Supramolecular interactions in cocrystals of benzoic acid derivatives with selective COX-2 inhibitor etoricoxib.

The structures of three 1:1 cocrystal forms of etoricoxib {ETR; systematic name: 5-chloro-2-(6-methylpyridin-3-yl)-3-[4-(methylsulfonyl)phenyl]pyridine, C18H15ClN2O2S} have been synthesized and characterized by single-crystal X-ray diffraction; these are etoricoxib-benzoic acid (1/1), C18H15ClN2O2S·C7H6O2 (ETR-Bz), etoricoxib-4-fluorobenzoic acid (1/1), C18H15ClN2O2S·C7H5FO2 (ETR-PFB), and etoricoxib-4-nitrobenzoic acid (1/1), C18H15ClN2O2S·C7H5NO4 (ETR-PNB). Powder X-ray diffraction and thermal differential scanning calorimetry-thermogravimetry (DSC-TG) techniques were also used to characterize these multicomponent systems. Due to the influence of the corresponding acids, ETR shows different conformations. Furthermore, the energetic contributions of the supramolecular motifs have been established by energy framework studies of the stabilizing interaction forces and are consistent with the thermal stability of the cocrystals.

Emerging insights into ethnic-specific TP53 germline variants.

The recent expansion of human genomics repositories has facilitated the discovery of novel TP53 variants in populations of different ethnic origins. Interpreting TP53 variants is a major clinical challenge because they are functionally diverse, confer highly variable predisposition to cancer (including elusive low-penetrance alleles), and interact with genetic modifiers that alter tumor susceptibility. Here, we discuss how a cancer risk continuum may relate to germline TP53 mutations on the basis of our current review of genotype-phenotype studies and an integrative analysis combining functional and sequencing datasets. Our study reveals that each ancestry contains a distinct TP53 variant landscape defined by enriched ethnic-specific alleles. In particular, the discovery and characterization of suspected low-penetrance ethnic-specific variants with unique functional consequences, including P47S (African), G334R (Ashkenazi Jewish), and rs78378222 (Icelandic), may provide new insights in terms of managing cancer risk and the efficacy of therapy. Additionally, our analysis highlights infrequent variants linked to milder cancer phenotypes in various published reports that may be underdiagnosed and require further investigation, including D49H in East Asians and R181H in Europeans. Overall, the sequencing and projected functions of TP53 variants arising within ethnic populations and their interplay with modifiers, as well as the emergence of CRISPR screens and AI tools, are now rapidly improving our understanding of the cancer susceptibility spectrum, leading toward more accurate and personalized cancer risk assessments.

VISTA as a ligand downregulates LPS-mediated inflammation in macrophages and neutrophils.

VISTA has been proposed to function both as a ligand and a receptor to dampen immune responses, although the role of VISTA as a ligand on myeloid cells has been largely ignored. We observed that a VISTA receptor is rapidly expressed on the surface of macrophages and neutrophils upon exposure to lipopolysaccharides (LPS). Importantly, treating LPS-stimulated macrophages and neutrophils ex vivo with a high-avidity agonist of the VISTA receptor (VISTA.COMP) results in the downregulation of pro-inflammatory cytokines and the increased expression of immunoregulatory genes. Finally, the in vivo administration of VISTA.COMP attenuated the rise in circulating TNFα, IL-6, and IL-12p40 in LPS-treated mice.

Agonistic nanobodies and antibodies to human VISTA.

The V-domain Ig Suppressor of T-cell Activation (VISTA) is an immune checkpoint regulator that suppresses immune responses and is readily expressed on human and murine myeloid cells and T cells. This immunosuppressive pathway can be activated using VISTA agonists. Here, we report the development of murine anti-human VISTA (anti-hVISTA) monoclonal antibodies (mAbs), anti-hVISTA nanobodies (Nbs), and cross-reactive rat anti-murine/human VISTA (anti-hmVISTA) mAbs. All mAbs and Nbs generated bound to VISTA (human and/or murine) with dissociation constants in the sub-nanomolar or low nanomolar range. Competition analysis revealed that the selected Nbs bound the same or a nearby epitope(s) as the human VISTA-specific mAbs. However, the cross-reactive mAbs only partially competed with Nbs for binding to hVISTA. All mAbs and one Nb (hVISTANb7) were able to strongly detect VISTA expression on primary human monocytes. Importantly, the murine anti-hVISTA mAbs 7E12 and 7G5 displayed strong agonistic activity in human peripheral blood mononuclear cell cultures, while Nb7 and rat anti-hmVISTA mAbs 3C3, 7C6, 7C7, and 7G1 also behaved as hVISTA agonists, albeit to a lesser extent. Cross-reactive mAbs 7C7 and 7G1 further displayed agonistic potential in murine splenocyte assays. Importantly, mAb 7G1 significantly reduced inflammation associated with the murine model of imiquimod-induced psoriasis. These agonistic VISTA mAbs may represent therapeutic leads to treat inflammatory disorders.

Microfluidic platform for studying osteocyte mechanoregulation of breast cancer bone metastasis.

Bone metastasis is a common, yet serious, complication of breast cancer. Breast cancer cells that extravasate from blood vessels to the bone devastate bone quality by interacting with bone cells and disrupting the bone remodeling balance. Although exercise is often suggested as a cancer intervention strategy and mechanical loading during exercise is known to regulate bone remodeling, its role in preventing bone metastasis remains unknown. We developed a novel in vitro microfluidic tissue model to investigate the role of osteocytes in the mechanical regulation of breast cancer bone metastasis. Metastatic MDA-MB-231 breast cancer cells were cultured inside a 3D microfluidic lumen lined with human umbilical vein endothelial cells (HUVECs), which is adjacent to a channel seeded with osteocyte-like MLO-Y4 cells. Physiologically relevant oscillatory fluid flow (OFF) (1 Pa, 1 Hz) was applied to mechanically stimulate the osteocytes. Hydrogel-filled side channels in-between the two channels allowed real-time, bi-directional cellular signaling and cancer cell extravasation over 3 days. The applied OFF was capable of inducing intracellular calcium responses in osteocytes (82.3% cells responding with a 3.71 fold increase average magnitude). Both extravasation distance and percentage of extravasated side-channels were significantly reduced with mechanically stimulated osteocytes (32.4% and 53.5% of control, respectively) compared to static osteocytes (102.1% and 107.3% of control, respectively). This is the first microfluidic device that has successfully integrated stimulatory bone fluid flow, and demonstrated that mechanically stimulated osteocytes reduced breast cancer extravasation. Future work with this platform will determine the specific mechanisms involved in osteocyte mechanoregulation of breast cancer bone metastasis, as well as other types of cancer metastasis and diseases.

OCY454 Osteocytes as an in Vitro Cell Model for Bone Remodeling Under Mechanical Loading.

Osteocytes' mechano-regulation of bone formation and resorption is key to maintaining appropriate bone health. Although extensive in vitro studies have explored osteocyte mechanobiology using the well-established MLO-Y4 cell model, the low amount of sclerostin secreted by this cell line renders it inadequate for studying cross-talk between osteocytes and osteoblasts under mechanical loading. Here, we investigated the potential of the sclerostin-expressing OCY454 osteocyte cell model in fulfilling this role. Fully differentiated OCY454 cells were tested for mechano-sensitivity by measuring changes in protein secretion, total adenosine triphosphate (ATP) content, and intracellular calcium in response to oscillatory fluid flow. Increases in sclerostin expression and total ATP content were observed. However, very low levels of receptor activator of the nuclear factor κ-B ligand were detected, and there was a great inconsistency in calcium response. Conditioned medium (CM) from OCY454 cells was then used to culture osteoblast and osteoclast precursors. Osteoblast activity was quantified with alkaline phosphatase (ALP) and Alizarin Red S stain, while osteoclast differentiation was quantified with tartrate-resistant acid phosphatase (TRAP) staining. We demonstrated that mechanically stimulated OCY454 cells released soluble factors that increased osteoblasts' ALP activity (p < 0.05) and calcium deposition (p < 0.05). There was also a significant decrease of large-sized TRAP-positive osteoclasts when osteoclast precursors were treated with CM from flow-stimulated OCY454 cells (p < 0.05). Results from this study suggest that OCY454 cells respond to mechanical loading with the release of key factors such as sclerostin to regulate downstream bone cells, thus demonstrating its potential as a novel cell model for in vitro osteocyte mechanobiology studies. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1681-1689, 2019.

Piezo1 mediates neuron oxygen-glucose deprivation/reoxygenation injury via Ca2+/calpain signaling.

OBJECTIVE: We investigated whether Piezo1 could regulate oxygen-glucose deprivation/reoxygenation injury of neurons through Ca2+/calpain signaling. METHODS: Piezo1 expression in rat brain cortex and PC12 cells were confirmed by immunohistochemistry, immunofluorescence and Western blotting. The effects of Yoda1 and GsMTx4 on OGD/R-induced decrease in cell viability, increase in cell apoptosis and activation of downstreaming Ca2+/calpain signaling were investigated. Furthermore, calpain signaling was inhibited by PD151746 to see whether Ca2+/calpain signaling participated in the neurotoxic effects of Piezo1 activation. RESULTS: Piezo1 expression was increased in rat cerebral cortex after ischemia/reperfusion and in PC12 cells after OGD/R. Activation of Piezo1 by Yoda1 enhanced OGD/R-induced cell viability inhibition, apoptosis, increase intracellular calcium levels and enhanced calpain activity while GsMTx4 showed the opposite effects. The effects of Piezo1 activation on cell viability and apoptosis were reversed by PD151746. CONCLUSION: Piezo1 could regulate neuron oxygen-glucose deprivation/reoxygenation injury via activation of Ca2+/calpain signaling.

Mechanically stimulated osteocytes reduce the bone-metastatic potential of breast cancer cells in vitro by signaling through endothelial cells.

Bone metastases occur in 65% to 75% of patients with advanced breast cancer and significantly worsen their survival and quality of life. We previously showed that conditioned medium (CM) from osteocytes stimulated with oscillatory fluid flow, mimicking bone mechanical loading during routine physical activities, reduced the transendothelial migration of breast cancer cells. Endothelial cells are situated at an ideal location to mediate signals between osteocytes in the bone matrix and metastasizing cancer cells in the blood vessels. In this study, we investigated the specific effects of flow-stimulated osteocytes on the interaction between endothelial cells and breast cancer cells in vitro. We observed that CM from flow-stimulated osteocytes reduced endothelial permeability by 15% and breast cancer cell adhesion onto endothelial monolayers by 18%. The difference in adhesion was abolished with anti-intercellular adhesion molecule 1 (ICAM-1) neutralizing antibodies. Furthermore, CM from endothelial cells conditioned in CM from flow-stimulated osteocytes significantly altered the gene expression in bone-metastatic breast cancer cells, as shown by RNA sequencing. Specifically, breast cancer cell expression of matrix metallopeptidase 9 (MMP-9) was downregulated by 62%, and frizzled-4 (FZD4) by 61%, when the osteocytes were stimulated with flow. The invasion of these breast cancer cells across Matrigel was also reduced by 47%, and this difference was abolished by MMP-9 inhibitors. In conclusion, we demonstrated that flow-stimulated osteocytes downregulate the bone-metastatic potential of breast cancer cells by signaling through endothelial cells. This provides insights into the capability of bone mechanical regulation in preventing bone metastases; and may assist in prescribing exercise or bone-loading regimens to patients with breast cancers.

Mechanical regulation of breast cancer migration and apoptosis via direct and indirect osteocyte signaling.

Bone metastases, the migration of cancers to bone, occur in 65-80% of patients with advanced breast cancer. Metastasized cancer cells interact with cells such as the bone-resorbing osteoclasts to alter bone remodeling. Exercise, often suggested as an intervention for cancer patients, regulates bone remodeling via osteocytes. Osteocytes also signal to endothelial cells, which may affect cancer cell extravasation. Therefore, we hypothesize that mechanically stimulated osteocytes can regulate processes in breast cancer bone metastasis. To test this, we exposed osteocytes to oscillatory fluid flow in vitro using parallel-plate flow chambers. We observed that conditioned medium from flow-stimulated osteocytes increased migration (by 45%) and reduced apoptosis (by 12%) of breast cancer cells. Conditioned medium from osteoclasts conditioned in flowed osteocytes' conditioned medium reduced migration (by 47%) and increased apoptosis (by 55%) of cancer cells. Cancer cell trans-endothelial migration was reduced by 34% toward flowed osteocytes' conditioned medium. This difference was abolished with ICAM-1 or IL-6 neutralizing antibodies. Conditioned medium from endothelial cells conditioned in flowed osteocytes' conditioned medium increased cancer cell apoptosis by 29%. To summarize, this study demonstrated mechanically stimulated osteocytes' potential to affect breast cancer cells not only through direct signaling, but also through osteoclasts and endothelial cells. The anti-metastatic potential of the indirect signalings is particularly exciting since osteocytes are further away from metastasizing cancer cells than osteoclasts and endothelial cells. Future studies into the effect of bone mechanical loading on metastases and its mechanism will assist in designing cancer intervention programs that lowers the risk for bone metastases.

Highly efficient electrochemiluminescence based on pyrazolecarboxylic metal organic framework.

A series of transition metal complexes with the ligands 3-pyrazoledicarboxylic acid (H2L(1)) and ethyl 1-(2-ethoxy-2-oxoethyl)-1H-pyrazole-3-carboxylate (epzc) have been synthesized. The epzc generated 1-(carboxymethyl)-1H-pyrazole-3-carboxylic acid (H2L(2)) by an in situ hydrothermal hydrolysis reaction, using a one-pot method. Simple mononuclear [Co(HL(1))2(H2O)2] (1) and [Ni(L(2))(H2O)4] (4), dinuclear [Ni2(L(1))2(H2O)6]·H2O (2) and [Cu2(L(2))2(H2O)4] (5) and 2D frameworks [Cu2(L(1))2]n (3), [Co2(L(2))2(H2O)4]n (6) have been isolated. The structures have been established by single-crystal X-ray diffraction, and the complexes characterized by FT-IR, thermogravimetric analysis (TGA), PRXD, UV-Vis spectroscopy, and fluorescent spectroscopy. 1, 2, 4 and 4 were further assembled to form a supramolecular structure by hydrogen-bonding interactions and/or π···π stacking. 3 and 6 both possess a 2D network structure that is further interlinked via intermolecular hydrogen-bonding interactions. Most importantly, the complexes demonstrated highly intense electrochemiluminescence (ECL) in DMF solution.

Anesthesia for stem cell transplantation in autistic children: A prospective, randomized, double-blind comparison of propofol and etomidate following sevoflurane inhalation.

The objective of the present study was to comparatively investigate the feasibility and safety of etomidate and propofol use following sevoflurane inhalation in autistic children during the intrathecal transplantation of stem cells. The patients selected were 60 autistic children with American Society of Anesthesiologists physical status I, who were aged between two and 12 years and scheduled for stem cell transplantation. The children received an inhalation induction of 8% sevoflurane, followed by intravenous injection of etomidate (0.2 mg/kg) in group E and propofol (2 mg/kg) in group P (n=30/group). Supplemental doses of 0.1 mg/kg etomidate or 1 mg/kg propofol were used until a deep sedation was obtained. The heart rate (HR), mean arterial pressure, oxygen saturation, respiratory rate, Ramsay sedation score (RSS) and recovery time were monitored continuously. Following anesthesia, blood pressure and HR measurements were significantly decreased in group P compared with the baseline (P<0.01) and group E values at the same time-points (P<0.05). The occurrence of adverse effects, such as respiratory depression, bradycardia, hypotension and pain on injection, was significantly higher in group P than that in group E, whereas the incidence of myoclonus in group E was significantly higher than that in group P (P<0.01). No significant differences in anesthesia induction, surgery duration, recovery time, RSS and physician satisfaction were observed between the two groups. In conclusion, sevoflurane-etomidate combinations resulted in more stable hemodynamic responses and relatively fewer adverse effects compared with propofol injection following sevoflurane inhalation and may therefore be more suitable for the induction of short-term anesthesia in autistic children during stem cell transplantation.

Circulating sclerostin associated with vertebral bone marrow fat in older men but not women.

CONTEXT: Osteocyte activity is crucial to the maintenance of bone quality. Sclerostin, an osteocyte product, inhibits bone formation, yet higher circulating sclerostin is associated with higher bone density. Bone marrow fat (MF) is associated with osteoporosis, but little is known about the relationship between osteocyte activity and MF. OBJECTIVE: Our objective was to assess the relationships between circulating sclerostin, vertebral MF, volumetric bone mineral density (vBMD), and other fat depots in older adults. DESIGN, SETTING, AND PARTICIPANTS: We conducted a cross-sectional study in the Age Gene/Environment Susceptibility-Reykjavik cohort. MAIN OUTCOME MEASURES: Outcome measures included vertebral MF (L1-L4) measured with magnetic resonance spectroscopy and vBMD (spine and hip) and abdominal fat measured with quantitative computed tomography. RESULTS: After excluding subjects with bone-active medication use (n = 50), inadequate serum (n = 2), or inadequate magnetic resonance spectroscopy (n = 1), analyses included 115 men and 134 women (mean age 79 y, mean body mass index 27.7 kg/m(2)). In men, but not women, vertebral MF was greater in those with higher serum sclerostin levels. MF was 52.2 % in the lowest tertile of serum sclerostin and 56.3% in the highest tertile in men (P for trend <.01) in models adjusted for age, body mass index, and diabetes. Sclerostin was positively associated with cortical and trabecular total hip vBMD, weight in men and women, and total fat mass in men but was not associated with total lean mass or abdominal fat depots. CONCLUSION: Circulating sclerostin levels are associated with higher vertebral marrow fat in men, suggesting a relationship between osteocyte function and marrow adipogenesis.

[Design, synthesis, and PPARgamma agonistic activity of novel indenone derivatives].

Agonists of peroxisome proliferator-activated receptor gamma (PPARgamma) are of interest as a treatment of type II diabetes, and indenone derivatives are a new class of non-TZD PPARgamma agonists. Based on existing indenone derivatives, a series of novel ones have been designed and synthesized. Meanwhile the structures have been comfirmed with 1H NMR and MS. Among them, 17b and 19 showed higher agonistic activities than rosiglitazone.

Evaluation of effects of Fc domain high-mannose glycan on antibody stability.

Elevated levels of CH2 domain N-linked high-mannose (HM) glycans are commonly observed in therapeutic monoclonal antibodies at various stages of the development. The effect of HM glycans on antibody stability was evaluated by using two approaches. In the first approach, immunoglobulin G (IgG) 1 material containing 21% HM was incubated at 29°C for 6 weeks and fractionated into monomeric and aggregate species by using size-exclusion chromatography (SEC). These fractions were analyzed for the levels of HM. No significant difference was observed in the amount of HM in aggregate and monomer fractions indicating that the HM-containing fractions did not have a greater tendency to form aggregates. In the second approach, both IgG1 material and IgG2 material were separated by Concanavalin-A affinity chromatography into a HM-enriched fraction and a HM-depleted fraction, respectively. Real-time and accelerated stability studies were carried out with these fractions together with untreated samples under standard formulation conditions. The stability of these fractions over time was monitored using SEC and cation-exchange chromatography. No significant difference was observed in rates of aggregation or charge variant formation. These data indicate that HM glycans had no effect on the IgG1 and IgG2 product's stability under the formulation conditions studied.

[Design, synthesis and biological assay of novel tripeptidic tetrazoles as inhibitors of 20S proteasome].

Ubiquitin-proteasome pathway (UPP) is one of the ways utilized for selective degradation of many proteins in cells, and the 20S proteasome takes the functional machinery where hydrolysis of targeted proteins takes place. Based on existing peptide inhibitors, a series of novel tripeptidic tetrazoles have been designed, synthesized, and the structures have been confirmed with 1H NMR, MS and elemental analysis. Among them, three compounds (6b, 6d and 6h) showed inhibitory activities of ChT-L of 20S proteasome.