Proceedings of the 2023 Viral Clearance Symposium, Session 6: Viral Inactivation.

The use of detergents or low pH hold are commonly employed techniques in biologics downstream processing to inactivate enveloped viruses. These approaches have been demonstrated to be robust and are detailed in ASTM E2888 (low pH) and ASTM E3042-16 (Triton X-100), accordingly. One of the recent challenges is the need for a replacement of Triton X-100 with a more environmentally friendly detergent with similar log10 reduction value (LRV) achieved. The presentations in this session focused on a detailed assessment of a range of detergents. The most well characterized and potentially robust detergents identified were TDAO (n-Tetradecyl-N,N-dimethylamine-N-oxide) and Simulsol SL 11 W. Key performance factors assessed (in direct comparison with the industry standard Triton X-100) were viral inactivation kinetics (total elapsed time to achieve equilibrium), LRV achieved of enveloped viruses, toxicity, potential impact on product quality and process performance, clearance of residual detergent in subsequent downstream steps, assays to support assessment with appropriate limit of quantification, and commercial supply of detergent of the appropriate quality standard. Both TDAO and Simulsol SL11 had similar overall LRV as Triton-100. In addition, for the low pH viral inactivation, reduced LRV was observed at pH > 3.70 and low salt concentration (outside of the ASTM range), which is a cautionary note when applying low pH inactivation to labile proteins.

Transient control of lytic activity via a non-equilibrium chemical reaction system.

The development of artificial non-equilibrium chemical reaction systems has recently attracted considerable attention as a new type of biomimetic. However, due to the lack of bioorthogonality, such reaction systems could not be linked to the regulation of any biological phenomena. Here, we have newly designed a non-equilibrium reaction system based on olefin metathesis to produce the Triton X-mimetic non-ionic amphiphile as a kinetic product. Using phospholipid vesicles encapsulating fluorescent dyes and red blood cells as cell models, we demonstrate that the developed chemical reaction system is applicable for transient control of the resulting lytic activity.

Intrinsic Lipid Curvature and Bilayer Elasticity as Regulators of Channel Function: A Comparative Single-Molecule Study.

Perturbations in bilayer material properties (thickness, lipid intrinsic curvature and elastic moduli) modulate the free energy difference between different membrane protein conformations, thereby leading to changes in the conformational preferences of bilayer-spanning proteins. To further explore the relative importance of curvature and elasticity in determining the changes in bilayer properties that underlie the modulation of channel function, we investigated how the micelle-forming amphiphiles Triton X-100, reduced Triton X-100 and the HII lipid phase promoter capsaicin modulate the function of alamethicin and gramicidin channels. Whether the amphiphile-induced changes in intrinsic curvature were negative or positive, amphiphile addition increased gramicidin channel appearance rates and lifetimes and stabilized the higher conductance states in alamethicin channels. When the intrinsic curvature was modulated by altering phospholipid head group interactions, however, maneuvers that promote a negative-going curvature stabilized the higher conductance states in alamethicin channels but destabilized gramicidin channels. Using gramicidin channels of different lengths to probe for changes in bilayer elasticity, we found that amphiphile adsorption increases bilayer elasticity, whereas altering head group interactions does not. We draw the following conclusions: first, confirming previous studies, both alamethicin and gramicidin channels are modulated by changes in lipid bilayer material properties, the changes occurring in parallel yet differing dependent on the property that is being changed; second, isolated, negative-going changes in curvature stabilize the higher current levels in alamethicin channels and destabilize gramicidin channels; third, increases in bilayer elasticity stabilize the higher current levels in alamethicin channels and stabilize gramicidin channels; and fourth, the energetic consequences of changes in elasticity tend to dominate over changes in curvature.

Unraveling the Biophysical Mechanisms of How Antiviral Detergents Disrupt Supported Lipid Membranes: Toward Replacing Triton X-100.

Triton X-100 (TX-100) is a membrane-disrupting detergent that is widely used to inactivate membrane-enveloped viral pathogens, yet is being phased out due to environmental safety concerns. Intense efforts are underway to discover regulatory acceptable detergents to replace TX-100, but there is scarce mechanistic understanding about how these other detergents disrupt phospholipid membranes and hence which ones are suitable to replace TX-100 from a biophysical interaction perspective. Herein, using the quartz crystal microbalance-dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS) techniques in combination with supported lipid membrane platforms, we characterized the membrane-disruptive properties of a panel of TX-100 replacement candidates with varying antiviral activities and identified two distinct classes of membrane-interacting detergents with different critical micelle concentration (CMC) dependencies and biophysical mechanisms. While all tested detergents formed micelles, only a subset of the detergents caused CMC-dependent membrane solubilization similarly to that of TX-100, whereas other detergents adsorbed irreversibly to lipid membrane interfaces in a CMC-independent manner. We compared these biophysical results to virus inactivation data, which led us to identify that certain membrane-interaction profiles contribute to greater antiviral activity and such insights can help with the discovery and validation of antiviral detergents to replace TX-100.

Highly effective and reusable Ni-Al oxide/Zn0.4Co0.6Fe2O4 superparamagnetic aerogel for oil-water separation.

Oily wastewater from the oil industry and oil spill accidents has become a serious environmental problem and has attracted worldwide attention. The present study reports on the successful preparation of a novel magnetic Ni-Al oxide/Zn0.4Co0.6F2O4 mesoporous aerogel (MNA) as a highly selective adsorbent for oil removal from water. Oleic acid (OA) and Triton X-100 (TX) were used as hydrophobic agents for MNA surface modification. It was found that the attached amount of OA on the mesoporous MNA aerogel is 3.5 times larger than that of TX, giving an advantage to MNA-OA in oil separation. The MNA-OA displayed superhydrophobicity (contact angle ∼150°) and superparamagnetism properties that allowed the adsorbent to be used selectively for oil removal. The MNA-OA was found to have a high oil removal efficiency of ∼97% with an adsorption capacity of ∼2 g/g. Furthermore, the produced magnetic adsorbent has high stability due to the strong chemical binding of OA, which is demonstrated by its good reusability performance. Throughout five separate runs, the MNA-OA was shown to be a very efficient and reusable adsorbent for oily wastewater.

Identification of protein components of the transformation system in the cell line of immortalized human keratinocytes HaCaT exposed to surfactants.

Using the method of shotgun mass spectrometry, we have evaluated changes in the proteomic profile of HaCat cells in response to the treatment with sodium dodecyl sulfate (anionic surfactant) and Triton-X100 (non-ionic surfactant) in two concentrations (12.5 µg/ml and 25.0 µg/ml). The study revealed induction of orphan CYP2S1 (biotransformation phase I) in response to Triton-X100. We have identified proteins of II (glutathione-S-transferases, GSTs) and III (solute carrier proteins, SLCs) biotransformation phases, as well as antioxidant proteins (peroxiredoxins, PRDXs; catalase, CAT; thioredoxin, TXN). Thus, proteins of all three xenobiotic detoxification phases were detected. The presented results suggest a new prospect of using HaCaT keratinocytes as a model of human epidermis for studying the metabolism of drugs/toxicants in human skin in vitro.

Decellularized tissue exhibits large differences of extracellular matrix properties dependent on decellularization method: novel insights from a standardized characterization on skeletal muscle.

Decellularized matrices are an attractive choice of scaffold in regenerative medicine as they can provide the necessary extracellular matrix (ECM) components, signals and mechanical properties. Various detergent-based protocols have already been proposed for decellularization of skeletal muscle tissue. However, a proper comparison is difficult due to differences in species, muscle origin and sample sizes. Moreover, a thorough evaluation of the remaining acellular matrix is often lacking. We compared an in-house developed decellularization protocol to four previously published methods in a standardized manner. Porcine skeletal muscle samples with uniform thickness were subjected to in-depth histological, ultrastructural, biochemical and biomechanical analysis. In addition, 2D and three-dimensional cytocompatibility experiments were performed. We found that the decellularization methods had a differential effect on the properties of the resulting acellular matrices. Sodium deoxycholate combined with deoxyribonuclease I was not an effective method for decellularizing thick skeletal muscle tissue. Triton X-100 in combination with trypsin, on the other hand, removed nuclear material but not cytoplasmic proteins at low concentrations. Moreover, it led to significant alterations in the biomechanical properties. Finally, sodium dodecyl sulphate (SDS) seemed most promising, resulting in a drastic decrease in DNA content without major effects on the ECM composition and biomechanical properties. Moreover, cell attachment and metabolic activity were also found to be the highest on samples decellularized with SDS. Through a newly proposed standardized analysis, we provide a comprehensive understanding of the impact of different decellularizing agents on the structure and composition of skeletal muscle. Evaluation of nuclear content as well as ECM composition, biomechanical properties and cell growth are important parameters to assess. SDS comes forward as a detergent with the best balance between all measured parameters and holds the most promise for decellularization of skeletal muscle tissue.

Cell counting to monitor swab efficiency.

Plastic bags, such as ziplock bags, have been used to transport illicit materials worldwide; however, very few studies have tried to optimize the recovery of DNA from these items. This study reports on the best combination of swabs and moistening solution for the greatest recovery of cellular material from ziplock bags. Five swabs, two different variations of Copan Diagnostics nylon 4N6FLOQSwabs, one Medical Wire rayon DRYSWAB, one IsoHelix rayon swab, and one Livingstone cotton swab, were evaluated with two moistening solutions, Triton X-100 in either distilled water or isopropanol. Fingermarks were deposited on ziplock bags and stained with Diamond™ Nucleic Acid Dye to allow visualization of the cells pre- and post-swabbing to determine the number of cells recovered. Based on cell counting data, swabs moistened with Triton X-100 in distilled water performed better than those moistened with isopropanol. Livingstone cotton swabs had the worst recovery of cellular material, while the other swabs tested had no significant difference in their respective solutions. A comparison of the best three swabs for cellular recovery yielded no differences in the DNA concentration extracted. A linear relationship was observed between the log number of cells recovered by swabbing and the DNA concentration following extraction and quantification. The process of monitoring cell collection using fluorescence microscopy on ziplock bags allowed evaluation of swabbing efficacy. Additionally, this study highlights the ability to evaluate cellular recovery independently of traditional extraction, quantification, or profiling techniques which may unequally affect samples.

[Effects of cerium oxide nanoenzyme-gelatin methacrylate anhydride hydrogel in the repair of infected full-thickness skin defect wounds in mice].

Objective: To investigate the effects of cerium oxide nanoenzyme-gelatin methacrylate anhydride (GelMA) hydrogel (hereinafter referred to as composite hydrogel) in the repair of infected full-thickness skin defect wounds in mice. Methods: This study was an experimental study. Cerium oxide nanoenzyme with a particle size of (116±9) nm was prepared by hydrothermal method, and GelMA hydrogel with porous network structure and good gelling performance was also prepared. The 25 µg/mL cerium oxide nanoenzyme which could significantly promote the proliferation of human skin fibroblasts and had high superoxide dismutase activity was screened out. It was added to GelMA hydrogel to prepare composite hydrogel. The percentage of cerium oxide nanoenzyme released from the composite hydrogel was calculated after immersing it in phosphate buffer solution (PBS) for 3 and 7 d. The red blood cell suspension of mice was divided into PBS group, Triton X-100 group, cerium oxide nanoenzyme group, GelMA hydrogel group, and composite hydrogel group, which were treated with corresponding solution. The hemolysis of red blood cells was detected by microplate reader after 1 h of treatment. The bacterial concentrations of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli were determined after being cultured with PBS, cerium oxide nanoenzyme, GelMA hydrogel, and composite hydrogel for 2 h. The sample size in all above experiments was 3. Twenty-four 8-week-old male BALB/c mice were taken, and a full-thickness skin defect wound was prepared in the symmetrical position on the back and infected with MRSA. The mice were divided into control group without any drug intervention, and cerium oxide nanoenzyme group, GelMA hydrogel group, and composite hydrogel group applied with corresponding solution, with 6 mice in each group. The wound healing was observed on 3, 7, and 14 d after injury, and the remaining wound areas on 3 and 7 d after injury were measured (the sample size was 5). The concentration of MRSA in the wound exudation of mice on 3 d after injury was measured (the sample size was 3), and the blood flow perfusion in the wound of mice on 5 d after injury was observed using a laser speckle flow imaging system (the sample size was 6). On 14 d after injury, the wound tissue of mice was collected for hematoxylin-eosin staining to observe the newly formed epithelium and for Masson staining to observe the collagen situation (the sample size was both 3). Results: After immersion for 3 and 7 d, the release percentages of cerium oxide nanoenzyme in the composite hydrogel were about 39% and 75%, respectively. After 1 h of treatment, compared with that in Triton X-100 group, the hemolysis of red blood cells in PBS group, GelMA hydrogel group, cerium oxide nanoenzyme group, and composite hydrogel group was significantly decreased (P<0.05). Compared with that cultured with PBS, the concentrations of MRSA and Escherichia coli cultured with cerium oxide nanoenzyme, GelMA hydrogel, and composite hydrogel for 2 h were significantly decreased (P<0.05). The wounds of mice in the four groups were gradually healed from 3 to 14 d after injury, and the wounds of mice in composite hydrogel group were all healed on 14 d after injury. On 3 and 7 d after injury, the remaining wound areas of mice in composite hydrogel group were (29±3) and (13±5) mm2, respectively, which were significantly smaller than (56±12) and (46±10) mm2 in control group and (51±7) and (38±8) mm2 in cerium oxide nanoenzyme group (with P values all <0.05), but was similar to (41±5) and (24±9) mm2 in GelMA hydrogel group (with P values both >0.05). On 3 d after injury, the concentration of MRSA on the wound of mice in composite hydrogel group was significantly lower than that in control group, cerium oxide nanoenzyme group, and GelMA hydrogel group, respectively (with P values all <0.05). On 5 d after injury, the volume of blood perfusion in the wound of mice in composite hydrogel group was significantly higher than that in control group, cerium oxide nanoenzyme group, and GelMA hydrogel group, respectively (P<0.05). On 14 d after injury, the wound of mice in composite hydrogel group basically completed epithelization, and the epithelization was significantly better than that in the other three groups. Compared with that in the other three groups, the content of collagen in the wound of mice in composite hydrogel group was significantly increased, and the arrangement was also more orderly. Conclusions: The composite hydrogel has good biocompatibility and antibacterial effect in vivo and in vitro. It can continuously sustained release cerium oxide nanoenzyme, improve wound blood perfusion in the early stage, and promote wound re-epithelialization and collagen synthesis, therefore promoting the healing of infected full-thickness skin defect wounds in mice.

Effect of chemical and physical agents on monkeypox virus infectivity and downstream research applications.

The 2022 global spread of Monkeypox Virus (MPXV) underlined the need to investigate safe-handling procedures of clinical and research samples. Here we evaluated the efficiency in reducing MPXV infectious titer of Triton X-100 (0.1 and 0.2%), UV-C irradiation (15 or 30 min), and heat (56 °C 30 min or 70 °C 5 min). The treatment of MPXV at 70 °C resulted in the strongest decrease of MPXV infectious titer (5.4 Log TCID50/mL), 56 °C and UV-C had a lighter impact (3.9 and 4.3Log), Triton X-100 was less efficient (1.8-2.5Log). Notably, SARS-CoV-2 was much more susceptible to Triton X-100 (4.0 Log decrease). UV-C had the highest impact on MPXV DNA detection by PCR (2.2-4.3 Ct value increase); protein detection by ELISA was dramatically impaired by heating. Overall, UV-C and heating were more effective in lowering MPXV infectious titer but their impact on nucleic acids or protein detection assays must be considered.

The effect of emulsifier type and oil inclusion on stress-related gene expression of Salmonella typhimurium in oil-in-water emulsion.

Salmonella has been associated with numerous outbreaks from contaminated food products, including emulsions. Emulsions are influenced by emulsifier type and oil presence, which can have varying degrees of stress or protection on bacteria. Although our previous research has shown that emulsifier solutions, rather than emulsions, provide a protective effect on Salmonella typhimurium after thermal treatment, the underlying mechanism remains unclear. This study selected S. typhimurium as the model microorganism and utilized the same emulsifiers (Tween 20, Tween 80, Triton X-100) to create emulsifier solutions and emulsions with the same oil fraction (60% (v/v)) to examine their effect on the expression of nine selected genes (rpoE, rpoH, otsB, proV, fadA, fabA, dnaK, ibpA, ompC) associated with stress response. Specifically, the study observed variations in gene expression under normal and thermal stress at 55°C. After 20-h incubation, Triton X-100 emulsion caused an upregulation of stress-related genes, rpoE, otsB, and fabA, suggesting stressful environment. After thermal treatment, S. typhimurium in Triton X-100 solution showed a longer 5-log reduction time with increased proV and decreased fabA and ompC expression, suggesting enhanced thermal protection compared to its emulsion. Conversely, Tween 80 solution increased fabA and ompC expression, indicating greater membrane fluidity and passive diffusion, potentially reducing thermal resistance. However, according to the upregulation of ibpA, this effect was likely mitigated by the overproduction of heat shock proteins. Notably, Triton X-100 environments exhibited the most significant gene expression changes after heat treatment, whereas Tween 80 without oil was the most inhospitable for bacterial survival. These findings inform bacterial responses under various conditions, aiding food safety strategies.

Preparing for another Ebola Outbreak: The impact of viral inactivation methods on commonly measured biochemistry analytes in plasma and urine.

INTRODUCTION: Infectious specimens containing viruses like Ebola require sample manipulation to ensure the safety of laboratory staff, which may negatively impact biochemistry test results. We evaluated the impact of viral inactivation methods on 25 biochemistry analytes in plasma, and seven biochemistry analytes in urine. METHODS: Fifteen lithium heparinized plasma specimens with and without gel underwent the following viral inactivation methods: 1) untreated, 2) Triton X-100 treatment, 2) heated for 60 min then Triton X-100 treatment, 3) heated for 60 min, 4) heated for 75 min, and 5) heated for 90 min. Electrolytes, protein, enzymes, glucose, as well as hepatic and renal markers were measured on the Roche Cobas e601, c502 or c702. Urinalysis analytes were measured on the Siemens CLINITEK. Acceptable recovery was based on Institute for Quality Management in Healthcare 2021 guidelines or ± 1 for urinalysis. RESULTS: Potassium and lactate dehydrogenase were impacted by the presence of gel. Viral inactivation with Triton X-100 had minimal impact on the biochemistry results. Heat inactivation resulted in significant negative bias in alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, creatinine, total protein, amylase, lactate dehydrogenase and creatine kinase. Positive bias in phosphate, aspartate transaminase, total bilirubin, and uric acid were observed after heat inactivation. CONCLUSION: Reliable results for commonly measured electrolytes, enzymes and proteins can be obtained after viral inactivation by Triton X-100 treatment at room temperature. However, heat inactivation has significant negative impact on routine biochemistry enzymes and alternative testing processes should be explored.

Engineering Yarrowia lipolytica for sustainable ricinoleic acid production: A pathway to free fatty acid synthesis.

Ricinoleic acid (C18:1-OH, RA) is a valuable hydroxy fatty acid with versatile applications. The current industrial source of RA relies on the hydrolysis of castor bean oil. However, the coexistence of the toxic compound ricin and the unstable supply of this plant have led to an exploration of promising alternatives: generating RA in heterologous plants or microorganisms. In this study, we engineered the oleaginous yeast Yarrowia lipolytica to produce RA in the form of free fatty acids (FFA). First, we overexpressed fungal Δ12 oleate hydroxylase gene (CpFAH12) from Claviceps purpurea while deleting genes related to fatty acid degradation (MEF1 and PEX10) and oleic acid desaturation (FAD2). Since Δ12 oleate hydroxylase converts oleic acid (C18:1) located at the sn-2 position of phosphatidylcholine (PC), we next focused on increasing the PC pool containing oleic acid. This objective was achieved thorough implementing metabolic engineering strategies designed to enhance the biosynthesis of PC and C18 fatty acids. To increase the PC pool, we redirected the flux towards phospholipid biosynthesis by deleting phosphatidic acid phosphatase genes (PAH1 and APP1) and diacylglycerol acyltransferase gene (DGA1), involved in the production of diacylglycerol and triacylglycerol, respectively. Furthermore, the PC biosynthesis via the CDP-DAG pathway was enhanced through the overexpression of CDS1, PSD1, CHO2, and OPI3 genes. Subsequently, to increase the oleic acid content within PC, we overexpressed the heterologous fatty acid elongase gene (MaC16E) involved in the conversion of C16 to C18 fatty acids. As RA production titer escalated, the produced RA was mainly found in the FFA form, leading to cell growth inhibition. The growth inhibition was mitigated by inducing RA secretion via Triton X-100 treatment, a process that simultaneously amplified RA production by redirecting flux towards RA synthesis. The final engineered strain JHYL-R146 produced 2.061 g/L of free RA in a medium treated with 5% Triton X-100, constituting 74% of the total FFAs produced. Generating free RA offers the added benefit of bypassing the hydrolysis stage required when employing castor bean oil as an RA source. This achievement represents the highest level of RA synthesis from glucose reported thus far, underscoring the potential of Y. lipolytica as a host for sustainable RA production.

Efficient removal of PAHs from peanut oil using coconut shell-based activated charcoal decorated by cationic (CTAB), anionic (SDS), non-ionic surfactant (Triton X-100).

The coconut shell-based activated charcoal was decorated by three different electronegativities of surfactants (CTAB, SDS, and Triton X-100) through the impregnation method, and the decorated activated charcoal adsorbents were used for the removal of PAHs from peanut oil, respectively. The influence of surfactant decoration on the adsorption and detoxification effect of coconut shell-based activated charcoal was discussed. The thermodynamic and kinetic behaviors of PAHs adsorption on the surfactant-modified activated charcoal were investigated, and the adsorption mechanism was analyzed in-depth. Notably, the prepared modified coconut shell activated charcoal could not only remove more than 90% of PAHs from the peanut oil but also keep the cytotoxicity of the treated peanut oil low. Meanwhile, the detoxification procedure has little effect on the nutritional quality and flavor of the peanut oil. The results of this fundamental study demonstrate that the low-cost surfactant-modified coconut shell-based activated charcoal was effective and feasible.

Adsorption of Organic Pollutants on Carbonaceous Adsorbents Prepared by Direct Activation of Sweet Cherry Stones.

The main objective of this study was to assess the usefulness of the sweet cherry stones for the production of carbonaceous adsorbents by means of direct physical activation method, using conventional and microwave variant of heating. The adsorbents were characterized in terms of textural parameters, acidic-basic character of the surface, electrokinetic properties and their suitability for drinking water purification. Adsorption tests were carried out against three organic compounds - Triton X-100 (surfactant), bovine serum albumin (protein) and methylene blue (synthetic dye). Depending on the variant of heating applied during activation procedure, the obtained activated biochars differed significantly in terms of the elemental composition, acidic-basic properties as well as degree of specific surface development and the type of porous structure generated. Adsorption tests have showed that the efficiency of organic pollutants removal from aqueous solutions depends significantly not only on the type of the adsorbent and adsorbate applied, but also on the temperature and pH of the system. The sample prepared by microwave-assisted direct activation proved to be very effective in terms of all tested organic pollutants adsorption. The maximum sorption capacity toward Triton X-100, bovine serum albumin and methylene blue reached the level of 86.5, 23.4 and 81.1 mg/g, respectively.

Effects of chemical factors on the infectivity of Decapod iridescent virus 1 (DIV1).

The effects of chemical factors on the infectivity of DIV1 have not been fully accessed yet. In order to investigate the stability of DIV1 to strong brine, pH, and other chemical conditions, we conducted a bioassay using clinically healthy Penaeus vannamei individuals. DIV1 inoculum was exposed to various chemical conditions, and the infectivity of DIV1 was determined through intramuscular injection. The results showed that DIV1 lost its infectivity when exposed to strong brine, specifically in a 3 mol/L NaCl solution for a duration of 1 h. Moreover, DIV1 was found to be inactivated within 1 h when subjected to pH levels below 3.1 or above 9.6. Additionally, both Triton X-100 and 1 % formaldehyde demonstrated the ability to inactivate DIV1. These results provide valuable insights into the tolerance of DIV1 towards certain chemical factors, serving as a reference for the establishment of biosecurity measures against DIV1.

Neuroprotective effects of isatin and afobazole in rats with rotenone-induced Parkinsonism are accompanied by increased brain levels of Triton X-100 soluble alpha-synuclein.

Effects of the endogenous neuroprotector isatin and the pharmacological drug afobazole (exhibiting neuroprotective properties) on behavioral reactions and quantitative changes in the brain proteomic profile have been investigated in rats with experimental rotenone Parkinsonism. A single dose of isatin (100 mg/kg subcutaneously on the last day of a 7-day course of rotenone administration) improved the motor activity of rats with rotenone-induced Parkinsonism in the open field test (horizontal movements) and the rotating rod test. Afobazole (10 mg/kg intraperitoneally, daily during the 7-day course of rotenone administration) reduced the manifestations of rigidity and postural instability. Proteomic analysis, performed using brain samples obtained the day after the last administration of rotenone and neuroprotectors, revealed similar quantitative changes in the brain of rats with rotenone Parkinsonism. An increase in the relative content of 65 proteins and a decrease in the relative content of 21 proteins were detected. The most pronounced changes - an almost ninety-fold increase in the alpha-synuclein content - were found in the brains of rats treated with isatin. In animals of the experimental groups treated with "Rotenone + Isatin", as well as "Rotenone + Afobazole", the increase in the relative content of this protein in the brain was almost 60 and 50 times higher than the control values. Taking into consideration the known data on the physiological role of alpha-synuclein, an increase in the content of this protein in the brain upon administration of neuroprotectors to animals with rotenone Parkinsonism may represent a compensatory reaction, at least in the early stages of this disease and the beginning of its treatment.

The effect of emulsifier type and oil fraction on Salmonella Typhimurium growth and thermal inactivation in oil-in-water emulsion.

High water activity oil-in-water emulsions can promote survival and growth of Salmonella Typhimurium. Nevertheless, the precise effect of emulsifier type and oil content on bacterial growth and inactivation is not fully understood. Here, emulsions were prepared using different emulsifiers (Tween 20, Tween 80, and Triton X-100) and different oil fractions (20%, 40%, and 60% (v/v)). TSB (control), emulsifier solutions, and emulsions were inoculated with S. Typhimurium. Bacterial growth rate was measured at 7, 22, and 37°C, whereas thermal inactivation was performed at 55°C. Growth and inactivation data was fitted into Logistic and Weibull models, respectively. At an incubation temperature of 37°C, the presence of high amount of oil (60%) in Tween 20 and Triton X stabilized emulsions extended the lag phase (5.83 ± 2.20 and 9.43 ± 1.07 h, respectively, compared to 2.28 ± 1.54 h for TSB, p < 0.05), whereas individual emulsifiers had no effect on growth behavior compared to TSB. This effect was also prevalent but attenuated at 22°C, whereas no growth was observed at 7°C. In thermal inactivation, we observed protective effect in Tween 80 and Triton X-100 solutions, where time required for five-log reduction was 1914.70 ± 706.35 min and 795.34 ± 420.09 min, respectively, compared to 203.89 ± 10.18 min for TSB (p < 0.05). Interestingly, the presence of high amount of oil did not offer protective effect during thermal inactivation. We hypothesize that oleic acid in Tween 80 and lower hydrophobicity value of Triton X-100 help maintain membrane integrity and improve the resistance of bacteria to heat inactivation.

Descelularización y regeneración de tráquea porcina: un acercamiento a la ingeniería tisular / Decellularization and Regeneration of Porcine Trachea: an Approach to Tissue Engineering

Resumen Antecedentes: la ingeniería tisular permite obtener órganos como injertos a partir de tejidos descelularizados, regenerados con células autólogas. Objetivo: descelularizar y regenerar tráqueas porcinas. Material y métodos: se descelularizaron tráqueas porcinas colocándolas cada una en el epiplón de cuatro cerdos Yorkshire para su regeneración in vivo. Una tráquea desce-lularizada con tritón (DT), descelularizada con desoxicolato (DD), descelularizada con desoxicolato y reforzada con un polímero y células epiteliales (DDR), y una nativa crio-preservada (NC). Después de 8 días se obtuvieron la DD, NC y DDR; y al día 15, la DT. Se las evaluó mecánica e histológicamente, se realizó el análisis casuístico. Resultados: las tráqueas descelularizadas conservaron la integridad del cartílago, sin diferencias mecánicas, excepto la DDR con mayor rigidez. Las tráqueas regeneradas presentaron menor rigidez, excepto la DDR que además perdió el epitelio y la vascula-ridad. Las DT, DD mostraron epitelio no respiratorio, fibrosis y vasculogénesis con in-flamación. Conclusiones: las matrices conservaron sus características mecánicas. La regenera-ción in vivo ofrece ventajas como la esterilidad, interacción celular, nutrientes; es senci-llo, factible y económico, pero no hay control del crecimiento celular y vascularización, y los tejidos presentaron alteraciones mecánicas e histológicas. El polímero impidió la re-epitelialización y revascularización. Este estudio abre la posibilidad de mejorar las me-todologías de ingeniería tisular aplicadas al tejido traqueal.

Abstract Introduction: tissue engineering makes it possible to obtain organs as grafts from de-cellularized tissues, regenerated with autologous cells.Objective: decellularize and regenerate porcine tracheas.ARTÍCULO ORIGINAL | Respirar, 2023; 15(3): 188-199 | ISSN 2953-3414 | https://doi.org/10.55720/respirar.15.3.5RECIBIDO: 9 agosto 2023ACEP TADO: 31 agosto 2023 Elisa Barrera-Ramírezhttps://orcid.org/0000-0002-2778-0882Rubén Efraín Garrido-Cardonahttps://orcid.org/0000-0001-6083-5403Alejandro Martínez-Martínezhttps://orcid.org/0000-0003-3448-910XLuis Fernando Plenge-Tellecheahttps://orcid.org/0000-0002-1619-5004Edna Rico-Escobarhttps://orcid.org/0000-0002-0933-0220Esta revista está bajo una licencia de Creative Commons Reconocimiento 4.0 Internacional. Respirar 2023; 15 (3): 189ARTÍCULO ORIGINAL / E. Barrera-Ramírez, R.E. Garrido-Cardona, A. Martínez-Martínez, L.F. Plenge-Tellechea, E. Rico-EscobarDescelularización y regeneración de tráqueaISSN 2953-3414Materials and Methods: Porcine tracheas were decellularized by placing each one in the omentum of four Yorkshire pigs for regeneration in vivo. A trachea decellularized with triton (DT), decellularized with deoxycholate (DD), decellularized with deoxycho-late and reinforced with a polymer, and epithelial cells (DDR), and a cryopreserved na-tive (NC). After 8 days, the DD, NC and DDR were obtained; and on day 15, the DT. The evaluation was mechanically and histologically, performing the case analysis.Results: the decellularized tracheas preserved the integrity of the cartilage, with no me-chanical differences, except for the DDR with greater rigidity. The regenerated trache-as presented less rigidity, except the DDR, which also lost the epithelium and vascular-ity. The DT, DD showed non-respiratory epithelium, fibrosis and vasculogenesis with inflammation.Conclusions: the matrices retained their mechanical characteristics, in vivo regenera-tion offers advantages such as sterility, cell interaction, nutrients; it is simple, feasible and economical, but there is no control of cell growth and vascularization, and the tis-sues presented mechanical and histological alterations. The polymer prevented re-epi-thelialization and revascularization. This study opens the possibility of improving tissue engineering methodologies applied to tracheal tissue.

Bioremediation of Automotive Residual Oil-Contaminated Soils by Biostimulation with Enzymes, Surfactant, and Vermicompost.

Contamination of soils by automotive residual oil represents a global environmental problem. Bioremediation is the technology most suitable to remove this contaminant from the medium. Therefore, this work aimed to evaluate the effectiveness of bioremediation of automotive residual oil-contaminated soils by biostimulation with enzymes, surfactant, and vermicompost. The bioremediation efficiency was examined using a factorial design of 24 to determine the effect of the time, pH and temperature conditions, biostimulation with enzyme-vermicompost, and biostimulation with enzyme-surfactant. Enzymes obtained from Ricinus communis L. seeds, commercial vermicompost, and Triton X-100 were used. Results showed that the highest removal efficiency (99.9%) was achieved at 49 days, with a pH of 4.5, temperature of 37 °C, and using biostimulation with enzyme-vermicompost (3% w/v-5% w/w). The addition of surfactant was not significant in increasing the removal efficiency. Therefore, the results provide adequate conditions to bioremediate automotive residual oil-contaminated soils by biostimulation using enzymes supported with vermicompost.