Berberine-loaded polylactic acid nanofiber scaffold as a drug delivery system: The relationship between chemical characteristics, drug-release behavior, and antibacterial efficiency.

Hydrophobic berberine powder (BBR) and hydrophilic BBR nanoparticles (BBR NPs) were loaded into an electrospun polylactic acid (PLA) nanofiber scaffold for modulating the release behavior of BBR in an aqueous medium. The BBR release from the BBR/PLA and BBR NPs/PLA nanofiber scaffolds was investigated in relation to their chemical characteristics, BBR dispersion into nanofibers, and wettability. The BBR release profiles strongly influenced the antibacterial efficiency of the scaffolds over time. When the BBR was loaded, the BBR/PLA nanofiber scaffold exhibited an extremely hydrophobic feature, causing a triphasic release profile in which only 9.8 wt % of the loaded BBR was released in the first 24 h. This resulted in a negligible inhibitory effect against methicillin-resistant Staphylococcus aureus bacteria. Meanwhile, the BBR NPs/PLA nanofiber scaffold had more wettability and higher concentration of BBR NPs dispersed on the surface of PLA nanofibers. This led to a sustained release of 75 wt % of the loaded BBR during the first 24 h, and consequently boosted the antibacterial effectiveness. Moreover, the cytotoxicity test revealed that the BBR NPs/PLA nanofiber scaffold did not induce any changes in morphology and proliferation of MA-104 cell monolayers. It suggests that the BBR/PLA and BBR NPs/PLA nanofiber scaffolds can be used in different biomedical applications, such as wound dressing, drug delivery systems, and tissue engineering, according to the requirement of BBR concentration for the desired therapeutic effects.

Fyn-mediated phosphorylation of Menin disrupts telomere maintenance in stem cells.

Telomeres protect chromosome ends and determine the replication potential of dividing cells. The canonical telomere sequence TTAGGG is synthesized by telomerase holoenzyme, which maintains telomere length in proliferative stem cells. Although the core components of telomerase are well-defined, mechanisms of telomerase regulation are still under investigation. We report a novel role for the Src family kinase Fyn, which disrupts telomere maintenance in stem cells by phosphorylating the scaffold protein Menin. We found that Fyn knockdown prevented telomere erosion in human and mouse stem cells, validating the results with four telomere measurement techniques. We show that Fyn phosphorylates Menin at tyrosine 603 (Y603), which increases Menin's SUMO1 modification, C-terminal stability, and importantly, its association with the telomerase RNA component (TR). Using mass spectrometry, immunoprecipitation, and immunofluorescence experiments we found that SUMO1-Menin decreases TR's association with telomerase subunit Dyskerin, suggesting that Fyn's phosphorylation of Menin induces telomerase subunit mislocalization and may compromise telomerase function at telomeres. Importantly, we find that Fyn inhibition reduces accelerated telomere shortening in human iPSCs harboring mutations for dyskeratosis congenita.

Complexes of Ag and ZnO nanoparticles with BBR for enhancement of gastrointestinal antibacterial activity through the impacts of size and composition.

This study introduces the bioformulations of Ag/BBR and ZnO/BBR complexes against pathogenic bacteria in the gastrointestinal tract. Without the use of toxic reduction agents, Ag and ZnO NPs were prepared using an electrochemical method and then facially mixed with BBR solution to form Ag/BBR and ZnO/BBR complexes. BBR molecules are strongly conjugated with Ag and ZnO NPs through coordinated bonding and electrostatic interaction. As a result, the presence of BBR significantly influenced the nanoparticle growth, resulting in the formation of core/shell structured Ag/BBR and ZnO/BBR NPs with small particle sizes. The antibacterial test showed that BBR, Ag, or ZnO components all contributed to the increase of antibacterial ability of Ag/BBR and ZnO/BBR NPs against both methicillin-resistant Staphylococcus aureus (MRSA) and Salmonella enteritidis (S. enteritidis). The bactericidal ability of Ag/BBR and ZnO/BBR complexes against MRSA was exhibited even at a concentration of four-fold dilution (corresponding to 1.25 g L-1 of BBR and 46.25 mg L-1 of Ag) and two-fold dilution (corresponding to 2.5 g L-1 of BBR and 10 mg L-1 of ZnO), respectively, while that of the Ag/BBR complex against S. enteritidis showed at a concentration of two-fold dilution corresponding to 2.5 g L-1 of BBR and 92.5 mg L-1 of Ag. The results obtained in this study support that Ag/BBR and ZnO/BBR complexes can be potential therapeutic agents against gastrointestinal infections.

Cryo-EM structures of human ABCA7 provide insights into its phospholipid translocation mechanisms.

Phospholipid extrusion by ABC subfamily A (ABCA) exporters is central to cellular physiology, although the specifics of the underlying substrate interactions and transport mechanisms remain poorly resolved at the molecular level. Here we report cryo-EM structures of lipid-embedded human ABCA7 in an open state and in a nucleotide-bound, closed state at resolutions between 3.6 and 4.0 Å. The former reveals an ordered patch of bilayer lipids traversing the transmembrane domain (TMD), while the latter reveals a lipid-free, closed TMD with a small extracellular opening. These structures offer a structural framework for both substrate entry and exit from the ABCA7 TMD and highlight conserved rigid-body motions that underlie the associated conformational transitions. Combined with functional analysis and molecular dynamics (MD) simulations, our data also shed light on lipid partitioning into the ABCA7 TMD and localized membrane perturbations that underlie ABCA7 function and have broader implications for other ABCA family transporters.

Structural basis for the interaction between human Npl4 and Npl4-binding motif of human Ufd1.

The heterodimer of human ubiquitin fusion degradation 1 (hUfd1) and human nuclear protein localization 4 (hNpl4) is a major cofactor of human p97 adenosine triphosphatase (ATPase). The p97-Ufd1-Npl4 complex translocates the ubiquitin-conjugated proteins from the endoplasmic reticulum membrane to the cytoplasm. Ubiquitinated proteins are then degraded by the proteasome. The structures of Npl4 and Ufd1-Npl4 (UN) complex in Saccharomyces cerevisiae have been recently reported; however, the structures of hNpl4 and the human UN complex remain unknown. Here, we report the crystal structures of the human UN complex at a resolution of 2.7 Å and hNpl4 at a resolution of 3.0 Å. We also present atomic details and characterization of the human UN complex. Crystallographic studies and site-directed mutagenesis of the hUfd1 residues involved in the interaction with hNpl4 revealed the atomic details of the two proteins.

Structures of the human peroxisomal fatty acid transporter ABCD1 in a lipid environment.

The peroxisomal very long chain fatty acid (VLCFA) transporter ABCD1 is central to fatty acid catabolism and lipid biosynthesis. Its dysfunction underlies toxic cytosolic accumulation of VLCFAs, progressive demyelination, and neurological impairments including X-linked adrenoleukodystrophy (X-ALD). We present cryo-EM structures of ABCD1 in phospholipid nanodiscs in a nucleotide bound conformation open to the peroxisomal lumen and an inward facing conformation open to the cytosol at up to 3.5 Å resolution, revealing details of its transmembrane cavity and ATP dependent conformational spectrum. We identify features distinguishing ABCD1 from its closest homologs and show that coenzyme A (CoA) esters of VLCFAs modulate ABCD1 activity in a species dependent manner. Our data suggest a transport mechanism where the CoA moieties of VLCFA-CoAs enter the hydrophilic transmembrane domain while the acyl chains extend out into the surrounding membrane bilayer. The structures help rationalize disease causing mutations and may aid ABCD1 targeted structure-based drug design.

A Simple and Straightforward Approach for Generating Small, Stable, Homogeneous, Unilamellar 1-Palmitoyl 2-Oleoyl Phosphatidylcholine (POPC) Bilayer Vesicles.

Various methods have been developed to generate phosphoglyceride liposomes. Approaches resulting in homogeneous populations of unilamellar bilayer vesicles are generally preferred to mimic various cell membrane situations, as well as to optimize aqueous solute trapping efficiency using the least amount of lipid for biotechnological purposes. Most are time-consuming, often tedious, or require specialized equipment, and produce vesicles with limited shelf-life at room temperature or in cold storage. Herein, we describe a straightforward approach that avoids the preceding complications and streamlines the construction of unilamellar bilayer vesicles from 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/dihexanoyl phosphatidylcholine (DHPC) bicelle mixtures at room temperature. The resulting vesicles are small (32-36 nm diameter), unilamellar, bilayer vesicles that are homogeneous, stable, and resistant to freeze-thaw alterations. Graphic abstract: Cryo-EM of POPC vesicles formed by dilution of 0.5 q-value POPC/DHPC bicelle mix.

Electronic cigarettes promotes the lung colonization of human breast cancer in NOD-SCID-Gamma mice.

Electronic cigarettes (E-cigs) smoking or vaping is an emerging problem to public health due to its popularity. While its multi-faceted detrimental effects on human health are being reported, no current study addresses the effect of E-cigs on tumor metastasis, the main cause of tumor mortality. Using a well-established human breast cancer cell line MDA MB-231, we first showed that E-cig vapor extract (nicotine 24 mg/ml, propylene glycol 50%, vegetable glycerin 50%, no flavorings) significantly enhanced tumor cell migration (P<0.0001), but showed no significant effect on tumor cell proliferation (P>0.05). To evaluate the metastasis-promoting effect of E-cigs in vivo, we used NOD-SCID-Gamma mice and introduced tumor cells to the mice by tail vein injection. Among these mice, 4-week E-cigs exposure (nicotine 24 mg/ml, propylene glycol 50%, vegetable glycerin 50%, no flavorings, 2 h/day, 5 days/week) almost doubled the tumor load in the exposed lungs compared to controls (P=0.0036). While E-cig exposure did not alter the proliferative index of tumor cells colonized in the lungs (P=0.7953), tumor cell apoptosis was significantly reduced (P<0.001). Taken together, our data for the first time, demonstrated the lung colonization-promoting effects of E-cigs on human breast cancer cells. These findings show the risks of E-cigs on the lung metastasis of various cancers, and warrant more studies on the underlying mechanisms.

Knock-down of the TIM/TIPIN complex promotes apoptosis in melanoma cells.

The Timeless (TIM) and it's interacting partner TIPIN protein complex is well known for its role in replication checkpoints and normal DNA replication processes. Recent studies revealed the involvement of TIM and TIPIN in human malignancies; however, no evidence is available regarding the expression of the TIM/TIPIN protein complex or its potential role in melanoma. Therefore, we investigated the role of this complex in melanoma. To assess the role of the TIM/TIPIN complex in melanoma, we analyzed TIM/TIPIN expression data from the publicly accessible TCGA online database, Western blot analysis, and RT-qPCR in a panel of melanoma cell lines. Lentivirus-mediated TIM/TIPIN knockdown in A375 melanoma cells was used to examine proliferation, colony formation, and apoptosis. A xenograft tumor formation assay was also performed. The TIM/TIPIN complex is frequently overexpressed in melanoma cells compared to normal melanocytes. We also discovered that the overexpression of TIM and TIPIN was significantly associated with poorer prognosis of melanoma patients. Furthermore, we observed that shRNA-mediated knockdown of TIM and TIPIN reduced cell viability and proliferation due to the induction of apoptosis and increased levels of γH2AX, a marker of DNA damage. In a xenograft tumor nude mouse model, shRNA-knockdown of TIM/TIPIN significantly reduced tumor growth. Our results suggest that the TIM/TIPIN complex plays an important role in tumorigenesis of melanoma, which might reveal novel approaches for the development of new melanoma therapies. Our studies also provide a beginning structural basis for understanding the assembly of the TIM/TIPIN complex. Further mechanistic investigations are needed to determine the complex's potential as a biomarker of melanoma susceptibility. Targeting TIM/TIPIN might be a potential therapeutic strategy against melanoma.

Measuring Lipid Transfer Protein Activity Using Bicelle-Dilution Model Membranes.

In vitro assessment of lipid intermembrane transfer activity by cellular proteins typically involves measurement of either radiolabeled or fluorescently labeled lipid trafficking between vesicle model membranes. Use of bilayer vesicles in lipid transfer assays usually comes with inherent challenges because of complexities associated with the preparation of vesicles and their rather short "shelf life". Such issues necessitate the laborious task of fresh vesicle preparation to achieve lipid transfer assays of high quality, precision, and reproducibility. To overcome these limitations, we have assessed model membrane generation by bicelle dilution for monitoring the transfer rates and specificity of various BODIPY-labeled sphingolipids by different glycolipid transfer protein (GLTP) superfamily members using a sensitive fluorescence resonance energy transfer approach. Robust, protein-selective sphingolipid transfer is observed using donor and acceptor model membranes generated by dilution of 0.5 q-value mixtures. The sphingolipid transfer rates are comparable to those observed between small bilayer vesicles produced by sonication or ethanol injection. Among the notable advantages of using bicelle-generated model membranes are (i) easy and straightforward preparation by means that avoid lipid fluorophore degradation and (ii) long "shelf life" after production (≥6 days) and resilience to freeze-thaw storage. The bicelle-dilution-based assay is sufficiently robust, sensitive, and stable for application, not only to purified LTPs but also for LTP activity detection in crude cytosolic fractions of cell homogenates.

Characteristics of PCDD/Fs in soil and sediment samples collected from A-So former airbase in Central Vietnam.

A-So airbase, located in A-Luoi Valley - Central Vietnam, is a former military base occupied by US Special Forces between 1963 and 1966. The storage of Agent Orange in A-So airbase during the Vietnam War now poses a high potential for PCDD/F contamination in soils and sediments. In order to evaluate the occurrence and characteristics of PCDD/Fs in A-So former airbase, which has been reserved for a long time and suffered almost no significant anthropogenic impacts, soil and sediment samples were collected from 40 sites of two adjacent zones A and B in an area of 160,000 m2. Seventeen 2,3,7,8-substituted PCDD/Fs were analyzed using HRCG/HRMS (US EPA method 1613). Results indicate that concentrations of PCDD/Fs measured in zone A ranged from 95.0 to 4534 ng kgdw-1 (4.58 to 746 ng TEQ kgdw-1), while those in zone B were in the range of 80.8-4150 ng kgdw-1 (2.70-89.0 ng TEQ kgdw-1). The concentrations of PCDD/Fs observed in zone A are higher than those in zone B, suggesting that PCDD/Fs could be transported from zone A to zone B through surface soil erosion and runoff events. The main contributor to the total TEQ concentration was 2,3,7,8-TCDD, which was the indicator of Agent Orange contamination, accounting for 91 ±â€¯9% and 72 ±â€¯17% of the total TEQ concentrations measured in zones A and B, respectively. Comparison of PCDD/F concentrations in different soil layers reveals that the topsoil layer (at depth < 1 m) contributed 81-95% to the total PCDD/Fs in the study area, indicating that future remediation projects should focus on this topsoil layer. Since PCDD/F contamination in A-So airbase has not significantly improved for the last 20 years, remediation projects are urgently needed in order to mitigate the negative impacts of PCDD/F contamination on human health and wellbeing.

Structural Details of Ufd1 Binding to p97 and Their Functional Implications in ER-Associated Degradation.

The hexameric ATPase p97 has been implicated in diverse cellular processes through interactions with many different adaptor proteins at its N-terminal domain. Among these, the Ufd1-Npl4 heterodimer is a major adaptor, and the p97-Ufd1-Npl4 complex plays an essential role in endoplasmic reticulum-associated degradation (ERAD), acting as a segregase that translocates the ubiquitinated client protein from the ER membrane into the cytosol for proteasomal degradation. We determined the crystal structure of the complex of the N-terminal domain of p97 and the SHP box of Ufd1 at a resolution of 1.55 Å. The 11-residue-long SHP box of Ufd1 binds at the far-most side of the Nc lobe of the p97 N domain primarily through hydrophobic interactions, such that F225, F228, N233 and L235 of the SHP box contact hydrophobic residues on the surface of the p97 Nc lobe. Mutating these key interface residues abolished the interactions in two different binding experiments, isothermal titration calorimetry and co-immunoprecipitation. Furthermore, cycloheximide chase assays showed that these same mutations caused accumulation of tyrosinase-C89R, a well-known ERAD substrate, thus implying decreased rate of protein degradation due to their defects in ERAD function. Together, these results provide structural and biochemical insights into the interaction between p97 N domain and Ufd1 SHP box.

Structural and biochemical basis for the inhibition of cell death by APIP, a methionine salvage enzyme.

APIP, Apaf-1 interacting protein, has been known to inhibit two main types of programmed cell death, apoptosis and pyroptosis, and was recently found to be associated with cancers and inflammatory diseases. Distinct from its inhibitory role in cell death, APIP was also shown to act as a 5-methylthioribulose-1-phosphate dehydratase, or MtnB, in the methionine salvage pathway. Here we report the structural and enzymatic characterization of human APIP as an MtnB enzyme with a Km of 9.32 µM and a Vmax of 1.39 µmol min(-1) mg(-1). The crystal structure was determined at 2.0-Å resolution, revealing an overall fold similar to members of the zinc-dependent class II aldolase family. APIP/MtnB exists as a tetramer in solution and exhibits an assembly with C4 symmetry in the crystal lattice. The pocket-shaped active site is located at the end of a long cleft between two adjacent subunits. We propose an enzymatic reaction mechanism involving Glu139* as a catalytic acid/base, as supported by enzymatic assay, substrate-docking study, and sequence conservation analysis. We explored the relationship between two distinct functions of APIP/MtnB, cell death inhibition, and methionine salvage, by measuring the ability of enzymatic mutants to inhibit cell death, and determined that APIP/MtnB functions as a cell death inhibitor independently of its MtnB enzyme activity for apoptosis induced by either hypoxia or etoposide, but dependently for caspase-1-induced pyroptosis. Our results establish the structural and biochemical groundwork for future mechanistic studies of the role of APIP/MtnB in modulating cell death and inflammation and in the development of related diseases.