Dasatinib Nanoemulsion and Nanocrystal for Enhanced Oral Drug Delivery

In this work, dasatinib (DAS) nanoemulsion and nanocrystal are produced by high-gravity technology that approaches to practical mass production. The drug nanoformulations were systematically characterized and evaluated. At a low high-gravity level (β) = 47, nanoemulsion droplets were 16.15 ± 0.42 nm with a PDI of 0.122 ± 0.021. The nanoemulsion’s size and active pharmaceutical ingredient (API) content remained stable at long-term (4 months) freeze–thaw and dilution experiments. At a high β = 188, the as-prepared nanocrystal was lamellar with a short diameter of about 200 nm and a long diameter of about 750 nm. In vitro performances demonstrated the nanoemulsion displayed higher cytotoxicity on MDA-MB-231 tumor cells, Caco-2 cell permeability and drug release than that of the nanocrystal, indicating that nanoemulsion should be an ideal alternative for dasatinib oral administration.

A Solvent-Mediated Excited-State Intermolecular Proton Transfer Fluorescent Probe for Fe3+ Sensing and Cell Imaging

Constructing excited-state intermolecular proton transfer (ESIPT-e) fluorophores represents significant challenges due to the harsh requirement of bearing a proton donor-acceptor (D-A) system and their matching proton donating-accepting ability in the same molecule. Herein, we synthesized a new-type ESIPT-e fluorophor (2-APC) using the “four-component one-pot” reaction. By the installing of a cyano-group on pyridine scaffold, the proton donating ability of -NH2 was greatly enhanced, enabling 2-APC to undergo ESIPT-e process. Surprisingly, 2-APC exhibited dual-emissions in protic solvents ethanol and normal fluorescence in aprotic solvents, which is vastly different from that of conventional ESIPT-a dyes. The ESIPT emission can be obviously suppressed by Fe3+ due to the coordination reaction of Fe3+ with the A-D system in 2-APC. From this basis, a highly sensitive and selective method was established using 2-APC as a fluorescent probe, which offers the sensitive detection of Fe3+ ranging from 0 to 13 μM with the detection limit of 7.5 nM. The recovery study of spiked Fe3+ measured by the probe showed satisfactory results (97.2103.4%) with the reasonable RSD ranging from 3.1 to 3.8%. Moreover, 2-APC can also exhibit aggregation-induced effect in poor solvent or solid-state, eliciting strong red fluorescence. 2-APC was also applied to cell-imaging, exhibiting good cell-permeability, biocompatibility and color rendering. This multi-mode emission of 2-APC is significant departure from that of conventional extended p-conjugated systems and ESIPT dyes based on a flat and rigid molecular design. The “one-pot synthesis” strategy for the construction of ESIPT molecules pioneered a new route to achieve tricolor-emissive fluorophores.

Midazolam Ameliorates Hyperglycemia-Induced Glomerular Endothelial Dysfunction by Inhibiting Transglutaminase 2 in Diabetes

Midazolam is an anesthetic widely used for anxiolysis and sedation; however, to date, a possible role for midazolam in diabetic kidney disease remains unknown. Here, we investigated the effect of midazolam on hyperglycemia-induced glomerular endothelial dysfunction and elucidated its mechanism of action in kidneys of diabetic mice and human glomerular microvascular endothelial cells (HGECs). We found that, in diabetic mice, subcutaneous midazolam treatment for 6 weeks attenuated hyperglycemia-induced elevation in urine albumin/creatinine ratios. It also ameliorated hyperglycemia-induced adherens junction disruption and subsequent microvascular leakage in glomeruli of diabetic mice. In HGECs, midazolam suppressed high glucose-induced vascular endothelial-cadherin disruption and endothelial cell permeability via inhibition of intracellular Ca2+ elevation and subsequent generation of reactive oxygen species (ROS) and transglutaminase 2 (TGase2) activation. Notably, midazolam also suppressed hyperglycemia-induced ROS generation and TGase2 activation in glomeruli of diabetic mice and markedly improved pathological alterations in glomerular ultrastructure in these animals. Analysis of kidneys from diabetic Tgm2−/− mice further revealed that TGase2 played a critical role in microvascular leakage. Overall, our findings indicate that midazolam ameliorates hyperglycemia-induced glomerular endothelial dysfunction by inhibiting ROS-mediated activation of TGase2.

Microtubule Associated Protein 4 Phosphorylation-Induced Epithelial-to-Mesenchymal Transition of Podocyte Leads to Proteinuria in Diabetic Nephropathy

Background Diabetic nephropathy (DN) involves various structural and functional changes because of chronic glycemic assault and kidney failure. Proteinuria is an early clinical manifestation of DN, but the associated pathogenesis remains elusive. This study aimed to investigate the role of microtubule associated protein 4 (MAP4) phosphorylation (p-MAP4) in proteinuria in DN and its possible mechanisms. Methods In this study, the urine samples of diabetic patients and kidney tissues of streptozotocin (STZ)-induced diabetic mice were obtained to detect changes of microtubule associated protein 4 (MAP4) phosphorylation. A murine model of hyperphosphorylated MAP4 was established to examine the effect of MAP4 phosphorylation in DN. Podocyte was applied to explore changes of kidney phenotypes and potential mechanisms with multiple methods. Results Our results demonstrated elevated content of p-MAP4 in diabetic patients’ urine samples, and increased kidney p-MAP4 in streptozocin (STZ)-induced diabetic mice. Moreover, p-MAP4 triggered proteinuria with aging in mice, and induced epithelial-to-mesenchymal transition (EMT) and apoptosis in podocytes. Additionally, p-MAP4 mice were much more susceptible to STZ treatment and showed robust DN pathology as compared to wild-type mice. In vitro study revealed high glucose (HG) triggered elevation of p-MAP4, rearrangement of microtubules and F-actin filaments with enhanced cell permeability, accompanied with dedifferentiation and apoptosis of podocytes. These effects were significantly reinforced by MAP4 hyperphosphorylation, and were rectified by MAP4 dephosphorylation. Notably, pretreatment of p38/MAPK inhibitor SB203580 reinstated all HG-induced pathological alterations. Conclusions The findings indicated a novel role for p-MAP4 in causing proteinuria in DN. Our results indicated the therapeutic potential of MAP4 in protecting against proteinuria and related diseases.

Loss of YhcB results in dysregulation of coordinated peptidoglycan, LPS and phospholipid synthesis during Escherichia coli cell growth

The cell envelope is essential for viability in all domains of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the best studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole-genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies, we report the comprehensive genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a ΔyhcB background. Subsequent analyses suggest that YhcB functions at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.

Mandipropamid as a chemical inducer of proximity for in vivo applications

Direct control of protein interactions by chemically induced protein proximity holds great potential for both cell and synthetic biology as well as therapeutic applications. Low toxicity, orthogonality and excellent cell permeability are important criteria for chemical inducers of proximity (CIPs), in particular for in vivo applications. Here, we present the use of the agrochemical mandipropamid (Mandi) as a highly efficient CIP in cell culture systems and living organisms. Mandi specifically induces complex formation between a sixfold mutant of the plant hormone receptor pyrabactin resistance 1 (PYR1) and abscisic acid insensitive (ABI). It is orthogonal to other plant hormone-based CIPs and rapamycin-based CIP systems. We demonstrate the applicability of the Mandi system for rapid and efficient protein translocation in mammalian cells and zebrafish embryos, protein network shuttling and manipulation of endogenous proteins.

Crossing of the Cystic Barriers of Toxoplasma gondii by the Fluorescent Coumarin Tetra-Cyclopeptide

FR235222 is a natural tetra-cyclopeptide with a strong inhibition effect on histone deacetylases, effective on mammalian cells as well as on intracellular apicomplexan parasites, such as Toxoplasma gondii, in the tachyzoite and bradyzoite stages. This molecule is characterized by two parts: the zinc-binding group, responsible for the binding to the histone deacetylase, and the cyclic tetrapeptide moiety, which plays a crucial role in cell permeability. Recently, we have shown that the cyclic tetrapeptide coupled with a fluorescent diethyl-amino-coumarin was able to maintain properties of cellular penetration on human cells. Here, we show that this property can be extended to the crossing of the Toxoplasma gondii cystic cell wall and the cell membrane of the parasite in its bradyzoite form, while maintaining a high efficacy as a histone deacetylase inhibitor. The investigation by molecular modeling allows a better understanding of the penetration mechanism.

Notch Ligand Delta-Like 1 Is Associated With Loss of Vascular Endothelial Barrier Function

Vascular leakage associated with vascular endothelial cell (vEC) dysfunction is a hallmark of sepsis. Causative for the decreased integrity of the vascular endothelium (vE) is a complex concurrence of pathogen components, inflammation-associated host factors, and the interaction of vECs and activated circulating immune cells. One signaling pathway that regulates the integrity of the vE is the Notch cascade, which is activated through the binding of a Notch ligand to its respective Notch receptor. Recently, we showed that the soluble form of the Notch ligand Delta-like1 (sDLL1) is highly abundant in the blood of patients with sepsis. However, a direct connection between DLL1-activated Notch signaling and loss of vEC barrier function has not been addressed so far. To study the impact of infection-associated sDLL1, we used human umbilical vein cells (HUVEC) grown in a transwell system and cocultured with blood. Stimulation with sDLL1 induced activation as well as loss of endothelial tight structure and barrier function. Moreover, LPS-stimulated HUVEC activation and increase in endothelial cell permeability could be significantly decreased by blocking DLL1-receptor binding and Notch signaling, confirming the involvement of the cascade in LPS-mediated endothelial dysfunction. In conclusion, our results suggest that during bacterial infection and LPS recognition, DLL1-activated Notch signaling is associated with vascular permeability. This finding might be of clinical relevance in terms of preventing vascular leakage and the severity of sepsis.