Plasma Membrane Integrity Research Articles

Effects of electron beam irradiation on the postharvest quality characteristics of winter jujube (Zizyphus jujuba mill. cv. Dalidongzao)

Winter jujube was irradiated with 0.5 kGy electron beam (EB) and stored at 0 °C for 60 days. The quality of winter jujube was analyzed every 10 days to explore the effect of EB on antioxidant activity of winter jujube. Results indicated that 0.5 kGy EB reduced the decay rate of winter jujube (25.95% the 60th day), maintained the hardness and cell membrane integrity, delayed the changes of total soluble solids (TSS) and ascorbic acid (AA) contents, moreover maintained high contents of total phenols (TPC) and flavonoids. Furthermore, the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) increased, and the free radical scavenging ability of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) cation radicals and the ferric ion reducing antioxidant power (FRAP) increased. Research demonstrated the potential of EB in the preservation of winter jujube and provided important information for the application of irradiation technology.

Antifungal Activity of Sesamol on Pestalotiopsis neglecta: Interfering with Cell Membrane and Energy Metabolism.

This paper investigated the inhibitory effect of Sesamol (Ses) on Pestalotiopsis neglecta. The potential inhibitory mechanisms were explored by observing changes in cell morphology, measuring alterations in cell membrane-related indices, as well as energy metabolism-related indices and changes in enzyme activities related to virulence. The results show that Ses completely inhibited the growth of P. neglecta at 600 μg/mL (minimum inhibitory concentration and minimum fungicidal concentration), with an EC50 of 142 ± 13.22 μg/mL. As observed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM), Ses treatment resulted in the breakage and crumpling of P. neglecta cell membrane and organelle lysis. Ergosterol content and the total lipid in P. neglecta treated with 300 μg/mL Ses was 91.52% and 54% of that in the control groups, respectively. In addition, spores were stained, increased leakage of intracellular constituents at 260 nm, and decreased extracellular pH. This suggests damage to the cell membrane integrity and permeability. Furthermore, Ses decreased the ATP levels and key enzymes in the tricarboxylic acid (TCA) cycle, indicating interference with the fungal energy metabolism. Moreover, the activities of polygalacturonase (PG) and endoglucanase (EG) of P. neglecta treated with 300 μg/mL of Ses were only 28.20% and 29.13% of that in the control groups, respectively, indicating that Ses can reduce the virulence of P. neglecta. In conclusion, our results show that Ses should be considered as a potential plant-derived fungicide due to its ability to disrupt the morphology of P. neglecta, damage cell membrane integrity and permeability in P. neglecta, interfere with energy metabolism, and reduce its virulence, ultimately affecting the fungal growth.

Postharvest biocontrol ability and involved mechanism of volatile organic compounds from Serratia nematodiphila BC-SKRU-1 against Penicillium digitatum tangerine fruit

Volatile organic compounds (VOCs) emitted by microorganisms offer a promising strategy for managing green mold disease in citrus fruits caused by Penicillium digitatum. This study evaluated the biocontrol efficacy of VOCs produced by Serratia nematodiphila BC-SKRU-1 (BC-SKRU-1 VOCs) against green mold in postharvest tangerine fruits, targeting P. digitatum NKP4321. Our findings demonstrated that BC-SKRU-1 VOCs exhibited a broad spectrum of activity against seven plant pathogens, with the highest efficacy observed against NKP4321 (68.21%). A concentration of 106 CFU mL−1 for VOC production by BC-SKRU-1 completely inhibited the mycelial growth of NKP4321, whereas a concentration of 107 CFU mL−1 was required to fully inhibit spore germination. The application of four plates of BC-SKRU-1 VOCs at 108 CFU mL−1 significantly reduced the severity of green mold disease, with optimal biocontrol achieved through continuous fumigation during storage. Notably, 2-nonanol, identified as the predominant component among the 20 VOCs using HS-SPME/GC-MS, exhibited superior antifungal activity both in vitro and in vivo. It effectively decreased ergosterol production and disrupted plasma membrane integrity in NKP4321 cells. Furthermore, 2-nonanol altered the cellular content of non-enzymatic antioxidants (e.g., GSH, GSSG) and enzymatic antioxidants (e.g., CAT, SOD), elucidating its comprehensive antifungal mechanism. These findings highlight the potential of BC-SKRU-1 VOCs, particularly 2-nonanol, as effective biocontrol agents for enhancing postharvest fruit preservation by combating fungal pathogens.

Novel mandelic acid derivatives containing piperazinyls as potential candidate fungicides against Monilinia fructicola: Design, synthesis and mechanism study

Brown rot of stone fruit, a disease caused by the ascomycete fungus Monilinia fructicola, has caused significant losses to the agricultural industry. In order to explore and discover potential fungicides against M. fructicola, thirty-one novel mandelic acid derivatives containing piperazine moieties were designed and synthesized based on the amide skeleton. Among them, target compound Z31 exhibited obvious in vitro antifungal activity with the EC50 value of 11.8 mg/L, and significant effects for the postharvest pears (79.4 % protective activity and 70.5 % curative activity) at a concentration of 200 mg/L. Antifungal activity for the target compounds was found to be significantly improved by the large steric hindrance of the R1 groups and the electronegative of the piperazines in the molecular structure, according to a three-dimensional quantitative structure–activity relationship (3D-QSAR) analysis. Further mechanism studies have demonstrated that the compound Z31 can disrupt cell membrane integrity, resulting in increased membrane permeability, release of intracellular electrolytes, and affect the normal growth of hyphae. Additional, morphological study also indicated that Z31 may disrupt the integrity of the membrane by inducing generate excess endogenous reactive oxygen species (ROS) and resulting in the peroxidation of cellular lipids, which was further verified by the detection of malondialdehyde (MDA) content. These studies have provided the basis for the creation of novel fungicides to prevent brown rot in stone fruits.

Influences of bisphenol A on hydrogen production from food waste by thermophilic dark fermentation

The extensive use of plastic products in food packaging and daily life makes them inevitably enter the treatment process of food waste (FW). Plasticizer as a new pollutant is threatening the dark fermentation of FW. Our study showed that bisphenol A (BPA) at > 250 mg/L had a significant inhibition on hydrogen production from FW by thermophilic dark fermentation. The endogenous ATP content and lactate dehydrogenase (LDH) release showed that high level of BPA not only inhibited the growth of hydrogen-producing consortium, but also led to cell death. In addition, BPA mainly affects the hydrogen-producing consortium by reducing cell membrane fluidity, damaging cell membrane integrity and reducing cell membrane potential, resulting in cell death. This study provides some new insights into the mechanism of the effect of BPA on hydrogen production from FW by thermophilic dark fermentation, and lays the foundation on the utilization of FW.

Luteolin exhibits antimicrobial actions against Salmonella Typhimurium and Escherichia coli: Impairment of cell adhesion, membrane integrity, and energy metabolism

There has been an increasing interest in phytochemicals with antimicrobial properties in food safety management. However, the mechanisms of action of phytochemicals remain mostly unknown, preventing their selective applications against biofilms of specific pathogenic bacteria. The objectives of the present study were to understand the antibiofilm and antibacterial properties of luteolin (LUT) and its modes of action against two detrimental foodborne pathogens, Salmonella enterica ser. Typhimurium and enterohemorrhagic Escherichia coli. The focus encompasses biofilm inhibition, interference with cell adhesion, disruption of membrane integrity, and dysfunctions of membrane-linked bacterial electron transport systems and energy metabolism. Our findings evidence that LUT prevents pathogenic biofilm formation on both biotic (eggshell) and abiotic (stainless steel and silicon rubber) surfaces by resisting irreversible cell adhesion and interfering with hydrophobic interactions between bacteria and contact surfaces. Our comprehensive biochemical and visual analyses further reveal that LUT mainly exhibits bactericidal activities by imposing oxidative stress on cells, leading to severe cellular structure damage, inhibition of metabolic and respiratory activities, and dysfunctions of energy metabolism, ultimately resulting in bacterial cell death. This study suggests that LUT has the potential to serve as a solution for developing effective, novel antimicrobials for improving food safety management.

Phase Angle as Surrogate Marker of Muscle Weakness in Kidney Transplant Candidates Referred to Prehabilitation.

Phase angle (PhA), a marker of nutritional status obtained by bioelectrical impedance analysis (BIA), is associated with the integrity of cell membranes. Damage to muscle fiber membranes can impact muscle strength, which is related to adverse outcomes in adults with advanced chronic kidney disease (CKD). The main objective of this study was to determine the usefulness of the PhA in identifying muscle weakness in candidates for kidney transplants (KTs). Secondly, it aimed to examine the associations of PhA with other parameters of body composition, exercise performance, and muscle structure. Sensitivity, specificity, and area under the receiver operating characteristics curve were used to evaluate the PhA (index test) as a biomarker of muscle weakness. Muscle strength was estimated with maximal voluntary isometric contraction of the quadriceps (MVCI-Q) of the dominant side. Muscle weakness was defined as MVIC-Q < 40% of body weight. A total of 119 patients were evaluated (mean age 63.7 years, 75.6% men). A phase angle cut-off of 5.1° was identified to classify men with a higher likelihood of having low muscle strength in upper limbs (MVIC-Q 40% of their body weight). Male KT candidates with PhA < 5.1° had poorer exercise capacity, lower muscle strength, less muscle mass, and smaller muscle size. A PhA < 5.1° was significantly associated with an eight-fold higher muscle weakness risk (OR = 8.2, 95%CI 2.3-29.2) in a binary regression model adjusted by age, frailty, and hydration status. Remarkably, PhA is an easily obtainable objective parameter in CKD patients, requiring no volitional effort from the individual. The associations of PhA with aerobic capacity, physical activity, muscle mass, and muscle size underscore its clinical relevance and potential utility in the comprehensive evaluation of these patients.

Uncovering the Antibacterial Potential of a Peptide-Rich Extract of Edible Bird's Nest against Staphylococcus aureus.

The diverse pharmacological properties of edible bird's nest (EBN) have been elucidated in recent years; however, investigations into its antibacterial effects are still limited. In the present study, we explored the antibacterial activity of a peptide-rich extract of EBN against Staphylococcus aureus, a notorious pathogen. The EBN extract (EEE) was prepared by soaking EBN in 80% ethanol for 2 days at 60°C. Biochemical analyses showed that peptides at the molecular weight range of 1.7-10 kDa were the major biochemical compounds in the EEE. The extract exhibited strong inhibition against S. aureus at a minimum inhibitory concentration (MIC) of 125 μg/ml and a minimum bactericidal concentration (MBC) of 250 μg/ml. This activity could be attributed to the impact of the extract on cell membrane integrity and potential, biofilm formation, and reactive oxidative species (ROS) production. Notably, the expression of biofilm- and ROS-associated genes, including intercellular adhesion A (icaA), icaB, icaC, icaD, and superoxide dismutase A (sodA), were deregulated in S. aureus upon the extract treatment. Our findings indicate a noteworthy pharmacological activity of EBN that could have potential application in the control of S. aureus.

MAP3K1, SPEF2 and PLCZ1 genes association with fertility and semen quality traits of AI bulls in Andhra Pradesh

Bull fertility plays an important role in improving the economic value of the herd and is measured in terms of semen quality traits, which is influenced by both genetic and non-genetic factors. Polymorphism of MAP3K1/ CviQI (rs463712269), SPEF2/HpyCH4V (rs722354121) and PLCZ1/AvaII (rs208019489) and their association with scrotal circumference, sperm motility and plasma membrane integrity, respectively were studied on 239 bulls of different breeds reared in different frozen semen bull stations. The MAP3K1/CviQ1 assay revealed that T allele was more frequent in Ongole and Murrah population. RFLP at SPEF2/ HpyCH4V locus indicated fixation of T allele in both the exotic purebred cattle (Holstein Friesian and Jersey) whereas selective advantage of C allele was observed in Murrah buffaloes. Except in Holstein Friesian crossbred, other cattle genetic groups were with CC genotype for PLCZ1/AvaII assay and the Murrah population had low genetic diversity and heterozygosity excess. The PLCZ1/ AvaII genotypes had significant influence on the plasma membrane integrity of sperm in Holstein Friesian, Jersey and Ongole bulls. The plasma membrane integrity of sperm was reported to be high in heterozygotes (CG) of Jersey and Ongole cattle. The present study, concluded the importance of PLCZ1 gene as a marker for semen quality assessment and selection in bulls.

Dexmedetomidine and argon in combination against ferroptosis through tackling TXNIP-mediated oxidative stress in DCD porcine livers

Graft availability from donation after circulatory death (DCD) is significantly limited by ischaemia reperfusion (IR) injury. Effective strategies to mitigate IR injury in DCD grafts are essential to improve graft quality and expand the donor pool. In this study, liver grafts from DCD pigs were preserved in the University of Wisconsin (UW) solution saturated with 0.1 nM dexmedetomidine (Dex) and various concentrations of noble gases Argon (Ar) and/or Xenon (Xe) at 4 °C for 24 or 72 h. The combined 50% Ar and Dex provided maximum protection to liver grafts by reducing morphological damage, apoptosis, necroptosis, ferroptosis, hepatocyte glycogen depletion, reticulin framework collapse, iron deposition, and oxidative stress. In vitro, human liver Hep G2 cells were preserved in the UW solution saturated with 0.1 nM Dex and 50% Ar in combination at 4 °C for 24 h, followed by recovery in medium at 37 °C for up to 48 h to mimic clinical IR injury. This treatment significantly increased the expression of anti-oxidative stress proteins by promoting the translocation of thioredoxin-interacting protein (TXNIP) to mitochondria, thereby inhibiting ferroptosis, increasing plasma membrane integrity, and maintaining cell viability.In summary, The combination of 0.1 nM Dex and 50% Ar may be a promising strategy to reduce ferroptosis and other form cell death, and preserve liver grafts.

Comparison of Nanoparticles and Single-Layer Centrifugation for Separation of Dead from Live Stallion Spermatozoa.

The goal of this study was to compare the efficacy of coated iron-core nanoparticles and single-layer centrifugation for separation of dead from live stallion spermatozoa. Our hypothesis was that nanoparticles would bind to dead sperm and allow for separation from live sperm using a magnet, resulting in a population of spermatozoa with a high percentage of total and progressive motility. Treatment Group 1 was an untreated control. Treatment Group 2 (nanoparticles, NP) utilized sperm incubated with nanoparticles followed by application of a magnet to remove dead sperm adhered to the coated nanoparticles. Treatment Group 3 (single-layer centrifugation, SLC) layered sperm above EquiPure™ followed by centrifugation. Semen samples were subsequently evaluated for sperm motility parameters, plasma membrane integrity, acrosome status, and morphology. The SLC technique yielded higher (p < 0.05) progressive motility (76 ± 9.2%) than the NP separation technique (59 ± 12.2%) or the untreated control (47.3 ± 5.1%). However, the total number of sperm recovered was higher (p < 0.05) in the NP technique (526.2 ± 96.6 × 106) than the SLC procedure (211.7 ± 70 × 106), yielding a higher total number of progressively motile sperm (317.6 ± 109 × 106) recovered using the NP technique than the SLC technique (157.8 ± 43.6 × 106). The percentage of live, acrosome intact sperm recovered was higher for SLC than NP. In summary, the SLC technique yielded a higher percentage of sperm motility, intact plasma membranes, and acrosome integrity, but yielded lower total sperm than with the nanoparticle separation technique.

Design, Synthesis, Bioactivity, and Tentative Exploration of Action Mode for Benzyl Ester-Containing Derivatives.

The active splicing strategy has witnessed improvement in bioactivity and antifungal spectra in pesticide discovery. Herein, a series of simple-structured molecules (Y1-Y53) containing chloro-substituted benzyl esters were designed using the above strategy. The structure-activity relationship (SAR) analysis demonstrated that the fatty acid fragment-structured esters were more effective than those containing an aromatic acid moiety or naphthenic acid part. Compounds Y36 and Y41, which featured a thiazole-4-acid moiety and trifluoromethyl aliphatic acid part, respectively, exhibited excellent in vivo curative activity (89.4%, 100 mg/L Y36) and in vitro fungicidal activity (EC50 = 0.708 mg/L, Y41) against Botrytis cinerea. Determination of antifungal spectra and analysis of scanning electron microscopy (SEM), membrane permeability, cell peroxidation, ergosterol content, oxalic acid pathways, and enzymatic assays were performed separately here. Compound Y41 is cost effective due to its simple structure and shows promise as a disease control candidate. In addition, Y41 might act on a novel target through a new pathway that disrupts the cell membrane integrity by inducing cell peroxidation.

Quercetin in semen extender curtails reactive oxygen and nitrogen species and improves functional attributes of cryopreserved buck semen

Cryopreservation of goat spermatozoa is challenging due to several factors, including one of the most essential, i.e., oxidative stress. It is particularly essential in goat semen due to its scanty ejaculate volume and high sperm concentration. This leaves a narrow sperm-to-seminal plasma ratio owing to marginal antioxidant support; moreover, semen extension further dilutes the antioxidant level, leading to an imbalance of oxidant-antioxidant equilibrium. The present study aimed to evaluate the effect of quercetin on curtailing oxidative stress and its reflection on the post-thaw survivability and membrane integrity of goat spermatozoa. For this study, six bucks were selected. Six ejaculates from each buck totaling 36 ejaculates were collected, which were then split into five parts; furthermore, each part was added with a semen extender having a particular concentration of additive. Group C without quercetin and T1 containing Vitamin E at 3 mmol/mL were considered the control and positive control respectively, whereas T2, T3, and T4 contain 10, 20, and 30 μmol/mL of Quercetin respectively. The final sperm concentration of each group was kept at 200 × 106 spermatozoa/mL. All groups were subjected to equilibration at 4 °C for 4 h, then filled in French mini (0.25 mL) straws, followed by sealing and cryopreservation. Samples after 72 h of cryopreservation were subjected to evaluation of plasma membrane integrity and viability through staining, acrosomal integrity, and mitochondrial membrane activity through flow cytometry. Evaluation of sperm kinematics as well as the oxidant-antioxidant status of sperm (ROS and nitric oxide) and seminal plasma (SOD, CAT, GPx, FRAP, and lipid peroxidation through MDA estimation) were also carried out. Quercetin, when supplemented at 20 μmol/mL in buck semen extender, significantly (p < 0.01) improved cryopreserved sperm functions in terms of plasma membrane integrity, viability, acrosomal integrity, mitochondrial membrane activity, and sperm kinematics of buck semen. Similarly, Quercetin supplementation at 20 μmol/mL significantly reduced reactive oxygen and nitrogen species (RONS) in sperm and improved the antioxidant status of seminal plasma, which was indicated by reduced oxidative damage and improved the antioxidant status of buck semen. In conclusion, Quercetin at 20 μmol/mL reduced oxidative stress, improved semen antioxidant status, and improved sperm membranes integrity and kinematics.

Antimicrobial peptide A20L: in vitro and in vivo antibacterial and antibiofilm activity against carbapenem-resistant Klebsiella pneumoniae.

A20L showed antibacterial and anti-infective efficacy in vitro and in vivo against Klebsiella pneumoniae. It can have an antibacterial effect by disrupting the integrity of cell membranes. A20L displayed anti-biofilm and anti-inflammatory activity against carbapenem-resistant K. pneumoniae and certain application potential in vivo, which provides a new idea for the clinical treatment of biofilm-associated infections.

Post-cooling sperm processing can rescue sperm quality of cooled-stored stallion semen

Poor sperm quality in cooled-shipped semen has been related to subpar fertility in horses. Therefore, this study aimed to evaluate the ability of post-cooling sperm processing to improve sperm parameters of cooled-stored stallion semen for artificial insemination. For all experiments, ejaculates were collected, processed, and diluted in skimmed milk-based (SM) medium and stored at 5 °C/24h. In all experiments an aliquot of unprocessed cooled semen was used as a control. In the first experiment (Exp 1.), cooled-stored semen from 16 stallions (n = 32) was processed by SpermFilter or centrifugation (600×g/10min) and resuspended in an egg yolk-based freezing medium containing permeating cryoprotectants (EY-C) for cryopreservation. Sperm recovery and motility parameters were immediately assessed after sperm resuspension in both groups and compared with unprocessed (Unp) samples. In Exp 2., cooled semen samples from six stallions (n = 18) were processed using SpermFilter and resuspended in SM or EY-C. Motility parameters and plasma membrane integrity were assessed in all groups (Unp, SM, and EY-C). In Exp 3, cooled semen from four stallions (n = 20) was processed by SpermFilter, resuspended in SM, EY-C, or egg yolk-based medium without cryoprotectants (EY-nC); and submitted to a thermoresistance test (37 °C/3h). Motility parameters, plasma membrane integrity and stability, mitochondrial membrane potential, mitochondrial superoxide generation, and DNA fragmentation index were evaluated in all groups. Finally, in Exp 4, 39 estrous cycles of 11 mares were inseminated with unprocessed (n = 6) cooled-stored semen or semen cooled at 5 °C/24h and then processed by SpermFilter and resuspended in SM (n = 5), EY-C (n = 11), EY-nC (n = 11), or centrifuged and resuspended in EY-C (n = 6). Overall, semen processing and resuspension in EY mediums (EY-C and EY-nC) improved sperm parameters compared with those of unprocessed semen (P < 0.05). Centrifugation (91 ± 5 %) recovered more sperm than SpermFilter (84 ± 9 %; P < 0.05). Sperm resuspended in EY-nC maintained better sperm parameters throughout the thermoresistance test than those in the other groups (P < 0.05). The fertility rates were similar between all groups (P > 0.05). In conclusion, processing and resuspension in EY medium can improve sperm parameters in post-cooled-stored stallion semen.

The contribution of different sperm parameters to better explain ram semen cryoresistance

In order to determine an effective procedure for explaining ram sperm cryoresistance and develop a new model for breeders classification, a retrospective study was conducted using sperm analysis data obtained over two consecutive years from a total of 82 sessions of ram semen cryopreservation. In each session, fresh ejaculates from eight males were collected via artificial vagina, pooled and frozen in liquid nitrogen vapors. After thawing, a total of 19,084 sperm tracks and 11,319 morphometric measurements were analysed. Clustering analyses were applied to establish motile and morphometric sperm subpopulations. Additionally, plasma and acrosome membrane integrity, as well mitochondrial activity using flow cytometry immediately after sperm thawing and following hypoosmotic shock test (HOST) was assessed. To develop a Ram Sperm Cryoresistance Index, Principal Component Analyses (PCA) using 22 variables were conducted. In the first PCA, the parameters that best explain cryoresistance include total motility (TM), motile subpopulation 2 (motSP2, which groups slow, very linear spermatozoa with low lateral head displacement), morphometric subpopulation 1 (morphSP1, grouping spermatozoa with the smallest head size and lowest shape values), sperm plasma membrane integrity immediately after thawing and following hypoosmotic shock test. These parameters collectively account for 77.34 % of the accumulated variance. To emphasize their importance, a second PCA was performed, revealing significant higher weighting coefficients for the quantity (TM) and quality (motSP2) of sperm movement after thawing, compared to the head size and shape of the thawed sperm (morphSP1). Furthermore, HOST Viability played a more decisive role than what was observed under isotonic conditions.

A Highly Charged Positive Cage Causes Simultaneous Enhancement of Type‐II and O2‐Independent‐Type‐I Photodynamic Therapy via One‐/Two‐Photon Stimulation and Tumor Immunotherapy via PANoptosis and Ferroptosis

To solve the oxygen dependence problem of photodynamic therapy (PDT), it is critical to explore photosensitizers that do not rely on O2 molecule to generate reactive oxygen species (ROS). Herein, a stable cationic metal‐organic cage [Pd6(RuLoz3)8](BF4)28 (MOC‐88) that possesses high +28 charges is designed. The cage‐confined positive microenvironment enables efficient generation of hydroxyl radicals and improved yield of the singlet oxygen under one‐/two‐photon excitation, showing excellent performance to concurrently enhance Type‐II and O2‐independent‐Type‐I PDT. Moreover, the effective ROS production and robust lipid peroxidation trigger a series of signaling pathways (inflammasome, cyclic guanosine monophosphate–adenosine monophosphate synthase stimulator of interferon genes, and NF‐κB) to evoke PANoptosis and ferroptosis in tumor cells, enabling MOC‐88 to simultaneously cause the loss of cell membrane integrity, release a series of inflammatory cytokines and damage‐associated molecular patterns, stimulate the maturation and antigen presentation ability of dendritic cells, and ultimately activate T‐cell‐dependent adaptive immunity in vivo to inhibit tumor growth.

Effects of Copper on Legionella pneumophila Revealed via Viability Assays and Proteomics.

Cu is an antimicrobial that is commonly applied to premise (i.e., building) plumbing systems for Legionella control, but the precise mechanisms of inactivation are not well defined. Here, we applied a suite of viability assays and mass spectrometry-based proteomics to assess the mechanistic effects of Cu on L. pneumophila. Although a five- to six-log reduction in culturability was observed with 5 mg/L Cu2+ exposure, cell membrane integrity only indicated a <50% reduction. Whole-cell proteomic analysis revealed that AhpD, a protein related to oxidative stress, was elevated in Cu-exposed Legionella relative to culturable cells. Other proteins related to cell membrane synthesis and motility were also higher for the Cu-exposed cells relative to controls without Cu. While the proteins related to primary metabolism decreased for the Cu-exposed cells, no significant differences in the abundance of proteins related to virulence or infectivity were found, which was consistent with the ability of VBNC cells to cause infections. Whereas the cell-membrane integrity assay provided an upper-bound measurement of viability, an amoebae co-culture assay provided a lower-bound limit. The findings have important implications for assessing Legionella risk following its exposure to copper in engineered water systems.

Subtle membrane changes in cryopreserved bull spermatozoa when modified temperature drop rates are used during the first phase of freezing

BACKGROUND: Cryopreservation of spermatozoa involves reduction of temperature to a subzero level, leading to increased longevity. However, temperature reduction has a significant effect on sperm membranes. OBJECTIVE: To evaluate the impact of the rate of temperature drop during the first phase of freezing on subtle membrane changes in cryopreserved bull spermatozoa. MATERIALS AND METHODS: Thirty-two ejaculates from four bulls (eight ejaculates/bull) were collected using artificial vagina while keeping a 3 to 4 days gap between two collections. Diluted semen samples were equilibrated at 5°C for 4 hours. The samples were then placed in a pre-programmed semen freezer. The first phase of freezing, that is, 5°C till −10°C was subjected to three different temperature drop rates: accelerated (F1), moderate (F2), and slow (F3), at 20°C per min, 10°C per min and 5°C per min, respectively. After thawing, spermatozoa were assessed for percentage live, plasma, and acrosomal membrane integrity, along with the external appearance of phosphatidyl serine, indicating apoptosis. RESULTS: A significant difference (p&lt;0.05) in viability, plasma membrane integrity (HOS test), and acrosome membrane integrity (PSA test) was observed between F3 and the other groups. However, the parameters did not significantly differ between F1 and F2. The annexin V-PI assay (AN/PI) categorized four types of sperm populations: non-apoptotic and viable (AN−/PI−), apoptotic and viable (AN+/PI−), non-apoptotic and non-viable (AN−/PI+), and apoptotic and non-viable (AN+/PI+). The proportion of spermatozoa with (AN−/PI−) and (AN+/PI+) differed significantly (p&lt;0.05) between F3 and the other groups. The values for apoptotic and viable (AN+/PI−) and non-apoptotic and non-viable (AN−/PI+) sperm were not significantly different among all freezing categories. CONCLUSION: A slower temperature drop rate (freezing rate) during the first phase of freezing results in less damaging, subtle membrane changes.

Synthesis, characterization, antioxidant and bacteriostasis in preservation of isoorientin loaded Zein/GA nanoparticles

Isoorientin (Iso) is a natural flavone with multiple activities. In the present study, the partial chemical properties and activities of Iso were improved by the nanoparticle loading technique. Zein/GA nanoparticles were successfully synthesized with the antisolvent precipitation method, and the structure and stability of the Zein/GA nanoparticles loaded with Iso (Zein/GA-Iso nanoparticles) were characterized by FTIR, UV–vis spectroscopy and zeta-sizer analysis. Results showed that Zein/GA-Iso nanoparticles possessed greater stability, light stability, hydrophilicity and antioxidant activity. Furthermore, Zein/GA-Iso nanoparticles exerted notable antibacterial activity against E. coli, S. aureus, and P. aeruginosa by destroying the permeability and integrity of cell membrane. On this basis, Zein/GA-Iso nanoparticles do have a bacteriostatic effect on pork. In conclusion, Zein/GA-Iso nanoparticles had better stability and pork preservation for applications in the food processing field as a new type of antiseptic and freshening agent.