Zintegrowana analiza FFT sygnałów sił skrawania i profili chropowatości powierzchni generowanych w precyzyjnym toczeniu ostrzami z CBN

The paper presents an original approach to the prediction of deteriorations of the surface profiles produced on hardened steel parts using CBN tools. The experimental investigations involve recording of surface profiles and corresponding measurements of cutting forces resulting from variable feed rates of 0,05-0,1 mm/rev. As a result, machined surfaces with the Ra parameter ranging from 0,05 μm to 0,3 μm were produced. Both signals of recorded surface profiles and relevant cutting forces were processed using the FFT signal processing technique. The frequency characteristics including the signal amplitude and the wavelength were estimated from the power spectral density (PSD) spectra. A methodology for the prediction of surface profile deterioration based on the relation between the differences of the Rz (Ra) roughness parameter and signal amplitude is proposed.Artykuł przedstawia oryginalne rozwiązanie w przewidywaniu zniekształcenia profili powierzchni wytwarzanych elementów maszyn ze stali utwardzonej z użyciem narzędzi skrawających z PCBN. Badania doświadczalne obejmują pomiary profili powierzchni i określenia wartości składowych sił skrawania dla zmiany posuwu w zakresie 0,05-0,1 mm/obr. Powierzchnie po obróbce cechuje parametr chropowatości Ra od 0,05 μm do 0,3 μm. Zarówno sygnały zapisywanych profili powierzchni jak również i mierzonych składowych sił skrawania przetwarzano z użyciem techniki FFT. Także charakterystyki częstotliwościowe amplituda sygnału i długość fali były wyznaczane na podstawie gęstości widmowej mocy (GWM). Zaproponowano metodologię przewidywania zniekształcenia profili powierzchni uwzględniającą korelację zmiany parametru chropowatości powierzchni Rz i amplitudy sygnału

Formulacja nanocząstek polilaktyd/poli(kwas mlekowy-co-glikolowy) z nimesulidem oraz ocena kinetyki ich uwalniania

Polymeric nanoparticles containing nimesulide (NIM) were prepared by the emulsion solvent evaporation method. Biodegradable polymers were used as materials for nanoparticle formulations with the application of studies for the drug delivery sector in mind. Two analytical methods, scanning electron microscopy (SEM) and particle size analysis, were applied to investigate the obtained nanoparticles. Characteristic parameters were calculated to describe the amount of active pharmaceutical ingredient trapped in nanoparticles. The biological active substance release process was investigated under different conditions. The impact of various parameters [kind of polymer (PLA or PLGA), speed of mixing and especially of pH] is presented across our research.Polimerowe nanocząstki zawierające nimesulid (NIM) otrzymano metodą emulsyjną z odparowaniem rozpuszczalnika. Do ich wytworzenia wykorzystano biodegradowalne polimery. Wytworzone nanocząstki zbadano za pomocą skaningowej mikroskopii elektronowej (SEM) oraz analizatora wielkości cząstek. Obliczono charakterystyczne parametry opisujące ilość substancji farmaceutycznie aktywnej uwięzionej wewnątrz nanocząstek. Oceniono wpływ rodzaju polimeru stosowanego do kapsułkowania, szybkości mieszania suspensji, w szczególności pH środowiska na proces uwalniania substancji biologicznie czynnej

Structures of ABCB4 provide insight into phosphatidylcholine translocation

ABCB4 is expressed in hepatocytes and translocates phosphatidylcholine into bile canaliculi. The mechanism of specific lipid recruitment from the canalicular membrane, which is essential to mitigate the cytotoxicity of bile salts, is poorly understood. We present cryogenic electron microscopy structures of human ABCB4 in three distinct functional conformations. An apo-inward structure reveals how phospholipid can be recruited from the inner leaflet of the membrane without flipping its orientation. An occluded structure reveals a single phospholipid molecule in a central cavity. Its choline moiety is stabilized by cation-π interactions with an essential tryptophan residue, rationalizing the specificity of ABCB4 for phosphatidylcholine. In an inhibitor-bound structure, a posaconazole molecule blocks phospholipids from reaching the central cavity. Using a proteoliposome-based translocation assay with fluorescently labeled phosphatidylcholine analogs, we recapitulated the substrate specificity of ABCB4 in vitro and confirmed the role of the key tryptophan residue. Our results provide a structural basis for understanding an essential translocation step in the generation of bile and its sensitivity to azole drugs. Keywords: ABC transporter; cryo-EM; hepatocyte; membrane transport; phosphatidylcholine

Structures of ABCB4 provide insight into phosphatidylcholine translocation

ABCB4 is expressed in hepatocytes and translocates phosphatidylcholine into bile canaliculi. The mechanism of specific lipid recruitment from the canalicular membrane, which is essential to mitigate the cytotoxicity of bile salts, is poorly understood. We present cryogenic electron microscopy structures of human ABCB4 in three distinct functional conformations. An apo-inward structure reveals how phospholipid can be recruited from the inner leaflet of the membrane without flipping its orientation. An occluded structure reveals a single phospholipid molecule in a central cavity. Its choline moiety is stabilized by cation-π interactions with an essential tryptophan residue, rationalizing the specificity of ABCB4 for phosphatidylcholine. In an inhibitor-bound structure, a posaconazole molecule blocks phospholipids from reaching the central cavity. Using a proteoliposomebased translocation assay with fluorescently labeled phosphatidylcholine analogs, we recapitulated the substrate specificity of ABCB4 in vitro and confirmed the role of the key tryptophan residue. Our results provide a structural basis for understanding an essential translocation step in the generation of bile and its sensitivity to azole drugs.ISSN:0027-8424ISSN:1091-649

Structural basis of bile salt extrusion and small-molecule inhibition in human BSEP.

BSEP (ABCB11) is an ATP-binding cassette transporter that is expressed in hepatocytes and extrudes bile salts into the canaliculi of the liver. BSEP dysfunction, caused by mutations or induced by drugs, is frequently associated with severe cholestatic liver disease. We report the cryo-EM structure of glibenclamide-bound human BSEP in nanodiscs, revealing the basis of small-molecule inhibition. Glibenclamide binds the apex of a central binding pocket between the transmembrane domains, preventing BSEP from undergoing conformational changes, and thus rationalizing the reduced uptake of bile salts. We further report two high-resolution structures of BSEP trapped in distinct nucleotide-bound states by using a catalytically inactivated BSEP variant (BSEP <sub>E1244Q</sub> ) to visualize a pre-hydrolysis state, and wild-type BSEP trapped by vanadate to visualize a post-hydrolysis state. Our studies provide structural and functional insight into the mechanism of bile salt extrusion and into small-molecule inhibition of BSEP, which may rationalize drug-induced liver toxicity

Structure and drug resistance of the Plasmodium falciparum transporter PfCRT

10.1038/s41586-019-1795-xNATURE5767786315-

Antiviral HIV-1 SERINC restriction factors disrupt virus membrane asymmetry

The host proteins SERINC3 and SERINC5 are HIV-1 restriction factors that reduce infectivity when incorporated into the viral envelope. The HIV-1 accessory protein Nef abrogates incorporation of SERINCs via binding to intracellular loop 4 (ICL4). Here, we determine cryoEM maps of full-length human SERINC3 and an ICL4 deletion construct, which reveal that hSERINC3 is comprised of two α-helical bundles connected by a ~ 40-residue, highly tilted, “crossmember” helix. The design resembles non-ATP-dependent lipid transporters. Consistently, purified hSERINCs reconstituted into proteoliposomes induce flipping of phosphatidylserine (PS), phosphatidylethanolamine and phosphatidylcholine. Furthermore, SERINC3, SERINC5 and the scramblase TMEM16F expose PS on the surface of HIV-1 and reduce infectivity, with similar results in MLV. SERINC effects in HIV-1 and MLV are counteracted by Nef and GlycoGag, respectively. Our results demonstrate that SERINCs are membrane transporters that flip lipids, resulting in a loss of membrane asymmetry that is strongly correlated with changes in Env conformation and loss of infectivity

Structural basis of lipopolysaccharide maturation by the WaaL O-antigen ligase

The outer membrane of Gram-negative bacteria has an external leaflet that is largely composed of lipopolysaccharide, which provides a selective permeation barrier, particularly against antimicrobials1. The final and crucial step in the biosynthesis of lipopolysaccharide is the addition of a species-dependent O-antigen to the lipid A core oligosaccharide, which is catalysed by the O-antigen ligase WaaL2. Here we present structures of WaaL from Cupriavidus metallidurans, both in the apo state and in complex with its lipid carrier undecaprenyl pyrophosphate, determined by single-particle cryo-electron microscopy. The structures reveal that WaaL comprises 12 transmembrane helices and a predominantly α-helical periplasmic region, which we show contains many of the conserved residues that are required for catalysis. We observe a conserved fold within the GT-C family of glycosyltransferases and hypothesize that they have a common mechanism for shuttling the undecaprenyl-based carrier to and from the active site. The structures, combined with genetic, biochemical, bioinformatics and molecular dynamics simulation experiments, offer molecular details on how the ligands come in apposition, and allows us to propose a mechanistic model for catalysis. Together, our work provides a structural basis for lipopolysaccharide maturation in a member of the GT-C superfamily of glycosyltransferases