Activation Of Phospholipase Research Articles

A fungal phospholipase C involved in the degradation of plant glycosylinositol phosphorylceramides during Arabidopsis/Botrytis interaction

This study investigates the presence and significance of phosphorylated oligosaccharides that accumulate during the interaction between Arabidopsis thaliana and Botrytis cinerea, a necrotrophic fungus that poses a major threat to crops worldwide. While previous research has extensively characterized cell wall-derived molecules during fungal infection, the role of plasma membrane-derived ones remains unclear. Here, we reveal the discovery of inositol phosphate glycans (IPGs) released during infection, originating from plant sphingolipids, specifically glycosylinositol phosphorylceramides (GIPC). Advanced chromatography, mass spectrometry techniques and molecular biology were employed to identify these IPGs, and determine their origins. In addition to the well-characterized role of B. cinerea in releasing cell wall-degrading enzymes, this research suggests that B. cinerea’s enzymatic machinery may also target the degradation of the plant plasma membrane. As a consequence of this, IPGs identical to those generated by the host plant are released, most likely due to activity of a putative phospholipase C that acts on GIPC plasma membrane lipids. These insights could pave the way for developing new strategies to enhance crop resistance by focusing on membrane integrity in addition to cell wall fortification.

Characterization of Yeast Isolated from the Gut Microbiota of Tunisian Children with Autism Spectrum Disorder

Research on the microbiota–gut–brain axis in autism has primarily focused on bacteria, with limited attention to fungi. There is a growing interest in understanding the involvement of fungi, particularly Candida, in patients with autism spectrum disorder. The aim of this study was to assess the prevalence, antifungal susceptibility profiles and virulence factors of Candida isolates from the guts of Tunisian children with autism. Twenty-eight children with autism and forty-six controls were enrolled. Candida isolates from the faecal samples were identified using biochemical and molecular methods; antifungal susceptibility testing was determined by the EUCAST broth microdilution method and virulence factors, including biofilm formation, cell surface hydrophobicity and phospholipase and proteinase activities, were assessed in vitro. As a result, Candida was detected in 13 children with autism (46.4%) and 14 control children (30.4%). Candida albicans was found to be the most common species isolate in the faeces of both groups of children. Antifungal susceptibility profiles showed that one Candida isolate was resistant to amphotericin B and anidulafungin (3.7%), six were resistant to micafungin (22.2%) and five were resistant to fluconazole (18.5%). All Candida isolates were biofilm producers. Of the twenty-seven isolates, only four showed phospholipase activity (14.8%), eight showed aspartyl-proteinase activity (29.6%) and nine were hydrophobic (33.3%). These results highlight the presence of Candida in the guts of children with autism, as well as the ability to express multiple virulence factors and the antifungal resistance, and they emphasize the need for further studies to confirm intestinal Candida colonization and its potential role in autism.

Phase-Specific Immobilization of Phospholipase D as an Efficient Pickering Emulsion Interfacial Catalyst for Converting Antarctic Krill Oil Phospholipid.

Phosphatidylserine (PS), a rare phospholipid in Antarctic krill oil (AKO) critical for brain development, can be produced from the abundant phosphatidylcholine (PC) using phospholipase D (PLD) in Pickering emulsion interfacial catalysis (PIC) systems. However, the exposure of PLD to organic solvent around the emulsion interface diminished PLD activity, limiting the conversion efficiency of PS. In this study, we proposed a strategy to fabricate a PIC system with high efficiency and stability by immobilizing PLD in a specific phase on the emulsion interface, based on investigating the effect of the interfacial microenvironment on PLD activity. Janus-poly(acrylic acid)/polystyrene (JPP) and Janus-polyethylenimine/octadecane (JPO) particles were fabricated as carriers to realize the specific-phase immobilization of PLD. The highest activity was observed when PLD was immobilized on the hydrophilic side of JPP (PLD@JPP(W)), 1.9-fold that of free PLD. The catalytic efficiency of PLD@JPP(W) was 1.7-fold that of free PLD, confirmed by the kcat/Km value enhancement. Immobilization on the hydrophilic side also enhanced the thermal stability of PLD. The half-lives of PLD were extended from 4 to 36 h at 40 °C and from 6 to 28 days at 4 °C. Importantly, PLD@JPP(W) showed excellent catalytic efficiency as a PIC system, achieving a PS productivity of 93% within a short time of 2 h at an enzyme dosage of 0.05 mg. PLD@JPP(W) exhibited a 3.6 times higher yield than free PLD in the production of PS from PC rich in Antarctic krill oil. The strategy in this work could also be applied to other lipases, providing a promising method for the efficient conversion of functional lipids.

Advanced glycation end-product crosslinking activates a type VI secretion system phospholipase effector protein

Advanced glycation end-products (AGE) are a pervasive form of protein damage implicated in the pathogenesis of neurodegenerative disease, atherosclerosis and diabetes mellitus. Glycation is typically mediated by reactive dicarbonyl compounds that accumulate in all cells as toxic byproducts of glucose metabolism. Here, we show that AGE crosslinking is harnessed to activate an antibacterial phospholipase effector protein deployed by the type VI secretion system of Enterobacter cloacae. Endogenous methylglyoxal reacts with a specific arginine-lysine pair to tether the N- and C-terminal α-helices of the phospholipase domain. Substitutions at these positions abrogate both crosslinking and toxic phospholipase activity, but in vitro enzyme function can be restored with an engineered disulfide that covalently links the N- and C-termini. Thus, AGE crosslinking serves as a bona fide post-translation modification to stabilize phospholipase structure. Given the ubiquity of methylglyoxal in prokaryotic and eukaryotic cells, these findings suggest that glycation may be exploited more generally to stabilize other proteins. This alternative strategy to fortify tertiary structure could be particularly advantageous in the cytoplasm, where redox potentials preclude disulfide bond formation.

Loss of Carbamoyl Phosphate Synthetase 1 Potentiates Hepatocellular Carcinoma Metastasis by Reducing Aspartate Level.

Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide. Numerous studies have shown that metabolic reprogramming is crucial for the development of HCC. Carbamoyl phosphate synthase 1 (CPS1), a rate-limiting enzyme in urea cycle, is an abundant protein in normal hepatocytes, however, lacking systemic research in HCC. It is found that CPS1 is low-expressed in HCC tissues and circulating tumor cells, negatively correlated with HCC stage and prognosis. Further study reveals that CPS1 is a double-edged sword. On the one hand, it inhibits the activity of phosphatidylcholine-specific phospholipase C to block the biosynthesis of diacylglycerol (DAG), leading to the downregulation of the DAG/protein kinase C pathway to inhibit invasion and metastasis of cancer cells. On the other hand, CPS1 promotes cell proliferation by increasing intracellular S-adenosylmethionin to enhance the m6A modification of solute carrier family 1 member 3 mRNA, a key transporter for aspartate intake. Finally, CPS1 overexpressing adeno-associated virus can dampen HCC progression. Collectively, this results uncovered that CPS1 is a switch between HCC proliferation and metastasis by increasing intracellular aspartate level.

Diverse pathways in GPCR-mediated activation of Ca2+ mobilization in HEK293 cells

G protein-coupled receptors (GPCRs) transduce extracellular stimuli into intracellular signaling. Ca2+ is a well-known second messenger that can be induced by GPCR activation through the primary canonical pathways involving Gαq- and Gβγ-mediated activation of phospholipase C-β (PLCβ). While some Gs-coupled receptors are shown to trigger Ca2+ mobilization, underlying mechanisms remain elusive. Here we evaluated whether Gs-coupled receptors including the β2-adrenergic receptor (β2AR) and the prostaglandin EP2 and EP4 receptors (EP2R and EP4R) that are endogenously expressed in HEK293 cells utilize common pathways for mediating Ca2+ mobilization. For the β2AR, we found an essential role for Gq in agonist-promoted Ca2+ mobilization while genetic or pharmacological inhibition of Gs or Gi had minimal effect. β-agonist-promoted Ca2+ mobilization was effectively blocked by the Gq-selective inhibitor YM-254890 and was not observed in ΔGαq/11 or ΔPLCβ cells. Bioluminescence resonance energy transfer analysis also suggests agonist-dependent association of the β2AR with Gq. For the EP2R, which couples to Gs, agonist treatment induced Ca2+ mobilization in a pertussis toxin (PTX)-sensitive but YM-254890-insensitive manner. In contrast, EP4R, which couples to Gs and Gi, exhibited Ca2+ mobilization that was sensitive to both PTX and YM-254890. Interestingly, both EP2R and EP4R were largely unable to induce Ca2+ mobilization in ΔGαs or ΔPLCβ cells, supporting a strong dependency on Gs signaling in HEK293 cells. Taken together, we identify differences in the signaling pathways that are utilized to mediate Ca2+ mobilization in HEK293 cells where the β2AR primarily utilizes Gq, EP2R uses Gs and Gi, and EP4R utilizes Gs, Gi and Gq.

Ascorbic Acid Enhances the Inhibitory Effect of Theasaponins against Candida albicans

Candida albicans (C. albicans) is a main cause of hospital-acquired fungal infections. Combination therapy is promising as a novel anti-C. albicans strategy because of its better efficacy. Theasaponins are pentacyclic triterpenes in the Camellia genus with multiple biological activities. Our previous studies prove that theasaponins display inhibitory activity against C. albicans. Ascorbic acid (VC) is a vitamin found in many plants that shows potential in combination therapy. However, whether VC enhances the activity of theasaponins remains unclear. In this study, the checkerboard micro-dilution method was used to assess the effect of VC (0–80 mmol/L) on the anti-C. albicans effect of theasaponins (0–1000 μg/mL). Then, the effects of theasaponins (31.25 μg/mL), VC (80 mmol/L), and theasaponins (31.25 μg/mL) + VC (80 mmol/L) on C. albicans planktonic cells and different stages of biofilm formation were assessed. Transcriptomic analysis was conducted to investigate the molecular mechanisms. According to the results, VC enhanced the anti-planktonic and anti-biofilm effect of theasaponins against C. albicans. The minimum inhibitory concentration of theasaponins was significantly decreased and the fungicidal efficiency was increased with the addition of VC. VC remarkably aggravated the suppression of theasaponins with regard to various virulence factors of C. albicans, including adhesion, early biofilm formation, mature biofilm, cell surface hydrophobicity, and phospholipase activity. Compared with the theasaponins or VC groups, the level of intracellular reactive oxygen species was higher, while the levels of mitochondrial membrane potential and adenosine triphosphate were lower in the combination group, suggesting more severe oxidative stress, mitochondrial injury, and energy deficiency. Transcriptomic analysis revealed that the combination predominantly suppressed the pathways of glycolysis, glycerophospholipid metabolism, glutathione metabolism, and cysteine and methionine metabolism. This implied that energy deficiency and redox imbalance were associated with the anti-C. albicans activity of the combination. These results prove that VC enhances the inhibitory effect of theasaponins against C. albicans and that the combination has the potential to be used as a topical antifungal therapy or disinfectant.

Characterization of Photobacterium damselae subsp. damselae isolated from diseased Asian seabass (Lates calcarifer) and the preliminary development of a formalin-killed cell vaccine.

Asian seabass (Lates calcarifer) is an economically important fish species that is widely cultivated in Thailand. However, aquaculture of Asian seabass is limited by infectious diseases. One of the most serious diseases is photobacteriosis, caused by Photobacterium damselae. Vaccination is recognized as an efficient disease prevention and pathogen control method for strengthening the aquaculture industry. To promote vaccine development, the characterization of pathogenic bacteria and their pathogenesis is required. In this study, isolates of P. damselae were obtained from commercial aquaculture farms in Thailand during 2019-2021. Analyses of 16S rRNA and the urease subunit alpha genes identified the isolates as P. damselae subsp. damselae (Phdd). Antibiotic susceptibility analyses showed that all Phdd isolates were resistant to amoxicillin (10 μg). Haemolysis and phospholipase activities were used to categorize P. damselae into three groups based on their biological activities. The pathogenicity of four candidates (SK136, PD001, PD002 and T11L) was tested in Asian seabass. Isolate SK136 showed the highest virulence, with a lethal dose (LD50) of 1.47 × 105 CFU/fish, whereas isolate PD001 did not show any virulence. Genotypic characterization, based on multi-locus sequence typing analysis, demonstrated that all candidates were novel strains with new sequence types (64, 65, 66 and 67). Preliminary vaccination using formalin-killed cells (FKCs) protected Asian seabass from artificial challenges. Taken together, these results provide fundamental knowledge for vaccine development against Phdd infection in Asian seabass.

Evaluation of Microbial Phospholipase and Lipase Activity Through the Chromatographic Analysis of Crude, Degummed, and Transesterified Soybean Oil for Biodiesel Production.

The present study aimed at synthesizing fatty acid methyl esters in a combined enzymatic method by applying degumming and transesterification of soybean oil. A soluble lipase from Serratia sp. W3 and a recombinant phosphatidylcholine-preferring phospholipase C (PC-PLC) from Bacillus thuringiensis were used in a consecutive manner for phosphorus removal and conversion into methyl esters. By applying 1% of recombinant PC-PLC almost 83% of phosphorus was removed (final content of 21.01mg/kg). Moreover, a sensitive and selective high-performance liquid chromatography method coupled to tandem mass spectrometry was applied to obtain a comprehensive lipid profile for the simultaneous evaluation of phospholipids removal and diacylglycerol (DAG) increase. A significant increase for all the monitored DAG species, up to 138.42%, was observed by using the enzymatic degumming, in comparison to the crude sample, resulting in an increased oil yield. Serratia sp. W3 lipase was identified as a suitable biocatalyst for biodiesel production, converting efficiently the acylglycerols. The results regarding the physical-chemical characteristics show that the cetane level, density and pour point of the obtained biodiesel are close to current regulation requirements. These findings highlight the potential of a two-step process implementation, based on the combination of lipase and phospholipase, as a suitable alternative for biodiesel production.

Effects of phospholipase D1 inhibitory peptide on the growth and metastasis of gastric cancer cells

Phospholipase D1 (PLD1) contributes to cancer development and progression through its effects on cell proliferation, survival, invasion, metastasis, angiogenesis, drug resistance, and modulation of the tumor microenvironment. Its central role in these processes makes it a promising target for novel cancer treatments aimed at inhibiting its activity and disrupting the signaling pathways it regulates. In this study, we aimed to investigate the effect of PLD1 inhibition on gastric cancer cell growth using a novel peptide inhibitor, TAT-TVTSP. PLD1, which plays a role in cancer progression, catalyzes the conversion of phosphatidylcholine into choline and phosphatidic acid through hydrolysis. To effectively target PLD1 in cells, we engineered TAT-TVTSP by fusing a PLD1-inhibitory peptide (TVTSP) with a cell-penetrating peptide (TAT). We observed that TAT-TVTSP effectively inhibited PLD1 activity in AGS gastric cancer cells. Moreover, TAT-TVTSP significantly inhibited the mammalian target of the rapamycin signaling pathway, including the phosphorylation of key downstream targets such as S6K1, AKT, S473, glycogen synthase kinase-3b, and forkhead box O1. TAT-TVTSP did not induce cell death, but it triggered cell cycle arrest by activating p21 and p27 via AKT phosphorylation. Functional assays revealed that TAT-TVTSP significantly impaired the colony-forming ability of AGS cells, thus inhibiting cell proliferation. Transwell and wound-healing assays revealed that this peptide disrupted the cellular behaviors critical to cancer progression, such as migration and invasion. In vivo, TAT-TVTSP significantly reduced tumor growth in the xenograft model of gastric cancer without any toxicity. Overall, our results suggest that TAT-TVTSP is a novel therapeutic agent for PLD1-mediated cancers.

Assessment of Potential Probiotic Yeasts Isolated from The Small Intestines of Cattle in Thailand

Y easts are widely studied and considered good candidates for probiotics due to their numerous benefits. This study aimed to investigate potential probiotic attributes of yeasts isolated from the small intestines of cattle in Thailand. Growth at body temperature, resistance to low pH, auto-aggregation capability, hydrophobicity, growth under gastrointestinal tract (GIT) conditions, safety profiles, antimicrobial activity, and extracellular enzyme production of yeast strains were evaluated in vitro. The results of sequential screening indicated that 829 out of 1,025 yeast strains could grow at both 37 and 39 °C, 682 strains could resist acidity at pH 2 for 24 h, 252 strains revealed greater than 83% auto-aggregation capability, and 97 strains showed higher than 90% hydrophobicity. Sixty strains grew well under simulated GIT conditions in the presence of pepsin, pancreatin, and bile salts. Thirteen strains of Diutina rugosa showed negative results for hemolytic, gelatinase, and phospholipase activities, while seven strains of Pichia kudriavzevii also showed negative results for the above activities, except for phospholipase. Neither D. rugosa nor P. kudriavzevii inhibited Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Additionally, 12 strains of D. rugosa and four strains of P. kudriavzevii produced phytase and cellulase, while only D. rugosa PCD3-6 exhibited protease and phytase activities. Therefore, D. rugosa (strains PCD3-6, PCD93-19, PYJ93-7, PYJ93-16, PCJ94-4, PCJ94-6, PCJ94-10, PYD100-8, CTD93-1, CTD93-2, CTJ93-5, CTJ93-6, and YTJ97-4) and P. kudriavzevii (strains PYI3-3, PCI3-4, PCJ90-7, and PYI91-15) were proposed as potential probiotic yeasts. They may be beneficially applied as feed additives to improve nutritional value and quality, thereby enhancing animal performance and health. Moreover, this work indicated useful preliminary results that could be a baseline for future research in human probiotic applications.

Crystal structure of Alzheimer's disease phospholipase D3 provides a molecular basis for understanding its normal and pathological functions.

Human 5'-3' exonuclease PLD3, a member of the phospholipase D family of enzymes, has been validated as a therapeutic target for treating Alzheimer's disease. Here, we have determined the crystal structure of the luminal domain of the enzyme at 2.3 Å resolution, revealing a bilobal structure with a catalytic site located between the lobes. We then compared the structure with published crystal structures of other human PLD family members which revealed that a number of catalytic and lipid recognition residues, previously shown to be key for phospholipase activity, are not conserved or, are absent. This led us to test whether the enzyme is actually a phospholipase. We could not measure any phospholipase activity but the enzyme shows robust nuclease activity. Finally, we have mapped key single nucleotide polymorphisms onto the structure which reveals plausible reasons as to why they have an impact on Alzheimer's disease.

Cannabinol modulates the endocannabinoid system and shows TRPV1-mediated anti-inflammatory properties in human keratinocytes.

Cannabinol (CBN) is a secondary metabolite of cannabis whose beneficial activity on inflammatory diseases of human skin has attracted increasing attention. Here, we sought to investigate the possible modulation by CBN of the major elements of the endocannabinoid system (ECS), in both normal and lipopolysaccharide-inflamed human keratinocytes (HaCaT cells). CBN was found to increase the expression of cannabinoid receptor 1 (CB1) at gene level and that of vanilloid receptor 1 (TRPV1) at protein level, as well as their functional activity. In addition, CBN modulated the metabolism of anandamide (AEA) and 2-arachidonoylglicerol (2-AG), by increasing the activities of N-acyl phosphatidylethanolamines-specific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH)-the biosynthetic and degradative enzyme of AEA-and that of monoacylglycerol lipase (MAGL), the hydrolytic enzyme of 2-AG. CBN also affected keratinocyte inflammation by reducing the release of pro-inflammatory interleukin (IL)-8, IL-12, and IL-31 and increasing the release of anti-inflammatory IL-10. Of note, the release of IL-31 was mediated by TRPV1. Finally, the mitogen-activated protein kinases (MAPK) signaling pathway was investigated in inflamed keratinocytes, demonstrating a specific modulation of glycogen synthase kinase 3β (GSK3β) upon treatment with CBN, in the presence or not of distinct ECS-directed drugs. Overall, these results demonstrate that CBN modulates distinct ECS elements and exerts anti-inflammatory effects-remarkably via TRPV1-in human keratinocytes, thus holding potential for both therapeutic and cosmetic purposes.

Antivenom Properties and Chemical Profiling via Molecular Networking of Mimosa gracilis Extracts.

The species Mimosa gracilis var. capillipes (Benth.) Barneby is used for its antivenom properties in the Coqueiros community, municipality of Catalão, state of Goiás. This study focused on three varieties: M. gracilis Benth. var. gracilis, M. gracilis var. capillipes (Benth.) Barneby, and M. gracilis var. invisiformis Barneby. The chemical profiles of extracts from these varieties were analysed using molecular networking through liquid chromatography with tandem mass spectrometry. Additionally, the study investigated the inhibitory potential of these three varieties against the proteolytic, coagulant, and phospholipase activities of Bothrops and Crotalus venoms. In vitro results confirmed the antivenom potential of nine extracts. Remarkably, the ethanolic extracts of roots from M. gracilis var. capillipes (Benth.) Barneby and the leaves from M. gracilis Benth. var. gracilis exhibited 100 % inhibition of the tested activities. The study also revealed 19 annotated compounds through molecular networking, reported for the first time in the species M. gracilis.

Characterization of a GDS(L)-like hydrolase from Pleurotus sapidus with an unusual SGNH motif

The GDS(L)-like lipase from the Basidiomycota Pleurotus sapidus (PSA_Lip) was heterologously expressed using Trichoderma reesei with an activity of 350 U L−1. The isoelectric point of 5.0 was determined by isoelectric focusing. The novel PSA_Lip showed only 23.8–25.1%, 25.5%, 26.6% and 28.4% identity to the previously characterized GDSL-like enzymes phospholipase, plant lipase, acetylcholinesterase and acetylxylan esterase, from the carbohydrate esterase family 16, respectively. Therefore, the enzyme was purified from the culture supernatant and the catalytic properties and the substrate specificity of the enzyme were investigated using different assays to reveal its potential function. While no phospholipase, acetylcholinesterase and acetylxylan esterase activities were detected, studies on the hydrolysis of ferulic acid methyl ester (~ 8.3%) and feruloylated carbohydrate 5-O-transferuloyl-arabino-furanose (~ 0.8%) showed low conversions of these substrates. By investigating the hydrolytic activity towards p-nitrophenyl-(pNP)-esters with various chain-lengths, the highest activity was determined for medium chain-length pNP-octanoate at 65 °C and a pH value of 8, while almost no activity was detected for pNP-hexanoate. The enzyme is highly stable when stored at pH 10 and 4 °C for at least 7 days. Moreover, using consensus sequence analysis and homology modeling, we could demonstrate that the PSA_Lip does not contain the usual SGNH residues in the actives site, which are usually present in GDS(L)-like enzymes.

OsPLDα1 mediates cadmium stress response in rice by regulating reactive oxygen species accumulation and lipid remodeling

Lipid remodeling is crucial for various cellular activities and the stress tolerance of plants; however, little is known about the lipid dynamics induced by the heavy metal cadmium (Cd). In this study, we investigated the phospholipid profiles in rice (Oryza sativa) under Cd exposure. We observed a significant decline in the total amounts of phosphatidylcholine and phosphatidylserine, contrasted with an elevation in phosphatidic acid (PA) due to Cd stress. Additionally, Cd stress prompted the activation of phospholipase D (PLD) and induced the expression of PLDα1. OsPLDα1 knockout mutants (Ospldα1) showed increased sensitivity to Cd, characterized by a heightened accumulation of hydrogen peroxide in roots and diminished PA production following Cd treatment. Conversely, PLDα1-overexpressing (OsPLDα1-OE) lines demonstrated enhanced tolerance to Cd, with suppressed transcription of the respiratory burst oxidase homolog (Rboh) genes. The transcription levels of genes associated with Cd uptake and transport were accordingly modulated in Ospldα1 and OsPLDα1-OE plants relative to the wild-type. Taken together, our findings underscore the pivotal role of OsPLDα1 in conferring tolerance to Cd by modulating reactive oxygen species homeostasis and lipid remodeling in rice.

Evaluation of Epidemiological Pattern of Candida Species Associated with Candidemia from A Tertiary Care Facility in South India

Candidemia ranks the 4th most prevalence cause of bloodstream infections and stands out as the primary cause of invasive fungal infections among hospitalized patients. Its incidence varies globally from 0.33 to 6.51 episodes per 1000 admissions, representing a major public health burden due to its increasing incidence and high mortality rates. The present research work has been conducted to identify the distribution of Candida species among septicemic patients and to determine the patterns of antifungal susceptibility of Candida species isolates from them in a tertiary care center in South India. Among the 88 Candida isolates, 13 (14.8%) were speciated and identified as C. albicans and 75 (85.2%) were Candida non-albicans. Of them, C. tropicalis (42%) ranks more prevalent. The distribution of virulence factors among 88 Candida isolates revealed that 49 isolates (55.7%) exhibited phospholipase activity, hemolysin production was detected in 68.2% of isolates, biofilm production was demonstrated in 73.9% isolates and coagulase activity was observed in 46.7% isolates. In the present study, Candida species were most sensitive to Amphotericin B (94.3%), which is followed by Caspofungin (93.2%), Voriconazole (92%), Micafungin (90.9%), and the least was observed with Flucytosine (78.4%) and Fluconazole (71.5%). Thus, in order to improve treatment responses, the insights acquired from this research will aid in clinical management and the development of antifungal stewardship recommendations.

An Investigation of Volatile Flavor Compounds and Lipolysis-Oxidation in Coppa as Affected by the Inoculation of Coagulase-Negative Staphylococcus during the Air-Drying Stage.

This study aimed to explore the effects of coagulase-negative Staphylococcus inoculation on flavor generation and lipolysis-oxidation in Coppa. Acid lipase, neutral lipase, phospholipase, and lipoxygenase (LOX) activities, as well as free fatty acids, volatile compounds, and sensory evaluation, were determined during the fermentation and air-drying processes of Coppa over 40 days. Staphylococcus carnosus and Staphylococcus xylosus or a combination of both strains were selected for this study, and natural fermentation was treated as a control. The results showed that Staphylococcus inoculation significantly enhanced lipase and LOX activities, and mixed strains had a superior effect. Palmitic acid, stearic acid, linoleic acid, and oleic acid were identified as the predominant free fatty acids in Coppa, with the mixed fermentation group exhibiting the highest contents. Acids, aldehydes, alcohols, ketones, esters, and phenols were found for the volatile compounds in Coppa. These findings thus suggested a positive role of Staphylococcus inoculation in activating lipolysis-oxidation and contributing to the flavor formation of Coppa during the air-drying stage.

Interplay of mitochondrial calcium signalling and reactive oxygen species production in the brain.

Intracellular communication and regulation in brain cells is controlled by the ubiquitous Ca2+ and by redox signalling. Both of these independent signalling systems regulate most of the processes in cells including the cell surviving mechanism or cell death. In physiology Ca2+ can regulate and trigger reactive oxygen species (ROS) production by various enzymes and in mitochondria but ROS could also transmit redox signal to calcium levels via modification of calcium channels or phospholipase activity. Changes in calcium or redox signalling could lead to severe pathology resulting in excitotoxicity or oxidative stress. Interaction of the calcium and ROS is essential to trigger opening of mitochondrial permeability transition pore - the initial step of apoptosis, Ca2+ and ROS-induced oxidative stress involved in necrosis and ferroptosis. Here we review the role of redox signalling and Ca2+ in cytosol and mitochondria in the physiology of brain cells - neurons and astrocytes and how this integration can lead to pathology, including ischaemia injury and neurodegeneration.

Preparation and Application of Clostridium perfringens Alpha Toxin Nanobodies.

All subtypes of Clostridium perfringens (C. perfringens) produce the alpha toxin (CPA), which can cause enteritis or enterotoxemia in lambs, cattle, pigs, and horses, as well as traumatic clostridial myonecrosis in humans and animals. CPA acts on cell membranes, ultimately leading to endocytosis and cell death. Therefore, the neutralization of CPA is crucial for the prevention and treatment of diseases caused by C. perfringens. In this study, utilizing CPA as an antigen, a nanobody (CPA-VHH) with a half-life of 2.9 h, an affinity constant (KD) of 0.9 nmol/L, and good stability below 60 °C was prepared from a natural nanobody library from alpacas. The biological activity analysis of CPA-VHH revealed its ability to effectively neutralize the phospholipase and hemolytic activity of CPA at a 15-fold ratio. In Vero cells, 9.8 μg/mL CPA-VHH neutralized the cytotoxicity of CPA at two times the half-maximal inhibitory concentration (IC50). In a mouse model, 35.7 ng/g body weight (BW) of CPA-VHH neutralized 90% of the lethality caused by a 2× median lethal dose (LD50) of CPA. It was found that CPA-VHH protected 80% of mice within 30 min at 2 × LD50 CPA, but this dropped below 50% after 2 h and to 0% after 4 h. Rescue trials indicated that using CPA-VHH within 30 min post-infection with 2 × LD50 CPA achieved an 80% rescue rate, which decreased to 10% after 2 h. Furthermore, CPA-VHH effectively mitigated the reduction in the expression levels of zonula occludens-1 (ZO-1), Occludin, and Claudin-1, while also attenuating the upregulation of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), and interferon-γ (IFN-γ) induced by CPA infection. Overall, this study has identified a specific nanobody, CPA-VHH, that effectively neutralizes CPA toxins in vitro and in animal models, providing a new tool for inhibiting the pathogenicity resulting from these toxins and laying an important foundation for the development of new anti-C. perfringens toxin-related therapeutic products.