Deep learning with mixup augmentation for improved pore detection during additive manufacturing.

In additive manufacturing (AM), process defects such as keyhole pores are difficult to anticipate, affecting the quality and integrity of the AM-produced materials. Hence, considerable efforts have aimed to predict these process defects by training machine learning (ML) models using passive measurements such as acoustic emissions. This work considered a dataset in which keyhole pores of a laser powder bed fusion (LPBF) experiment were identified using X-ray radiography and then registered both in space and time to acoustic measurements recorded during the LPBF experiment. Due to AM's intrinsic process controls, where a pore-forming event is relatively rare, the acoustic datasets collected during monitoring include more non-pores than pores. In other words, the dataset for ML model development is imbalanced. Moreover, this imbalanced and sparse data phenomenon remains ubiquitous across many AM monitoring schemes since training data is nontrivial to collect. Hence, we propose a machine learning approach to improve this dataset imbalance and enhance the prediction accuracy of pore-labeled data. Specifically, we investigate how data augmentation helps predict pores and non-pores better. This imbalance is improved using recent advances in data augmentation called Mixup, a weak-supervised learning method. Convolutional neural networks (CNNs) are trained on original and augmented datasets, and an appreciable increase in performance is reported when testing on five different experimental trials. When ML models are trained on original and augmented datasets, they achieve an accuracy of 95% and 99% on test datasets, respectively. We also provide information on how dataset size affects model performance. Lastly, we investigate the optimal Mixup parameters for augmentation in the context of CNN performance.

The estimation of pore size distribution of electroporated MCF-7 cell membrane.

The size of the pores created by external electrical pulses is important for molecule delivery into the cell. The size of pores and their distribution on the cell membrane determine the efficiency of molecule transport into the cell. There are very few studies visualizing the presence of electropores. In this study, we aimed to investigate the size distribution of electropores that were created by high intensity and short duration electrical pulses on MCF-7 cell membrane. Scanning Electron Microscopy (SEM) was used to visualize and characterize the membrane pores created by the external electric field. Structural changes on the surface of the electroporated cell membrane was observed by Atomic Force Microscopy (AFM). The size distribution of pore sizes was obtained by measuring the radius of 500 electropores. SEM imaging showed non-uniform patterning. The average radius of the electropores was 12 nm, 51.60% of pores were distributed within the range of 5 to 10 nm, and 81% of pores had radius below 15 nm. These results showed that microsecond (µs) high intensity electrical pulses cause the creation of heterogeneous nanopores on the cell membrane.

Electroporation is a phenomenon in which permeability of the cell membrane to molecules and ions is increased due to externally applied high electric field pulses. The externally applied electric field pulses create pores on the cell membrane, allowing ions and molecules that normally can not pass through the membrane. The transport of molecules into the cell is related to the size and distribution of the pores created on the membrane. Studies visualizing the presence of electropores are very limited. In this study, we aimed to visualize pores and determine the size distribution of pores created due to the application of external electric field pulses on the cell membrane of human breast cancer cells. The membrane pores created by external electric field were visualized and characterized by different imaging techniques. The size distribution of pores was obtained by measuring the radius of 500 pores created on the cell membrane due to the applied electric fields. The surface of the electropermeabilized cells were very rough due to deformation during electroporation. We observed heterogeneous pore populations that were formed due to application of external electrical pulses on the surface of cell membrane. The average radius of the pores was found to be 12 nm.

Conformational Switch of a Peptide Provides a Novel Strategy to Design Peptide Loaded Porous Organic Polymer for Pyroptosis Pathway Mediated Cancer Therapy.

While peptide-based drug development is extensively explored, this strategy has limitations due to rapid excretion from the body (or shorter half-life in the body) and vulnerability to protease-mediated degradation. To overcome these limitations, a novel strategy for the development of a peptide-based anticancer agent is introduced, utilizing the conformation switch property of a chameleon sequence stretch (PEP1) derived from a mycobacterium secretory protein, MPT63. The selected peptide is then loaded into a new porous organic polymer (PG-DFC-POP) synthesized using phloroglucinol and a cresol derivative via a condensation reaction to deliver the peptide selectively to cancer cells. Utilizing ensemble and single-molecule approaches, this peptide undergoes a transition from a disordered to an alpha-helical conformation, triggered by the acidic environment within cancer cells that is demonstrated. This adopted alpha-helical conformation resulted in the formation of proteolysis-resistant oligomers, which showed efficient membrane pore-forming activity selectively for negatively charged phospholipids accumulated in cancer cell membranes. The experimental results demonstrated that the peptide-loaded PG-DFC-POP-PEP1 exhibited significant cytotoxicity in cancer cells, leading to cell death through the Pyroptosis pathway, which is established by monitoring numerous associated events starting from lysosome membrane damage to GSDMD-induced cell membrane demolition. This novel conformational switch-based drug design strategy is believed to have great potential in endogenous environment-responsive cancer therapy and the development of future drug candidates to mitigate cancers.

Glucose and Oxygen Levels Modulate the Pore-Forming Effects of Cholesterol-Dependent Cytolysin Pneumolysin from Streptococcus pneumoniae.

A major Streptococcus pneumoniae pathogenic factor is the cholesterol-dependent cytolysin pneumolysin, binding membrane cholesterol and producing permanent lytic or transient pores. During brain infections, vascular damage with variable ischemia occurs. The role of ischemia on pneumolysin's pore-forming capacity remains unknown. In acute brain slice cultures and primary cultured glia, we studied acute toxin lysis (via propidium iodide staining and LDH release) and transient pore formation (by analyzing increases in the intracellular calcium). We analyzed normal peripheral tissue glucose conditions (80 mg%), normal brain glucose levels (20 mg%), and brain hypoglycemic conditions (3 mg%), in combinations either with normoxia (8% oxygen) or hypoxia (2% oxygen). At 80 mg% glucose, hypoxia enhanced cytolysis via pneumolysin. At 20 mg% glucose, hypoxia did not affect cell lysis, but impaired calcium restoration after non-lytic pore formation. Only at 3 mg% glucose, during normoxia, did pneumolysin produce stronger lysis. In hypoglycemic (3 mg% glucose) conditions, pneumolysin caused a milder calcium increase, but restoration was missing. Microglia bound more pneumolysin than astrocytes and demonstrated generally stronger calcium elevation. Thus, our work demonstrated that the toxin pore-forming capacity in cells continuously diminishes when oxygen is reduced, overlapping with a continuously reduced ability of cells to maintain homeostasis of the calcium influx once oxygen and glucose are reduced.

α-Latrotoxin Tetramers Spontaneously Form Two-Dimensional Crystals in Solution and Coordinated Multi-Pore Assemblies in Biological Membranes.

α-Latrotoxin (α-LTX) was found to form two-dimensional (2D) monolayer arrays in solution at relatively low concentrations (0.1 mg/mL), with the toxin tetramer constituting a unit cell. The crystals were imaged using cryogenic electron microscopy (cryoEM), and image analysis yielded a ~12 Å projection map. At this resolution, no major conformational changes between the crystalline and solution states of α-LTX tetramers were observed. Electrophysiological studies showed that, under the conditions of crystallization, α-LTX simultaneously formed multiple channels in biological membranes that displayed coordinated gating. Two types of channels with conductance levels of 120 and 208 pS were identified. Furthermore, we observed two distinct tetramer conformations of tetramers both when observed as monodisperse single particles and within the 2D crystals, with pore diameters of 11 and 13.5 Å, suggestive of a flickering pore in the middle of the tetramer, which may correspond to the two states of toxin channels with different conductance levels. We discuss the structural changes that occur in α-LTX tetramers in solution and propose a mechanism of α-LTX insertion into the membrane. The propensity of α-LTX tetramers to form 2D crystals may explain many features of α-LTX toxicology and suggest that other pore-forming toxins may also form arrays of channels to exert maximal toxic effect.

Poptosis or Peptide-Induced Transmembrane Pore Formation: A Novel Way to Kill Cancer Cells without Affecting Normal Cells.

Recent advances in cancer treatment like personalized chemotherapy and immunotherapy are aimed at tumors that meet certain specifications. In this review, we describe a new approach to general cancer treatment, termed peptide-induced poptosis, in which specific peptides, e.g., PNC-27 and its shorter analogue, PNC-28, that contain the segment of the p53 transactivating 12-26 domain that bind to HDM-2 in its 1-109 domain, bind to HDM-2 in the membranes of cancer cells, resulting in transmembrane pore formation and the rapid extrusion of cancer cell contents, i.e., tumor cell necrosis. These peptides cause tumor cell necrosis of a wide variety of solid tissue and hematopoietic tumors but have no effect on the viability and growth of normal cells since they express at most low levels of membrane-bound HDM-2. They have been found to successfully treat a highly metastatic pancreatic tumor as well as stem-cell-enriched human acute myelogenous leukemias in nude mice, with no evidence of off-target effects. These peptides also are cytotoxic to chemotherapy-resistant cancers and to primary tumors. We performed high-resolution scanning immuno-electron microscopy and visualized the pores in cancer cells induced by PNC-27. This peptide forms 1:1 complexes with HDM-2 in a temperature-independent step, followed by dimerization of these complexes to form transmembrane channels in a highly temperature-dependent step parallel to the mode of action of other membranolytic but less specific agents like streptolysin. These peptides therefore may be effective as general anti-cancer agents.

Formation of pores and bubbles and their impacts on the quality attributes of processed foods: A review.

Pores and bubbles significantly influence the physical attributes (like texture, density, and structural integrity), organoleptic properties, and shelf life of processed foods. Hence, the quality of foods and their acceptance by the consumers could be influenced by the properties and prevalence of pores and bubbles within the food structure. Considering the importance of pores, this review aimed to comprehensively discuss the factors and mechanisms involved in the generation of pores and bubbles during the processing of different food products. Moreover, the characteristics and effects of pores on the properties of chocolates, cheeses, cereal-based foods (like cake, puffed grains, and pasta), dried, and fried products were discussed. The impacts of bubbles on the quality of foam-based products, foam creamers, and beverages were also explored. This review concludes that intrinsic factors (like food compositions, initial moisture content, and porosity) and extrinsic factors (like applied technologies, processing, and storage conditions) affect various properties of the pores and bubbles including their number, size, orientation, and distribution. These factors collectively shape the overall structure and quality of processed food products such as density, texture (hardness, cohesiveness, chewiness), and water holding capacity. The desirability or undesirability of pores and their characteristics depends on the type of products; hence, some practical hints were provided to mitigate their adverse effects or to enhance their formation in foods. For example, pores could increase the nutrient digestion and reduce the shelf life of the products by enhancing the risk of fat oxidation and microbial growth. In conclusion, this study provides a valuable resource for food scientists and industry professionals by discussing the effects of pores on food preservation, heat, and mass transfer (including oxygen, moisture, flavors, and nutrients). Understanding the dynamic changes in porosity during processing will be effective in customization of final product quality with desired attributes, ensuring tailored outcomes for specific applications.

Analytical and numerical solutions of pore formation in elastic food materials during dehydration.

In this paper, we describe a model for pore formation in food materials during drying. As a proxy for fruits and vegetables, we take a spherical hydrogel, with a stiff elastic skin, and a central cavity filled with air and water vapour. The model describes moisture transport coupled to large deformation mechanics. Both stress and chemical potential are derived from a free energy functional, following the framework developed by Suo and coworkers. We have compared Finite Volume and Finite Element implementations and analytical solutions with each other, and we show that they render similar solutions. The Finite Element solver has a larger range of numerical stability than the Finite Volume solver, and the analytical solution also has a limited range of validity. Since the Finite Element solver operates using the mathematically intricate weak form, we introduce the method in a tutorial manner for food scientists. Subsequently, we have explored the physics of the pore formation problem further with the Finite Element solver. We show that the presence of an elastic skin is a prerequisite for the growth of the central cavity. The elastic skin must have an elastic modulus of at least 10 times that of the hydrogel. An initial pore with 10% of the size of the gel can grow to 5 times its initial size. Such an increase in porosity has been reported in the literature on drying of vegetables, if a dense hard skin is formed, known as case hardening. We discuss that models as presented in this paper, where moisture transport is strongly coupled to large deformation mechanics, are required if one wants to describe pore/structure formation during drying and intensive heating (as baking and frying) of food materials from first principles.

Synthetic antimicrobial peptides: activity against vancomycin-resistant Enterococcus faecalis and modulation of chloramphenicol antibacterial activity.

AIMS: Enterococcus faecalis (E. faecalis) is a leading cause of nosocomial infection and presents a wide spectrum of antibiotic resistance, being vancomycin-resistant Enterococcus (VRE) one of the most relevant. Synthetic antimicrobial peptides (SAMPs) are currently a promising option to overcome antimicrobial resistance. Thus, the purpose of this study was to assess the effect of eight SAMPs against vancomycin-resistant E. faecalis, as well as to investigate their mechanism of action and synergy with conventional antibiotics. METHODS AND RESULTS: Here, eight SAMPs, Mo-CBP3-PepI, Mo-CBP3-PepII, Mo-CBP3-PepIII, RcAlb-PepI, RcAlb-PepII, RcAlb-PepIII, PepGAT, and PepKAA, were tested for antibacterial activity in vitro against E. faecalis (ATCC® 51299) through broth microdilution. A maximum of 48% of E. faecalis growth inhibition was achieved by treatment with SAMPs alone. However, when these peptides were combined with the antibiotic chloramphenicol, assessed by checkerboard method, the inhibition increased to 55%-76% of inhibition, two to three-folds of increase if compared to the effects of the compounds alone. Microscopic analysis showed that E. faecalis cells treated with a combination of SAMPs and chloramphenicol resulted in bacterial membrane damage. The biofilm inhibition maximum was 22% for SAMPs alone, when combined with chloramphenicol, the maximum increased to 33%. CONCLUSIONS: SAMPs and their combination with chloramphenicol demonstrate antibacterial activity against E. faecalis, possibly by inducing bacterial membrane damage.

One-Step Construction of Closed Pores Enabling High Plateau Capacity Hard Carbon Anodes for Sodium-Ion Batteries: Closed-Pore Formation and Energy Storage Mechanisms.

Closed pores play a crucial role in improving the low-voltage (<0.1 V) plateau capacity of hard carbon anodes for sodium-ion batteries (SIBs). However, the lack of simple and effective closed-pore construction strategies, as well as the unclear closed-pore formation mechanism, has severely hindered the development of high plateau capacity hard carbon anodes. Herein, we present an effective closed-pore construction strategy by one-step pyrolysis of zinc gluconate (ZG) and elucidate the corresponding mechanism of closed-pore formation. The closed-pore formation mechanism during the pyrolysis of ZG mainly involves (i) the precipitation of ZnO nanoparticles and the ZnO etching on carbon under 1100 °C to generate open pores of 0.45-4 nm and (ii) the development of graphitic domains and the shrinkage of the partial open pores at 1100-1500 °C to convert the open pores to closed pores. Benefiting from the considerable closed-pore content and suitable microstructure, the optimized hard carbon achieves an ultrahigh reversible specific capacity of 481.5 mA h g-1 and an extraordinary plateau capacity of 389 mA h g-1 for use as the anode of SIBs. Additionally, some in situ and ex situ characterizations demonstrate that the high-voltage slope capacity and the low-voltage plateau capacity stem from the adsorption of Na+ at the defect sites and Na-cluster formation in closed pores, respectively.

Exploring conventional and emerging dehydration technologies for slurry/liquid food matrices and their impact on porosity of powders: A comprehensive review.

The contribution of dehydration to the growing market of food powders from slurry/liquid matrices is inevitable. To overcome the challenges posed by conventional drying technologies, several innovative approaches have emerged. However, industrial implementation is limited due to insufficient information on the best-suited drying technologies for targeted products. Therefore, this review aimed to compare various conventional and emerging dehydration technologies (such as active freeze, supercritical, agitated thin-film, and vortex chamber drying) based on their fundamental principles, potential applications, and limitations. Additionally, this article reviewed the effects of drying technologies on porosity, which greatly influence the solubility, rehydration, and stability of powder. The comparison between different drying technologies enables informed decision-making in selecting the appropriate one. It was found that active freeze drying is effective in producing free-flowing powders, unlike conventional freeze drying. Vortex chamber drying could be considered a viable alternative to spray drying, requiring a compact chamber than the large tower needed for spray drying. Freeze-dried, spray freeze-dried, and foam mat-dried powders exhibit higher porosity than spray-dried ones, whereas supercritical drying produces nano-porous interconnected powders. Notably, several factors like glass transition temperature, drying technologies, particle aggregation, agglomeration, and sintering impact powder porosity. However, some binders, such as maltodextrin, sucrose, and lactose, could be applied in controlled agglomeration to enhance powder porosity. Further investigation on the effect of emerging technologies on powder properties and their commercial feasibility is required to discover their potential in liquid drying. Moreover, utilizing clean-label drying ingredients like dietary fibers, derived from agricultural waste, presents promising opportunities.

Processes and mechanisms underlying burst of giant unilamellar vesicles induced by antimicrobial peptides and compounds.

To clarify the damage of lipid bilayer region in bacterial cell membrane caused by antimicrobial peptides (AMPs) and antimicrobial compounds (AMCs), their interactions with giant unilamellar vesicles (GUVs) of various lipid compositions have been examined. The findings revealed two main causes for the leakage: nanopore formation in the membrane and burst of GUVs. Although GUV burst has been explained previously based on the carpet model, the supporting evidence is limited. In this review, to better clarify the mechanism of GUV burst by AMPs, AMCs, and other membrane-active peptides, we described current knowledge of the conditions, characteristics, and detailed processes of GUV burst and the changes in the shape of the GUVs during burst. We identified several physical factors that affect GUV burst, such as membrane tension, electrostatic interaction, structural changes of GUV membrane such as membrane folding, and oil in the membrane. We also clarified one of the physical mechanisms underlying the instability of lipid bilayers that are associated with leakage in the carpet model. Based on these results, we propose a mechanism underlying some types of GUV burst induced by these substances: the growth of a nanopore to a micropore, resulting in GUV burst.

Membrane damage by MBP-1 is mediated by pore formation and amplified by mtDNA.

Eosinophils play a crucial role in host defense while also contributing to immunopathology through the release of inflammatory mediators. Characterized by distinctive cytoplasmic granules, eosinophils securely store and rapidly release various proteins exhibiting high toxicity upon extracellular release. Among these, major basic protein 1 (MBP-1) emerges as an important mediator in eosinophil function against pathogens and in eosinophil-associated diseases. While MBP-1 targets both microorganisms and host cells, its precise mechanism remains elusive. We demonstrate that formation of small pores by MBP-1 in lipid bilayers induces membrane permeabilization and disrupts potassium balance. Additionally, we reveal that mitochondrial DNA (mtDNA) present in eosinophil extracellular traps (EETs) amplifies MBP-1 toxic effects, underscoring the pivotal role of mtDNA in EETs. Furthermore, we present evidence indicating that absence of CpG methylation in mtDNA contributes to the regulation of MBP-1-mediated toxicity. Taken together, our data suggest that the mtDNA scaffold within extracellular traps promotes MBP-1 toxicity.

Estimates of differential toxin expression governing heterogeneous intracellular lifespans of Streptococcus pneumoniae.

Following invasion of the host cell, pore-forming toxins secreted by pathogens compromise vacuole integrity and expose the microbe to diverse intracellular defence mechanisms. However, the quantitative correlation between toxin expression levels and consequent pore dynamics, fostering the intracellular life of pathogens, remains largely unexplored. In this study, using Streptococcus pneumoniae and its secreted pore-forming toxin pneumolysin (Ply) as a model system, we explored various facets of host-pathogen interactions in the host cytosol. Using time-lapse fluorescence imaging, we monitored pore formation dynamics and lifespans of different pneumococcal subpopulations inside host cells. Based on experimental histograms of various event timescales such as pore formation time, vacuolar death or cytosolic escape time and total degradation time, we developed a mathematical model based on first-passage processes that could correlate the event timescales to intravacuolar toxin accumulation. This allowed us to estimate Ply production rate, burst size and threshold Ply quantities that trigger these outcomes. Collectively, we present a general method that illustrates a correlation between toxin expression levels and pore dynamics, dictating intracellular lifespans of pathogens.

Effect of membrane tension on antimicrobial peptide PGLa-induced pore formation in lipid bilayers.

The osmotic pressure (Π) method has recently been developed to quantitatively examine the effect of membrane tension (σ) on pore formation in giant unilamellar vesicles (GUVs) induced by antimicrobial peptides (AMPs). Here, we used the Π method to reveal the effect of σ on the interaction of an AMP, PGLa, with lipid bilayers comprising dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (4/6). PGLa induced leakage of fluorescent probes from single GUVs under Π, indicating nanopore formation. Membrane tension did not transform a PGLa-induced nanopore into a micropore nor cause GUV burst up to 3.4 mN/m, which is in contrast with the effect of σ on another AMP, magainin 2-induced pore formation, where lower σ resulted in GUV burst. The fraction of leaking GUVs at a specific time increased with increasing σ, indicating that the rate of PGLa-induced pore formation increases with increasing σ. The rate of transfer of fluorescent probe-labeled PGLa across the lipid bilayer without pore formation also increased with increasing σ. PGLa-induced pore formation requires a symmetric distribution of peptides in both leaflets of the GUV bilayer, and thus we infer that the increase in the rate of PGLa transfer from the outer leaflet to the inner leaflet underlies the increase in the rate of pore formation with increasing σ. On the basis of these results, we discuss the difference between the effect of σ on nanopore formation in GUV membranes induced by PGLa and that by magainin 2.

The Designed Pore-Forming Antimicrobial Peptide C14R Combines Excellent Activity against the Major Opportunistic Human Pathogen Pseudomonas aeruginosa with Low Cytotoxicity.

The diminishing portfolio of mankind's available antibiotics urges science to develop novel potent drugs. Here, we present a peptide fitting the typical blueprint of amphipathic and membrane-active antimicrobial peptides, denominated C14R. This 2 kDa peptide consists of 16 amino acid residues, with seven being either hydrophobic, aromatic, or non-polar, and nine being polar or positively charged, strictly separated on opposite sides of the predicted α-helix. The affinity of the peptide C14R to P. aeruginosa membranes and its intrinsic tendency to productively insert into membranes of such composition were analyzed by dynamic simulations. Its biological impact on the viability of two different P. aeruginosa reference strains was demonstrated by determining the minimal inhibitory concentrations (MICs), which were found to be in the range of 10-15 µg/mL. C14R's pore-forming capability was verified in a permeabilization assay based on the peptide-triggered uptake of fluorescent dyes into the bacterial cells. Finally, the peptide was used in radial diffusion assays, which are commonly used for susceptibility testing of antimicrobial peptides in clinical microbiology. In comparison to reference strains, six clinical P. aeruginosa isolates were clearly affected, thereby paving the way for further in-depth analyses of C14R as a promising new AMP drug in the future.

COBL7 is required for stomatal formation via regulation of cellulose deposition in Arabidopsis.

As a key regulator of plant photosynthesis, water use efficiency and immunity, stomata are specialized cellular structures that adopt defined shapes. However, our knowledge about the genetic players of stomatal pore formation and stomatal morphogenesis remains limited. Forward genetic screening, positional cloning, confocal and electron microscopy, physiological and pharmacological assays were employed for isolation and characterization of mutants and genes. We identified a mutant, dsm1, with impaired cytokinesis and deformed stomata. DSM1 is highly expressed in guard mother cells and guard cells, and encodes COBRA-LIKE 7 (COBL7), a plant-specific glycosylphosphatidylinositol (GPI)-anchored protein. COBRA-LIKE 7 and its closest homologue, COBL8, are first enriched on the forming cell plates during cytokinesis, and then their subcellular distribution and abundance change are correlated with the progressive stages of stomatal pore formation. Both COBL7 and COBL8 possess an ability to bind cellulose. Perturbing the expression of COBL7 and COBL8 leads to a decrease in cellulose content and inhibition of stomatal pore development. Moreover, we found that COBL7, COBL8 and CSLD5 have synergistic effects on stomatal development and plant growth. Our findings reveal that COBL7 plays a predominant and functionally redundant role with COBL8 in stomatal formation through regulating cellulose deposition and ventral wall modification in Arabidopsis.

Channel Formation in Cry Toxins: An Alphafold-2 Perspective.

Bacillus thuringiensis (Bt) strains produce pore-forming toxins (PFTs) that attack insect pests. Information for pre-pore and pore structures of some of these Bt toxins is available. However, for the three-domain (I-III) crystal (Cry) toxins, the most used Bt toxins in pest control, this crucial information is still missing. In these Cry toxins, biochemical data have shown that 7-helix domain I is involved in insertion in membranes, oligomerization and formation of a channel lined mainly by helix α4, whereas helices α1 to α3 seem to have a dynamic role during insertion. In the case of Cry1Aa, toxic against Manduca sexta larvae, a tetrameric oligomer seems to precede membrane insertion. Given the experimental difficulty in the elucidation of the membrane insertion steps, we used Alphafold-2 (AF2) to shed light on possible oligomeric structural intermediates in the membrane insertion of this toxin. AF2 very accurately (<1 Å RMSD) predicted the crystal monomeric and trimeric structures of Cry1Aa and Cry4Ba. The prediction of a tetramer of Cry1Aa, but not Cry4Ba, produced an 'extended model' where domain I helices α3 and α2b form a continuous helix and where hydrophobic helices α1 and α2 cluster at the tip of the bundle. We hypothesize that this represents an intermediate that binds the membrane and precedes α4/α5 hairpin insertion, together with helices α6 and α7. Another Cry1Aa tetrameric model was predicted after deleting helices α1 to α3, where domain I produced a central cavity consistent with an ion channel, lined by polar and charged residues in helix α4. We propose that this second model corresponds to the 'membrane-inserted' structure. AF2 also predicted larger α4/α5 hairpin n-mers (14 ≤n ≤ 17) with high confidence, which formed even larger (~5 nm) pores. The plausibility of these models is discussed in the context of available experimental data and current paradigms.

Lipid-Centric Approaches in Combating Infectious Diseases: Antibacterials, Antifungals and Antivirals with Lipid-Associated Mechanisms of Action.

One of the global challenges of the 21st century is the increase in mortality from infectious diseases against the backdrop of the spread of antibiotic-resistant pathogenic microorganisms. In this regard, it is worth targeting antibacterials towards the membranes of pathogens that are quite conservative and not amenable to elimination. This review is an attempt to critically analyze the possibilities of targeting antimicrobial agents towards enzymes involved in pathogen lipid biosynthesis or towards bacterial, fungal, and viral lipid membranes, to increase the permeability via pore formation and to modulate the membranes' properties in a manner that makes them incompatible with the pathogen's life cycle. This review discusses the advantages and disadvantages of each approach in the search for highly effective but nontoxic antimicrobial agents. Examples of compounds with a proven molecular mechanism of action are presented, and the types of the most promising pharmacophores for further research and the improvement of the characteristics of antibiotics are discussed. The strategies that pathogens use for survival in terms of modulating the lipid composition and physical properties of the membrane, achieving a balance between resistance to antibiotics and the ability to facilitate all necessary transport and signaling processes, are also considered.

Structure, Function, and Physicochemical Properties of Pore-forming Antimicrobial Peptides.

Antimicrobial peptides (AMPs), a class of antimicrobial agents, possess considerable potential to treat various microbial ailments. The broad range of activity and rare complete bacterial resistance to AMPs make them ideal candidates for commercial development. These peptides with widely varying compositions and sources share recurrent structural and functional features in mechanisms of action. Studying the mechanisms of AMP activity against bacteria may lead to the development of new antimicrobial agents that are more potent. Generally, AMPs are effective against bacteria by forming pores or disrupting membrane barriers. The important structural aspects of cytoplasmic membranes of pathogens and host cells will also be outlined to understand the selective antimicrobial actions. The antimicrobial activities of AMPs are related to multiple physicochemical properties, such as length, sequence, helicity, charge, hydrophobicity, amphipathicity, polar angle, and also self-association. These parameters are interrelated and need to be considered in combination. So, gathering the most relevant available information will help to design and choose the most effective AMPs.