Transduction System Research Articles

The inhibitory effect of Gremlin-2 on adipogenesis suppresses breast cancer cell growth and metastasis

BackgroundGremlin-1 (GREM1) and Gremlin-2 (GREM2) are bone morphogenetic protein antagonists that play important roles in organogenesis, tissue differentiation, and tissue homeostasis. Although GREM1 has been reported to be involved in promoting various cancers, little has been reported about effects of GREM2 on cancer. Recently, it has been reported that GREM2 can inhibit adipogenesis in adipose-derived stromal/stem cells. However, as an inhibitor of adipogenesis, the role of GREM2 in cancer progression is not well understood yet.MethodsPre-adipocyte 3T3-L1 cells overexpressing mock or Grem2 were established using a lentiviral transduction system and differentiated into adipocytes-mock and adipocytes-Grem2, respectively. To investigate the effect of adipocyte-Grem2 on breast cancer cells, we analyzed the proliferative and invasion abilities of spheroids using a 3D co-culture system of breast cancer cells and adipocytes or conditioned medium (CM) of adipocytes. An orthotopic breast cancer mouse model was used to examine the role of adipocytes-Grem2 in breast cancer progression.ResultsGrem2 overexpression suppressed adipogenesis of 3T3-L1 cells. Proliferative and invasion abilities of spheroids formed by co-culturing MTV/TM-011 breast cancer cells and adipocytes-Grem2 were significantly reduced compared to those of spheroids formed by co-culturing MTV/TM-011 cells and adipocytes-mock. Compared to adipocytes-mock, adipocytes-Grem2 showed decreased mRNA expression of several adipokines, notably IL-6. The concentration of IL-6 in the CM of these cells was also decreased. Proliferative and invasive abilities of breast cancer cells reduced by adipocytes-Grem2 were restored by IL-6 treatment. Expression levels of vimentin, slug, and twist1 in breast cancer cells were decreased by treatment with CM of adipocytes-Grem2 but increased by IL-6 treatment. In orthotopic breast cancer mouse model, mice injected with both MTV/TM-011 cells and adipocytes-Grem2 showed smaller primary tumors and lower lung metastasis than controls. However, IL-6 administration increased both the size of primary tumor and the number of metastatic lung lesions, which were reduced by adipocytes-Grem2.ConclusionsOur study suggests that GREM2 overexpression in adipocytes can inhibit adipogenesis, reduce the expression and secretion of several adipokines, including IL-6, and ultimately inhibit breast cancer progression.

A novel highly selective allosteric inhibitor of tyrosine kinase 2 (TYK2) can block inflammation- and autoimmune-related pathways

BackgroundAs a member of the Janus kinase (JAK) family, which includes JAK1, JAK2 and JAK3, tyrosine kinase 2 (TYK2) plays an important role in signal transduction and immune system regulation. Moreover, it is also involved in the development of many types of inflammatory and autoimmune diseases, such as psoriasis and systemic lupus erythematosus (SLE). TYK2 is an attractive therapeutic target, and selective inhibition of TYK2 over other JAK family members is critical for the development of TYK2 small molecule inhibitors. However, targeting the catalytic region of the TYK2 ATP-binding site is a major challenge due to the high structural homology between the catalytic regions of the JAK family proteins.ResultsIn this study, we developed a novel small molecule inhibitor (QL-1200186) by targeting the pseudokinase regulatory domain (Janus homology 2, JH2) of the TYK2 protein. The binding sites of QL-1200186 were predicted and screened by molecular docking. The inhibitory effects on IFNα, IL-12 and IL-23 signaling were tested in cell lines, human peripheral blood cells and human whole blood. The pharmacokinetic (PK) and pharmacodynamic properties of QL-1200186 were verified in mice. QL-1200186 showed high affinity for TYK2 JH2 and had no apparent selectivity for the TYK2 and JAK homologous kinase domains; these effects were demonstrated using biochemical binding, signaling pathway transduction (JAK1/2/3) and off-target effect assays. More importantly, we revealed that QL-1200186 was functionally comparable and selectivity superior to two clinical-stage TYK2 inhibitors (BMS-986165 and NDI-034858) in vitro. In the PK studies, QL-1200186 exhibited excellent exposure, high bioavailability and low clearance rates in mice. Oral administration of QL-1200186 dose-dependently inhibited interferon-γ (IFNγ) production after interleukin-12 (IL-12) challenge and significantly ameliorated skin lesions in psoriatic mice.ConclusionThese findings suggest that QL-1200186 is a highly selective and potent inhibitor of TYK2. QL-1200186 could be an appealing clinical drug candidate for the treatment of psoriasis and other autoimmune diseases.-PuiM22MyQoZC1Ksigy-wDVideo

Topoisomerase Inhibitors Increase Episomal DNA Expression by Inducing the Integration of Episomal DNA in Hepatic Cells.

Gene therapy is a promising strategy to treat and cure most inherited metabolic liver disorders. Viral vectors such as those based on adeno-associated viruses (AAVs) and lentiviruses (LVs) are used as vehicles to deliver functional genes to affected hepatocytes. Adverse events associated with the use of high vector doses have motivated the use of small molecules as adjuvants to reduce the dose. In this study, we showed that a one-hour treatment with topoisomerase inhibitors (camptothecin and etoposide) prior to viral transduction is enough to increase AAV and LV reporter expression in non-dividing hepatic cells in culture. Topoisomerase inhibitors increased both integration-competent (ICLV) and integration-deficient (IDLV) LV-derived expression, with a much stronger increase in the IDLV transduction system. In agreement with that, topoisomerase inhibitors increased viral genome integration in both strains, with a greater impact on the IDLV strain, supporting the idea that topoisomerase inhibitors increased episomal DNA integration, especially when viral integrase activity is abolished. These effects correlated with an increase in the DNA damage response produced by the treatments. Our study highlights the need to monitor DNA damage and undesired integration of viral episomal DNAs into the host genome when studying chemical compounds that increase viral transduction.

Chromosomal gene of hybrid multisensor histidine kinase is involved in motility regulation in the rhizobacterium Azospirillum baldaniorum Sp245 under mechanical and water stress.

Azospirillum alphaproteobacteria, which live in the rhizosphere of many crops, are used widely as biofertilizers. Two-component signal transduction systems (TCSs) mediate the bacterial perception of signals and the corresponding adjustment of behavior facilitating the adaptation of bacteria to their habitats. In this study, we obtained the A. baldaniorum Sp245 mutant for the AZOBR_150176 gene, which encodes the TCS of the hybrid histidine kinase/response sensory regulator (HSHK-RR). Inactivation of this gene affected bacterial morphology and motility. In mutant Sp245-HSHKΔRR-Km, the cells were still able to synthesize a functioning polar flagellum (Fla), were shorter than those of strain Sp245, and were impaired in aerotaxis, elaboration of inducible lateral flagella (Laf), and motility in semiliquid media. The mutant showed decreased transcription of the genes encoding the proteins of the secretion apparatus, which ensures the assembly of Laf, Laf flagellin, and the repressor protein of translation of the Laf flagellin's mRNA. The study examined the effects of polyethylene glycol 6000 (PEG 6000), an agent used to simulate osmotic stress and drought conditions. Under osmotic stress, the mutant was no longer able to use collective motility in semiliquid media but formed more biofilm biomass than did strain Sp245. Introduction into mutant cells of the AZOBR_150176 gene as part of an expression vector led to recovery of the lost traits, including those mediating bacterial motility under mechanical stress induced by increased medium density. The results suggest that the HSHK-RR under study modulates the response of A. baldaniorum Sp245 to mechanical and osmotic/water stress.

MiST 4.0: a new release of the microbial signal transduction database, now with a metagenomiccomponent.

Signal transduction systems in bacteria and archaea link environmental stimuli to specific adaptive cellular responses. They control gene expression, motility, biofilm formation, development and other processes that are vital to survival. The microbial signal transduction (MiST) database is an online resource that stores tens of thousands of genomes and allows users to explore their signal transduction profiles, analyze genomes in bulk using the database application programming interface (API) and make testable hypotheses about the functions of newly identified signaling systems. However, signal transduction in metagenomes remained completely unexplored. To lay the foundation for research in metagenomic signal transduction, we have prepared a new release of the MiST database, MiST 4.0, which features over 10000 metagenome-assembled genomes (MAGs), a scaled representation of proteins and detailed BioSample information. In addition, several thousands of new genomes have been processed and stored in the database. A new interface has been developed that allows users to seamlessly switch between genomes and MAGs. MiST 4.0 is freely available at https://mistdb.com; metagenomes and MAGs can also be explored using the API available on the same page.

Phosphorus starvation response and PhoB-independent utilization of organic phosphate sources by Salmonella enterica.

Phosphorus (P) is the fifth most abundant element in living cells. This element is acquired mainly as inorganic phosphate (Pi, PO4 3-). In enteric bacteria, P starvation activates a two-component signal transduction system which is composed of the membrane sensor protein PhoR and its cognate transcription regulator PhoB. PhoB, in turn, promotes the transcription of genes that help maintain Pi homeostasis. Here, we characterize the P starvation response of the bacterium Salmonella enterica. We determine the PhoB-dependent and independent transcriptional changes promoted by P starvation and identify proteins enabling the utilization of a range of organic substrates as sole P sources. We show that transcription and activity of a subset of these proteins are independent of PhoB and Pi availability. These results establish that Salmonella enterica can maintain Pi homeostasis and repress PhoB/PhoR activation even when cells are grown in medium lacking Pi.

The Mechanism of Bushen Zhuyun Prescription in Regulating Luteal Phase Deficiency via Intervention of the Kiss1/GPR54 System in the Hypothalamus

Luteal phase deficiency (LPD) is a significant contributor to infertility and miscarriage. Bushen Zhuyun Prescription (BSZY) is a traditional Chinese medicine formula that has potential to treat LPD. To investigate the effect of BSZY in treating LPD, SD rats with complete estrous cycles were divided into blank, model, dydrogesterone (DYD), high-dose BSZY (BSZY-HD), and low-dose BSZY (BSZY-LD) groups. All the groups were received mifepristone gavage except for the blank group. The pathological changes were observed in the hypothalamus, and the analysis of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), progesterone (P), gonadotropin-releasing hormone (GnRH), and cyclic adenosine monophosphate (cAMP) levels in serum. Additionally, the mRNA and their protein expression levels of estrogen receptor α (ERα), ERβ, G protein-coupled receptor 30 (GPR30), kisspeptin 1 (Kiss1), and GPR54 were assessed; and the key molecules expression in cAMP/protein kinase A (PKA) pathway were analyzed. The results revealed that mifepristone treatment caused a decrease in FSH and cAMP levels and an increase in E2 and LH levels, which were normalised in the BSZY treatment groups. The expression of ERα and ERβ mRNA and protein in the hypothalamus was higher in the BSZY groups compared to the blank group. BSZY also resulted in increased expression of Kiss-1, GPR54, and PKA, as well as decreased expression of cAMP-response element binding protein (CREB), c-Fos, phospho-CREB (p-CREB), and p-c-Fos, compared to model group. No significant difference observed in GPR30 expression between the mifepristone and BSZY groups. The mRNA and protein of Kiss1 and GPR54 were decreased in model group and increased after BSZY treatment, while the mRNA and protein expression of PKA, CREB, c-Fos, p-CREB, and p-c-Fos were also decreased significantly. In conclusion, the findings demonstrate that BSZY could effectively regulate hypothalamic ER, interfere with the Kiss1/GPR54 signal transduction system, activate the cAMP/PKA pathway, and regulate key transduction molecules expression of PKA, CREB, c-Fos, there by effecting the level of reproductive hormones and has the potential to treat LPD-related infertility.

Intra-Rater and Test–Retest Reliability of Barbell Force, Velocity, and Power during the Landmine Punch Throw Test Assessed by the GymAware Linear Transducer System

Background: Velocity-based training (VBT) requires measurement of the velocity at which the barbell is moved in the concentric phase with regard to different resistance exercises, which provides accurate, indirect estimations of 1 RM. However, for assessing punch performance, no study has been carried out to date. The purpose of this study was to analyse the reliability of the GymAware linear transducer for the measurement of barbell velocity during the landmine push throw (LPT) test using four loads. Methods: Twenty-five healthy, physically active male students, aged 24.13 ± 2.82 years, volunteered to take part in this study. The reliability of the LPT test was measured at two separate visits, with a 2-day interval between them. One series of the test protocol included four parts of the LPT test with progressively increasing loads (20, 25, 30, and 35 kg) and 5 min intervals for rests between loads. Results: For all four loads, excellent intra-rater and test–retest reliability was noted for the mean force variable (ICC = 0.97–0.99). Additionally, very strong and significant correlations were established between measurements (r = 0.96–0.99). Poor reliability was observed for barbell height and total work (ICC below 0.5). A trend of decreasing reliability was detected with increasing barbell load. Furthermore, measurements without the barbell throw were more reliable than those with it. Conclusions: These results support the use of the GymAware linear transducer to track barbell velocity during the LPT test. This device may have valuable practical applications for strength and conditioning coaches. Therefore, we suggest that the LPT assessed with the GymAware linear transducer may be a useful method for evaluating upper limb strength and power during boxing punches.

Intrinsic disorder and conformational coexistence in auxin coreceptors

AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) transcriptional repressor proteins and the TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB) proteins to which they bind act as auxin coreceptors. While the structure of TIR1 has been solved, structural characterization of the regions of the Aux/IAA protein responsible for auxin perception has been complicated by their predicted disorder. Here, we use NMR, CD and molecular dynamics simulation to investigate the N-terminal domains of the Aux/IAA protein IAA17/AXR3. We show that despite the conformational flexibility of the region, a critical W-P bond in the core of the Aux/IAA degron motif occurs at a strikingly high (1:1) ratio of cis to trans isomers, consistent with the requirement of the cis conformer for the formation of the fully-docked receptor complex. We show that the N-terminal half of AXR3 is a mixture of multiple transiently structured conformations with a propensity for two predominant and distinct conformational subpopulations within the overall ensemble. These two states were modeled together with the C-terminal PB1 domain to provide the first complete simulation of an Aux/IAA. Using MD to recreate the assembly of each complex in the presence of auxin, both structural arrangements were shown to engage with the TIR1 receptor, and contact maps from the simulations match closely observations of NMR signal-decreases. Together, our results and approach provide a platform for exploring the functional significance of variation in the Aux/IAA coreceptor family and for understanding the role of intrinsic disorder in auxin signal transduction and other signaling systems.

3-Iodothyronamine, a trace amine-associated receptor agonist, regulates intracellular Ca2+ increases via CaMK II through Epac2 in rat cerebral arterioles.

Trace amines (TAs) in the nervous system bind to TA-associated receptors (TAARs) and are involved in the regulation of monoaminergic functions. Among TAAR subtypes, TAAR1 has been implicated in the development of neurological disorders, such as schizophrenia. The present study investigated the effects of the TAAR1 agonist, 3-iodothyronamine (T1AM) on cerebral arterioles using fluctuations in the intracellular concentration of Ca2+ ([Ca2+]i) as an index of contractile responses. In cerebral arterioles, most of the TAAR agonists did not increase [Ca2+]i, while only T1AM elevated [Ca2+]i in vascular smooth muscle cells. This increase involved extracellular Ca2+ influx through T-type Ca2+ channels and inositol trisphosphate- and ryanodine-receptor-mediated Ca2+ release from intracellular stores. The inhibition of the cAMP sensor, exchange protein directly activated by cAMP (Epac) 2, and calmodulin kinase (CaMK) II strongly inhibited Ca2+ elevations. The present study revealed that T1AM acted not only on the TAAR1 receptor as previously suggested, but also on other G-protein coupled receptors and/or signal transduction systems to increase intracellular Ca2+ in cerebral arteriole smooth muscle cells. These results suggest that when using T1AM in clinical practice, attention should be paid to the early rise in blood pressure.

A computational study for understanding the impact of p120-catenin on the cis-dimerization of cadherin.

A prototype of cross-membrane signal transduction is that extracellular binding of cell surface receptors to their ligands induces intracellular signalling cascades. However, much less is known about the process in the opposite direction, called inside-out signalling. Recent studies show that it plays a more important role in regulating the functions of many cell surface receptors than we used to think. In particular, in cadherin-mediated cell adhesion, recent experiments indicate that intracellular binding of the scaffold protein p120-catenin (p120ctn) can promote extracellular clustering of cadherin and alter its adhesive function. The underlying mechanism, however, is not well understood. To explore possible mechanisms, we designed a new multiscale simulation procedure. Using all-atom molecular dynamics simulations, we found that the conformational dynamics of the cadherin extracellular region can be altered by the intracellular binding of p120ctn. More intriguingly, by integrating all-atom simulation results into coarse-grained random sampling, we showed that the altered conformational dynamics of cadherin caused by the binding of p120ctn can increase the probability of lateral interactions between cadherins on the cell surface. These results suggest that p120ctn could allosterically regulate the cis-dimerization of cadherin through two mechanisms. First, p120ctn controls the extracellular conformational dynamics of cadherin. Second, p120ctn oligomerization can further promote cadherin clustering. Therefore, our study provides a mechanistic foundation for the inside-out signalling in cadherin-mediated cell adhesion, while the computational framework can be generally applied to other cross-membrane signal transduction systems.

In vivo direct cell-penetrating peptide mediated protein transduction system in Acyrthosiphon pisum

ObjectiveThe principal delivery method for CRISPR-based genome editing in insects is now based on microinjection into single cells or embryos. The direct protein transduction systems cannot be employed in aphids because oogenesis occurs without apparent vitellogenesis. Given the limited timing of injection into the embryonic stage in oviparous aphids, a protein delivery system from the hemolymph to the germline and embryos would be a useful tool for genome editing. This study reports a newly developed direct protein delivery system for aphids using cell-penetrating peptides (CPPs). CPPs are short peptides that translocate across the plasma membrane when bound to cargo proteins.ResultsPenetratin (PEN), a widely conserved CPP among insects, was identified in this study. We used mVenus, a recombinant fluorescent protein, as a visual marker for CPP availability assessments, and fused it with PEN by bacterial protein expression. The mVenus-PEN recombinant proteins were introduced into the hemolymph of adult unwinged Acyrthosiphon pisum females using a nanoinjector. Fluorescence emitted by mVenus-PEN was observed in various tissues, such as the gut, trachea, bacteriocytes, and their progeny. This study shows that PEN can deliver exogenously expressed proteins into tissues in vivo, indicating that CPPs are powerful tools for protein transduction.

Targeted Delivery of Chimeric Antigen Receptor into T Cells via CRISPR-Mediated Homology-Directed Repair with a Dual-AAV6 Transduction System.

CAR-T cell therapy involves genetically engineering T cells to recognize and attack tumour cells by adding a chimeric antigen receptor (CAR) to their surface. In this study, we have used dual transduction with AAV serotype 6 (AAV6) to integrate an anti-CD19 CAR into human T cells at a known genomic location. The first viral vector expresses the Cas9 endonuclease and a guide RNA (gRNA) targeting the T cell receptor alpha constant locus, while the second vector carries the DNA template for homology-mediated CAR insertion. We evaluated three gRNA candidates and determined their efficiency in generating indels. The AAV6 successfully delivered the CRISPR/Cas9 machinery in vitro, and molecular analysis of the dual transduction showed the integration of the CAR transgene into the desired location. In contrast to the random integration methods typically used to generate CAR-T cells, targeted integration into a known genomic locus can potentially lower the risk of insertional mutagenesis and provide more stable levels of CAR expression. Critically, this method also results in the knockout of the endogenous T cell receptor, allowing target cells to be derived from allogeneic donors. This raises the exciting possibility of "off-the-shelf" universal immunotherapies that would greatly simplify the production and administration of CAR-T cells.

The implication of viability and pathogenicity by truncated lipopolysaccharide in Yersinia enterocolitica.

The fast envelope stress responses play a key role in the transmission and pathogenesis of Yersinia enterocolitica, one of the most common foodborne pathogens. Our previous study showed that deletion of the waaF gene, essential for the biosynthesis of lipopolysaccharide (LPS) core polysaccharides, led to the formation of a truncated LPS structure and induced cell envelope stress. This envelope stress may disturb the intracellular signal transduction, thereby affecting the physiological functions of Y. enterocolitica. In this study, truncated LPS caused by waaF deletion was used as a model of envelope stress in Y. enterocolitica. We investigated the mechanisms of envelope stress responses and the cellular functions affected by truncated LPS. Transcriptome analysis and phenotypic validation showed that LPS truncation reduced flagellar assembly, bacterial chemotaxis, and inositol phosphate metabolism, presenting lower pathogenicity and viability both in vivo and in vitro environments. Further 4D label-free phosphorylation analysis confirmed that truncated LPS perturbed multiple intracellular signal transduction pathways. Specifically, a comprehensive discussion was conducted on the mechanisms by which chemotactic signal transduction and Rcs system contribute to the inhibition of chemotaxis. Finally, the pathogenicity of Y. enterocolitica with truncated LPS was evaluated in vitro using IPEC-J2 cells as models, and it was found that truncated LPS exhibited reduced adhesion, invasion, and toxicity of Y. enterocolitica to IPEC-J2 cells. Our research provides an understanding of LPS in the regulation of Y. enterocolitica viability and pathogenicity and, thus, opening new avenues to develop novel food safety strategies or drugs to prevent and control Y. enterocolitica infections. KEY POINTS: • Truncated LPS reduces flagellar assembly, chemotaxis, and inositol phosphate metabolism in Y. enterocolitica. • Truncated LPS reduces adhesion, invasion, and toxicity of Y. enterocolitica to IPEC-J2 cells. • Truncated LPS regulates intracellular signal transduction of Y. enterocolitica.

Bio-mimetic selectivity in Hg2+ sensing developed via electro-copolymerized PEDOT and benzothiazole-Au nanoparticles composite.

To eliminate the potential health risksof mercury, development of stable and selective mercury sensor with high sensitivity is the need of the hour. To address this, a novel PEDOT-AA-BTZ-Au-based Hg2+ selective, hybrid electrochemical sensor has been designed by following a simple protocol for electrode fabrication. The electrode was designed by carefully optimizing the onset oxidation potential of supramolecule 2-(anthracen-9-yl)benzo[d]thiazole (AA-BTZ) and conducting polymer poly-(3,4-ethylenedioxythiophene) (PEDOT), using copolymerization approach followed by dropcasting of gold nanoparticles (AuNPs). The designed electrode offered synergistic effects thus augmenting the electrical conductivity and adsorption capacity as depicted by its porous surface morphology. The highly sensitive analytical signal was generated by sulphur pockets present in AA-BTZ and PEDOT conducting framework. This is further complemented by the selectivity offered by the soft interactions between AuNPs and Hg2+ resulting in a low detection limit of 0.60 nM. The prepared system was further utilized for sensing Hg2+ ion in real systems including lake water and cosmetic samples. Lowinterference from other ions and better reproducibility further established the suitability of the designed transducer system for future on-site sensing.

Folding and Unfolding of a Fully Synthetic Transmembrane Receptor for ON/OFF Signal Transduction.

Signal transduction processes in living organisms are mainly transmitted through conformational changes in transmembrane protein receptors. So far, the development of signal transduction models induced by artificial simulation of conformational changes remains limited. We herein report a new artificial receptor that achieves controllable "ON/OFF" signal transduction through conformational changes between the folding and unfolding of a transmembrane foldamer moiety. The receptor contains three functional modules: a lipid-anchored cholic acid headgroup, a foldamer transmembrane moiety, and a precatalyst tailgroup. After inserting in the lipid membrane, the addition of Zn2+ induces unfolding of the foldamer, which changes the molecular conformation and activates the tailgroup to enter the cavity to perform its catalytic task, resulting in signal transduction in an "ON" state. By further adding a competitive ligand to bind Zn2+, the transduction can be turned "OFF". External signals can be used to reversibly switch intravesicular catalysis on and off, which provides a new model for constructing artificial signal transduction systems.

Tuberous sclerosis complex 1 targeted depletion in macrophages promotes osteogenesis by modulating secretion of Oncostatin M in the inflammatory stage of bone healing

In bone healing, earlier bone formation benefits bone repair. The first process of repair following bone injury involves the interaction between macrophage polarization and osteogenic activation of osteoblast linage cells, but the radical difference between the contributions of classically-activated M1 macrophages and alternatively-activated M2 macrophages to osteogenesis remains obscure. To test our hypothesis that M1 macrophages promote bone healing, we generated transgenic mice with myeloid lineage-specific TSC1 deletion (TSC1KO) to investigate the functional roles of M1 macrophages in the process of bone defect healing. We demonstrated that constitutive activation of mammalian target of rapamycin complex 1 (mTORC1) enhances M1 macrophage polarization during bone healing. By creating tibial bone defect as a model of bone repair in TSC1KO mice and their littermates, we surprisingly found osteogenic responses in the defective bone region of TSC1KO mice, where repair occurred by intramembranous ossification (IO) in the mice was promoted due to the enhanced M1-polarized macrophage polarization. We propose that Oncostatin M (OSM) secreted by M1-polarized macrophages but not M2 macrophages likely functions as a paracrine factor in this promoted repair process, as verified by the induction of osteoblastic differentiation and matrix mineralization. Interestingly, the expression level of the OSM receptor (OSMR) was continually upregulated in osteoblast linage cells with M1 medium. Additionally, OSMR activated the signaling transduction system of JAK/STAT/RUNX2 in MSCs, which in turn stimulates the recruitment of osteoblast lineage cells and activates IO. These results indicate that TSC1 targeted depletion in macrophages promotes bone healing by inducing secretion of OSM. This study highlights that regulation of M1 macrophage polarization is a novel basis for the improvement of bone regeneration and that regulation of macrophage polarization can be a potential therapeutic strategy to treat defects in the repair phase of bone healing.

Delamination imaging in composites using cross-correlation method by non-contact air-coupled Lamb waves

This paper presents a cross-correlation damage detection technique using damage scattered waves of all directions cross-correlate with the incident waves for delamination imaging in carbon fiber reinforced polymer (CFRP) composite plate. The presented imaging can not only detect the delamination but also precisely quantify the damage with locations and sizes. First, a CFRP composite sample with interlaminar delamination by inserting the Teflon layer is designed and manufactured in house using the hot-press machine. And a three-dimensional model of composite sample is simulated using the finite element method. Next, the cross-correlation imaging algorithm is introduced in detail, and the cross-correlation imaging proof-of-concept study is conducted with the simulated Lamb waves in the composite sample. Finally, a fully non-contact air-coupled transducer and scanning laser Doppler vibrometer system with a single-mode Lamb wave method is established to actuate and sense the interrogating Lamb waves in the structure. The imaging method is experimentally implemented for the one delamination and two delaminations imaging and quantification.

Staphylococcus aureus susceptibility to complestatin and corbomycin depends on the VraSR two-component system.

The overuse of antibiotics in humans and livestock has driven the emergence and spread of antimicrobial resistance and has therefore prompted research on the discovery of novel antibiotics. Complestatin (Cm) and corbomycin (Cb) are glycopeptide antibiotics with an unprecedented mechanism of action that is active even against methicillin-resistant and daptomycin-resistant Staphylococcus aureus. They bind to peptidoglycan and block the activity of peptidoglycan hydrolases required for remodeling the cell wall during growth. Bacterial signaling through two-component transduction systems (TCSs) has been associated with the development of S. aureus antimicrobial resistance. However, the role of TCSs in S. aureus susceptibility to Cm and Cb has not been previously addressed. In this study, we determined that, among all 16 S. aureus TCSs, VraSR is the only one controlling the susceptibility to Cm and Cb. Deletion of vraSR increased bacterial susceptibility to both antibiotics. Epistasis analysis with members of the vraSR regulon revealed that deletion of spdC, which encodes a membrane protein that scaffolds SagB for cleavage of peptidoglycan strands to achieve physiological length, in the vraSR mutant restored Cm and Cb susceptibility to wild-type levels. Moreover, deletion of either spdC or sagB in the wild-type strain increased resistance to both antibiotics. Further analyses revealed a significant rise in the relative amount of peptidoglycan and its total degree of cross-linkage in ΔspdC and ΔsagB mutants compared to the wild-type strain, suggesting that these changes in the cell wall provide resistance to the damaging effect of Cm and Cb. IMPORTANCE Although Staphylococcus aureus is a common colonizer of the skin and digestive tract of humans and many animals, it is also a versatile pathogen responsible for causing a wide variety and number of infections. Treatment of these infections requires the bacteria to be constantly exposed to antibiotic treatment, which facilitates the selection of antibiotic-resistant strains. The development of new antibiotics is, therefore, urgently needed. In this paper, we investigated the role of the sensory system of S. aureus in susceptibility to two new antibiotics: corbomycin and complestatin. The results shed light on the cell-wall synthesis processes that are affected by the presence of the antibiotic and the sensory system responsible for coordinating their activity.

Ag-CuO integrated polyaniline based impedimetric detection of cancer biomarker MUC1

MUC 1 is majorly over expressed in multiple malignancies especially breast cancer. Therefore, the reliable, cost effective and convenient detection of MUC1 is of great essence in the health care sector. In this regard, biosensors are the promising tool since it not only effectively mitigate the inherent setbacks of conventional techniques but also provides additional advantage of point-of-care diagnosis. With this aim, herein an impedimetric immunosensor based on silver doped copper oxide conjugated polyaniline (Ag-CuO@PANI) nanocomposite for sensitive detection of MUC1 has been developed. The facile synthesis of Ag-CuO@PANI was carried out by using hydrothermal method. Further, the Ag-CuO@PANI was deposited onto the FTO platform followed by the incubation of bi-functional linker cysteamine which permitted covalent and directional binding of the bio-recognition element i.e. antibody specific to MUC1 onto the Cys/Ag-CuO@PANI/FTO. After thorough characterization and optimization studies, the synthesized Ab/Cys/Ag-CuO@PANI/FTO based sensing platform was employed for the detection of MUC1 using electrochemical impedance spectroscopy (EIS) based transduction system in [Fe(CN)6]3-/4- redox couple. The immunosensor generated a satisfactory analytical outcome with linear dynamic range of 1–100 pg mL−1 and 1–100 ng mL−1 along with a low detection limit of 3.23 pg mL−1 and ultra-sensitivity of 0.68 kΩ pg mL−1 cm−2. Also, the adequate reproducibility (RSD ≤ 0.84%), high selectivity, long shelf life of 25 days at 4 °C storage condition and satisfactory performance in spike-in analysis (Recovery % within 96.4–102.7% with ≤ 2.48% RSD value) further emphasize the analytical metrics of the immunosensor.