Membrane Disintegration Research Articles

Selenium ameliorates oxidized phospholipid-mediated testicular dysfunction and epididymal sperm abnormalities following Bisphenol A exposure in adult Wistar rats

Bisphenol A (BPA) is an endocrine-disrupting compound extensively utilized in the production of polycarbonate polymers and epoxy resins that, upon exposure, pose a significant threat to male reproductive health because of its estrogenic properties. Accumulating evidence suggests that BPA exposure disrupts the normal process of spermatogenesis, alters testicular morphology and function, and interferes with testicular steroidogenesis and hormonal signaling. However, the precise mechanism by which BPA affects testicular function remains unclear. In this study, we explored the mechanism underlying BPA-induced testicular abnormalities and evaluated the protective effects of Selenium (Se). Thirty-two adult male albino Wistar rats were divided into four groups, and BPA was administered at 50 mg/kg body weight, with or without Se supplementation, for 30 days. Se supplementation (2.5 mg/kg body weight) was initiated 1 week before BPA administration. BPA administration resulted in alterations in testicular architecture, characterized by basement membrane disintegration in the seminiferous tubules, reduced spermatogenic cell counts, and increased interstitial tubule noncellular space. Furthermore, BPA exposure increased the levels of oxidized phospholipids, lipid peroxides, and hydroxyl radicals and decreased the activities of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. In addition, BPA significantly reduced the activities of 3β- and 17β-hydroxysteroid dehydrogenases, interfering with testicular steroidogenesis. In rats, coadministration of Se and BPA reduced the levels of oxidized phospholipids and increased the activities of antioxidant enzymes, leading to improved testicular function and epididymal sperm parameters, suggesting that Se plays a critical role in alleviating endocrine disruptor-induced testicular dysfunctions in rats.

Hemin promotes platelet activation and plasma membrane disintegration regulated by the subtilisin-like proprotein convertase furin.

Platelet activation plays a critical role in thrombosis and hemostasis. Several pathophysiological situations lead to hemolysis, resulting in the liberation of free ferric iron-containing hemin. Hemin has been shown to activate platelets and induce thrombo-inflammation. Classical antiplatelet therapy failed to prevent hemin-induced platelet activation. Thus, the aim of the present study was to characterize the mechanism of hemin-induced platelet death (ferroptosis). We evaluated the invitro effect of hemin on platelet activation, signaling, oxylipins, and plasma membrane destruction using light transmission aggregometry, exvivo thrombus formation, multiparametric flow cytometry, micro-UHPLC mass spectrometry for oxylipin profiling, and scanning ion conductance microscopy (SICM). We found that hemin induces platelet cell death indicated by increased ROS levels, phosphatidyl serine (PS) exposure, and loss of mitochondrial membrane potential (ΔΨm). Further, hemin causes lipid peroxidation and generation of distinct oxylipins, which strongly affects plasma membrane integrity leading to generation of platelet-derived microvesicles. Interestingly, hemin-dependent platelet death (ferroptosis) is specifically regulated by the subtilisin-like proprotein convertase furin. In summary, platelet undergo a non-apoptotic cell death mediated by furin. Inhibition of furin may offer a therapeutic strategy to control hemin-induced thrombosis and thrombo-inflammation at a site of hemolysis.

Conjunctival squamous metaplasia on amniotic membrane in Stevens-Johnson syndrome: a case report

BackgroundTo present a case of conjunctival growth on the amniotic membrane and subsequent pathology revealing conjunctival squamous metaplasia in a patient with Stevens-Johnson syndrome.Case presentationA 21-year-old female presented with painful, blurred vision in both eyes for two weeks. She was diagnosed with Stevens-Johnson syndrome 5 weeks before. Due to bilateral corneal epithelial defects, ProKera®, an amniotic membrane corneal bandage with a polycarbonate ring, was placed in both eyes. However, three weeks later, a slit-lamp examination revealed vascularized tissue growth from the palpebral conjunctiva to the amniotic membrane, along with symblepharon formation in the left eye. The patient underwent conjunctival biopsy, amniotic membrane removal, and symblepharon release. Pathology report showed the growth of squamous epithelium on the acellular amniotic membrane. Immunohistochemistry further supported the diagnosis, revealing squamous markers through p40 staining and highlighting the presence of the amniotic membrane using trichrome stain. Three months later, the patient’s visual acuity had improved to 20/25 and no symblepharon was noted.ConclusionsThis is the first case of conjunctival squamous metaplasia on amniotic membrane associated with Stevens-Johnson syndrome. Our case indicates that, despite the anti-inflammatory properties of amniotic membrane, conjunctival squamous metaplasia may arise after amniotic membrane grafting due to intense inflammation in Stevens-Johnson syndrome. Clinicians should conduct regular monitoring before amniotic membrane dissolution to preclude the development of conjunctival squamous metaplasia on the membrane and potential invasion into the cornea.

Antibacterial peptides from black soldier fly (Hermetia illucens) larvae: mode of action and characterization

Antibacterial peptides from black soldier fly larvae extract were prepared using Flash column chromatography. Three out of five fractions (F2, F3 and F4) showed antibacterial activity against Listeria monocytogenes DMST 17303 with a minimum inhibitory concentration (MIC) of 1 mM, followed by Salmonella enterica Enteritidis DMST 15679 and Escherichia coli O157:H7 DMST 12743 with a MIC ranging from 4 to 8 mM. Due to the higher yield, F2 and F3 were further analyzed on their mode of action against L. monocytogenes DMST 17303. Both fractions, particularly F2, exerted antibacterial activity through inducing bacterial cell membrane disintegration and interaction with intracellular compounds including fatty acids, proteins, and nucleic acids. The F3 did not show significant hemolytic activity up to 4 mM, while F2 showed lower than 5% hemolysis up to 8 mM. Time-to-kill analysis revealed that F2 was more effective and exerted a sustainable killing effect after 2 and 4 h, depending on the concentration of 1 and 2×MIC, respectively, while the F3 at 2×MIC could completely kill the test bacteria within 24 h. Among the identified peptides in the fractions, those with charged, either positively or negatively, and moderate hydrophobicity of ranging 6.68–15.70, namely CGPPRQGPFPR, HLEEELK, LEEAEERAD, TEELEEAKKK, and KGNSELEEAKKK, are potential antimicrobial peptides.

Unveiling abiotic stress-induced cell death modalities in the heterocytous cyanobacterium Anabaena sp. PCC 7120

Regulated cell death (RCD) in cyanobacteria is a post-stress phenomenon necessary for stress adaptability and eco-physiology. However, the sequential modulations in cellular processes conferring regulated forms of death remained elusive. In this study, we evaluated morpho-physiological and biochemical determinants of cell death in a time-dependent manner to understand the sequential cellular events during the process. The death was instigated by exposing heterocytous cyanobacterium Anabaena sp. PCC 7120 to 1mM H2O2, 250mM NaCl and sulfur-starvation. A significant changes in the sequences of morpho-physiological and biochemical events were observed in different stress conditions. Upon H2O2 treatment rapid oxidation of cytosol and disintegration of cell envelop was observed prior to DNA fragmentation. On the other hand, salt-stress and sulfur-deprivation induced phosphatidylserine (PS) externalisation, gradual rise in redox potential, and DNA fragmentation prior to membrane disintegration. Furthermore, we analysed the time-dependent expression of five orthocaspases, the putative executioner of cyanobacterial cell death. Out of five, only two orthocaspases anaOC2 and anaOC6 exhibit expression pattern synergistic and coherent with DNA fragmentation in salt treated and sulfur-deficient cells. However, no orthocaspases expression was evident during H2O2 treatment. Considering these parameters, H2O2 induced death was regarded as accidental cell death (ACD), whereas in salt and sulfur stresses mediated two different RCD subroutines, which were orthocaspases-dependent. Overall for the first time, we demonstrated the existence of three death modalities in cyanobacteria based on sequential cellular events and relative expression of orthocaspases.

Early intervention in viper bites: A potential strategy to mitigate acute kidney injury and its long-term consequences.

Dear Editor, By this letter we intend to highlight the close association between snake bites and acute kidney injury, as the causative snakes are common natives of Tropical regions and a common cause of death because of being poorly managed. Snake bites are a significant cause of acute kidney injury in tropical regions, particularly in Eastern Pakistan, where Russell's viper, a common cause, is prevalent. The farmlands of Sindh and Balochistan are predisposed to snake bites due to their favorable weather and vast fields. A 2020 study found 454 snakebite cases in Southern Sindh, with 6 deaths. The most common mechanisms are venom-mediated consumption coagulopathy (VMCC) and endothelial damage, leading to systemic bleeding diathesis and decreased perfusion of end organs. Long-term effects of snake bites include worsening renal conditions and progression towards end-stage renal disease. Eastern Pakistan commonly harbors Russell’s viper- a frequent cause of snakebite-associated mortality, which belongs to the Viperidae family and is famously known to produce vasculotoxic venom. (1) The farmlands of Sindh and Balochistan are predisposed to snake bites as the weather of these regions and the vast fields allow the vipers to flourish and thrive well. A study conducted in 2020, representing the population of Southern Sindh, demonstrated a total of 454 cases of snakebite in a span of 1 year, out of which 6 victims died. (2) Since the prevalence of vipers is common in the district of Sindh, the deaths could be attributable to the hemorrhagic manifestations of vipers' bites. The most significant mechanisms underlying viper bite presentations are venom-mediated consumption coagulopathy (VMCC) and endothelial damage resulting in systemic bleeding diathesis, which together contribute to decreased perfusion of end organs like the heart, kidneys, brain, or gastrointestinal system. (3) VMCC is a major contributor to acute kidney injury (AKI), a common outcome of vasculotoxic snake bites, along with enzymatic dissolution of the glomerular basement membrane (BM) by the venom metalloproteinases. (4) The hemorrhagic deterioration of the vessels leads to ischemic nephropathy, and the impaired BM allows proteinuria, hemoglobinuria, and increased creatinine, often predisposing the victim to timely dialysis. A study concluded that individuals with co-morbidities are more susceptible to developing long-term effects of snake bites like worsening renal conditions such as new-onset hypertension, dialysis dependence, and end-stage renal disease. (5) ---Continue

The exploitation of local electric field generated by oxygen vacancies on BiOIO3 nano-antibacterial agents for enhanced photocatalytic sterilisation

Methicillin-resistant Staphylococcus aureus (MRSA) is a typical Gram-positive foodborne pathogen, and its infection has been reported since its discovery. Photocatalysis has emerged as a safe and efficient new sterilization method in clinical and food sectors. In this study, we developed an oxygen vacancy-rich BiOIO3 nano-antibacterial agent using a hydrothermal method, which was successfully synthesized and confirmed through various characterization techniques. Notably, the BiOIO3 nano-antibacterial agent exhibits almost complete inactivation of 107 CFU/mL MRSA within 2 h under visible light irradiation. Furthermore, with increasing exposure duration to irradiation, gradual shrinkage and disintegration of the MRSA cell membrane were observed along with inhibition of the intracellular enzyme activity. Haemolysis tests were performed to prove the biological safety of the agent. Mechanistic investigations revealed that surface oxygen vacancies of BiOIO3 facilitated adsorption binding of water and oxygen within a local electric field promoting reactive oxygen species (ROS) generation. This leads to the rapid accumulation of ROS, which attacks the cell membrane causing significant damage, disrupting normal physiological metabolism of the bacteria, and ultimately killing MRSA. The present study is expected to provide a favourable solution for developing novel efficient and green nano-antibacterial agents through surface modifications.

Model of Selectivity of Membrane Processes and Dissolution of Impurities in a Membrane Pore in a Medium with Surface-Active Micelles

Model of Selectivity of Membrane Processes and Dissolution of Impurities in a Membrane Pore in a Medium with Surface-Active Micelles

Fermencin B2: A promising heat-stable cationic peptide for targeted inhibition of Staphylococcus aureus

In this study, a novel strain of L. fermentum JF9 with bacteriocin production capacity was firstly isolated from Chinese fermented vinegar Pei, and the significant antibacterial potential and safety of L. fermentum JF9 were confirmed by three-generation whole genome sequencing. The bacteriocin Fermencin B2, secreted by L. fermentum JF9, was purified by series chromatographic separation and LC-MS/MS. Fermencin B2, a cationic peptide containing the HLVRNP amino acid sequence, exhibited a stable β-folded structure and antibacterial activity at different temperatures. Meanwhile, Fermencin B2 had low cytotoxicity and was not hemolytic. The inhibitory activity and mechanisms of Fermencin B2 against S. aureus were investigated using SEM and TEM analysis, molecular docking analysis, assays for membrane proteins and ion levels, nucleic acids, LDH leakage, as well as flow cytometry. Fermencin B2 caused severe damage to the cell membrane of S. aureus, resulting in the disruption of ATPase activity, alteration of ion transport, increased membrane permeability, and disintegration of the cell membrane. This ultimately led to the leakage of nucleic acids and LDH, resulting in bacterial death. Overall, Fermencin B2 could be a novel antibacterial agent and food preservative for targeted inhibition of S. aureus.

Enhanced biosafety, anticancerand antibacterial photodynamic activities using silver-pyropheophorbide-a nanoconjugates.

Aim: A study of the enhancement of photodynamic activities of pyropheophorbide-a using PG-Ag-PPa nanoconjugates.Materials & methods: The nanoconjugates were formulated from silver nanoparticles and PPa via amide linkage, then characterized, and their photodynamic activities were examined.Results: The nanoconjugates displayed a higher rate of reactive oxygen species generation, commendable cellular uptake by Eca-109 cancer cells, higher photocytotoxicity toward the cancer cellsand better bio-safety. They revealed strong antibacterial activity against Escherichia coli following internal reactive oxygen species generation and membrane disintegration. The in vivo anticancer studies confirmed higher cytotoxicity of thenanoconjugatestoward cancer cells and better safety than PPa.Conclusion: Therefore, PG-Ag-PPa nanoconjugates could be considered potential nano photosensitizers for photodynamic therapy of tumors and bacterial infection with good bio-safety.

Calcium ions do not influence the Aβ(25–35) triggered morphological changes of lipid membranes

We have studied the effect of calcium ions (Ca2+) at various concentrations on the structure of lipid vesicles in the presence of amyloid-beta peptide Aβ(25–35). In particular, we have investigated the influence of calcium ions on the formation of recently documented bicelle-like structures (BLSs) emerged as a result of Aβ(25–35) triggered membrane disintegration. First, we have shown by using small-angle X-ray and neutron scattering that peptide molecules rigidify the lipid bilayer of gel phase DPPC unilamellar vesicles (ULVs), while addition of the calcium ions to the system hinders this effect of Aβ(25–35). Secondly, the Aβ(25–35) demonstrates a critical peptide concentration at which the BLSs reorganize from ULVs due to heating and cooling the samples through the lipid main phase transition temperature (Tm). However, addition of calcium ions does not affect noticeably the Aβ-induced formation of BLSs and their structural parameters, though the changes in peptide's secondary structure, e.g. the increased α-helix fraction, has been registered by circular dichroism spectroscopy. Finally, according to 31P nuclear magnetic resonance (NMR) measurements, calcium ions do not affect the lipid-peptide arrangement in BLSs and their ability to align in the magnetic field of NMR spectrometer. The influences of various concentrations of calcium ions on the lipid-peptide interactions may prove biologically important because their local concentrations vary widely in in-vivo conditions. In the present work, calcium ions were investigated as a possible tool aimed at regulating the lipid-peptide interactions that demonstrated the disruptive effect of Aβ(25–35) on lipid membranes.

Norgestrel causes oxidative damage to the digestive gland of the clam Mactra veneriformis

Increasingly, progestins are being detected in aquatic environments, especially in aquaculture areas, and concern about their adverse effects on aquatic organisms is growing. However, the mechanisms underlying progestin-induced oxidative stress and the antioxidant response of aquatic invertebrates to progestin exposure are unknown. Researchers recently reported that norgestrel (NGT) likely causes oxidative stress in the clam Mactra veneriformis. In this study, we assessed reactive oxygen species (ROS) levels, antioxidant enzyme activity, malonaldehyde content, ultrastructure, and DNA damage in the digestive gland of M. veneriformis exposed to 7.69 or 756.62 ng/L of NGT for 21 days. We also conducted tandem mass tag-based quantitative proteomic analysis to detect the protein response and gain insights into response mechanisms in clams exposed to NGT. We found that NGT enhanced oxidative phosphorylation via the wingless/integrated signaling pathway and triggered ROS generation. The alterations of antioxidant enzyme activities suggested that these enzymes are involved in scavenging NGT-induced ROS. NGT weakened antioxidant defense by suppressing the antioxidant synthesis-related AMP-activated protein kinase/forkhead box O and sirtuin 1/forkhead box O pathways. Additionally, cell membrane dissolution, mitochondrial cristae rupture, DNA damage, elevation of malonaldehyde content, and oxidation of lipid, protein, and glucose occurred due to compromised redox homeostasis. Our results highlight the oxidative stress, antioxidant response, and oxidative damage induced by NGT in M. veneriformis and shed light on ecological risks of progestins to aquatic invertebrates.

Decreased susceptibility to viscosin in Streptococcus pneumoniae.

Growing numbers of infections caused by antibiotic-resistant Streptococcus pneumoniae strains are a major concern for healthcare systems that will require new antibiotics for treatment as well as preventative measures that reduce the number of infections. Lipopeptides are antimicrobial molecules, of which some are used as antibiotics, including the last resort antibiotics daptomycin and polymyxins. Here we have studied the antimicrobial effect of the cyclic lipopeptide viscosin on S. pneumoniae growth and morphology. Most lipopeptides function as surfactants that create pores in membrane layers, which is regarded as their main antimicrobial activity. We show that viscosin can inhibit growth of S. pneumoniae without disintegration of the cytoplasmic membrane. Instead, the cells developed abnormal shapes and misplaced new division sites. The cell wall of these bacteria appeared less dense in electron microscopy images, suggesting that viscosin interfered with normal cell wall synthesis. Corroborating this observation, a luciferase reporter assay was used to show that the two-component systems LiaFSR and CiaRH, which are known to be activated upon cell wall stress, were strongly induced by viscosin. Furthermore, a mutant displaying 1.8-fold decreased susceptibility to viscosin was generated by sequential exposure to increasing concentrations of the lipopeptide. The mutant suffered from significant fitness loss and had mutations in genes involved in fatty acid synthesis, teichoic acid synthesis, and cell wall synthesis as well as transcription and translation. How these mutations might be linked to decreased viscosin susceptibility is discussed.IMPORTANCEStreptococcus pneumoniae is a leading cause of bacterial pneumonia, sepsis, and meningitis in children, and the incidence of infections caused by antibiotic-resistant strains is increasing. Development of new antibiotics is therefore necessary to treat these types of infections in the future. Here, we have studied the activity of the antimicrobial lipopeptide viscosin on S. pneumoniae and show that in addition to having the typical membrane destabilizing activity of lipopeptides, viscosin inhibits pneumococcal growth by obstructing normal cell wall synthesis. This suggests a more specific mode of action than just the surfactant activity. Furthermore, we show that S. pneumoniae does not easily acquire resistance to viscosin, which makes it a promising molecule to explore further, for example, by synthesizing less toxic derivates that can be tested for therapeutic potential.

Formulation of silver-loaded zeolitic imidazole framework-67 and its antibacterial coating on titanium surface for bioimplant applications

Artificial bioimplant infections caused by bacteria have been a persistent problem that requires re-surgery and is harmful to human tissue. A facile, inexpensive and stable antibacterial agent coating can be a significant alternative. The present study attempted to construct titanium (Ti) implant coated with silver loaded zeolitic imidazole framework (ZIF)-67 (Ag@ZIF-67)-chitosan composite through a casting technique. The as-prepared nanocomposite and its coating were subjected to various characterization techniques, including XPS, XRD, SEM, and TEM to understand the material properties. Antibacterial activity was assessed through a standard spread-plate method, and the cytocompatibility of the fabricated materials was examined upon the skin fibroblast (L929) cell line. Corrosion studies were performed by using anodic polarization and impedance methods. The bactericidal efficacy of the coating was validated in vivo by utilizing a subcutaneous mouse model. The nanocomposite and coating exhibited a significant antibacterial efficiency of above 95 % against S. aureus and E. coli as the combined release of Co2+ and Ag+ ions entailed cellular membrane disintegration. The Ag@ZIF-67 composite revealed negligible cytotoxicity and its coating exhibited strong bone cell attachment. In contrast to pure Ti surface, the Ag@ZIF-67-chitosan coated Ti exhibited a minor reduction in corrosion resistance and modifications to its passivation properties as a result of the formation of a galvanic cell. A remarkable antibacterial effect was evinced through the in vivo experiments demonstrating a complete disinfection with 99 % of S. aureus lysis with no bacterial growth in the nearby healthy tissues. In conclusion, the current results demonstrated that the fabricated Ag@ZIF-67 could be applied as an antibacterial coating material of Ti for bioimplant applications.

Zinc modulates hypothalamo-pituitary-gonadal-liver axis to impair reproduction in female Mystus vittatus (Bloch, 1794).

The present study investigated the effects of zinc on the hypothalamo-pituitary-gonadal-liver (HPGL) axis of the bagrid catfish Mystus vittatus. Female fish (pre-ovulatory and ovulatory phases) were exposed to zinc sulphate at 1/10th of LC50 (5.62mg/L) for 60days and sacrificed at every 15-day interval to collect tissues. Zinc concentration in all tissues was significantly higher in the metal-exposed group at all exposure durations compared to control for both phases. Metallothionein (MT) levels increased in the brain, liver and ovary of fish from both phases with exposure duration. Reactive oxygen species (ROS) generation in the brain, liver and ovary tissues increased with exposure duration at both reproductive phases while serum cortisol levels in ovulatory fish increased significantly compared to pre-ovulatory. Condition factor, gonadosomatic index and hepatosomatic index decreased in Zn-exposed fish. Brain GnRH and kisspeptin levels decreased significantly in the Zn-exposed group for both phases. GnIH was significantly higher in Zn-exposed fish. Serum FSH levels in pre-ovulatory and LH levels in ovulatory fish decreased gradually with an increase in the duration of exposure. Zn exposure reduced vitellogenin (Vtg) and estradiol (E2) in the liver and ovary with an increase in duration from both phases. Ovary maturation-inducing hormone (MIH) levels showed a decrease with exposure duration in ovulatory fish. Moreover, Zn-exposed ovulatory fish showed a degenerated oocyte nucleus due to the disintegration of the nuclear membrane. It might be inferred that Zn altered the HPGL regulatory system of M. vittatus reproduction at both the pre-ovulatory and ovulatory phases.

Brassinosteroid-induced transcriptomic rearrangements unveiled the physiological mechanism of chromium stress tolerance in Brassica napus

Brassinosteroid (BR), a plant hormone regulating growth, development, and stress responses, emerges as a promising tool for maintaining agricultural production under abiotic stress conditions. In this study, we conducted RNA-seq profiling and morpho-physiological analysis to investigate the molecular cross-talk involved in 24-epibrassinolide (EBR) mediating alleviation of chromium (Cr) stress. EBR inhibited Cr accumulation and reversed Cr-induced phytotoxicity, thereby promoting plant growth. The photosynthetic pigments, chlorophyll fluorescence a, electron transport rate (ETR) and non-photochemical quenching (NPQ) were significantly higher in EBR+Cr treated plants compared to Cr alone. EBR application facilitated the recovery from Cr-induced structural deformities, including the disintegration of cell walls and membranes. Furthermore, under Cr stress, EBR application reduced malondialdehyde (MDA) and reactive oxygen species (ROS) production and accumulation. The levels of glutathione reductase (GR) and the activities of antioxidant enzymes were notably higher in plants subjected to EBR application following Cr stress. In addition, we established a transcriptomic database comprising 2345 differentially expressed genes (DEGs) (1255 upregulated and 1090 downregulated) as a result of EBR application under Cr stress. The transcriptome analysis unveiled key DEGs and the associated pathways, emphasizing the importance of defense responses, genes encoding photosystem I and II, jasmonate signaling, aquaporins, ABC transporters, and cell wall biogenesis-related genes in the response of EBR to Cr stress.

Cellular Distribution and Ultrastructural Changes in HaCaT Cells, Induced by Podophyllotoxin and Its Novel Fluorescent Derivative, Supported by the Molecular Docking Studies.

Podophyllotoxin (PPT) is an active pharmaceutical ingredient (API) with established antitumor potential. However, due to its systemic toxicity, its use is restricted to topical treatment of anogenital warts. Less toxic PPT derivatives (e.g., etoposide and teniposide) are used intravenously as anticancer agents. PPT has been exploited as a scaffold of new potential therapeutic agents; however, fewer studies have been conducted on the parent molecule than on its derivatives. We have undertaken a study of ultrastructural changes induced by PPT on HaCaT keratinocytes. We have also tracked the intracellular localization of PPT using its fluorescent derivative (PPT-FL). Moreover, we performed molecular docking of both PPT and PPT-FL to compare their affinity to various binding sites of tubulin. Using the Presto blue viability assay, we established working concentrations of PPT in HaCaT cells. Subsequently, we have used selected concentrations to determine PPT effects at the ultrastructural level. Dynamics of PPT distribution by confocal microscopy was performed using PPT-FL. Molecular docking calculations were conducted using Glide. PPT induces a time-dependent cytotoxic effect on HaCaT cells. Within 24 h, we observed the elongation of cytoplasmic processes, formation of cytoplasmic vacuoles, progressive ER stress, and shortening of the mitochondrial long axis. After 48 h, we noticed disintegration of the cell membrane, progressive vacuolization, apoptotic/necrotic vesicles, and a change in the cell nucleus's appearance. PPT-FL was detected within HaCaT cells after ~10 min of incubation and remained within cells in the following measurements. Molecular docking confirmed the formation of a stable complex between tubulin and both PPT and PPT-FL. However, it was formed at different binding sites. PPT is highly toxic to normal human keratinocytes, even at low concentrations. It promptly enters the cells, probably via endocytosis. At lower concentrations, PPT causes disruptions in both ER and mitochondria, while at higher concentrations, it leads to massive vacuolization with subsequent cell death. The novel derivative of PPT, PPT-FL, forms a stable complex with tubulin, and therefore, it is a useful tracker of intracellular PPT binding and trafficking.

Optimisation of garlic mustard oil macerate with respect to its antifungal activity against Candida albicans MTCC 183 and in-silico molecular docking of the volatile compounds with N-myristoyltransferase

Candida albicans infections are widespread in people and cause cutaneous and systemic infections. Optimisation of garlic mustard oil macerate (GMM) based on antifungal activity against C. albicans was done using agar diffusion method. Upon vapour diffusion assay, the volatile organic compounds of both GMM and MO were found to eradicate C. albicans. During agar diffusion, MO did not inhibit fungal growth, while undiluted GMM oil demonstrated a 26.33 ± 0.33 mm zone of inhibition. The minimum inhibitory concentration and minimum fungicidal concentration against C. albicans were 12.5%, v/v of GMM oil and 25%, v/v of GMM oil, respectively. Scanning electron microscopy analysis showed cell membrane disintegration of fungal cells by 50%, v/v of GMM oil, and MO caused no cell wall damage. In-silico analysis revealed strong binding affinity of sinigrin, ajoene, dithiin with N-myristoyltransferase. In conclusion, the optimised GMM preparation can be a potential antifungal agent against tropical C. albicans infections.

Unlocking roles of cationic and aromatic residues in peptide amphiphiles in treating drug-resistant gram-positive pathogens

Multidrug resistance (MDR) is a rising threat to global health because the number of essential antibiotics used for treating MDR infections is increasingly compromised. In this work we report a group of new amphiphilic peptides (AMPs) derived from the well-studied G3 (G(IIKK)3I-NH2) to fight infections from Gram-positive bacteria including susceptible Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), focusing on membrane interactions. Time-dependent killing experiments revealed that substitutions of II by WW (GWK), II by FF (GFK) and KK by RR (GIR) resulted in improved bactericidal efficiencies compared to G3 (GIK) on both S. aureus and MRSA, with the order of GWK > GIR > GFK > GIK. Electronic microscopy imaging revealed structural disruptions of AMP binding to bacterial cell walls. Fluorescence assays including AMP binding to anionic lipoteichoic acids (LTA) in cell-free and cell systems indicated concentration and time-dependent membrane destabilization associated with bacterial killing. Furthermore, AMP’s binding to anionic plasma membrane via similar fluorescence assays revealed a different extent of membrane depolarization and leakage. These observations were supported by the penetration of AMPs into the LTA barrier and the subsequent structural compromise to the cytoplasmic membrane as revealed from SANS (small angle neutron scattering). Both experiments and molecular dynamics (MD) simulations revealed that GWK and GIR could make the membrane more rigid but less effective in diffusive efficiency than GIK and GFK through forming intramembrane peptide nanoaggregates associated with hydrophobic mismatch and formation of fluidic and rigid patches. The reported peptide-aggregate-induced phase-separation emerged as a crucial factor in accelerated membrane disintegration and fast bacterial killing. This work has demonstrated the importance of membrane interactions to the development of more effective AMPs and the relevance of the approaches as reported in assisting this area of research.

Evaluation of the mechanistic basis for the antibacterial activity of ursolic acid against Staphylococcus aureus.

The antibiotics are generally regarded as the first choice approach to treat dairy mastitis, targeting the public health problems associated with the food safety and the emergence of antibioticresistant bacteria. The objective of the study was to evaluate the antibacterial efficacy of ursolic acid (UA) when used to treat Staphylococcus aureus and other isolates associated with bovine mastitis and to clarify the mechanistic basis for these effects. The bacteriostatic properties of UA extracted from Rosmarinus officinalis L. at four different purity levels were assessed by calculating minimum inhibitory concentration (MIC) values, while the synergistic effects of combining 98% UA with antibiotics were evaluated by measuring the fractional inhibitory concentration index (FICI). Changes in biofilm formation and the growth curves of the clinical isolates were assessed to clarify the bacteriostatic effect of UA. Furthermore, the cell wall integrity, protein synthesis, and reactive oxygen species (ROS) production were assessed to determine the antibacterial mechanism of UA treatment. Ultimately, UA was revealed to exhibit robust activity against Gram-positive bacteria including S. aureus (ATCC 25923), Streptococcus dysgalactiae (ATCC27957), Streptococcus agalactiae (ATCC13813), Enterococcus faecalis (ATCC29212), and Streptococcus mutans (ATCC25175). However, it did not affect Escherichia coli (ATCC 25922). The MIC values of UA preparations that were 98, 50, 30, and 10% pure against S. aureus were 39, 312, 625, and 625 μg/mL, respectively, whereas the corresponding MIC for E. coli was >5,000 μg/mL. The minimum bactericidal concentrations of 98% UA when used to treat three clinical S. aureus isolates (S4, S5, and S6) were 78, 78, and 156 μg/mL, respectively. Levels of biofilm formation for clinical S. aureus isolates decreased with increasing 98% UA concentrations. Above the MIC dose, UA treatment resulted in the dissolution of bacterial cell walls and membranes, with cells becoming irregularly shaped and exhibiting markedly impaired intracellular protein synthesis. S. aureus treated with 98% UA was able to rapidly promote intracellular ROS biogenesis. Together, these data highlight the promising utility of UA as a compound that can be used together with other antibiotics for the treatment of infections caused by S. aureus.