Visualization of the mechanosensitive ion channel MscS under membrane tension.

Mechanosensitive channels sense mechanical forces in cell membranes and underlie many biological sensing processes1-3. However, how exactly they sense mechanical force remains under investigation4. The bacterial mechanosensitive channel of small conductance, MscS, is one of the most extensively studied mechanosensitive channels4-8, but how it is regulated by membrane tension remains unclear, even though the structures are known for its open and closed states9-11. Here we used cryo-electron microscopy to determine the structure of MscS in different membrane environments, including one that mimics a membrane under tension. We present the structures of MscS in the subconducting and desensitized states, and demonstrate that the conformation of MscS in a lipid bilayer in the open state is dynamic. Several associated lipids have distinct roles in MscS mechanosensation. Pore lipids are necessary to prevent ion conduction in the closed state. Gatekeeper lipids stabilize the closed conformation and dissociate with membrane tension, allowing the channel to open. Pocket lipids in a solvent-exposed pocket between subunits are pulled out under sustained tension, allowing the channel to transition to the subconducting state and then to the desensitized state. Our results provide a mechanistic underpinning and expand on the 'force-from-lipids' model for MscS mechanosensation4,11.

Detergent-induced quantitatively limited formation of diacyl phosphatidylinositol dimannoside in Mycobacterium smegmatis.

Mycobacterial plasma membrane, together with the peptidoglycan-arabinogalactan cell wall and waxy outer membrane, creates a robust permeability barrier against xenobiotics. The fact that several antituberculosis drugs target plasma membrane-embedded enzymes underscores the importance of the plasma membrane in bacterial physiology and pathogenesis. Nevertheless, its accurate phospholipid composition remains undefined, with conflicting reports on the abundance of phosphatidylinositol mannosides (PIMs), physiologically important glycolipids evolutionarily conserved among mycobacteria and related bacteria. Some studies indicate cardiolipin, phosphatidylethanolamine, and phosphatidylinositol as dominant structural phospholipids. Conversely, some suggest PIMs dominate the plasma membrane. A striking example of the latter is the use of reverse micelle extraction, showing diacyl phosphatidylinositol dimannoside (Ac2PIM2) as the most abundant phospholipid in a model organism, Mycobacterium smegmatis. Our recent work reveals a rapid response mechanism to membrane-fluidizing stress in mycobacterial plasma membrane: monoacyl phosphatidylinositol dimannoside and hexamannoside (AcPIM2 and AcPIM6) are converted to diacyl forms (Ac2PIM2 and Ac2PIM6). Given the dynamic nature of PIMs, we aimed to resolve the conflicting data in the literature. We show that unstressed M. smegmatis lacks an Ac2PIM2-dominated plasma membrane. Ac2PIM2 accumulation is induced by experimental conditions involving sodium docusate, a component of the reverse micellar solution. Using chemically synthesized PIMs as standards, we accurately quantified phospholipid ratio in M. smegmatis through liquid chromatography-mass spectrometry, revealing that mycobacterial plasma membrane is dominated by cardiolipin, phosphatidylethanolamine, and phosphatidylinositol. PIMs are quantitatively minor but responsive to environmental stresses in M. smegmatis. Our study paves the way for accurate modeling of mycobacterial plasma membrane.

Solubilization, purification, and characterization of the hexameric form of phosphatidylserine synthase from Candida albicans.

Phosphatidylserine (PS) synthase from Candida albicans, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals against systemic candidiasis. Rational drug design or small molecule screening are effective ways to identify specific inhibitors of Cho1, but both will be facilitated by protein purification. Due to the transmembrane nature of Cho1, methods were needed to solubilize and purify the native form of Cho1. Here, we used six non-ionic detergents and three styrene maleic acids (SMAs) to solubilize an HA-tagged Cho1 protein from the total microsomal fractions. Blue native PAGE and immunoblot analysis revealed a single band corresponding to Cho1 in all detergent-solubilized fractions, while two bands were present in the SMA2000-solubilized fraction. Our enzymatic assay suggests that digitonin- or DDM-solubilized enzyme has the most PS synthase activity. Pull-downs of HA-tagged Cho1 from the digitonin-solubilized fraction reveal an apparent MW of Cho1 consistent with a hexamer. Furthermore, negative-staining electron microscopy analysis and AlphaFold2 structure prediction modeling suggest the hexamer is composed of a trimer of dimers. We purified Cho1 protein to near-homogeneity as a hexamer using affinity chromatography and TEV protease treatment, and optimized Cho1 enzyme activity for manganese and detergent concentrations, temperature (24 °C), and pH (8.0). The purified Cho1 has a Km for its substrate CDP-diacylglycerol of 72.20 µM with a Vmax of 0.079 nmol/(µg∗min) while exhibiting a sigmoidal kinetic curve for its other substrate serine, indicating cooperative binding. Purified hexameric Cho1 can potentially be used in downstream structure determination and small drug screening.

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.

Purification, and characterization of detergent-compatible serine protease from Bacillussafensis strain PRN1: A sustainable alternative to hazardous chemicals in detergent industry.

Owing to vast therapeutic, commercial, and industrial applications of microbial proteases microorganisms from different sources are being explored. In this regard, the gut microbiota of Monopteruscuchia were isolated and examined for the production of protease. All the isolates were primarily and secondarily screened on skim milk and gelatin agar plates. The protease-positive isolates were characterized morphologically, biochemically, and molecularly. Out of the 20 isolated strains,6 belonging to five different genera viz.Bacillus,Priestia,Aeromonas,Staphylococcus, and Serratia demonstrated proteolytic activity. Bacillussafensis strain PRN1 demonstrated the highest protease production and, thus, the largest hydrolytic clear zones in both skim milk agar (15 ± 1 mm) and gelatin (16 ± 1 mm) plates. The optimized parameters (time, pH, temperature, carbon, nitrogen) for highest protease activity and microbial growth of B.safensis strain PRN1 includes 72 h (OD600 = 0.56,1303 U/mL), pH 8 (OD600 = 0.83, 403.29 U/mL), 40 °C (OD600 = 1.75, 1849.11 U/mL), fructose (OD600 = 1.22, 1502 U/mL), and gelatin (OD600 = 1.88, 1015.33 U/mL). The enzyme was purified to homogeneity using salt-precipitation and gel filtration chromatography. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the purified enzyme was a monomer of a molecular weight of ∼33 kDa. The protease demonstrated optimal activity at pH 8 and 60 °C. It was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), demonstrating that it belongs to the serine-proteases family. The compatibility of the enzyme with surfactants and commercial detergents demonstrates its potential use in the detergent industry. Furthermore, the purified enzyme showed antibacterial and blood-stain removal properties.

A comprehensive approach for evaluating the virucidal performance of domestic laundry detergents under practical conditions.

AIMS: We aimed to develop a method to assess the virucidal performance of domestic laundry in a lab-scale washing machine (Rotawash) based on EN 17658. METHODS AND RESULTS: For method development, virus recovery was investigated after drying on cotton carriers for three test viruses murine norovirus (MNV), modified vaccinia virus Ankara (MVA), and bovine coronavirus (BCoV), followed by washing simulations in flasks and Rotawash. MNV and MVA demonstrated sufficient recovery from carriers after drying and washing (up to 40°C and 60 min). BCoV exhibited lower recovery, indicating less relevance as a test virus. Rotawash efficacy tests conducted with MNV, a resistant, non-enveloped virus, showed limited efficacy of a bleach-free detergent, aligning with results from a domestic washing machine. Rotawash washes achieved higher reductions in infectious virus titers than suspension tests, indicating the role of washing mechanics in virus removal. CONCLUSIONS: This study established a practical method to test the virucidal efficacy of laundry detergents in Rotawash, simulating domestic washing.

Auto-induction, biochemical characterization and application of a novel thermo-alkaline and detergent-stable lipase (S9 peptidase domain) from Thermotoga petrophila as cleaning additive and degrading oil/fat wastes.

A peptidase S9 prolyl oligopeptidase domain from Thermotoga petrophila RKU-1T (TpS9) was over-expressed as an active, soluble and hyperstable lipolytic enzyme in the mesophilic host system. The sequence analysis demonstrated, TpS9 is an esterase/lipase-like protein belongs to alpha/beta (α/ß)-hydrolase superfamily with a well-conserved penta-peptide (GLSAG) motif and α/ß-hydrolase fold. Various approaches (induction and cultivation) were employed to enrich TpS9 production, 6.04- and 7.26-fold increment was observed with IPTG (0.4 mM) and lactose (200 mM) in the modified 4ZB medium (pH 7.0), but with IPTG-independent auto-induction strategy 9.02-fold augmentation was achieved after 16 h incubation at 24 °C (150 rev min-1). Purified TpS9 showed optimal activity in McIlvaine buffer (pH 6.5) at 80-85 °C, and revealed great thermal (30-85 °C) and pH (6.0-9.0) for 8 h. No obvious constraint was perceived with various metal ions, surfactants, commercial laundry detergents, and chemical modulators. Whereas, TpS9 activity was improved with Ca2+, Mn2+, and Mg2+ by 210 %, 142.5 %, and 134.3 %, respectively. With 2.5 M NaCl (215 %), 50 % (v/v) methanol (140 %), 50 % (v/v) ethanol (126.6 %), 50 % (v/v) n-butanol (122.3 %), 50 % (v/v) isopropanol (120.4 %), 50 % (v/v) acetone (118.6 %) and 50 % (v/v) glycerol (113.2 %) TpS9 activity was also enriched. TpS9 demonstrated great affinity toward natural oils and p-nitrophenyl ester substrates, but showed peak activity with p-nitrophenyl palmitate (3160 U mg-1). Km, Vmax, kcat, Vmax Km-1 and kcat Km-1 of TpS9 with pNPP were 0.421 mM, 4015 µmol mg-1 min-1, 906.4 s-1, 9536.8 min-1, and 2152.96 mM-1 s-1, respectively. Moreover, TPS9 has notable ability to clean stains (5 min) and degrade the animals' fat (3 h). Hence, TpS9 is a favorable candidate as cleaning bio-additive in detergent formulation, fat degradation and various other applications.

The outer membrane is an essential load-bearing element in Gram-negative bacteria.

Gram-negative bacteria possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan cell wall and an outer membrane. The envelope is a selective chemical barrier1 that defines cell shape2 and allows the cell to sustain large mechanical loads such as turgor pressure3. It is widely believed that the covalently cross-linked cell wall underpins the mechanical properties of the envelope4,5. Here we show that the stiffness and strength of Escherichia coli cells are largely due to the outer membrane. Compromising the outer membrane, either chemically or genetically, greatly increased deformation of the cell envelope in response to stretching, bending and indentation forces, and induced increased levels of cell lysis upon mechanical perturbation and during L-form proliferation. Both lipopolysaccharides and proteins contributed to the stiffness of the outer membrane. These findings overturn the prevailing dogma that the cell wall is the dominant mechanical element within Gram-negative bacteria, instead demonstrating that the outer membrane can be stiffer than the cell wall, and that mechanical loads are often balanced between these structures.

Sodium dodecylbenzene sulphonate (SDBS) present in detergents: action on the gills, skin, and blood of D. rerio fish.

1. Sodium dodecylbenzene sulphonate (SDBS) is one of the surfactants used worldwide in detergents which, due to high residual discharges, has great potential to cause ecotoxicological impacts. Therefore, the sublethal effects of SDBS on the gills and skin of male Danio rerio fish were investigated.2. The fish were distributed into three groups: GC (control), GT1 (0.25 mg/L of SDBS), and GT2 (0.5 mg/L of SDBS) and exposed for 21 days. After the experiment, histopathological analyses of the gills, histochemical analyses (counting of mucous cells), and biochemical analyses (antioxidant defense enzyme analysis, SOD, and CAT) were conducted.3. A significant increase (p < 0.05) in the incidence of circulatory disorders, progressive, and regressive alterations occurred in the GT1 and GT2 groups. Due to these changes, the total histopathological index of the gills was higher in these groups. Mucous cells in the gills and skin increased. There was an increase in SOD activity and a reduction in CAT activity in these groups. Haematology revealed neutrophilia and lymphocytosis in the blood of GT1 and GT2.4. The results clearly demonstrate that a 21-day exposure to SDBS causes severe morphophysiological damage to the gills, skin, and blood of D. rerio fish.

Multiscale mechanics and temporal evolution of vimentin intermediate filament networks.

The cytoskeleton, an intricate network of protein filaments, motor proteins, and cross-linkers, largely determines the mechanical properties of cells. Among the three filamentous components, F-actin, microtubules, and intermediate filaments (IFs), the IF network is by far the most extensible and resilient to stress. We present a multiscale approach to disentangle the three main contributions to vimentin IF network mechanics-single-filament mechanics, filament length, and interactions between filaments-including their temporal evolution. Combining particle tracking, quadruple optical trapping, and computational modeling, we derive quantitative information on the strength and kinetics of filament interactions. Specifically, we find that hydrophobic contributions to network mechanics enter mostly via filament-elongation kinetics, whereas electrostatics have a direct influence on filament-filament interactions.

Effects of nanoselenium supplementation on lactation performance, nutrient digestion and mammary gland development in dairy cows.

The objective of this experiment was to evaluate the influence of nanoselenium (NANO-Se) addition on milk production, milk fatty acid synthesis, the development and metabolism regulation of mammary gland in dairy cows. Forty-eight Holstein dairy cows averaging 720 ± 16.8 kg of body weight, 66.9 ± 3.84 d in milk (dry matter intake [DIM]) and 35.2 ± 1.66 kg/d of milk production were divided into four treatments blocked by DIM and milk yields. Treatments were control group, low-Se (LSe), medium-Se (MSe) and high-Se (HSe) with 0, 0.1, 0.2 and 0.3 mg Se, respectively, from NANO-Se per kg dietary dry matter (DM). Production of energy- and fat-corrected milk (FCM) and milk fat quadratically increased (p < 0.05), while milk lactose yields linearly increased (p < 0.05) with increasing NANO-Se addition. The proportion of saturated fatty acids (SFAs) linearly decreased (p < 0.05), while proportions of monounsaturated fatty acids (MUFAs) linearly increased and polyunsaturated fatty acids (PUFAs) quadratically increased. The digestibility of dietary DM, organic matter (OM), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF) quadratically increased (p < 0.05). Ruminal pH quadratically decreased (p < 0.01), while total VFA linearly increased (p < 0.05) with increasing NANO-Se addition. The acetic to propionic ratio decreased (p < 0.05) linearly due to the unaltered acetic molar percentage and a quadratical increase in propionic molar percentage. The activity of CMCase, xylanase, cellobiase and pectinase increased linearly (p < 0.05) following NANO-Se addition. The activity of α-amylase increased linearly (p < 0.01) with an increase in NANO-Se dosage. Blood glucose, total protein, estradiol, prolactin, IGF-1 and Se linearly increased (p < 0.05), while urea nitrogen concentration quadratically decreased (p = 0.04). Moreover, the addition of Se at 0.3 mg/kg from NANO-Se promoted (p < 0.05) mRNA and protein expression of PPARγ, SREBP1, ACACA, FASN, SCD, CCNA2, CCND1, PCNA, Bcl-2 and the ratios of p-ACACA/ACACA and BCL2/BAX4, but decreased (p < 0.05) mRNA and protein expressions of Bax, Caspase-3 and Caspase-9. The results suggest that milk production and milk fat synthesis increased by NANO-Se addition by stimulating rumen fermentation, nutrients digestion, gene and protein expressions concerned with milk fat synthesis and mammary gland development.

Evaluation of biofilm formation and expression of psl, pel, alg genes of Pseudomonas aeruginosa in exposure to detergents.

Pseudomonas aeruginosa has been in the center of attention for several years as an opportunistic human pathogen implicated in many severe acute and chronic infections particularly in immunocompromised patients. Its high persistence and resistance against many antimicrobial agents are mostly attributed to biofilm formation. Biofilms are microbial communities mainly consisting of extracellular polymeric substances that encapsulate bacteria together and protect them from extracellular stresses. This cell aggregation is a stress response that P. aeruginosa employes as a survival strategy during growth with the toxic detergents. This process has shown to involve several operons such as psl, pel, and alg. Here we used P. aeruginosa strain PAO1 in control group, 40 P. aeruginosa strains from sink and 40 strains from surface of public places. Biofilm formation and gene expression were measured before and after exposure to sub minimum inhibitory concentration (sub-MIC) of biocides chlorhexidine diacetate and benzalkonium chloride. The qRT-PCR and biofilm formation results demonstrated an increase in biofilm formation ability and gene expression of pslA/B and pelA/B in two groups collected from sink and surface in contrast to the control group. A remarkable increase was observed in the biofilm formation and expression of pslA in the bacterial strain collected from the sink after exposure to biocides chlorhexidine diacetate. Both Pel and Psl appeared to have redundant functions as structural scaffolds in biofilms. Sub-MIC levels of detergents can improve biofilm formation ability of P. aeruginosa and therefore trigger resistance.

Antibacterial effect of Softsoap used to disinfect dentures.

PURPOSE: To evaluate the in vitro antibacterial effect of Softsoap and Efferdent used as solutions to disinfect Lucitone 199 poly(methyl methacrylate) (PMMA) resin used for dentures. METHODS: S. mutans and plaque bacteria were grown for 24 hours, and suspended to a concentration of 1x106 cells/ml. Bacterial suspensions (0.2 mL) were added to the decontaminated PMMA discs placed in a 48-well culture plate and incubated for 3 days at 37°C. The discs were rinsed to remove the unbound bacterial cells and then incubated for 60 minutes with 5% and 1% dilutions (triplicates) of each of the detergent solutions (0.3 ml). Discs were rinsed and then MTT reagent (0.2 ml) was added and incubated for 2 hours, then overnight with a solubilizing agent. An aliquot from each well (0.1 ml) was transferred to a 96-well flat bottom plate and absorbance was measured to OD @ 595 nm (MTT) of four samples for each data point. Normalized data was compared and statistically analyzed using a three-way ANOVA with Student-Newman-Keuls on Rank data with P< 0.05 for significance. Additionally, data were double-checked with the Holm-Sidak test. RESULTS: There was no statistically significant difference between testing media for C. albicans and mixed plaque (P= 0.078) or testing duration in time at 24 hours and 21 days (P= 0.07). Statistically significant differences were found between all treatment solutions group combinations (P< 0.001) except for 30% Softsoap versus Efferdent (P= 0.056). CLINICAL SIGNIFICANCE: There was no statistically significant difference between testing media for C. albicans and mixed plaque (P= 0.078) or testing duration at 24 hours and 21 days (P= 0.07). Statistically significant differences were noted between all treatment solutions group combinations (P< 0.001), However, there was no difference between 30% Softsoap and Efferdent (P= 0.056).

UV mutagenesis for lipase overproduction from Bacillus cereus ATA179, nutritional optimization, characterization and its usability in the detergent industry.

In this study, the wild-type Bacillus cereus ATA179 was mutagenized by random UV mutagenesis to increase lipase production. The mutant with maximum lipolytic activity was named Bacillus cereus EV4. The mutant strain (10.6 U/mL at 24 h) produced 60% more enzyme than the wild strain (6.6 U/mL at 48 h). Nutritional factors on lipase production were investigated. Sucrose was the best carbon source, (NH4)2HPO4 was the best nitrogen source and CuSO4 was the best metal ion source. Mutant EV4 showed a 32% increase in lipase production in the modified medium. The optimum temperature and pH were found to be 60 °C and 7.0, respectively. CuSO4, CaCl2, LiSO4, KCl, BaCl2, and Tween 20 had an activating effect on the enzyme. Vmax and Km values were found to be 17.36 U/mL and 0.036 mM, respectively. The molecular weight was determined as 28.2 kDa. The activity of lipase was found to be stable up to 60 days at 20 °C, 75 days at 4 °C, and 90 days at -20 °C. The potential of lipase in the detergent industry was investigated. The enzyme was not affected by detergent additives but was effective in removing stains in fabrics contaminated with oily substances.

Detergent Chemistry Modulates the Transgression of Planetary Boundaries including Antimicrobial Resistance and Drug Discovery.

Detergent chemistry enables applications in the world today while harming safe operating spaces that humanity needs for survival. Aim of this review is to support a holistic thought process in the design of detergent chemistry. We harness the planetary boundary concept as a framework for literature survey to identify progresses and knowledge gaps in context with detergent chemistry and five planetary boundaries that are currently transgressed, i.e., climate, freshwater, land system, novel entities, biosphere integrity. Our survey unveils the status of three critical challenges to be addressed in the years to come, including (i) the implementation of a holistically, climate-friendly detergent industry; (ii) the alignment of materialistic and social aspects in creating technical solutions by means of sustainable chemistry; (iii) the development of detergents that serve the purpose of applications but do not harm the biosphere in their role as novel entities. Specifically, medically relevant case reports revealed that even the most sophisticated detergent design cannot sufficiently accelerate drug discovery to outperform the antibiotic resistance development that detergents simultaneously promote as novel entities. Safe operating spaces that humanity needs for its survival may be secured by directing future efforts beyond sustainable chemistry, resource efficiency, and net zero emission targets.

Phospholipid cofactor solubilization inhibits formation of native prions.

Cofactor molecules are required to generate infectious mammalian prions in vitro. Mouse and hamster prions appear to have different cofactor preferences: Whereas both mouse and hamster prions can use phosphatidylethanolamine (PE) as a prion cofactor, only hamster prions can also use single-stranded RNA as an alternative cofactor. Here, we investigated the effect of detergent solubilization on rodent prion formation in vitro. We discovered that detergents that can solubilize PE (n-octylglucoside, n-octylgalactoside, and CHAPS) inhibit mouse prion formation in serial protein misfolding cyclic amplification (sPMCA) reactions using bank vole brain homogenate substrate, whereas detergents that are unable to solubilize PE (Triton X-100 and IPEGAL) have no effect. For all three PE-solubilizing detergents, inhibition of RML mouse prion formation was only observed above the critical micellar concentration (CMC). Two other mouse prion strains, Me7 and 301C, were also inhibited by the three PE-solubilizing detergents but not by Triton X-100 or IPEGAL. In contrast, none of the detergents inhibited hamster prion formation in parallel sPMCA reactions using the same bank vole brain homogenate substrate. In reconstituted sPMCA reactions using purified substrates, n-octylglucoside inhibited hamster prion formation when immunopurified bank vole PrPC substrate was supplemented with brain phospholipid but not with RNA. Interestingly, phospholipid cofactor solubilization had no effect in sPMCA reactions using bacterially expressed recombinant PrP substrate, indicating that the inhibitory effect of solubilization requires PrPC post-translational modifications. Overall, these in vitro results show that the ability of PE to facilitate the formation of native but not recombinant prions requires phospholipid bilayer integrity, suggesting that membrane structure may play an important role in prion formation in vivo.

Biochemical and Genetic Analysis Identify CSLD3 as a beta-1,4-Glucan Synthase That Functions during Plant Cell Wall Synthesis.

In plants, changes in cell size and shape during development fundamentally depend on the ability to synthesize and modify cell wall polysaccharides. The main classes of cell wall polysaccharides produced by terrestrial plants are cellulose, hemicelluloses, and pectins. Members of the cellulose synthase (CESA) and cellulose synthase-like (CSL) families encode glycosyltransferases that synthesize the ß-1,4-linked glycan backbones of cellulose and most hemicellulosic polysaccharides that comprise plant cell walls. Cellulose microfibrils are the major load-bearing component in plant cell walls and are assembled from individual ß-1,4-glucan polymers synthesized by CESA proteins that are organized into multimeric complexes called CESA complexes, in the plant plasma membrane. During distinct modes of polarized cell wall deposition, such as in the tip growth that occurs during the formation of root hairs and pollen tubes or de novo formation of cell plates during plant cytokinesis, newly synthesized cell wall polysaccharides are deposited in a restricted region of the cell. These processes require the activity of members of the CESA-like D subfamily. However, while these CSLD polysaccharide synthases are essential, the nature of the polysaccharides they synthesize has remained elusive. Here, we use a combination of genetic rescue experiments with CSLD-CESA chimeric proteins, in vitro biochemical reconstitution, and supporting computational modeling and simulation, to demonstrate that Arabidopsis (Arabidopsis thaliana) CSLD3 is a UDP-glucose-dependent ß-1,4-glucan synthase that forms protein complexes displaying similar ultrastructural features to those formed by CESA6.

Susceptibility of Cu(I) transport ATPases to sodium dodecyl sulfate. Relevance of the composition of the micellar phase.

Sodium dodecyl sulfate (SDS) is a well-known protein denaturing agent. A less known property of this detergent is that it can activate or inactivate some enzymes at sub-denaturing concentrations. In this work we explore the effect of SDS on the ATPase activity of a hyper-thermophilic and a mesophilic Cu(I) ATPases reconstituted in mixed micelles of phospholipids and a non-denaturing detergent. An iterative procedure was used to evaluate the partition of SDS between the aqueous and the micellar phases, allowing to determine the composition of micelles prepared from phospholipid/detergent mixtures. The incubation of enzymes with SDS in the presence of different amounts of phospholipids reveals that higher SDS concentrations are required to obtain the same degree of inactivation when the initial concentration of phospholipids is increased. Remarkably, we found that, if represented as a function of the mole fraction of SDS in the micelle, the degree of inactivation obtained at different amounts of amphiphiles converges to a single inactivation curve. To interpret this result, we propose a simple model involving active and inactive enzyme molecules in equilibrium. This model allowed us to estimate the Gibbs free energy change for the inactivation process and its derivative with respect to the mole fraction of SDS in the micellar phase, the latter being a measure of the susceptibility of the enzyme to SDS. Our results showed that the inactivation free energy changes are similar for both proteins. Conversely, susceptibility to SDS is significantly lower for the hyperthermophilic ATPase, suggesting an inverse relation between thermophilicity and susceptibility to SDS.

Establishment of decellularized extracellular matrix scaffold derived from caprine pancreas as a novel alternative template over porcine pancreatic scaffold for prospective biomedical application.

In this study, the caprine pancreas has been presented as an alternative to the porcine organ for pancreatic xenotransplantation with lesser risk factors. The obtained caprine pancreas underwent a systematic cycle of detergent perfusion for decellularization. It was perfused using anionic (0.5% w/v sodium dodecyl sulfate) as well as non-ionic (0.1% v/v triton X-100, t-octyl phenoxy polyethoxy ethanol) detergents and washed intermittently with 1XPBS supplemented with 0.1% v/v antibiotic and nucleases in a gravitation-driven set-up. After 48 h, a white decellularized pancreas was obtained, and its extracellular matrix (ECM) content was examined for scaffold-like properties. The ECM content was assessed for removal of cellular content, and nuclear material was evaluated with temporal H&E staining. Quantified DNA was found to be present in a negligible amount in the resultant decellularized pancreas tissue (DPT), thus prohibiting it from triggering any immunogenicity. Collagen and fibronectin were confirmed to be preserved upon trichrome and immunohistochemical staining, respectively. SEM and AFM images reveal interconnected collagen fibril networks in the DPT, confirming that collagen was unaffected. sGAG was visualized using Prussian blue staining and quantified with DMMB assay, where DPT has effectively retained this ECM component. Uniaxial tensile analysis revealed that DPT possesses better elasticity than NPT (native pancreatic tissue). Physical parameters like tensile strength, stiffness, biodegradation, and swelling index were retained in the DPT with negligible loss. The cytocompatibility analysis of DPT has shown no cytotoxic effect for up to 72 h on normal insulin-producing cells (MIN-6) and cancerous glioblastoma (LN229) cells in vitro. The scaffold was recellularized using isolated mouse islets, which have established in vitro cell proliferation for up to 9 days. The scaffold received at the end of the decellularization cycle was found to be non-toxic to the cells, retained biological and physical properties of the native ECM, suitable for recellularization, and can be used as a safer and better alternative as a transplantable organ from a xenogeneic source.

Ionic Detergent Under Pressure-Vacuum as an Innovative Strategy to Generate Canine Tracheal Scaffold for Organ Engineering.

Decellularized scaffolds applied in tissue engineering offer improvements, supplying the elevated necessity for organs and tissues for replacement. However, obtaining a functional trachea for autotransplantation or allotransplantation is tricky due to the organ anatomical and structural complexity. Most tracheal decellularization protocols are lengthy, expensive, and could damage the tracheal extracellular matrix (ECM) architecture and functionality. Here, we aimed to evaluate the effectiveness of 3 different decellularization protocols combined with chemical and physical methods to obtain acellular canine tracheal scaffolds. Six adult dog tracheas were incised (tracheal segments) resulting in 28 rings for control tissue and 84 rings for decellularization (5-7 mm thick). Subsequently, decellularized tracheal scaffolds were microscopically/macroscopically characterized by histological analysis (Hematoxylin-Eosin, Masson's trichrome, Picrosirius red, Alcian blue, and Safranin O), immunohistochemistry for ECM components, scanning electron microscopy, and genomic DNA quantification. After decellularization, the tracheal tissue revealed reduced genomic DNA, and maintenance of ECM components preserved (structural proteins, adhesive glycoproteins, glycosaminoglycans and proteoglycans), suggesting ECM integrity and functionality. Comparatively, the combined ionic detergent with high vacuum pressure decellularization protocol revealed superior genomic DNA decrease (13.5 ng/mg) and improvement on glycosaminoglycans and proteoglycans preservation regarding the other decellularized trachea scaffolds and native tissue. Our results indicate that the 3 chemical/physical protocols reduce the decellularization time without ECM proteins damage. Notwithstanding, the use of ionic detergent under vacuum pressure was able to generate an innovative strategy to obtain acellular canine tracheal scaffolds with the highest levels of adhesive proteins that support its potentiality for recellularization and future tissue engineering application.