Presence Of Detergent Research Articles

Increased efficiency of peripheral nerve regeneration using supercritical carbon dioxide-based decellularization in acellular nerve graft

Acellular nerve grafts (ANGs) are a promising therapeutic for patients with nerve defects caused by injuries. Conventional decellularization methods utilize a variety of detergents and enzymes. However, these methods have disadvantages, such as long processing times and the presence of detergents that remain on the graft. In this study, we aimed to reduce process time and minimize the risks associated with residual detergents by replacing them with supercritical carbon dioxide (scCO2) and compared the effectiveness to Hudson’s decellularization method, which uses several detergents. The dsDNA and the expression of MHC1 and 2 were significantly reduced in both decellularized groups, which confirmed the effective removal of cellular debris. The extracellular matrix proteins and various factors were found to be better preserved in the scCO2 ANGs compared to the detergent-ANGs. We conducted behavioral tests and histological analyses to assess the impact of scCO2 ANGs on peripheral nerve regeneration in animal models. Compared with Hudson’s method, the scCO2 method effectively improved the efficacy of peripheral nerve regeneration. Therefore, the decellularization method using scCO2 is not only beneficial for ANG synthesis, but it may also be helpful for therapeutics by enhancing the efficacy of peripheral nerve regeneration.

Fluorescence labeling-based differential scanning fluorimetry, an effective method for protein thermal stability and protein-compound binding analysis

Differential scanning fluorimetry (DSF) is widely used to assess protein thermal stability and protein-ligand interaction. However, its utility is often limited by the presence of detergents, which can affect hydrophobic binding. To tackle this issue, we developed an effective fluorescence-labeled DSF (FL-DSF) technique that tracks protein denaturation by monitoring the labeling fluorescence decrease, thus overcoming challenges typically encountered with traditional DSF methods. In this research, FL-DSF was first validated using Peroxisome Proliferators-Activated Receptor γ (PPARγ), Retinoid X Receptor α (RXRα), and Lysozyme, confirming its accuracy in determining melting curves. Expectedly, FL-DSF also exhibited strong compatibility with detergents in our investigations. Besides this, a new calculation method was proposed to characterize the protein denaturation process and evaluate protein-ligand binding. This mathematical model goes beyond traditional approaches, which simply treated the melting temperature (TM) shift as a concentration-dependent variable. Instead, it comprehensively incorporates the influence of irreversible denaturation-induced native protein loss on the equilibrium of protein-ligand binding. This methodology was successfully applied into the evaluation of binding affinity for 2 classical binding systems of PPARγ-Rosiglitazone and RXRα-CD3254. It was also utilized for the following binding screening studies, leading to the discovery of promising ligands for PPARγ, RXRα, and Lysozyme.

Assessing the Role of Detergents in Pond Ecosystem.

Detergents are used as a part of our daily life routine. Though they are widely used but their active ingredients which are highly toxic and persist in the environment for long are an important cause of environmental pollution. In our current work, we have studied the harmful effects of a combination of some commonly used detergents which find their way into the water bodies especially the pond ecosystem through everyday activities like washing clothes, utensils, and bathing in water. This water is the home to many flora and fauna especially the fishes like Cirrhinus mrigala. In our work, we have analysed the levels of the hepatic enzymes Alanine Transaminase and Aspartate Aminotransferase as well as the histology of gill and liver tissues. We have also analysed the presence of micronucleus in the fish blood. It was observed that the presence of detergents has increased the enzyme level as well as resulted in destruction of gill and liver tissue morphology. Detergents also increased the presence of micronucleus in fish blood. These results are indicators of deterioration of fish health due to detergent pollution.

Insights on the G protein-coupled receptor helix 8 solution structure and orientation using a neurotensin receptor 1 peptide.

G-protein coupled receptors (GPCRs) are the largest class of membrane proteins encoded in the human genome with high pharmaceutical relevance and implications to human health. These receptors share a prevalent architecture of seven transmembrane helices followed by an intracellular, amphipathic helix 8 (H8) and a disordered C-terminal tail (Ctail). Technological advancements have led to over 1000 receptor structures in the last two decades, yet frequently H8 and the Ctail are conformationally heterogeneous or altogether absent. Here we synthesize a peptide comprising the neurotensin receptor 1 (NTS1) H8 and Ctail (H8-Ctail) to investigate its structural stability, conformational dynamics, and orientation in the presence of detergent and phospholipid micelles, which mimic the membrane. Circular dichroism (CD) and nuclear magnetic resonance (NMR) measurements confirm that zwitterionic 1,2-diheptanoyl-sn-glycero-3-phosphocholine is a potent stabilizer of H8 structure, whereas the commonly-used branched detergent lauryl maltose neopentyl glycol (LMNG) is unable to completely stabilize the helix - even at amounts four orders of magnitude greater than its critical micellar concentration. We then used NMR spectroscopy to assign the backbone chemical shifts. A series of temperature and lipid titrations were used to define the H8 boundaries as F376-R392 from chemical shift perturbations, changes in resonance intensity, and chemical-shift-derived phi/psi angles. Finally, the H8 azimuthal and tilt angles, defining the helix orientation relative of the membrane normal were measured using paramagnetic relaxation enhancement NMR. Taken together, our studies reveal the H8-Ctail region is sensitive to membrane physicochemical properties and is capable of more adaptive behavior than previously suggested by static structural techniques.

Molecular characterization of the PhiKo endolysin from Thermus thermophilus HB27 bacteriophage phiKo and its cryptic lytic peptide RAP-29.

In the era of increasing bacterial resistance to antibiotics, new bactericidal substances are sought, and lysins derived from extremophilic organisms have the undoubted advantage of being stable under harsh environmental conditions. The PhiKo endolysin is derived from the phiKo bacteriophage infecting Gram-negative extremophilic bacterium Thermus thermophilus HB27. This enzyme shows similarity to two previously investigated thermostable type-2 amidases, the Ts2631 and Ph2119 from Thermus scotoductus bacteriophages, that revealed high lytic activity not only against thermophiles but also against Gram-negative mesophilic bacteria. Therefore, antibacterial potential of the PhiKo endolysin was investigated in the study presented here. Enzyme activity was assessed using turbidity reduction assays (TRAs) and antibacterial tests. Differential scanning calorimetry was applied to evaluate protein stability. The Collection of Anti-Microbial Peptides (CAMP) and Antimicrobial Peptide Calculator and Predictor (APD3) were used to predict regions with antimicrobial potential in the PhiKo primary sequence. The minimum inhibitory concentration (MIC) of the RAP-29 synthetic peptide was determined against Gram-positive and Gram-negative selected strains, and mechanism of action was investigated with use of membrane potential sensitive fluorescent dye 3,3'-Dipropylthiacarbocyanine iodide (DiSC3(5)). The PhiKo endolysin is highly thermostable with melting temperature of 91.70°C. However, despite its lytic effect against such extremophiles as: T. thermophilus, Thermus flavus, Thermus parvatiensis, Thermus scotoductus, and Deinococcus radiodurans, PhiKo showed moderate antibacterial activity against mesophiles. Consequently, its protein sequence was searched for regions with potential antibacterial activity. A highly positively charged region was identified and synthetized (PhiKo105-133). The novel RAP-29 peptide lysed mesophilic strains of staphylococci and Gram-negative bacteria, reducing the number of cells by 3.7-7.1 log units and reaching the minimum inhibitory concentration values in the range of 2-31 μM. This peptide is unstructured in an aqueous solution but forms an α-helix in the presence of detergents. Moreover, it binds lipoteichoic acid and lipopolysaccharide, and causes depolarization of bacterial membranes. The RAP-29 peptide is a promising candidate for combating bacterial pathogens. The existence of this cryptic peptide testifies to a much wider panel of antimicrobial peptides than thought previously.

Phycoremediation potential and agar yield of red macroalgae (Gracilaria corticata) against HEDP (hydroxyethylidene diphosphonic acid) and CAPB (cocoamidopropyl betaine) detergents and the heavy metal pollutants.

The discharge of raw industrial, agricultural, and domestic wastes leads to an increase in heavy metal (HM) burden and detergents in aquatic environs, which can have destructive effects on aquatic organisms. Agarophyte Gracilaria corticata, a major component of seaweed flora of the southern coast of Iran (Bushehr) that contains agar and red pigments, is one of the economically valuable red marine algae. Agar is one of the important polysaccharides with high economic value, widely used in pharmaceutical, medicinal, and cosmetic product manufacturing industries. The aim of this work was to investigate the effect of 5 HMs and two common surfactants in household and industrial detergents on the agar yield, appearance color, and the red algae's phycoremediation potential against HMs. The metal ions were Zn(II), Cu(II), Ni(II), Mn(II), and Cr(VI), and the surfactants were HEDP and CAPB. The analysis results of samples cultured for 60 days in seawater and polluted environments showed that G. corticata can accumulate copper and nickel. In the presence of detergents without HMs, the amount of extracted agar significantly increased compared to the control sample with no change in algae color. But with increasing concentration of HMs, the amount of agar in seaweed samples decreased significantly, and the algae discolored from red to dark green or yellowish-green color (signs of death in the algae). These results show that increasing of HM pollution and detergents can lead to toxicological effects and reduce the species diversity of red seaweeds in the future.

Perspective: The unexplored dimensions behind the foam formation in River Yamuna, India.

For nearly two years, a persistent foam cover has been observed during the post-monsoon season in the Yamuna River beneath the barrage near Okhla in Delhi, India. This affair has been a matter of public concern now, after the gigantic appearance of foam in November 2021, as the visibility of foam has awakened people's environmental 'conscience' over the 'concealed' chemical pollution. The mechanisms of agents responsible for foaming in rivers, particularly surfactants and phosphates, have received wide attention in the dynamic community of river pollution. Many studies in the past, around the globe, have evidently provided different rationales behind the dense foam formation in rivers, yet the Concerned Govt. Authorities have highlighted the cause of foam formation in the river Yamuna is associated with the presence of detergents and phosphates as foaming agents. Despite this, an aperture with copious unaccounted factors or underlying agents still exists to rationalize the foam formation and persistence. In this article, we outline these unaccounted factors which might be responsible for the foam formation and stabilization and give indications for future research directives towards the emergence of studies regarding the dense foam formation in river Yamuna.

Elongated Bacterial Pili as a Versatile Alignment Medium for NMR Spectroscopy

AbstractIn NMR spectroscopy, residual dipolar couplings (RDCs) have emerged as one of the most exquisite probes of biological structure and dynamics. The measurement of RDCs relies on the partial alignment of the molecule of interest, for example by using a liquid crystal as a solvent. Here, we establish bacterial type 1 pili as an alternative liquid‐crystalline alignment medium for the measurement of RDCs. To achieve alignment at pilus concentrations that allow for efficient NMR sample preparation, we elongated wild‐type pili by recombinant overproduction of the main structural pilus subunit. Building on the extraordinary stability of type 1 pili against spontaneous dissociation and unfolding, we show that the medium is compatible with challenging experimental conditions such as high temperature, the presence of detergents, organic solvents or very acidic pH, setting it apart from most established alignment media. Using human ubiquitin, HIV‐1 TAR RNA and camphor as spectroscopic probes, we demonstrate the applicability of the medium for the determination of RDCs of proteins, nucleic acids and small molecules. Our results show that type 1 pili represent a very useful alternative to existing alignment media and may readily assist the characterization of molecular structure and dynamics by NMR.

Elongated Bacterial Pili as a Versatile Alignment Medium for NMR Spectroscopy.

In NMR spectroscopy, residual dipolar couplings (RDCs) have emerged as one of the most exquisite probes of biological structure and dynamics. The measurement of RDCs relies on the partial alignment of the molecule of interest, for example by using a liquid crystal as a solvent. Here, we establish bacterial type 1 pili as an alternative liquid-crystalline alignment medium for the measurement of RDCs. To achieve alignment at pilus concentrations that allow for efficient NMR sample preparation, we elongated wild-type pili by recombinant overproduction of the main structural pilus subunit. Building on the extraordinary stability of type 1 pili against spontaneous dissociation and unfolding, we show that the medium is compatible with challenging experimental conditions such as high temperature, the presence of detergents, organic solvents or very acidic pH, setting it apart from most established alignment media. Using human ubiquitin, HIV-1 TAR RNA and camphor as spectroscopic probes, we demonstrate the applicability of the medium for the determination of RDCs of proteins, nucleic acids and small molecules. Our results show that type 1 pili represent a very useful alternative to existing alignment media and may readily assist the characterization of molecular structure and dynamics by NMR.

Bacillus thuringiensis toxin Cyt2Aa forms filamentous oligomers when exposed to lipid membranes or detergents

The bacterium Bacillus thuringiensis (Bt) produces insecticidal proteins during the sporulation phase. These proteins are located in parasporal crystals consisting of two delta-endotoxin classes, crystal (Cry) and cytolytic (Cyt) toxins. In vitro, Cyt toxins show cytolytic activity against bacterial and a variety of insect and mammalian cells. They bind to cell membranes with unsaturated phospholipids and sphingomyelin. Although Bt and its parasporal crystals containing both Cry and Cyt toxins have been successfully used as bioinsecticides, the molecular mechanism of action of Cyt toxins is not yet fully understood. To address this, we exposed Cyt2Aa to lipid membranes and visualized membrane disruption process using cryo-electron microscopy. We observed two types of Cyt2Aa oligomers. First, Cyt2Aa forms smaller curved oligomers on the membrane surface that become linear over time, and detach when the membrane ruptures. Similar linear filamentous oligomers were also formed by Cyt2Aa in the presence of detergents without prior exposure to lipid membranes, which exhibited attenuated cytolytic activity. Furthermore, our data suggest that Cyt2Aa adopts different conformations between its monomeric and oligomeric forms. Overall, our results provide new evidence for a detergent-like mechanism of action of Cyt2Aa rather than the pore-forming model of target membrane disruption of this important class of insecticidal proteins.

Detergent modulates the conformational equilibrium of SARS-CoV-2 Spike during cryo-EM structural determination

The Spike glycoprotein of SARS-CoV-2 mediates viral entry into the host cell via the interaction between its receptor binding domain (RBD) and human angiotensin-converting enzyme 2 (ACE2). Spike RBD has been reported to adopt two primary conformations, a closed conformation in which the binding site is shielded and unable to interact with ACE2, and an open conformation that is capable of binding ACE2. Many structural studies have probed the conformational space of the homotrimeric Spike from SARS-CoV-2. However, how sample buffer conditions used during structural determination influence the Spike conformation is currently unclear. Here, we systematically explored the impact of commonly used detergents on the conformational space of Spike. We show that in the presence of detergent, the Spike glycoprotein predominantly occupies a closed conformational state during cryo-EM structural determination. However, in the absence of detergent, such conformational compaction was neither observed by cryo-EM, nor by single-molecule FRET designed to visualize the movement of RBD in solution in real-time. Our results highlight the highly sensitive nature of the Spike conformational space to buffer composition during cryo-EM structural determination, and emphasize the importance of orthogonal biophysical approaches to validate the structural models obtained.

Role of surfactants in electron cryo-microscopy film preparation

Single-particle electron cryo-microscopy (cryo-EM) has become an effective and straightforward approach to determine the structure of membrane proteins. However, obtaining cryo-EM grids of sufficient quality for high-resolution structural analysis remains a major bottleneck. One of the difficulties arises from the presence of detergents, which often leads to a lack of control of the ice thickness. Amphipathic polymers such as amphipols (APols) are detergent substitutes, which have proven to be valuable tools for cryo-EM studies. In this work, we investigate the physico-chemical behavior of APol- and detergent-containing solutions and show a correlation with the properties of vitreous thin films in cryo-EM grids. This study provides new insight on the potential of APols, allowing a better control of ice thickness while limiting protein adsorption at the air-water interface, as shown with the full-length mouse serotonin 5-HT3A receptor whose structure has been solved in APol. These findings may speed up the process of grid optimization to obtain high-resolution structures of membrane proteins.

Cryo-EM structure of the trehalose monomycolate transporter, MmpL3, reconstituted into peptidiscs

Mycobacteria have an atypical thick and waxy cell wall. One of the major building blocks of such mycomembrane is trehalose monomycolate (TMM). TMM is a mycolic acid ester of trehalose that possesses long acyl chains with up to 90 carbon atoms. TMM represents an essential component of mycobacteria and is synthesized in the cytoplasm, and then flipped over the plasma membrane by a specific transporter known as MmpL3. Over the last decade, MmpL3 has emerged as an attractive drug target to combat mycobacterial infections. Recent three-dimensional structures of MmpL3 determined by X-ray crystallography and cryo-EM have increased our understanding of the TMM transport, and the mode of action of inhibiting compounds. These structures were obtained in the presence of detergent and/or in a lipidic environment. In this study, we demonstrate the possibility of obtaining a high-quality cryo-EM structure of MmpL3 without any presence of detergent through the reconstitution of the protein into peptidiscs. The structure was determined at an overall resolution of 3.2 Å and demonstrates that the overall structure of MmpL3 is preserved as compared to previous structures. Further, the study identified a new structural arrangement of the linker that fuses the two subdomains of the transmembrane domain, suggesting the feature may serve a role in the transport process.

Reusability of face masks: Influence of washing and comparison of performance between medical face masks and community face masks

Reusability of masks is a rising environmental concern. Community face masks present a more eco-friendly option as they can be washed and reused several times. The potential reuse of single use medical face masks is also of interest. In this work, the impact of 20 wash cycles (at 60 °C with detergent and air-drying) on the particle filtration efficiency (particle size range 0.1–6.​ 5 μ m ) and breathability of masks was studied. With regards to the performance of new masks, the medical mask had breathability of 31 Pa.cm −2 whilst it ranged from 9 to 59 Pa.cm −2 for the community face masks. Additionally, for 3 μ m particles, the filtration efficiency was 99% for the medical mask and ranged from 28%–89% for the community face masks. Mixed fabric masks were able to maintain their performance up to 20 washing cycles. The washed 100% cotton masks were less breathable but with higher filtration efficiency because of shrinkage of the fabric. The washed medical face mask was able to maintain its breathability and filtration efficiency for sizes ≥ 3 μ m , however the filtration efficiency was significantly reduced for the submicron particles. This was because the presence of detergent caused the loss of electrostatic charges and led to a decrease of the particle collection by the electrostatic forces. All things considered, both the medical face mask and community face masks can be reused several times, with performances in terms of breathability and filtration efficiency (for particle sizes ≥ 3 μ m ) that remain globally little changed after washing. • Reusability of medical and community face masks (cotton/mixed fabric) investigated. • Filtration efficiency (0.1-6.5 μ m) and breathability analyzed after 20 wash cycles. • Masks generally maintain their breathability after washing. • Particle filtration efficiency of community face masks depends on type of fabric. • Washing reduces filtration efficiency of medical face masks for submicron particles.

Targeted dose delivery of Mycobacterium tuberculosis in mice using silicon antifoaming agent via aerosol exposure system.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that forms aggregates (clumps) on solid agar plates and in liquid media. Detergents such as Tween 80/Tyloxapol are considered the gold standard to disrupt clump formation in Mtb cultures. The presence of detergent, however, may generate foam and hinder Mtb aerosolization thus requiring addition of an antifoam agent for optimal Mtb aerosol-based procedures. Aerosol inhalation can be technically challenging, in particular to achieve a reproducible inhaled target dose. In this study, the impact of an antifoam, the silicon antifoaming agent (SAF), on Mtb aerosolization and whole-body mouse aerosol infection was investigated. A comparative study using SAF in a liquid suspension containing Mycobacterium bovis BCG (M. bovis BCG) or Mtb H37Rv did not cause any adverse effect on bacterial viability. Incorporation of SAF during mycobacteria inhalation procedures revealed that aerosolized mycobacterial strains were maintained under controlled environmental conditions such as humidity, temperature, pressure, and airflow inside the aerosol chamber. In addition, environmental factors and spray factors were not affected by the presence of SAF in mycobacterial cultures during aerosolization. Spray factor was significantly less during aerosol procedures with a low-input dose of mycobacteria in comparison to high-dose, as predicted. The mycobacterial load recovered in the biosampler (AGI) was ~2–3 logs lower than nebulizer or input bacterial load. A consistent Mtb bacillary load determined in mouse lungs indicates that SAF does not affect mycobacteria aerosolization during the aerosol generation process. These data confirmed that 1) SAF prevents formation of excessive foam during aerosolization, 2) SAF had no negative impact on mycobacterial viability within aerosol droplets, 3) Mtb droplets within aerosol-generated particles are well within the range required for reaching and depositing deep into lung tissue, and 4) SAF had no negative impact on achieving a target dose in mice exposed to Mtb aerosol.

Cloning and Co-Expression of Lipase and its Specific Foldase from Ralstonia pickettii BK6

This study aimed to obtain a functional lipase (LipRM) from Ralstonia pickettii BK6 through a co-expression involving its foldase. The ORF of the LipRM was 999 bp while the gene encoding of lipase-specific foldase (LifRM) was 1,030 bp. LipRM and LifRM genes were cloned into a plasmid and were successfully co-expressed in Escherichia coli strains to produce functional LipRM. Enzyme activity from partially purified enzymes showed quite surprising results, LipRM activity in E. coli BL21 (DE3) was 25.84 U/ml, while the other strains (DH5α, HB101, S17-1λpir) were 628.98 U/ml, 761 U/ml, and 1206.46 U/ml, respectively. The highest relative activity of LipRM was found at 50-55°C and pH 7-8 with pNP-laurate (C12) as the preferred substrate specificity. LipRM activity was enhanced sharply in the presence of 30% organic solvents (methanol and ethanol) but decreased by more than 50% in the presence of detergents. This study was the first to report heterologous expression of Ralstonia pickettii lipase employing its native foldase resulting in functional lipase from subfamily I.2.

Cloning, Expression and Biochemical Characterization of the Recombinant α-amylase from Bacillus subtilis YX48

Background: Amylase used in the market is mostly medium-temperature enzyme or high-temperature enzyme and has poor enzyme activity under low-temperature environment. Acid α-amylase can be used to develop digestion additives in the pharmaceutical and healthcare industries. The amino acid sequence and structural differences among α-amylases obtained from various organisms are high enough to confer interesting biochemical diversity to the enzymes. However, low- temperature (0-50°C) amylase, with an optimum temperature and heat sensitivity, has a greater potential value than medium (50-80°C) and high (80-110°C) temperature amylases. Methodology: The gene amy48 from encoding extracellular α-amylase in Bacillus subtilis YX48 was successfully cloned into the pET30a (+) vector and expressed in Escherichia coli BL21 (DE3) for biochemical characterization. Results and Conclusion: The molecular weight of α-amylase was 75 kDa. The activity of α-amylase was not affected by Ca2+, and Amy48 had the best activity at pH 5.0 and 37°C. AMY48 has high stability over a narrow pH and temperature range (5.0-8.0 and 30-45°C). Amylase activity was strongly inhibited by Zn2+, Mn2+, Cu2+, and Fe2+ ions, but Na+, K+, and Co2+ ions stimulate its activity slightly. The purified enzyme showed gradually reduced activity in the presence of detergents. However, it was remarkably stable against EDTA and urea.

Monitoring SARS-CoV-2 Surrogate TGEV Individual Virions Structure Survival under Harsh Physicochemical Environments.

Effective airborne transmission of coronaviruses via liquid microdroplets requires a virion structure that must withstand harsh environmental conditions. Due to the demanding biosafety requirements for the study of human respiratory viruses, it is important to develop surrogate models to facilitate their investigation. Here we explore the mechanical properties and nanostructure of transmissible gastroenteritis virus (TGEV) virions in liquid milieu and their response to different chemical agents commonly used as biocides. Our data provide two-fold results on virus stability: First, while particles with larger size and lower packing fraction kept their morphology intact after successive mechanical aggressions, smaller viruses with higher packing fraction showed conspicuous evidence of structural damage and content release. Second, monitoring the structure of single TGEV particles in the presence of detergent and alcohol in real time revealed the stages of gradual degradation of the virus structure in situ. These data suggest that detergent is three orders of magnitude more efficient than alcohol in destabilizing TGEV virus particles, paving the way for optimizing hygienic protocols for viruses with similar structure, such as SARS-CoV-2.

Quantification and morphological characterization of microfibers emitted from textile washing

Microplastics are a subject of growing interest as they are a potential threat for living organisms. Textile microfibers (MFs) are an important microplastics sub-group that have been reported as a major source of microplastics release into the environment. This pollution occurs mainly during the washing of synthetic garments. However, standardized methods to quantify and characterize these MFs are scarce. This study proposes a new analytical protocol to characterize these MFs in number and size by means of filtration techniques, optical and electronic microscopy and automatic image post-processing. This approach was developed and validated on effluents from washing machines produced in different conditions (5 different garments, sequential cycles, and presence or not of detergent). Among the analyzed effluents, it was found that 40 to 75% of microfibers have a length comprised between 50 and 200 μm, with average microfiber diameters ranging from 8 to 17 μm depending on the type of textile. The emission range of microfibers was estimated to be between 220,000 to 2,820,000 microfibers per kg of textile depending on the type of garment and the washing conditions. The counting method developed is adapted to a certain range of textiles, such as 100% polyester fleece jackets (PET-1), 100% smooth polyester T-shirt (PET-2) and 100% acrylic sweater (PAN), and is not affected by the presence of detergent. The proposed method of characterization of these MFs lengths can also be extrapolated to the counting of other objects that have a similar morphology to the analyzed fibers. Hence, it can be helpful to develop new testing capture technologies and, thus, contribute to the enhancement of filtering techniques of several pollutants.

RfaH May Oppose Silencing by H-NS and YmoA Proteins during Transcription Elongation.

Nucleoid-associated proteins (NAPs) silence xenogenes by blocking RNA polymerase binding to promoters and hindering transcript elongation. In Escherichia coli, H-NS and its homolog SptA interact with YmoA proteins Hha and YdgT to assemble nucleoprotein filaments that facilitate transcription termination by Rho, which acts in synergy with NusG. Countersilencing during initiation is facilitated by proteins that exclude NAPs from promoter regions, but auxiliary factors that alleviate silencing during elongation are not known. A specialized NusG paralog, RfaH, activates lipopolysaccharide core biosynthesis operons, enabling survival in the presence of detergents and antibiotics. RfaH strongly inhibits Rho-dependent termination by reducing RNA polymerase pausing, promoting translation, and competing with NusG. We hypothesize that RfaH also acts as a countersilencer of NAP/YmoA filaments. We show that deletions of hns and hha+ydgT suppress the growth defects of ΔrfaH by alleviating Rho-mediated polarity within the waa operon. The absence of YmoA proteins exacerbates cellular defects caused by reduced Rho levels or Rho inhibition by bicyclomycin but has negligible effects at a strong model Rho-dependent terminator. Our findings that the distribution of Hha and RfaH homologs is strongly correlated supports a model in which they comprise a silencing/countersilencing pair that controls expression of chromosomal and plasmid-encoded xenogenes. IMPORTANCE Horizontally acquired DNA drives bacterial evolution, but its unregulated expression may harm the recipient. Xenogeneic silencers recognize foreign genes and inhibit their transcription. However, some xenogenes, such as those encoding lipo- and exopolysaccharides, confer resistance to antibiotics, bile salts, and detergents, necessitating the existence of countersilencing fitness mechanisms. Here, we present evidence that Escherichia coli antiterminator RfaH alleviates silencing of the chromosomal waa operon and propose that plasmid-encoded RfaH homologs promote dissemination of antibiotic resistance genes through conjugation.