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Discusses the 2.0-A resolution crystal structure of the complex between ethyl ester of epoxysuccinyl-ile-pro-OH (CA030) and human cathepsin, a lysosomal cysteine protease. Electron density appearance of CA030; Structure of CA030; Sunstratelike interaction in CA030 sites; Role of residues His 110 and His 111 as receptors of a peptidic substrate C-terminal group.

To examine the relationship between transvaginal ultrasound cervical changes and pregnancy outcome and ultrasound elastography strain measurements of cervical stiffness in women at risk of cervical incompetency and preterm delivery Strain measurements values > 0,90 were associated with cervical incompetency and strain values > 0,98 were associated with preterm delivery. A shortened cervix with decreased thicknes of the anterior wall of lower uterine segment and dilatated endocervical canal together with ultrasound elastography strain measurements of cervical stiffness shows a strong association with cervical incompetency and risk for preterm delivery. [Extracted from the article]

For early amniocentesis we obtained amniotic fluid (3-5ml) by amnio-vacucentesis method using 21-22 G needle, and for TA-CVS using 20 G needle, both with continuous ultrasound guidance. TA-CVS and EA are a safe method of prenatal diagnosis for high-risk couples and does not significantly affect the pregnancy. EA obtained by amnio-vacucentesis method between 12 to 14 weeks is not associated with a greater risk of spontaneous miscarriage, neonatal talipes and fetal anomalies compared to TA-CVS. [Extracted from the article]

This study comprises data from 7055 (45,4%) women allocated to early transabdominal CVS (TA-CVS) and 8500 (54,6%) women allocated to late CVS (placental biopsy). The spontaneous abortion rate was lower among cases allocated to TA-CVS after 11 weeks of gestation. Mosaicisms was found in 87 (1,2%) cases after early CVS and 95 (1,1%) cases after late CVS and there were no significant differences. [Extracted from the article]

The purpose of this article was to evaluate the association between fetal cystic hygroma detected in the first trimester of pregnancy and cytogenetic abnormalities, and the long-term prognosis. We studied the outcome of fetuses in whom cystic hygroma was diagnosed in the first and early second-trimester of pregnancy using transabdominal and transvaginal ultrasonography (3D/4D ultrasonography). 357 consecutive fetuses between 8,0 and 14,0 weeks of gestation diagnosed as having a nuchal hygroma were evaluated ultrasonographically and karyotyped with transabdominal chorionic vilus sampling. [Extracted from the article]

EP04.07: Cell-free DNA testing for common chromosomal aneuploidies: data from a single centre in a routine screening population Non- invasive prenatal testing (NIPT) by analysis of cell-free DNA (cfDNA) from maternal blood has shown promise for highly accurate detection of common fetal trisomies. Among 22 (1,9%) high risk results there were 13 Trisomies 21, 2 Trisomies 18, 1 Trisomy 13, 1 45X0, 1 CriDu Chat, 1 Trisomy 5, 1 Trisomy 2 and 1 duplication 9 chr. [Extracted from the article]

An abstract of the article "Early detection (8 + 2 weeks) of sonographic markers that are predictive of fetal Turner syndrome," by P. Podobnik Brleč ić, B. Gebauer Vuković , F. Stipoljev, I. Brlečić, and M. Podobnik is presented.

An abstract of the conference paper "Twin reversed arterial perfusion (TRAP) sequence: Case report of conservative management and good pregnancy outcome," by P. Podobnik and colleagues is presented.

An abstract of the article "Late CVS: Experience with 5800 cases," by M. Podobnik and colleagues, is presented.

Objective: To determine the feasibility of correctly identifying fetal gender from 11 to 13 weeks of gestation. Methods: Fetal gender assessment by ultrasound was prospectively carried out in 425 singleton pregnancies at 11–13 weeks of gestation (confirmed by crown-rump length or biparietal diameter) immediately before chorionic villus sampling for karyo-typing. A total of 425 women underwent a detailed assessment of fetal anatomy at 11–13 weeks of gestation by means of transabdominal and transvaginal sonography. Fetal gender was identified in transverse and sagital planes, and was confirmed by chorionic villus sampling. Results: The accuracy of sex determination increased with gestation from 91.7% at 11 weeks, to 97.2% at 12 weeks and 100% at 13 weeks. Male fetus were wrongly assigned as female in 5.5% of cases in 11 weeks, 2.4% at 12 weeks and 0% at 13 weeks. The accuracy of correctly identifying fetal gender increased with gestational age. Conclusion: Whilst the accuracy of sonographic determination of fetal gender at 11–13 weeks is good, our decision on invasive testing for sex-linked conditions should be undertaken only after 12 weeks of gestation. [ABSTRACT FROM AUTHOR]

Background: The aim of this study is to confirm the relationship between the gestational age and quantitative assessment of ultrasonic signs of placental tissue, fetal lungs and liver tissue and to correlate Doppler parameters from main stems of the pulmonary arteries for determining fetal lung maturity in normal pregnancy and pregnancy with pre-eclampsia and diabetes. Methods: The placenta, fetal lungs and fetal liver in 300 normal pregnancies, 100 pre-eclamptic pregnancies and 100 diabetic pregnancy were examined by ultrasound at 30–41 weeks of gestation. The coefficients of variation (the standard deviation divided by the mean value) were used to characterize the tissue in different groups during pregnancy. Doppler velocimetry was performed in the main stems of pulmonary arteries in normal pregnancies and pregnancies with pre-eclampsia and diabetes. Results: The coefficients of variation in mature fetuses were greater than 29% for placentas in vivo, greater than 34% for placentas in vitro, greater than 28% for liver tissue and greater than 30% for lung tissue. Placental, lung and liver tissue of diabetic pregnancy tended to have lower coefficients of variation throughout their pregnancies. We found in mature fetuses mean peak systolic velocity higher than 40 cm/s and PI lower than 3.00. In mature fetuses with pre-eclampsia, we found higher mean peak systolic velocity in compared with normotensive patients. Conclusions: The coefficient of variation values for placentas in vivo and in vitro, and fetal lungs and liver increase during pregnancy in normal and pre-eclamptic patients with increasing gestational age and decrease in diabetic patients. [ABSTRACT FROM AUTHOR]

Background: To examine the relationship between transvaginal ultrasound cervical changes and pregnancy outcome in women at risk of cervical incompetence and preterm delivery. Methods: In 100 pregnancies with clinical and ultrasonic signs of cervical incompetence, the length of the cervix, the thickness of the anterior wall of a lower uterine segment and the width of the endo-cervical canal have been evaluated ultrasonically. These parameters were followed longitudinally from the 10 to 36 week gestation in the healthy pregnancies and pregnancies at risk for cervical incompetence and preterm delivery. Results: In patients from 10 to 14 weeks, the cervix is significantly longer than in the 25–36 week group and the anterior wall of the lower uterine segment in the 10–14 week group is significantly thicker than in the 20–36 week group. In pregnancies at risk for cervical incompetence, cervical lengths and wall thickness, were significantly different from those in comparable controls. Forty-five percent of the patients in the at-risk group, with cervical cerclage, delivered at 37.5 weeks and 8.5% of pregnancies ended in abortion when the amniotic membrane herniated into the cervical canal. The frequency of preterm delivery was 60, 38 and 15% for cervical length of <10, 10–20, and 20–30 mm. Conclusions: A shortened cervix with decreased thickness of the anterior wall of lower uterine segment and dilated endo-cervical canal shows a strong association with cervical incompetence and preterm birth. [ABSTRACT FROM AUTHOR]

The crucial molecular factors that shape the interfaces of lipid-binding proteins with their target ligands and surfaces remain unknown due to the complex makeup of biological membranes. Cholesterol, the major modulator of bilayer structure in mammalian cell membranes, is recognized by various proteins, including the well-studied cholesterol-dependent cytolysins. Here, we use in vitro evolution to investigate the molecular adaptations that preserve the cholesterol specificity of perfringolysin O, the prototypical cholesterol-dependent cytolysin from Clostridium perfringens. We identify variants with altered membrane-binding interfaces whose cholesterol-specific activity exceeds that of the wild-type perfringolysin O. These novel variants represent alternative evolutionary outcomes and have mutations at conserved positions that can only accumulate when epistatic constraints are alleviated. Our results improve the current understanding of the biochemical malleability of the surface of a lipid-binding protein., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Membrane lipids and proteins form dynamic domains crucial for physiological and pathophysiological processes, including viral infection. Many plasma membrane proteins, residing within membrane domains enriched with cholesterol (CHOL) and sphingomyelin (SM), serve as receptors for attachment and entry of viruses into the host cell. Among these, human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use proteins associated with membrane domains for initial binding and internalization. We hypothesized that the interaction of lipid-binding proteins with CHOL in plasma membrane could sequestrate lipids and thus affect the efficiency of virus entry into host cells, preventing the initial steps of viral infection. We have prepared CHOL-binding proteins with high affinities for lipids in the plasma membrane of mammalian cells. Binding of the perfringolysin O domain four (D4) and its variant D4 E458L to membrane CHOL impaired the internalization of the receptor-binding domain of the SARS-CoV-2 spike protein and the pseudovirus complemented with the SARS-CoV-2 spike protein. SARS-CoV-2 replication in Vero E6 cells was also decreased. Overall, our results demonstrate that the integrity of CHOL-rich membrane domains and the accessibility of CHOL in the membrane play an essential role in SARS-CoV-2 cell entry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Gas vesicles (GVs) are large cylindrical gas-filled protein assemblies found in diverse aquatic bacteria that enable their adaptation of buoyancy. GVs have already been used as ultrasound contrasting agents. Here, we investigate GVs derived from Bacillus megaterium , aiming to minimize the number of accessory Gvps within the GV gene cluster and demonstrate the use of GVs as enhancers of acoustic radiation force administered by ultrasound. Three ( GvpR , GvpT , and GvpU ) out of 11 genes in the cluster were found to be dispensable for functional GV formation, and their omission resulted in narrower GVs. Two essential proteins GvpJ and GvpN were absent from recently determined GV structures, but GvpJ was nevertheless found to be tightly bound to the cylindrical part of GVs in this study. Additionally, the N-terminus of GvpN was observed to play an important role in the formation of mature GVs. The binding of engineered GvpC from Anabaena flos-aquae to HEK293 cells via integrins enhanced the acoustic force delivered by ultrasound and resulted in an increased Ca 2+ influx into cells. Coupling with a synthetic Ca 2+ -dependent signaling pathway GVs efficiently enhanced cell stimulation by ultrasound, which expands the potentials of noninvasive sonogenetics cell stimulation.

The zebrafish (Danio rerio) is an important model organism for basic as well as applied bio-medical research. One main advantage is its genetic tractability, which was greatly enhanced by the introduction of the CRISPR/Cas method a decade ago. The generation of loss-of-function alleles via the production of small insertions or deletions in the coding sequences of genes with CRISPR/Cas systems is now routinely achieved with high efficiency. The method is based on the error prone repair of precisely targeted DNA double strand breaks by non-homologous end joining (NHEJ) in the cell nucleus. However, editing the genome with base pair precision, by homology-directed repair (HDR), is by far less efficient and therefore often requires large-scale screening of potential carriers by labour intensive genotyping. Here we confirm that the Cas9 protein variant SpRY, with relaxed PAM requirement, can be used to target some sites in the zebrafish genome. In addition, we demonstrate that the incorporation of an artificial nuclear localisation signal (aNLS) into the Cas9 protein variants not only enhances the efficiency of gene knockout but also the frequency of HDR, thereby facilitating the efficient modification of single base pairs in the genome. Our protocols provide a guide for a cost-effective generation of versatile and potent Cas9 protein variants and efficient gene editing in zebrafish., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The structural diversity and tunability of the capsid proteins (CPs) of various icosahedral and rod-shaped viruses have been well studied and exploited in the development of smart hybrid nanoparticles. However, the potential of CPs of the wide-spread flexuous filamentous plant viruses remains to be explored. Here, we show that we can control the shape, size, RNA encapsidation ability, symmetry, stability and surface functionalization of nanoparticles through structure-based design of CP from potato virus Y (PVY). We provide high-resolution insight into CP-based self-assemblies, ranging from large polymorphic or monomorphic filaments to smaller annular, cubic or spherical particles. Furthermore, we show that we can prevent CP self-assembly in bacteria by fusion with a cleavable protein, enabling controlled nanoparticle formation in vitro. Understanding the remarkable structural diversity of PVY CP not only provides possibilities for the production of biodegradable nanoparticles, but may also advance future studies of CP's polymorphism in a biological context., (© 2024. The Author(s).)

Listeriosis is one of the most serious foodborne diseases caused by the intracellular bacterium Listeria monocytogenes. Its two major virulence factors, broad-range phospholipase C (LmPC-PLC) and the pore-forming toxin listeriolysin O (LLO), enable the bacterium to spread in the host by destroying cell membranes. Here, we determine the crystal structure of LmPC-PLC and complement it with the functional analysis of this enzyme. This reveals that LmPC-PLC has evolved several structural features to regulate its activity, including the invariant position of the N-terminal tryptophan (W1), the structurally plastic active site, Zn 2+ -dependent activity, and the tendency to form oligomers with impaired enzymatic activity. We demonstrate that the enzymatic activity of LmPC-PLC can be specifically inhibited by its propeptide added in trans. Furthermore, we show that the phospholipase activity of LmPC-PLC facilitates the pore-forming activity of LLO and affects the morphology of LLO oligomerization on lipid membranes, revealing the multifaceted synergy of the two virulence factors., (© 2023. Springer Nature Limited.)

The bacterium Bacillus thuringiensis (Bt) produces insecticidal proteins during the sporulation phase. These proteins are located in parasporal crystals consisting of two delta-endotoxin classes, crystal (Cry) and cytolytic (Cyt) toxins. In vitro, Cyt toxins show cytolytic activity against bacterial and a variety of insect and mammalian cells. They bind to cell membranes with unsaturated phospholipids and sphingomyelin. Although Bt and its parasporal crystals containing both Cry and Cyt toxins have been successfully used as bioinsecticides, the molecular mechanism of action of Cyt toxins is not yet fully understood. To address this, we exposed Cyt2Aa to lipid membranes and visualized membrane disruption process using cryo-electron microscopy. We observed two types of Cyt2Aa oligomers. First, Cyt2Aa forms smaller curved oligomers on the membrane surface that become linear over time, and detach when the membrane ruptures. Similar linear filamentous oligomers were also formed by Cyt2Aa in the presence of detergents without prior exposure to lipid membranes, which exhibited attenuated cytolytic activity. Furthermore, our data suggest that Cyt2Aa adopts different conformations between its monomeric and oligomeric forms. Overall, our results provide new evidence for a detergent-like mechanism of action of Cyt2Aa rather than the pore-forming model of target membrane disruption of this important class of insecticidal proteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Teleost fish of the genus Danio are excellent models to study the genetic and cellular bases of pigment pattern variation in vertebrates. The two sister species Danio rerio and Danio aesculapii show divergent patterns of horizontal stripes and vertical bars that are partly caused by the divergence of the potassium channel gene kcnj13. Here, we show that kcnj13 is required only in melanophores for interactions with xanthophores and iridophores, which cause location-specific pigment cell shapes and thereby influence colour pattern and contrast in D. rerio. Cis-regulatory rather than protein coding changes underlie kcnj13 divergence between the two Danio species. Our results suggest that homotypic and heterotypic interactions between the pigment cells and their shapes diverged between species by quantitative changes in kcnj13 expression during pigment pattern diversification., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Small particles in natural sources are a subject of interest for their potential role in intercellular, inter-organism, and inter-species interactions, but their harvesting and assessment present a challenge due to their small size and transient identity. We applied a recently developed interferometric light microscopy (ILM) to assess the number density and hydrodynamic radius (R h ) of isolated small cellular particles (SCPs) from blood preparations (plasma and washed erythrocytes) (B), spruce needle homogenate (S), suspension of flagellae of microalgae Tetraselmis chuii (T), conditioned culture media of microalgae Phaeodactylum tricornutum (P), and liposomes (L). The aliquots were also assessed by flow cytometry (FCM), dynamic light scattering (DLS), ultraviolet-visible spectrometry (UV-vis), and imaging by cryogenic transmission electron microscopy (cryo-TEM). In R h , ILM showed agreement with DLS within the measurement error in 10 out of 13 samples and was the only method used here that yielded particle density. Cryo-TEM revealed that representative SCPs from Tetraselmis chuii flagella (T) did not have a globular shape, so the interpretation by R h of the batch methods was biased. Cryo-TEM showed the presence of thin filaments in isolates from Phaeodactylum tricornutum conditioned culture media (P), which provides an explanation for the considerably larger R h obtained by batch methods than the sizes of particles observed by cryo-TEM images. ILM proved convenient for assessment of number density and R h of SCPs in blood preparations (e.g., plasma); therefore, its use in population and clinical studies is indicated.

Pore-forming toxins are an important component of the venom of many animals. Actinoporins are potent cytolysins that were first detected in the venom of sea anemones; however, they are occasionally found in animals other than cnidarians and are expanded in a few predatory gastropods. Here, we report the presence of 27 unique actinoporin-like genes with monophyletic origin in Mytilus galloprovincialis, which we have termed mytiporins. These mytiporins exhibited a remarkable level of molecular diversity and gene presence-absence variation, which warranted further studies aimed at elucidating their functional role. We structurally and functionally characterized mytiporin-1 and found significant differences from the archetypal actinoporin fragaceatoxin C. Mytiporin-1 showed weaker permeabilization activity, no specificity towards sphingomyelin, and weak activity in model lipid systems with negatively charged lipids. In contrast to fragaceatoxin C, which forms octameric pores, functional mytiporin-1 pores on negatively charged lipid membranes were hexameric. Similar hexameric pores were observed for coluporin-26 from Cumia reticulata and a conoporin from Conus andremenezi. This indicates that also other molluscan actinoporin-like proteins differ from fragaceatoxin C. Although the functional role of mytiporins in the context of molluscan physiology remains to be elucidated, the lineage-specific gene family expansion event that characterizes mytiporins indicates that strong selective forces acted on their molecular diversification. Given the tissue distribution of mytiporins, this process may have broadened the taxonomic breadth of their biological targets, which would have important implications for digestive processes or mucosal immunity., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Pore-forming proteins perforate lipid membranes and consequently affect their integrity and cell fitness. Therefore, it is not surprising that many of these proteins from bacteria, fungi, or certain animals act as toxins. While pore-forming proteins have also been found in plants, there is little information about their molecular structure and mode of action. Bryoporin is a protein from the moss Physcomitrium patens, and its corresponding gene was found to be upregulated by various abiotic stresses, especially dehydration, as well as upon fungal infection. Based on the amino acid sequence, it was suggested that bryoporin was related to the actinoporin family of pore-forming proteins, originally discovered in sea anemones. Here, we provide the first detailed structural and functional analysis of this plant cytolysin. The crystal structure of monomeric bryoporin is highly similar to those of actinoporins. Our cryo-EM analysis of its pores showed an actinoporin-like octameric structure, thereby revealing a close kinship of proteins from evolutionarily distant organisms. This was further confirmed by our observation of bryoporin's preferential binding to and formation of pores in membranes containing animal sphingolipids, such as sphingomyelin and ceramide phosphoethanolamine; however, its binding affinity was weaker than that of actinoporin equinatoxin II. We determined bryoporin did not bind to major sphingolipids found in fungi or plants, and its membrane-binding and pore-forming activity was enhanced by various sterols. Our results suggest that bryoporin could represent a part of the moss defense arsenal, acting as a pore-forming toxin against membranes of potential animal pathogens, parasites, or predators., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

We studied the efficiency of three culture series of the microalgae Phaeodactylum tricornutum ( P. tricornutum ) and bacteria Thalassospira sp. (axenic microalgae, bacterial culture and co-culture of the two) in removing bisphenols (BPs) from their growth medium. Bacteria were identified by 16S ribosomal RNA polymerase chain reaction (16S rRNA PCR). The microorganism growth rate was determined by flow cytometry. Cultures and isolates of their small cellular particles (SCPs) were imaged by scanning electron microscopy (SEM) and cryogenic transmission electron microscopy (Cryo-TEM). BPs were analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). Our results indicate that some organisms may have the ability to remove a specific pollutant with high efficiency. P. tricornutum in axenic culture and in mixed culture removed almost all (more than 99%) of BPC2. Notable differences in the removal of 8 out of 18 BPs between the axenic, mixed and bacterial cultures were found. The overall removals of BPs in axenic P. tricornutum , mixed and bacterial cultures were 11%, 18% and 10%, respectively. Finding the respective organisms and creating microbe societies seems to be key for the improvement of wastewater treatment. As a possible mediating factor, numerous small cellular particles from all three cultures were detected by electron microscopy. Further research on the mechanisms of interspecies communication is needed to advance the understanding of microbial communities at the nano-level.

Tumor growth and metastasis strongly rely on cell-cell communication. One of the mechanisms by which tumor cells communicate involves the release and uptake of lipid membrane encapsulated particles full of bioactive molecules, called extracellular vesicles (EVs). EV exchange between cancer cells may induce phenotype changes in the recipient cells. Our work investigated the effect of EVs released by teratocarcinoma cells on glioblastoma (GBM) cells. EVs were isolated by differential centrifugation and analyzed through Western blot, nanoparticle tracking analysis, and electron microscopy. The effect of large EVs on GBM cells was tested through cell migration, proliferation, and drug-sensitivity assays, and resulted in a specific impairment in cell migration with no effects on proliferation and drug-sensitivity. Noticeably, we found the presence of the EGF-CFC founder member CRIPTO on both small and large EVs, in the latter case implicated in the EV-mediated negative regulation of GBM cell migration. Our data let us propose a novel route and function for CRIPTO during tumorigenesis, highlighting a complex scenario regulating its effect, and paving the way to novel strategies to control cell migration, to ultimately improve the prognosis and quality of life of GBM patients.

Microbial plant pathogens secrete a range of effector proteins that damage host plants and consequently constrain global food production. Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) are produced by numerous phytopathogenic microbes that cause important crop diseases. Many NLPs are cytolytic, causing cell death and tissue necrosis by disrupting the plant plasma membrane. Here, we reveal the unique molecular mechanism underlying the membrane damage induced by the cytotoxic model NLP. This membrane disruption is a multistep process that includes electrostatic-driven, plant-specific lipid recognition, shallow membrane binding, protein aggregation, and transient pore formation. The NLP-induced damage is not caused by membrane reorganization or large-scale defects but by small membrane ruptures. This distinct mechanism of lipid membrane disruption is highly adapted to effectively damage plant cells.

Amphibole asbestos is related to lung fibrosis and several types of lung tumors. The disease-triggering mechanisms still challenge our diagnostic capabilities and are still far from being fully understood. The literature focuses primarily on the role and formation of asbestos bodies in lung tissues, but there is a distinct lack of studies on amphibole particles that have been internalized by alveolar epithelial cells (AECs). These internalized particles may directly interact with the cell nucleus and the organelles, exerting a synergistic action with asbestos bodies (AB) from a different location. Here we document the near-atomic- to nano-scale transformations induced by, and taking place within, AECs of three distinct amphiboles (anthophyllite, grunerite, "amosite") with different Fe-content and morphologic features. We show that: (i) an Fe-rich layer is formed on the internalized particles, (ii) particle grain boundaries are transformed abiotically by the internal chemical environment of AECs and/or by a biologically induced mineralization mechanism, (iii) the Fe-rich material produced on the particle surface does not contain large amounts of P, in stark contrast to extracellular ABs, and (iv) the iron in the Fe-rich layer is derived from the particle itself. Internalized particles and ABs follow two distinct formation mechanisms reaching different physicochemical end-states., (© 2022. The Author(s).)

Unprotected dipeptides are attractive building blocks for environmentally friendly hydrogel biomaterials by virtue of their low-cost and ease of preparation. This work investigates the self-assembling behaviour of the distinct stereoisomers of Ile-Phe and Phe-Ile in phosphate buffered saline (PBS) to form hydrogels, using transmission electron microscopy (TEM), attenuated total reflectance infrared spectroscopy (ATR-IR), circular dichroism (CD), and oscillatory rheometry. Each peptide purity and identity was also confirmed by 1 H- and 13 C-NMR spectroscopy and HPLC-MS. Finally, single-crystal XRD data allowed the key interactions responsible for the supramolecular packing into amphipathic layers or water-channels to be revealed. The presence of the latter in the crystal structure is a distinctive feature of the only gelator of this work that self-organizes into stable hydrogels, with fast kinetics and the highest elastic modulus amongst its structural isomers and stereoisomers., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

In this study, we utilized human DNA topoisomerase IIα as a model target to outline a dynophore-based approach to catalytic inhibitor design. Based on MD simulations of a known catalytic inhibitor and the native ATP ligand analog, AMP-PNP, we derived a joint dynophore model that supplements the static structure-based-pharmacophore information with a dynamic component. Subsequently, derived pharmacophore models were employed in a virtual screening campaign of a library of natural compounds. Experimental evaluation identified flavonoid compounds with promising topoisomerase IIα catalytic inhibition and binding studies confirmed interaction with the ATPase domain. We constructed a binding model through docking and extensively investigated it with molecular dynamics MD simulations, essential dynamics, and MM-GBSA free energy calculations, thus reconnecting the new results to the initial dynophore-based screening model. We not only demonstrate a new design strategy that incorporates a dynamic component of molecular recognition, but also highlight new derivates in the established flavonoid class of topoisomerase II inhibitors.

Extracellular vesicles (EVs) are gaining increasing amounts of attention due to their potential use in diagnostics and therapy, but the poor reproducibility of the studies that have been conducted on these structures hinders their breakthrough into routine practice. We believe that a better understanding of EVs stability and methods to control their integrity are the key to resolving this issue. In this work, erythrocyte EVs (hbEVs) were isolated by centrifugation from suspensions of human erythrocytes that had been aged in vitro. The isolate was characterised by scanning (SEM) and cryo-transmission electron microscopy (cryo-TEM), flow cytometry (FCM), dynamic/static light scattering (LS), protein electrophoresis, and UV-V spectrometry. The hbEVs were exposed to various conditions (pH (4-10), osmolarity (50-1000 mOsm/L), temperature (15-60 °C), and surfactant Triton X-100 (10-500 μM)). Their stability was evaluated by LS by considering the hydrodynamic radius ( R h ), intensity of scattered light ( I ), and the shape parameter ( ρ ). The morphology of the hbEVs that had been stored in phosphate-buffered saline with citrate (PBS-citrate) at 4 °C remained consistent for more than 6 months. A change in the media properties (50-1000 mOsm/L, pH 4-10) had no significant effect on the R h (=100-130 nm). At pH values below 6 and above 8, at temperatures above 45 °C, and in the presence of Triton X-100, hbEVs degradation was indicated by a decrease in I of more than 20%. Due to the simple preparation, homogeneous morphology, and stability of hbEVs under a wide range of conditions, they are considered to be a suitable option for EV reference material.

Fungi are the most common pathogens of insects and thus important regulators of their populations. Lipid-binding aegerolysin proteins, which are commonly found in the fungal kingdom, may be involved in several biologically relevant processes including attack and defense against other organisms. Aegerolysins act alone or together with membrane-attack-complex/perforin (MACPF)-like proteins to form transmembrane pores that lead to cell lysis. We performed an in-depth bioinformatics analysis of aegerolysins in entomopathogenic fungi and selected a candidate aegerolysin, beauveriolysin A (BlyA) from Beauveria bassiana . BlyA was expressed as a recombinant protein in Escherichia coli , and purified to further determine its functional and structural properties, including lipid-binding ability. Aegerolysins were found to be encoded in genomes of entomopathogenic fungi, such as Beauveria , Cordyceps , Metarhizium and Ophiocordyceps . Detailed bioinformatics analysis revealed that they are linked to MACPF-like genes in most genomes. We also show that BlyA interacts with an insect-specific membrane lipid. These results were placed in the context of other fungal and bacterial aegerolysins and their partner proteins. We believe that aegerolysins play a role in promoting the entomopathogenic and antagonistic activity of B. bassiana , which is an active ingredient of bioinsecticides.