The Wnt-dependent master regulator NKX1-2 controls mouse pre-implantation development.

Embryo size, specification, and homeostasis are regulated by a complex gene regulatory and signaling network. Here we used gene expression signatures of Wnt-activated mouse embryonic stem cell (mESC) clones to reverse engineer an mESC regulatory network. We identify NKX1-2 as a novel master regulator of preimplantation embryo development. We find that Nkx1-2 inhibition reduces nascent RNA synthesis, downregulates genes controlling ribosome biogenesis, RNA translation, and transport, and induces severe alteration of nucleolus structure, resulting in the exclusion of RNA polymerase I from nucleoli. In turn, NKX1-2 loss of function leads to chromosome missegregation in the 2- to 4-cell embryo stages, severe decrease in blastomere numbers, alterations of tight junctions (TJs), and impairment of microlumen coarsening. Overall, these changes impair the blastocoel expansion-collapse cycle and embryo cavitation, leading to altered lineage specification and developmental arrest.

Curcumin loaded gold nanoparticles-chitosan/sodium alginate nanocomposite for nanotheranostic applications.

A solvent casting technique was used for the preparation of biomimetic nanocomposites scaffolds at three various concentrations of Curcumin loaded gold nanoparticles (Cur-AuNPs-1, 1.5, and 2 ml) as filler materials with chitosan-sodium alginate composite. The physico-chemical properties of prepared Cu-Au NPs and biomimetic nanocomposites were analyzed using various characterization techniques. In vitro biocompatibility of biomimetic nanocomposites are determined using simulated body fluid for biomineralization property, HAp formation and phosphate buffer saline (PBS) for swelling property, protein adsorption. Antibacterial activity of Cur-Au NPs and their nanocomposites carried out against Escherichia coli (E. coli) and Staphylococcus aureus. In vitro cytotoxicity of Cur-AuNPs is identified against UC-6 and MDA-MB 231 cell lines. The use of above studies and activity of Cur-AuNPs with contain biomimetic nanocomposites can adoptable for nanotheranostics.

Biomimetic development of chitosan and sodium alginate-based nanocomposites contains zirconia for tissue engineering applications.

Nanostructured materials possess unique structural and functional properties that play a crucial position in tissue engineering applications. Present investigation is aimed to synthesize chitosan-sodium alginate (CS) nanocomposite using hydrothermally prepared zirconia nanoparticles. In this, three different weight percentages of (0.5, 1, and 1.5) zirconia nanoparticles are utilized for the preparation of biomimetic nanocomposite scaffolds (CSZ) employing 4 wt% of CS by a solvent casting technique. Physico-chemical and thermal behavior of the prepared nanoparticles and their CSZ scaffolds are comprehensively characterized. Bioactivity of the prepared zirconia nanoparticles and CSZ scaffolds are explored in terms of in vitro biocompatibility, protein absorption in simulated body fluid (SBF), and phosphate buffered saline (PBS). Agar disc diffusion method is employed to identify the antibacterial property against Staphylococcus aureus and Escherichia coli. In vitro cytotoxicity of zirconia nanoparticles and CSZ scaffolds is identified against human urothelial carcinoma (UC6) and osteosarcoma (MG-63) cells. These studies explore that zirconia nanoparticles are suitable for biomedical applications while it is interacted with chitosan and sodium alginate (CS) due to their promising biocompatibility. Biomimetically obtained chitosan/sodium alginate scaffold contain 1 wt% zirconia nanoparticles show higher biocompatibility amenable for tissue engineering applications.

Pharmacokinetic and pharmacodynamic evaluation of nasal liposome and nanoparticle based rivastigmine formulations in acute and chronic models of Alzheimer's disease.

With the increasing aging population and progressive nature of the disease, Alzheimer's disease (AD) poses to be an oncoming epidemic with limited therapeutic strategies. It is characterized by memory loss, behavioral instability, impaired cognitive function, predominantly, cognitive inability manifested due to the accumulation of ß-amyloid, with malfunctioned cholinergic system. Rivastigmine, a reversible dual cholinesterase inhibitor, is a more tolerable and widely used choice of drug for AD. However, rivastigmine being hydrophilic and undergoing the first-pass metabolism exhibits low CNS bioavailability. Nanoformulations including liposomes and PLGA nanoparticles can encapsulate hydrophilic drugs and deliver them efficiently to the brain. Besides, the nasal route is receiving considerable attention recently, due to its direct access to the brain. Therefore, the present study attempts to evaluate the pharmacokinetic and pharmacodynamic properties of nasal liposomal and PLGA nanoparticle formulations of rivastigmine in acute scopolamine-induced amnesia and chronic colchicine induced cognitive dysfunction animal models, and validate the best formulation by employing pharmacokinetic and pharmacodynamic (PK-PD) modeling. Nasal liposomal rivastigmine formulation showed the best pharmacokinetic features with rapid onset of action (Tmax = 5 min), higher Cmax (1489.5 ± 620.71), enhanced systemic bioavailability (F = 118.65 ± 23.54; AUC = 35,921.75 ± 9559.46), increased half-life (30.92 ± 8.38 min), and reduced clearance rate (Kel (1/min) = 0.0224 ± 0.006) compared to oral rivastigmine (Tmax = 15 min; Cmax = 56.29 ± 27.05; F = 4.39 ± 1.82; AUC = 1663.79 ± 813.54; t1/2 = 13.48 ± 5.79; Kel (1/min) = 0.0514 ± 0.023). Further, the liposomal formulation significantly rescued the memory deficit induced by scopolamine as well as colchicine superior to other formulations as assessed in Morris water maze and passive avoidance tasks. PK-PD modeling demonstrated a strong correlation between the pharmacokinetic parameters and acetylcholinesterase inhibition of liposomal formulation.

Effectiveness of fascial manipulation on pain and disability in musculoskeletal conditions. A systematic review.

OBJECTIVE: To review the effectiveness of Stecco's fascial manipulation technique in patients with musculoskeletal pain. DESIGN: Systematic review of interventional studies. METHODS: A systematic search of literatures was performed in the electronic databases: PubMed, Cochrane, Scopus, ScienceDirect, and Ovid from January 2005 to December 2019. Studies were included if they were followed stecco's fascial manipulation as an intervention for the musculoskeletal conditions. RCTs and Non RCTs both were included in this review. RESULTS: Thirteen studies met the eligibility criteria [RCTs-8, Pre-Post studies-3, Case reports-2]. Stecco's FM technique was followed in all the included studies. Five studies applied FM technique alone, while two studies added exercise along with FM and one study substitute session with manual therapy. CONCLUSION: This systematic review reported low to moderate quality evidence for the effect of FM in improving pain and disability in subjects with musculoskeletal pain conditions, when it is carried out as described in the Stecco's concept.

Comparative Study of Microbiological Profile and Management Outcomes of Acute Endophthalmitis after Microincision Vitrectomy Surgery versus Intravitreal Injections.

Purpose: The aim of this study was to evaluate and compare incidence and outcomes of management of acute endophthalmitis after microincision vitrectomy surgery (MIVS) and intravitreal injections (IVIs).Methods: Medical records were retrospectively reviewed from January 2012 to December 2017, and the incidence, clinical and microbiological profiles of acute endophthalmitis were documented.Results: Of 26,332 MIVS and 24,143 IVI performed, incidence of acute endophthalmitis in MIVS group was 0.027% (1 in 3761 cases) against 0.054% (1 in 1857 cases) in IVI. Gram-positive organisms were causative in post IVI group as against gram-negative organisms in MIVS group.Conclusion: Incidence of endophthalmitis after IVI is almost twice that after MIVS. A trend toward poorer outcomes in MIVS eyes was observed. Both MIVS and IVI being pars plana procedures warrant similar kind of aseptic precautions.

Real-world outcomes of intravitreal anti-vascular endothelial growth factor monotherapy in proliferative type 2 macular telangiectasia.

BACKGROUND: To study the effect of anti-VEGFs in proliferative MacTel 2 METHODS: Sixty-four eyes of 51 patients of MacTel 2 with subretinal neovascular membrane (SRNVM) undergoing intravitreal anti-VEGF monotherapy at our institution between January 2015 and December 2018 were evaluated for visual acuity, central macular thickness (CMT) and total macular volume (TMV) using optical coherence tomography (OCT). Repeat investigations were performed at the final follow-up. RESULTS: Location of SRNVM was foveal (F) in 65.6% eyes and extrafoveal (EF) in 34.4% eyes. Both CMT and TMV were lower significantly at baseline in EF-SRNVM. Fifty-six eyes received intravitreal bevacizumab and 8 eyes received ranibizumab. Average 2.14 ± 1.21 injections (range, 1-6) were administered. After a mean follow-up duration of 16.01 ± 12.96 months, the final visual acuity (VA) improved in 35.9% eyes, reduced in 28.1% and remained unchanged in 35.9% eyes. Significant improvement of VA was seen in F-SRNVM; however, overall, there was no significant improvement in visual acuity. Significant reduction in CMT was seen in F-SRNVM from 391.73 ± 152.45 µm to 293.33 ± 114.77 µm (p < 0.05) while EF-SRNVM did not show significant reduction. Total macular volume overall changed significantly from 8.79 ± 1.69 to 8.05 ± 1.27 (p < 0.05) and individually in F- and EF-SRNVM (p < 0.05). Intravitreal bevacizumab and ranibizumab monotherapy both had similar efficacy. CONCLUSIONS: Anti-VEGF agents can be successfully used to treat proliferative MacTels. Macular volume measurement on OCT may be a useful parameter for evaluating EF-SRNVM and as a prognostic marker of management outcomes.

The Master Regulator Protein BAZ2B Can Reprogram Human Hematopoietic Lineage-Committed Progenitors into a Multipotent State.

Bi-species, fusion-mediated, somatic cell reprogramming allows precise, organism-specific tracking of unknown lineage drivers. The fusion of Tcf7l1-/- murine embryonic stem cells with EBV-transformed human B cell lymphocytes, leads to the generation of bi-species heterokaryons. Human mRNA transcript profiling at multiple time points permits the tracking of the reprogramming of B cell nuclei to a multipotent state. Interrogation of a human B cell regulatory network with gene expression signatures identifies 8 candidate master regulator proteins. Of these 8 candidates, ectopic expression of BAZ2B, from the bromodomain family, efficiently reprograms hematopoietic committed progenitors into a multipotent state and significantly enhances their long-term clonogenicity, stemness, and engraftment in immunocompromised mice. Unbiased systems biology approaches let us identify the early driving events of human B cell reprogramming.

ß-Catenin safeguards the ground state of mousepluripotency by strengthening the robustness of the transcriptional apparatus.

Mouse embryonic stem cells cultured with MEK (mitogen-activated protein kinase kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) more closely resemble the inner cell mass of preimplantation blastocysts than those cultured with SL [serum/leukemia inhibitory factor (LIF)]. The transcriptional mechanisms governing this pluripotent ground state are unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. We show that ß-catenin, stabilized by GSK3 inhibition in medium with 2i, supplies transcriptional coregulators at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by ß-catenin, and proliferation/self-renewal remains tightly controlled by Pol2 pause release under 2i conditions. Our findings explain how pluripotency is reinforced in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other contexts.

Improved in vitro and in vivo hepatoprotective effects of liposomal silymarin in alcohol-induced hepatotoxicity in Wistar rats.

BACKGROUND: Silymarin, a known hepatoprotectant, owing to its poor oral bioavailability, has limited pharmacological effects. The present study was designed to improve its in vitro and in vivo hepatoprotection and increase its oral bioavailability against alcohol intoxication by formulating it in four different liposomal formulations namely conventional, dicetyl phosphate, stearyl amine and PEGylated liposomes. METHOD: The liposomes were prepared using phosphatidylcholine, cholesterol, and silymarin in addition to dicetyl phosphate, stearyl amine and DSPE mPEG 2000 by film hydration method with 5% sucrose as a cryo-protectant. The optimized formulations were studied for their release profile at pH 1.2 and 6.8. Liposomes were studied for in vitro protection on Chang liver cells and efficacious liposomes were selected for in vivo hepatoprotection study. Further, conventional liposomes were studied for bioavailability in alcohol intoxicated Wistar rats. RESULTS: The conventional liposomes increased in vitro release profile at pH 1.2 and 6.8 and also showed better in vitro protection compared to silymarin alone. Conventional and PEGylated liposomes showed better improvement in liver function, better efficacy in combating inflammatory conditions, better improvement in antioxidant levels and reversal of histological changes compared to silymarin alone. Conventional also showed an almost fourfold increase in area under the curve compared to silymarin suspension. CONCLUSION: Conventional and PEGylated liposomes of silymarin were found to be more efficacious as hepatoprotective against alcohol-induced hepatotoxicity by its free radical scavenging and anti-inflammatory effects. Conventional liposomes showed enhanced bioavailability compared to silymarin alone.

Wnt/ß-catenin signaling pathway safeguards epigenetic stability and homeostasis of mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) are pluripotent and can differentiate into cells belonging to the three germ layers of the embryo. However, mESC pluripotency and genome stability can be compromised in prolonged in vitro culture conditions. Several factors control mESC pluripotency, including Wnt/ß-catenin signaling pathway, which is essential for mESC differentiation and proliferation. Here we show that the activity of the Wnt/ß-catenin signaling pathway safeguards normal DNA methylation of mESCs. The activity of the pathway is progressively silenced during passages in culture and this results into a loss of the DNA methylation at many imprinting control regions (ICRs), loss of recruitment of chromatin repressors, and activation of retrotransposons, resulting into impaired mESC differentiation. Accordingly, sustained Wnt/ß-catenin signaling maintains normal ICR methylation and mESC homeostasis and is a key regulator of genome stability.

Development of Mimokines, chemokine N terminus-based CXCR4 inhibitors optimized by phage display and rational design.

The chemokine receptor CXCR4 (C-X-C chemokine receptor type 4 also known as fusin or CD184 (cluster of differentiation 184)) is implicated in various biological and pathological processes of the hematopoietic and immune systems. CXCR4 is also one of the major coreceptors for HIV-1 entry into target cells and is overexpressed in many cancers, supporting cell survival, proliferation, and migration. CXCR4 is thus an extremely relevant drug target. Among the different strategies to block CXCR4, chemokine-derived peptide inhibitors hold great therapeutic potential. In this study, we used the N-terminus of vCCL2/vMIPII, a viral CXCR4 antagonist chemokine, as a scaffold motif to engineer and select CXCR4 peptide inhibitors, called Mimokines, which imitate the chemokine-binding mode but display an enhanced receptor affinity, antiviral properties, and receptor selectivity. We first engineered a Mimokine phage displayed library based on the first 21 residues of vCCL2, in which cysteine 11 and 12 were fully randomized and screened it against purified CXCR4 stabilized in liposomes. We identified Mimokines displaying up to 4-fold higher affinity for CXCR4 when compared to the reference peptide and fully protected MT-4 cells against HIV-1 infection. These selected Mimokines were then subjected to dimerization, D-amino acid, and aza-ß3-amino acid substitution to further enhance their potency and selectivity. Optimized Mimokines exhibited up to 120-fold enhanced CXCR4 binding (range of 20 nM) and more than 200-fold improved antiviral properties (≤ 1 µM) compared to the parental Mimokines. Interestingly, these optimized Mimokines also showed up to 25-fold weaker affinity for ACKR3/CXCR7 and may therefore serve as lead compounds for further development of more selective CXCR4 peptide inhibitors and probes.

Evasion of regulatory phosphorylation by an alternatively spliced isoform of Musashi2.

The Musashi family of RNA binding proteins act to promote stem cell self-renewal and oppose cell differentiation predominantly through translational repression of mRNAs encoding pro-differentiation factors and inhibitors of cell cycle progression. During tissue development and repair however, Musashi repressor function must be dynamically regulated to allow cell cycle exit and differentiation. The mechanism by which Musashi repressor function is attenuated has not been fully established. Our prior work indicated that the Musashi1 isoform undergoes site-specific regulatory phosphorylation. Here, we demonstrate that the canonical Musashi2 isoform is subject to similar regulated site-specific phosphorylation, converting Musashi2 from a repressor to an activator of target mRNA translation. We have also characterized a novel alternatively spliced, truncated isoform of human Musashi2 (variant 2) that lacks the sites of regulatory phosphorylation and fails to promote translation of target mRNAs. Consistent with a role in opposing cell cycle exit and differentiation, upregulation of Musashi2 variant 2 was observed in a number of cancers and overexpression of the Musashi2 variant 2 isoform promoted cell transformation. These findings indicate that alternately spliced isoforms of the Musashi protein family possess distinct functional and regulatory properties and suggest that differential expression of Musashi isoforms may influence cell fate decisions.

Insights into ligand stimulation effects on gastro-intestinal stromal tumors signalling.

Mutations in KIT or PDGFRA are responsible for >85% of gastrointestinal stromal tumors. The introduction of imatinib in the GIST therapy scheme revolutionized the patient outcome. Unfortunately, the therapy allows the disease stabilization instead of curation. Furthermore the resistance to the inhibitor arises in most cases within two first years of therapy. A thorough investigation of the signalling pathways activated by the major PDGFRA and KIT mutants encountered in the GIST landscape allowed to identify striking differences between the two receptor tyrosine kinases. PDGFRA mutants were not responsive to their ligand, PDGFAA, and displayed a high constitutive kinase activity. In contrast, all KIT mutants retained, in addition to their constitutive activation, the ability to be stimulated by their ligand. Kit mutants displayed a lower intrinsic kinase activity relative to PDGFRA mutants, while the KIT Exon 11 deletion mutant exhibited the highest intrinsic kinase activity among KIT mutants. At the transcriptomic level, the MAPK pathway was established as the most prominent activated pathway, which is commonly up-regulated by all PDGFRA and KIT mutants. Inhibition of this pathway, using the MEK inhibitor PD0325901, reduced the proliferation of GIST primary cells at nanomolar concentrations. Altogether, our data demonstrate the high value of MEK inhibitors for combination therapy in GIST treatment and more importantly the interest of evaluating the SCF expression profile in GIST patients presenting KIT mutations.

Data on quantification of signaling pathways activated by KIT and PDGFRA mutants.

The present data are related to the article entitled "Insights into ligand stimulation effects on gastro-intestinal stromal tumors signaling" (C. Bahlawane, M. Schmitz, E. Letellier, K. Arumugam, N. Nicot, P.V. Nazarov, S. Haan, 2016) [1]. Constitutive and ligand-derived signaling pathways mediated by KIT and PDGFRA mutated proteins found in gastrointestinal stromal tumors (GIST) were compared. Expression of mutant proteins was induced by doxycycline in an isogenic background (Hek293 cells). Kit was identified by FACS at the cell surface and found to be quickly degraded or internalized upon SCF stimulation for both Kit Wild type and Kit mutant counterparts. Investigation of the main activated pathways in GIST unraveled a new feature specific for oncogenic KIT mutants, namely their ability to be further activated by Kit ligand, the stem cell factor (scf). We were also able to identify the MAPK pathway as the most prominent target for a common inhibition of PDGFRA and KIT oncogenic signaling. Western blotting and micro-array analysis were applied to analyze the capacities of the mutant to induce an effective STATs response. Among all Kit mutants, only Kit Ex11 deletion mutant was able to elicit an effective STATs response whereas all PDGFRA were able to do so.

A novel nanoproliposomes of lercanidipine: Development, in vitro and preclinical studies to support its effectiveness in hypertension therapy.

AIM: The aim of the present study was to develop nanoproliposomes of lercanidipine, in order to overcome its poor biopharmaceutical properties and to improve its therapeutic efficacy in treating hypertension. MAIN METHODS: The nanoproliposomes were prepared using a modified thin-film hydration method, and the formula was optimized by varying the ratio of lipids and the types of cryoprotectants. This optimized formulation was characterized in terms of its particle size, solid-state, drug release, in-situ absorption, in-vivo pharmacokinetics, and in-vivo anti-hypertensive activity in DOCA-salt induced hypertensive rats. Finally, a PK-PD correlation was established in order to understand the clinical implications of the developed novel nanoproliposomes. KEY FINDINGS: The nanoproliposomes showed a particle size of 174.7nm and an entrapment efficiency of 85.4%. The in-vitro release displayed initial rapid release (19.33%) followed by a sustained release profile, releasing 88.37% of the encapsulated drug. The in-situ studies showed a significant increase in absorption rate across the rat intestinal membrane. The pharmacokinetics of this novel form indicated a 2.75-fold increase in the absolute bioavailability as compared to pure lercanidipine. In addition, the nanoproliposomes were found to be efficient in treating hypertension in DOCA-salt induced hypertensive rats. The PK-PD correlation demonstrated no time lag between effect and exposure, indicating that a direct PK-PD relationship can be expected in the clinic. SIGNIFICANCE: These findings suggest that nanoproliposomes are promising carriers in improving the oral bioavailability and bioactivity of lercanidipine, and can be an effective therapy in the management of hypertension.

Functional Integration of mRNA Translational Control Programs.

Regulated mRNA translation plays a key role in control of cell cycle progression in a variety of physiological and pathological processes, including in the self-renewal and survival of stem cells and cancer stem cells. While targeting mRNA translation presents an attractive strategy for control of aberrant cell cycle progression, mRNA translation is an underdeveloped therapeutic target. Regulated mRNAs are typically controlled through interaction with multiple RNA binding proteins (RBPs) but the mechanisms by which the functions of distinct RBPs bound to a common target mRNA are coordinated are poorly understood. The challenge now is to gain insight into these mechanisms of coordination and to identify the molecular mediators that integrate multiple, often conflicting, inputs. A first step includes the identification of altered mRNA ribonucleoprotein complex components that assemble on mRNAs bound by multiple, distinct RBPs compared to those recruited by individual RBPs. This review builds upon our knowledge of combinatorial control of mRNA translation during the maturation of oocytes from Xenopus laevis, to address molecular strategies that may mediate RBP diplomacy and conflict resolution for coordinated control of mRNA translational output. Continued study of regulated ribonucleoprotein complex dynamics promises valuable new insights into mRNA translational control and may suggest novel therapeutic strategies for the treatment of disease.

Immune responses to inhalant Mammalian allergens.

In Europe and the USA, at least one person in four is exposed every day to inhalant allergens of mammalian origin, a considerable number is regularly exposed for professional reasons and almost everyone is occasionally exposed to inhalant allergens from pets or domestic animals. The production of IgE to these inhalant allergens, often complicated by asthma and rhinitis, defines the atopic status. However, the immune response to these allergens largely imprints the cellular immune compartment and also drives non-IgE humoral immune responses in the allergic and non-allergic population. During the recent years, it has become clear that IgE antibodies recognize mammalian allergens that belong to three protein or glycoprotein families: the secretoglobins, the lipocalins, and the serum albumins. In this article, we review the humoral and cellular immune responses to the major members of these families and try to define common characteristics and also distinctive features.

Fish allergens at a glance: variable allergenicity of parvalbumins, the major fish allergens.

Fish is a common trigger of severe, food-allergic reactions. Only a limited number of proteins induce specific IgE-mediated immune reactions. The major fish allergens are the parvalbumins. They are members of the calcium-binding EF-hand protein family characterized by a conserved protein structure. They represent highly cross-reactive allergens for patients with specific IgE to conserved epitopes. These patients might experience clinical reactions with various fish species. On the other hand, some individuals have IgE antibodies directed against unique, species-specific parvalbumin epitopes, and these patients show clinical symptoms only with certain fish species. Furthermore, different parvalbumin isoforms and isoallergens are present in the same fish and might display variable allergenicity. This was shown for salmon homologs, where only a single parvalbumin (beta-1) isoform was identified as allergen in specific patients. In addition to the parvalbumins, several other fish proteins, enolases, aldolases, and fish gelatin, seem to be important allergens. New clinical and molecular insights advanced the knowledge and understanding of fish allergy in the last years. These findings were useful for the advancement of the IgE-based diagnosis and also for the management of fish allergies consisting of advice and treatment of fish-allergic patients.