of nano-therapeutic compounds based on the amoxicillin antibiotic drug

Nano-structure Cu(II) complex [Cu(AMAB)₂]Cl₂ with Schiff base (AMAB) derived from the condensation between 4-(dimethylamino)benzaldehyde and amoxicillin trihydrate was prepared. (AMAB) Schiff base and its Cu(II) complex were identified and confirmed by different physicochemical techniques. The Schiff base (AMAB) was coordinated to copper ion through carbonyl oxygen and imine nitrogen donor sites. X-ray powder diffraction shows a cubic crystal system of the Cu(II) complex. The density functional theory was used to optimize the structure geometries of the investigated compounds. The molecular docking of the active amino acids of the investigated proteins' interactions with the tested compounds was evaluated. The bactericidal or bacteriostatic effect of the compounds was screened against some bacterial strains. The activity of Cu-chelate against Gram-negative bacteria was mainly more effective than its (AMAB) ligand and vice versa in the case of Gram-positive bacteria. The biological activity of the prepared compounds with biomolecules calf thymus DNA (CT-DNA) was determined by electronic absorption spectra and DNA gel electrophoresis technique. All studies revealed that the Cu-chelate derivative exhibited better binding affinity to both CT-DNA than the AMAB and amoxicillin itself. The anti-inflammatory effect of the designed compounds was determined by testing their protein denaturation inhibitory activity spectrophotometrically. All obtained data supported that the designed nano-Cu(II) complex with Schiff base (AMAB) is a potent bactericide against H. pylori, and exhibits anti-inflammatory activity. The dual inhibition effects of the designed compound represent a modern therapeutic approach with extended spectrum of action. Therefore, it can act as good drug target in antimicrobial and anti-inflammtory therapies. Finally, H. pylori resistance to amoxicillin is absent or rare in many countries, thus amoxicillin nanoparticles may be beneficial for countries where amoxicillin resistance is reported.     

Acriflavine targets oncogenic STAT5 signaling in myeloid leukemia cells

Acriflavine (ACF) is an antiseptic with anticancer properties, blocking the growth of solid and haematopoietic tumour cells. Moreover, this compound has been also shown to overcome the resistance of cancer cells to chemotherapeutic agents. ACF has been shown to target hypoxia‐inducible factors (HIFs) activity, which are key effectors of hypoxia‐mediated chemoresistance. In this study, we showed that ACF inhibits the growth and survival of chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) cell lines in normoxic conditions. We further demonstrated that ACF down‐regulates STAT5 expression in CML and AML cells but activates STAT3 in CML cells in a HIF‐independent manner. In addition, we demonstrated that ACF suppresses the resistance of CML cells to tyrosine kinase inhibitors, such as imatinib. Our data suggest that the dual effect of ACF might be exploited to eradicate de novo or acquired resistance of myeloid leukaemia cells to chemotherapy.     

Protein characterization of a candidate mechanism SNP for Crohn's disease: the macrophage stimulating protein R689C substitution

High throughput genome wide associations studies (GWAS) are now identifying a large number of genome loci related to risk of common human disease. Each such locus presents a challenge in identifying the relevant underlying mechanism. Here we report the experimental characterization of a proposed causal single nucleotide polymorphism (SNP) in a locus related to risk of Crohn's disease and ulcerative colitis. The SNP lies in the MST1 gene encoding Macrophage Stimulating Protein (MSP), and results in an R689C amino acid substitution within the β-chain of MSP (MSPβ). MSP binding to the RON receptor tyrosine kinase activates signaling pathways involved in the inflammatory response. We have purified wild-type and mutant MSPβ proteins and compared biochemical and biophysical properties that might impact the MSP/RON signaling pathway. Surface plasmon resonance (SPR) binding studies showed that MSPβ R689C affinity to RON is approximately 10-fold lower than that of the wild-type MSPβ and differential scanning fluorimetry (DSF) showed that the thermal stability of the mutant MSPβ was slightly lower than that of wild-type MSPβ, by 1.6 K. The substitution was found not to impair the specific Arg483-Val484 peptide bond cleavage by matriptase-1, required for MSP activation, and mass spectrometry of tryptic fragments of the mutated protein showed that the free thiol introduced by the R689C mutation did not form an aberrant disulfide bond. Together, the studies indicate that the missense SNP impairs MSP function by reducing its affinity to RON and perhaps through a secondary effect on in vivo concentration arising from reduced thermodynamic stability, resulting in down-regulation of the MSP/RON signaling pathway.     

Evaluation of the technical and environmental performances of extraction and purification processes of arabinoxylans from wheat straw and bran

A process for hemicelluloses fractionation and purification from wheat straw and bran has been investigated and technical considerations (yields, purity) have been coupled to environmental characterizations (water consumption, carbon dioxide emissions) in order to develop an environment-friendly process. Extraction by twin-screw extrusion gave a yield in arabinoxylans equal to 8.5% (weight of (arabinose + xylose) in the extract after fractionation/dry weight of the destarched bran). The extraction of 86 kg of straw and bran (with a ratio 6.2:1) with 5.8 kg of NaOH in pellet form resulted in the production of a complex extract containing 1.0 kg of arabinoxylan polymer, which required concentration and purification steps. Evaporation (EV) followed by ethanol precipitation (P) and freeze-drying (FD), gave a yield in hemicellulosic powder of 36.5% (dry weight of powder/dry weight of extract after liquid/solid separation) with a total sugar content equal to 48.4% but also used a large amount of ethanol. The other studied purification process was based on a combination of ultrafiltration (UF), anion exchange chromatography (CHR) and spray-drying (SD). It gave a yield in hemicellulosic powders of 24.6% and a total sugar content equal to 28.7%. The technical performances of the second process appear to be less attractive but with a lower energetic and ethanol consumption. Thus secondly the environmental impacts (water consumption and CO₂ emission) of the ultrafiltration step were quantified. Life Cycle Assessment data (Ecoinvent) were used to convert materials used for the infrastructure and energy consumed during functioning into carbon dioxide emissions and water consumptions. Results have shown that environmental impacts due to the operating conditions are higher than those relative to raw material involved in the installation. The study showed that this kind of approach allows the determination of optimum conditions for the ultrafiltration step.     

Multivalent cations interactions with fluoroquinolones or tetracyclines: A cross-sectional study

IntroductionOral fluoroquinolones and tetracyclines are known to interact with divalent or trivalent cation-containing compounds (DTCCs) via chelation. The objective of this study is to describe the prevalence of these drug-drug interactions (DDIs) in an inpatient setting.MethodsA cross-sectional study of prospectively collected data were conducted at an academic tertiary care hospital. We included hospitalized adults who were receiving oral fluoroquinolones or tetracyclines with DTCCs in 2019. Our hospital uses electronic health records for medication ordering and handwritten medication administration records (MARs). The primary study outcome was the percentage of simultaneous administration of fluoroquinolones or tetracyclines with DTCCs, and the secondary outcome was the percentage of inappropriate separation time.ResultsAmong patients who received oral fluoroquinolones or tetracyclines, 47 patients (26.6%) were co-administered DTCCs and included in this study. Ciprofloxacin (n = 29; 61.7%) was the most commonly interacting antibiotic, followed by moxifloxacin (n = 12; 25.5%) and doxycycline (n = 6; 12.8%). The interacting DTCCs included iron-containing products and calcium-containing products, and half of the patients (n = 24; 51%) received DTCCs once daily. Most patients (n = 35; 74.5%) were found to receive oral fluoroquinolones or tetracyclines at the same time as DTCCs, while one (2.1%) received inappropriately separated DTCCs.ConclusionsDespite being a very known contraindicated DDI, the prevalence of simultaneous co-administration of oral fluoroquinolones or tetracyclines with polyvalent cations was extremely high in a hospital with handwritten MARs. Antimicrobial stewardship programs should target this DDI, and future studies should evaluate the impact of different practical solutions to this problem in different clinical settings.     

Antiproliferative and apoptotic potential of Glycyrrhizin against HPV16+ Caski cervical cancer cells: A plausible association with downreguation of HPV E6 and E7 oncogenes and Notch signaling pathway

Cervical cancer (CCa) is the second most frequent carcinoma in females and human papilloma virus (HPV) oncoproteins are regarded as one of the critical etiological agent. Despite recent advances in screening and management of CCa, still it remains the deadliest carcinoma as advanced and metastatic stages are mostly incurable. This urges for the development of newer therapeutic interventions. The current was aimed to investigate the antiproliferative and apoptotic potential of glycyrrhizin (Gly) against HPV16⁺ CaSki CCa cells. Our findings substantiated that Gly exerted antiproliferative effects on the CaSki cells by obstructing their proliferation rate. Gly substantially enhanced apoptosis in Caski cells in a dose-dependent manner via augmenting the generation of ROS, DNA fragmentation and disruption of the mitochondrial membrane potential. Gly mediated apoptosis in CaSki cells was found to be due to activation of caspase-8 and capsase-9 along with the modulation of pro-and anti-apoptotic gene expression. Moreover, Gly halts the progression of CaSki cells at G₀/G₁ phase which was found to be due to reduced expression of cyclin D1 and cyclin-dependent kinase 4 (CDK4) along with the enhanced expression of CDK inhibitor p21^Cip1. Further, Gly downregulates the expression of HPV oncoproteins (E6 & E7) along with the inhibition of Notch signaling pathway. Taken together, Gly represents as a potential therapeutic modality for CCa which could rapidly be translated for clinical studies.