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Objective: In tympanoplasty, surgical reconstruction of the tympanic membrane and ossicular chain is well-established; however, its hearing results still require improvement. Custom 3D printing of individualized ossicular prostheses seems to be an attractive solution for optimal prosthesis adjustment and better hearing results. Aim: The aim was to design a custom ossicular prosthesis using a 3D printing method based on Cone-beam Computed Tomography (CBCT) scans and assess the acoustic conduction properties of such prosthesis. Material and methods: A cadaver fresh frozen temporal bone was used. Based on CBCT images, a new incus prosthesis was designed and 3D printed. Next, canal wall-up tympanoplasty was performed. The intact ossicular chain and reconstructed 3D-printed prosthesis chain movements/vibrations were measured with Laser Doppler Vibrometer (LDV) system and analyzed in detail. Results: The CBCT scans provided enough information about the anatomical structures. For frequencies 500 and 1000 Hz and 80 dB SPL sound intensity, collected velocities were higher for the intact ossicular chain than the 3D-printed ossicular prosthesis. The intensity thresholds for movement at 500 and 1000 Hz were lower in the intact ossicular chain than in the 3D-printed ossicular prosthesis. At 2000 Hz, there was the same intensity threshold value in the two measured circumstances. Conclusion: It is possible to design a custom individually fitted ossicular prosthesis using a 3D printing method based on CBCT scans. The acoustic conduction properties of such 3D-printed prosthesis showed differences in movability pattern between the intact and reconstructed ossicular chain. More data are needed to analyze the acoustic properties of such designed prostheses in detail. The results of our experiment showed the 3D-printed prosthesis presents the potential to be an interesting option for conductive hearing loss treatment caused by chronic otitis media and the ossicular chain defects.

Journal of cell science 133(19), jcs239202 (1-15) - (2020). doi:10.1242/jcs.239202, Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the �� integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn$^{2+}$. Here we show that kindlin-1 can replace Mn2+ to mediate ��3 integrin clustering induced by the talin head, but not that induced by the F2���F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain. Further mutagenesis identified hydrophobic and acidic motifs in the F1 loop responsible for ��3 integrin clustering. Modeling, computational and cysteine crosslinking studies showed direct and catalytic interactions of the acidic F1 loop motif with the juxtamembrane domains of ��- and ��3-integrins, in order to activate the ��3 integrin heterodimer, further detailing the mechanism by which the talin���kindlin complex activates and clusters integrins. Moreover, the F1 loop interaction with the ��3 integrin tail required the newly identified compact FERM fold of the talin head, which positions the F1 loop next to the inner membrane clasp of the talin-bound integrin heterodimer., Published by Company of Biologists Limited, Cambridge

Cell walls of marine macroalgae are composed of diverse polysaccharides that provide abundant carbon sources for marine heterotrophic bacteria. Among them, Zobellia galactanivorans is considered as a model for studying algae–bacteria interactions. The degradation of typical algal polysaccharides, such as agars or alginate, has been intensively studied in this model bacterium, but the catabolism of plant-like polysaccharides is essentially uncharacterized. Here, we identify a polysaccharide utilization locus in the genome of Z. galactanivorans, induced by laminarin (β-1,3-glucans), and containing a putative GH5 subfamily 4 (GH5_4) enzyme, currently annotated as a endoglucanase (ZgEngAGH5_4). A phylogenetic analysis indicates that ZgEngAGH5_4 was laterally acquired from an ancestral Actinobacteria. We performed the biochemical and structural characterization of ZgEngAGH5_4 and demonstrated that this GH5 is, in fact, an endo-β-glucanase, most active on mixed-linked glucan (MLG). Although ZgEngAGH5_4 and GH16 lichenases both hydrolyze MLG, these two types of enzymes release different series of oligosaccharides. Structural analyses of ZgEngAGH5_4 reveal that all the amino acid residues involved in the catalytic triad and in the negative glucose-binding subsites are conserved, when compared with the closest relative, the cellulase EngD from Clostridium cellulovorans, and some other GH5s. In contrast, the positive glucose-binding subsites of ZgEngAGH5_4 are different and this could explain the preference for MLG, with respect to cellulose or laminarin. Molecular dynamics computer simulations using different hexaoses reveal that the specificity for MLG occurs through the +1 and +2 subsites of the binding pocket that display the most important differences when compared with the structures of other GH5_4 enzymes.

„Pogranicze” to słowo-klucz współczesnej humanistyki. Rozumiane szeroko (biorąc pod uwagę zarówno jego aspekt geograficzny, kulturowy jak i interpersonalny) dotyczy wielu paradygmatów współczesnej nauki. W niniejszym szkicu kategorię pogranicza powiążemy z myślą filozoficzną Michaiła Bachtina, Tzvetana Todorova, Martina Bubera i Emmanuela Levinasa. Pogranicze to spotkanie „ja” z innym (tym „ja” lub innym może być jednostka, grupa, kultura), nieuniknione i stanowiące o sensie życia, oparte na niewspółobecności (Bachtin, Todorov), odpowiedzialności (Buber, Levinas), pełnym oddaniu, obustronnym wzbogaceniu światopoglądowym (kulturowym), sprawiedliwości, próbie przezwyciężania różnic, rozbieżności w kwestiach fundamentalnych.

Stanisław Vincenz’s Na wysokiej połoninie in the context of the categories of frontier and geopoeticsStanisław Vincenz’s monumental work Na wysokiej połoninie [On the High Mountain Pastures] can be analysed in the context of civilisational transformations that took place at the turn of the 20th century in highland regions. Carpathian highlanders, including the Hutsuls, for centuries had cultivated traditions going way back in time, to the Hellenic herding culture values like subordination to the laws of nature and God, freedom, highland time, autumn knowledge, dialogue, unique understanding of space. The development of capitalism and influence of urban culture brought with them a possibility of coming into contact on a large scale with a worldview representing a different way of looking at issues fundamental to the individuals and entire community.Thus Vincenz’ tetralogy is set at the frontier of epochs, cultures and mentalities views of the world, impressions of the world. The present article aims at interpreting Vincenz’ work in the context of a category that is extremely popular in the humanities, namely frontier or borderland as well as Kenneth White’s category of geopoetics, which is attracting more and more “followers”.

It has been a long-standing question how the two leaflets in a lipid bilayer modulate each others' physical properties. In this paper, we discuss how this interaction may take place through interdigitation. We use atomistic molecular dynamics simulations to consider asymmetric lipid membrane models whose compositions are based on the lipidomics data determined for exosomes released by PC-3 prostate cancer cells. The simulations show interdigitation to be exceptionally strong for long-chain sphingomyelin (SM) molecules. In asymmetric membranes the amide-linked chain of SM is observed to extend deep into the opposing membrane leaflet. Interestingly, we find that the conformational order of the amide-linked SM chain increases the deeper it penetrates to the opposing leaflet. Analysis of this finding reveals that the amide-linked SM chain interacts favorably with the lipid chains in the opposite leaflet, and that cholesterol modulates the effect of SM interdigitation by influencing the conformational order of lipid hydrocarbon chains in the opposing (cytosolic) leaflet.

Cell walls of marine macroalgae are composed of diverse polysaccharides that provide abundant carbon sources for marine heterotrophic bacteria. Among them

peer-reviewed Transformation of cellulose into monosaccharides can be achieved by hydrolysis of the cellulose chains, carried out by a special group of enzymes known as cellulases. The enzymatic mechanism of cellulases is well described, but the role of non-enzymatic components of the cellulose-degradation machinery is still poorly understood, and difficult to measure using experiments alone. In this study, we use a comprehensive set of atomistic molecular dynamics simulations to probe the molecular details of binding of the family-3a carbohydrate-binding module (CBM3a) and the bacterial expansin protein (EXLX1) to a range of cellulose substrates. Our results suggest that CBM3a behaves in a similar way on both crystalline and amorphous cellulose, whereas binding of the dual-domain expansin protein depends on the substrate crystallinity, and we relate our computed binding modes to the experimentally measured features of CBM and expansin action on cellulose.

The conversion of cellulosic biomass into biofuels requires degradation of the biomass into fermentable sugars. The most efficient natural cellulase system for carrying out this conversion is an extracellular multi-enzymatic complex named the cellulosome. In addition to temperature and pH stability, mechanical stability is important for functioning of cellulosome domains, and experimental techniques such as Single Molecule Force Spectroscopy (SMFS) have been used to measure the mechanical strength of several cellulosomal proteins. Molecular dynamics computer simulations provide complementary atomic-resolution quantitative maps of domain mechanical stability for identification of experimental leads for protein stabilization. In this study, we used multi-scale steered molecular dynamics computer simulations, benchmarked against new SMFS measurements, to measure the intermolecular contacts that confer high mechanical stability to a family 3 Carbohydrate Binding Module protein (CBM3) derived from the archetypal Clostridium thermocellum cellulosome. Our data predicts that electrostatic interactions in the calcium binding pocket modulate the mechanostability of the cellulose-binding module, which provides an additional design rule for the rational re-engineering of designer cellulosomes for biotechnology. Our data offers new molecular insights into the origins of mechanostability in cellulose binding domains and gives leads for synthesis of more robust cellulose-binding protein modules. On the other hand, simulations predict that insertion of a flexible strand can promote alternative unfolding pathways and dramatically reduce the mechanostability of the carbohydrate binding module, which gives routes to rational design of tailormade fingerprint complexes for force spectroscopy experiments.

Integrins are major players in cell adhesion and migration, and malfunctions in controlling their activity are associated with various diseases. Nevertheless, the details of integrin activation are not completely understood, and the role of lipids in the process is largely unknown. Herein, we show using atomistic molecular dynamics simulations that the interplay of phosphatidylinositol 4,5-bisphosphate (PIP2) and talin may directly alter the conformation of integrin αIIbβ3. Our results provide a new perspective on the role of PIP2 in integrin activation and indicate that the charged PIP2 lipid headgroup can perturb a clasp at the cytoplasmic face of the integrin heterodimer.

Phosphatidylinositol-transfer proteins (PITPs) regulate phosphoinositide signaling in eukaryotic cells. The defining feature of PITPs is their ability to exchange phosphatidylinositol (PtdIns) molecules between membranes, and this property is central to PITP-mediated regulation of lipid signaling. However, the details of the PITP-mediated lipid exchange cycle remain entirely obscure. Here, all-atom molecular dynamics simulations of the mammalian StART-like PtdIns/phosphatidylcholine (PtdCho) transfer protein PITP alpha, both on membrane bilayers and in solvated systems, informed downstream biochemical analyses that tested key aspects of the hypotheses generated by the molecular dynamics simulations. These studies provided five key insights into the PITP alpha lipid exchange cycle: (i) interaction of PITP alpha with the membrane is spontaneous and mediated by four specific protein substructures; (ii) the ability of PITP alpha to initiate closure around the PtdCho ligand is accompanied by loss of flexibility of two helix/loop regions, as well as of the C-terminal helix; (iii) the energy barrier of phospholipid extraction from the membrane is lowered by a network of hydrogen bonds between the lipid molecule and PITP alpha; (iv) the trajectory of PtdIns or PtdCho into and through the lipidbinding pocket is chaperoned by sets of PITP alpha residues conserved throughout the StART-like PITP family; and (v) conformational transitions in the C-terminal helix have specific functional involvements in PtdIns transfer activity. Taken together, these findings provide the first mechanistic description of key aspects of the PITP alpha PtdIns/PtdCho exchange cycle and offer a rationale for the high conservation of particular sets of residues across evolutionarily distant members of the metazoan StART-like PITP family.

Proteins embedded in the plasma membrane mediate interactions with the cell environment and play decisive roles in many signaling events. For cell-cell recognition molecules, it is highly likely that their structures and behavior have been optimized in ways that overcome the limitations of membrane tethering. In particular, the ligand binding regions of these proteins likely need to be maximally exposed. Here we show by means of atomistic simulations of membrane-bound CD2, a small cell adhesion receptor expressed by human T-cells and natural killer cells, that the presentation of its ectodomain is highly dependent on membrane lipids and receptor glycosylation acting in apparent unison. Detailed analysis shows that the underlying mechanism is based on electrostatic interactions complemented by steric interactions between glycans in the protein and the membrane surface. The findings are significant for understanding the factors that render membrane receptors accessible for binding and signaling. publishedVersion

Mucormycosis is a rare disease caused by fungi of the genus Mucorales . It is most common in patients with inadequately controlled diabetes mellitus, in transplant recipients or in patients with cancer. A 41-year-old patient was referred to the ENT Department of Medical University of Warsaw with a diagnosis of sinusitis and blindness of the left eye. In an interview swelling tissue of the left orbital cavity, ptosis and loss of vision in the left eye was reported. Clinical CT and MRI revealed swelling of the left half of the face and left orbital fat tissue, massive mucosal thickening in the maxillary sinus and the sphenoid sinus on the left side, encephalitis of the left frontal lobe base, poor contrast enhancement in left cavernous sinus, and hard palate ulceration on the left side. After histopathological confirmation of fungal infection of the paranasal sinuses, the patient underwent endoscopic sphenoethmoidectomy with opening of the left orbit, limited maxillectomy. The patient was treated conservatively with liposomal form of amphotericin B. Two weeks after surgery, MRI revealed an abscess of the left frontal lobe base and the base of the skull. Sinus surgery with skull base and hard palate reconstruction and also left-sided enucleation was performed. Conservative treatment was continued which resulted in an improvement. Surgery is an independent factor for improving survival in patients with invasive fungal rhinosinusitis. Important factors for the patients’ survival are spread of the disease to the central nervous system and diabetes, which can cause a double increase in mortality.

Introduction Endoscopic-assisted microsurgery of the middle ear enables the field of view of the surgeon to be expanded during the removal of inflammatory tissue from the tympanic cavity and during myringo- and ossiculoplasty. Canal wall up tympanoplasty with posterior tympanotomy is a gold standard in surgical treatment of chronic otitis media. Most applications of endoscopy in middle ear surgery concern exclusively the endoscopic transcanal approach. Aim To determine the usefulness of endoscopic visualization during the standard surgical approach through the posterior tympanotomy. Material and methods The study compared the visualization of the elements of the middle ear through the posterior tympanotomy by endoscopes with 30° and 45° optics and a microscope. Posterior tympanotomy was performed in eleven temporal bones. Visualization of the tympanic recesses was assessed on a subjective scale. A microscope and 30° and 45° endoscopes were used for inspection of the hypotympanum, sinus tympani, Eustachian tube, Prussak's space and footplate. Friedman ANOVA test and Dunn's multiple comparisons test were used for statistical analysis of the data. Results Visualization of particular recesses by endoscopes, both 30° and 45°, was excellent, while the microscopic view was statistically significantly worse, especially for sinus tympani, Prussak's space and footplate. There were no significant differences in visibility of the middle ear spaces between the two types of endoscopic optics. Conclusions Additional application of the endoscopes during middle ear surgery provides valuable information due to excellent visualization of key recesses usually hidden from the microscope.

Introduction Intraoperative use of endoscopy in the treatment of chronic otitis media is one of the trends in otology. The aim of this study was to evaluate the use of the endoscope as an additional tool during canal wall up tympanoplasty with posterior tympanotomy ( endoscopy - assisted tympanoplasty) . Material and methods The study included eighty patients with chronic otitis media, the average age being 42.8 (+/-17.9) years. Intraoperative recordings endoscopic and microscopic camera were collected and visibility of anterior recess of epitympanum, sinus tympani, Eustachian tube, myryngo- and ossiculoplasty were compared on a visual analogue scale. The average value of air bone gap preoperative was compared to postoperative in main frequencies and statistical analysis by Wilcoxon test was performed. Results Optic of microscope allowed the partial visualization of anterior recess of epitympanum in 68% of the ears, while the endoscope allowed in 68% for total control of attic. Sinus tympani was completely visible in 64% of the ears in endoscopic optics, while it was not available for visual inspection in 80% of the ears in microscopic optic. Visibility of the Eustachian tube was poor in 78% of the ears in microscope, while in the endoscopic optics accurate insight into the Eustachian tube was achieved in 58% of the evaluable ears. An endoscope provided the total visibility of the tympanic membrane graft in 90% of the ears, while the microscope in 64% of the cases allowed only poor control of myryngoplasty. The endoscope has enabled the total evaluation of ossiculoplasty in 56% of the cases, and in further 24% of control was assessed on 4 points on 5-point scale, while the optics of microscope allowed to be exposed to 2 points in the adopted scale in 82% of ears. Average air bone gap postoperative statistically significant decreased in comparison to preoperative value (p = 0.0277). Conclusions Additional use of the endoscope through posterior tympanotomy during canal wall up tympanoplasty allows for much better visualization of recesses of the tympanic cavity and reconstruction of the conduction system of the middle ear.

In this Data in Brief article we provide a data package of GROMACS input files for atomistic molecular dynamics simulations of multicomponent, asymmetric lipid bilayers using the OPLS-AA force field. These data include 14 model bilayers composed of 8 different lipid molecules. The lipids present in these models are: cholesterol (CHOL), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidyl-ethanolamine (SOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (SOPS), N-palmitoyl-D-erythro-sphingosyl-phosphatidylcholine (SM16), and N-lignoceroyl-D-erythro-sphingosyl-phosphatidylcholine (SM24). The bilayers׳ compositions are based on lipidomic studies of PC-3 prostate cancer cells and exosomes discussed in Llorente et al. (2013) [1], showing an increase in the section of long-tail lipid species (SOPS, SOPE, and SM24) in the exosomes. Former knowledge about lipid asymmetry in cell membranes was accounted for in the models, meaning that the model of the inner leaflet is composed of a mixture of PC, PS, PE, and cholesterol, while the extracellular leaflet is composed of SM, PC and cholesterol discussed in Van Meer et al. (2008) [2]. The provided data include lipids׳ topologies, equilibrated structures of asymmetric bilayers, all force field parameters, and input files with parameters describing simulation conditions (md.mdp). The data is associated with the research article “Interdigitation of Long-Chain Sphingomyelin Induces Coupling of Membrane Leaflets in a Cholesterol Dependent Manner” (Róg et al., 2016) [3]., Graphical abstract

Introduction : The anterior epitympanum recess (AER) is a common place of the development of the cholesteatoma, which is why removal of the matrix from this area plays a key role in the surgical treatment of chronic otitis media. Aim : To evaluate the intraoperative visibility of AER in endoscopic optics in comparison to microscopic optics and to determine the prevalence of cholesteatoma in various types of construction of the AER. Study design: Retrospective analysis of intraoperative search. Material and methods : The study included 55 patients treated in the Department of Otolaryngology, Medical University of Warsaw within the years 2009–2011, who underwent endoscopy-assisted canal wall up tympanoplasty with posterior tympanotomy. The type of construction of the AER – cellular or dome-shaped – was determined. Results : Cellular type of recess was found intraoperatively in 32% of ears and dome-shaped in 68% of the study group. The population with chronic otitis media does not differ significantly compared to the general population in terms of the construction of the anterior epitympanum recess (p = 0.668108; χ 2 = 0.1838235, df = 1). Among the ears with cholesteatoma a cellular AER was found in 48.3% of cases and a dome-shaped AER was found in 51.7%. Conclusions : The cellular type of AER was significantly more frequent in ears with cholesteatoma (p < 0.01, χ 2 = 29.86492, df = 1). Level of evidence: 1b.

J. Neurochem. (2012) 122, 681–690. Abstract The interactions of the neurotransmitter dopamine, and its precursor l-dopa, with membrane lipids were investigated through a set of molecular dynamic simulations with all atom resolution. The results obtained indicate that both dopamine and l-dopa have a pronounced association with the lipid head groups, predominantly mediated through H-bonds. As a result the molecules are anchored to the interfacial region of the membrane. The strength of this interaction is dependent on lipid composition – the presence of phosphatidylserine leads to an increase in the strength of this interaction, resulting in an H-bond network with a lifetime much longer than the timescale of our simulations. Also, bilayers that include sphingomieline and cholesterol interact strongly with dopamine and l-dopa. We postulate that the high membrane association that we have observed for both dopamine and l-dopa could have the following effects: 1) when on the plasma membrane exterior, favour the availability of these compounds for cell membrane uptake processes and, 2) when on an internal membrane surface, accentuate the importance of membrane-bound metabolizing enzymes over their soluble counterparts.

We have combined Langmuir monolayer film experiments and all-atom molecular dynamics (MD) simulation of a bilayer to study the surface structure of a PEGylated liposome and its interaction with the ionic environment present under physiological conditions. Lipids that form both gel and liquid-crystalline membranes have been used in our study. By varying the salt concentration in the Langmuir film experiment and including salt at the physiological level in the simulation, we have studied the effect of salt ions present in the blood plasma on the structure of the poly(ethylene glycol) (PEG) layer. We have also studied the interaction between the PEG layer and the lipid bilayer in both the liquid-crystalline and gel states. The MD simulation shows two clear results: (a) The Na(+) ions form close interactions with the PEG oxygens, with the PEG chains forming loops around them and (b) PEG penetrates the lipid core of the membrane for the case of a liquid-crystalline membrane but is excluded from the tighter structure of the gel membrane. The Langmuir monolayer results indicate that the salt concentration affects the PEGylated lipid system, and these results can be interpreted in a fashion that is in agreement with the results of our MD simulation. We conclude that the currently accepted picture of the PEG surface layer acting as a generic neutral hydrophilic polymer entirely outside the membrane, with its effect explained through steric interactions, is not sufficient. The phenomena we have observed may affect both the interaction between the liposome and bloodstream proteins and the liquid-crystalline-gel transition and is thus relevant to nanotechnological drug delivery device design.

Transformation of cellulose into monosaccharides can be achieved in a chemical process performed by a special group of enzymes known as cellulases. We have used atomistic molecular dynamics simulations to study endoglucanase II (Cel5A) that is one of the proteins in this group. Based on the atomistic simulation results, we discuss how the Cel5A enzyme interacts with cellulose fibrils comprised of both crystalline and amorphous regions. We show that the enzyme’s carbohydrate-binding domain prefers to interact with crystalline regions of cellulose, while the catalytic domain has a high affinity to the amorphous regions of fibrils. In particular, through electrostatic interactions the catalytic domain attracts loose glucose chains to its catalytic cleft. The atomistic details of the enzyme–cellulose interaction are presented and the implications for practical applications are briefly discussed.

The epidermal growth factor receptor (EGFR) regulates several critical cellular processes and is an important target for cancer therapy. In lieu of a crystallographic structure of the complete receptor, atomistic molecular dynamics (MD) simulations have recently shown that they can excel in studies of the full-length receptor. Here we present atomistic MD simulations of the monomeric N-glycosylated human EGFR in biomimetic lipid bilayers that are, in parallel, also used for the reconstitution of full-length receptors. This combination enabled us to experimentally validate our simulations, using ligand binding assays and antibodies to monitor the conformational properties of the receptor reconstituted into membranes. We find that N-glycosylation is a critical determinant of EGFR conformation, and specifically the orientation of the EGFR ectodomain relative to the membrane. In the absence of a structure for full-length, posttranslationally modified membrane receptors, our approach offers new means to structurally define and experimentally validate functional properties of cell surface receptors in biomimetic membrane environments.

Long-chain sphingomyelin (SM) and glycolipids are not among the most abundant species of lipids, however they can be enriched in specific cells or organelles. For example, lipidomics data determined for exosomes released by PC-3 prostate cancer cells showed that exosomes are particularly enriched in long-chain SM (Biochim. Biophys.Acta, 2013, 1831, 1302–1309). In this study, we use atomistic molecular dynamics simulations to consider asymmetric lipid membrane models whose compositions are based on these data. The simulations show interdigitation of the long-chain SM to the opposite leaflet, which is quite expected, but interestingly we found out that interdigitation was particularly strong in asymmetric bilayers compared to symmetric ones. We observed that the conformational order of the amide-linked SM chain increases the deeper it penetrates to the opposing leaflet. We further showed that cholesterol modulates the effect of SM interdigitation by influencing the conformational order of lipid hydrocarbon chains in the opposing (cytosolic) leaflet.

The molecular lipid composition of exosomes is largely unknown. In this study, sophisticated shotgun and targeted molecular lipidomic assays were performed for in-depth analysis of the lipidomes of the metastatic prostate cancer cell line, PC-3, and their released exosomes. This study, based in the quantification of approximately 280 molecular lipid species, provides the most extensive lipid analysis of cells and exosomes to date. Interestingly, major differences were found in the lipid composition of exosomes compared to parent cells. Exosomes show a remarkable enrichment of distinct lipids, demonstrating an extraordinary discrimination of lipids sorted into these microvesicles. In particular, exosomes are highly enriched in glycosphingolipids, sphingomyelin, cholesterol, and phosphatidylserine (mol% of total lipids). Furthermore, lipid species, even of classes not enriched in exosomes, were selectively included in exosomes. Finally, it was found that there is an 8.4-fold enrichment of lipids per mg of protein in exosomes. The detailed lipid composition provided in this study may be useful to understand the mechanism of exosome formation, release and function. Several of the lipids enriched in exosomes could potentially be used as cancer biomarkers.

Glycosylation where glycans are enzymatically attached to proteins is a common post-translational modification among many membrane protein families. The glycans associated with glycosylation serve a variety of structural and functional roles in protein structures, such as recognition and cell-cell adhesion. Determination of glycosylation in membrane protein structures is a complicated matter, though, since due to methodological limitations and the soft nature of glycans, the structure of the crystallized membrane proteins does not include the information arising from glycosylation. Here we use extensive atomistic molecular dynamics simulations together with experimental ligand binding assays and antibodies (1) to show for the N-glycosylated human epidermal growth factor receptor (EGFR) in biomimetic lipid bilayers that N-glycosylation is a crucial determinant of EGFR conformation, and specifically the orientation of the EGFR ectodomain relative to the membrane. It is expected that similar conclusions as to the importance of glycosylation in protein conformational behavior can be drawn for a number of other membrane proteins, too.(1) K. Kaszuba et al., Proc. Natl. Acad. Sci. USA 112, 4334 (2015).

Fullerene C(70) is known to partition into lipid membranes and change their physical properties. Together with gallic acid (GA), C(70) induces cell contraction and cell death. How C(70) and GA-induced perturbations of lipid membranes affect cellular function and membrane protein activity is not understood, though. Meanwhile, fullerene is also known to interfere with the activity of potassium channel proteins, but the mechanisms of protein inhibition are not known. Here we consider the possibility that membrane protein function would be inhibited by C(70) and/or GA through direct contact or through lipid-mediated interactions. To this end, we use microsecond time scale atomistic simulations to explore (a) modifications of membrane properties in the presence of C(70) and/or GA, and (b) the possible conformational changes in Kv1.2, a voltage-gated potassium channel, upon exposure to C(70), or GA, or both. C(70) is found to have an observable effect on structural and elastic properties of protein-free membranes, while the effects of GA on the membrane are less evident. Fullerene–GA interaction is strong and affects significantly the partitioning of C(70) in the membrane, stabilizing C(70) in the aqueous phase. When Kv1.2 is exposed to the solutes, only small conformational changes are observed on the microsecond time scale – comparable to the fluctuations observed in the absence of any solute. Blocking of the channel entrance is not observed, as fullerene binds mainly to hydrophobic residues, both in the water-exposed loops and in the transmembrane helices. The tilt angle of transmembrane helices in the voltage-sensing domain appears to be affected by direct contact with fullerene, but a generic effect due to the small increase in membrane thickness might also play a role. A small rotation of the S3 and S4 helices in the voltage-sensing domain is noticed when C(70) is embedded in the membrane. The interpretation of the observed conformational changes is not straightforward due to the associated time scales, which are difficult to sample with state-of-the-art computing resources. We cannot exclude that both membrane-mediated interactions and specific protein–solute interactions affect the conformation of the protein.

The interactions of the neurotransmitter dopamine, and its precursor l-dopa, with membrane lipids were investigated through a set of molecular dynamic simulations with all atom resolution. The results obtained indicate that both dopamine and l-dopa have a pronounced association with the lipid head groups, predominantly mediated through H-bonds. As a result the molecules are anchored to the interfacial region of the membrane. The strength of this interaction is dependent on lipid composition - the presence of phosphatidylserine leads to an increase in the strength of this interaction, resulting in an H-bond network with a lifetime much longer than the timescale of our simulations. Also, bilayers that include sphingomieline and cholesterol interact strongly with dopamine and l-dopa. We postulate that the high membrane association that we have observed for both dopamine and l-dopa could have the following effects: 1) when on the plasma membrane exterior, favour the availability of these compounds for cell membrane uptake processes and, 2) when on an internal membrane surface, accentuate the importance of membrane-bound metabolizing enzymes over their soluble counterparts.

Theoretical studies predict hydrophobic matching between transmembrane domains of proteins and bilayer lipids to be a physical mechanism by which membranes laterally self-organize. We now experimentally study the direct consequences of mismatching of transmembrane peptides of different length with bilayers of different thicknesses at the molecular level. In both model membranes and simulations we show that cholesterol critically constrains structural adaptations at the peptide-lipid interface under mismatch. These constraints translate into a sorting potential and lead to selective lateral segregation of peptides and lipids according to their hydrophobic length.

We used atomistic simulations to study the membrane-bound form of catechol-O-methyltransferase (MB-COMT). In particular we investigated the 26-residue transmembrane a-helical segment of MB-COMT together with the 24-residue fragment that links the transmembrane component to the main protein unit that was not included in our model. In numerous independent simulations we observed the formation of a salt bridge between ARC 27 and GLU40. The salt bridge closed the flexible loop that formed in the linker and kept it in the vicinity of the membrane-water interface. All simulations supported this conclusion that the linker has a clear affinity for the interface and preferentially arranges its residues to reside next to the membrane, without a tendency to relocate into the water phase. Furthermore, an extensive analysis of databases for sequences of membrane proteins that have a single transmembrane helical segment brought about an interesting view that the flexible loop observed in our work can be a common structural element in these types of proteins. In the same spirit we close the article by discussing the role of salt bridges in the formation of three-dimensional structures of membrane proteins that exhibit a single transmembrane helix.

The aim of the study was to answer the questions about the quality of dialysis for the patients treated in the selected dialysis centres of the Lublin region in relation to a quantitative and qualitative breakthrough observed in the field of Polish dialysis therapy in recent years. The study including 288 patients in 7 medium-sized centres used the questionnaire filled in by interviewers. The present paper analyses the patients' answers to the questions about medical care provided in a particular centre. It is difficult to interpret the results explicity since there are no available standards of such management. We believe that this study could be used to define them. Nevertheless, the authors think that medical care for the patients treated in dialysis centres of our region is good, although only the patients' answers were analysed. To evaluate possible changes in the processes mentioned above, it should be worthy to broaden the studies including the remaining centres of our region and to continue them in the future.

The aim of the paper was to answer the questions about the quality of life of dialysis patients treated in the selected dialysis centres of the Lublin region in relation to the recent quantitative and qualitative breakthrough in dialysis therapy in Poland. The studies including 288 patients of 7 medium-sized centres were performed using the questionnaires filled in by interviewers. The present paper describes the results of analysis of the questions addressed to the centre staff but concerning the way of treatment of a particular patient. The results show that to follow the world-wide trends, chronic dialysis treatment should be of an optimum quality and the methods evaluating it widely propagated. Moreover, much is to be done in the field of the latest diagnostic procedures and therapy of dialysis patients. On the other hand, the technical aspects of dialysis therapy look quite good. To evaluate the changes in the aforementioned processes more accurately, it should be worthy to broaden the studies including the remaining centres of our region and to continue them in the future.

Atomistic molecular dynamics simulations were used to investigate the interactions between the neurotransmitter dopamine and its precursor L-dopa with membrane lipids. The results indicate that these molecules strongly interact with the lipid head groups e.g. via hydrogen bonds. These interactions anchor the dopamine and L-dopa to the membrane interfacial region. The strength of this bonding is dependent on lipids present in membrane. Presence of phosphatidylserine resulted in increased level of bonding strength with a lifetime longer than the timescale of our simulations.The high membrane association of dopamine and L-dopa both, extracellularly, might favour the availability of these compounds for cell membrane uptake processes and, intracellularly, can accentuate the importance of membrane-bound metabolizing enzymes over their soluble counterparts.We can also hypothesize that excessive association of dopamine and its precursor L-dopa with the membranes in the situation, when the concentration of phosphatidylserine is increased, may eventually limit the free use of dopamine as a synaptic transmitter what could possibly be a molecular level mechanism responsible for some of neurodegenerative disorders.View Large Image | View Hi-Res Image | Download PowerPoint Slide

A Sec14-nodulin protein model is used to identify the nodulin domain as a novel phosphoinositide effector module with a role in controlling lateral organization of phosphoinositide. The domain organization of Sec14-nodulin proteins suggests a versatile principle for the bit mapping of membrane surfaces into high-definition lipid-signaling screens., Polarized membrane morphogenesis is a fundamental activity of eukaryotic cells. This process is essential for the biology of cells and tissues, and its execution demands exquisite temporal coordination of functionally diverse membrane signaling reactions with high spatial resolution. Moreover, mechanisms must exist to establish and preserve such organization in the face of randomizing forces that would diffuse it. Here we identify the conserved AtSfh1 Sec14-nodulin protein as a novel effector of phosphoinositide signaling in the extreme polarized membrane growth program exhibited by growing Arabidopsis root hairs. The data are consistent with Sec14-nodulin proteins controlling the lateral organization of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) landmarks for polarized membrane morphogenesis in plants. This patterning activity requires both the PtdIns(4,5)P2 binding and homo-oligomerization activities of the AtSfh1 nodulin domain and is an essential aspect of the polarity signaling program in root hairs. Finally, the data suggest a general principle for how the phosphoinositide signaling landscape is physically bit mapped so that eukaryotic cells are able to convert a membrane surface into a high-definition lipid-signaling screen.