Protein Tryptophan Research Articles

Enhancing acid resistance of Escherichia coli based on directed morphology evolutionary of key transcription factor bolA

Succinic acid, as one of the important fermentation products, is commonly used as an acidulant, flavoring agent, and antimicrobial agent. However, the accumulation of organic acids in the later fermentation phase will restrict the production efficiency of strain. To address this issue, the directed evolution on the key transcription morphology factor bolA gene was performed to enhance acid resistance of Escherichia coli by ep-PCR library and CRISPR/Cas9. The best mutant strain 5-21-5 was screened from 4000 mutant colonies ep-PCR library, which showed higher cell survives in succinic acid stress. Its acid resistance ability was systematically illustrated from multiple aspects by transmission electron microscope (TEM), confocal laser scanning microscopy (CLSM), and three-dimensional excitation emission matrix (3D-EEM) analysis, respectively. Higher tyrosine and tryptophan protein level was beneficial to acid resistance, while cell tend to short rod shape. Subsequently, with the assistance of the CRISPR/Cas9 gene editing technology knocking the mutated sequences into the genome, the succinic acid yield of strain M-K10 reached 9.8 g/L, representing a 15.29% increase compared to the Escherichia coli MG1655 without gene editing.

Understanding Bacillus response to salt stress: Growth inhibition, enhanced EPS secretion, and molecular adaptation mechanisms

This study investigates the secretion pattern of extracellular polymeric substances (EPS) by Bacillus sp. under varying salt concentrations and elucidates the molecular mechanisms governing EPS synthesis and secretion. Salt stress inhibited cell proliferation, while optimal salt stimulation promoted EPS secretion, resulting in increased viscosity of the culture medium and the formation of bacterial clusters. Fourier infrared spectrum analysis revealed functional groups such as C-O-C and N-H within the EPS. Soluble-EPS (S-EPS) contained sulfur and phosphorus groups associated with heavy metal ions adsorption. The study also identified a novel polysaccharide formed through bonding EPS (B-EPS). High salt concentrations correlated with elevated levels of tryptophan protein and its derivatives, increased tyrosine polysaccharide derivatives, and decreased aromatic polysaccharides. B-EPS exhibited higher levels of aromatic polysaccharides, with Na+ promoting detachment of B-EPS from the cell surface. Transcriptome sequencing (RNA-seq) analysis under salt stress revealed significant expression of spore kinase (KinD) and response regulatory protein Spo0A in the phosphoric acid relay system. Key transcriptional regulatory factors, including OmpR and exopolysaccharide biosynthesis, were closely associated with EPS synthesis and secretion. This study establishes a theoretical foundation for the industrial production and practical application of EPS by elucidating the molecular mechanisms underlying Bacillus' response to salt stress.

True Digestibility of Tryptophan in Plant and Animal Protein

BackgroundProtein quality, evaluated using Digestible Indispensable Amino Acid Score (DIAAS), requires ileal digestibility values of individual indispensable amino acids (IAAs) in each protein. However, true tryptophan (Trp) digestibility has rarely been quantified in humans. ObjectiveTo measure the true Trp digestibility and DIAAS of 2H-intrinsically labeled plant and animal protein sources in humans, using the dual isotope tracer technique. MethodsThe true Trp digestibility of 2H intrinsically labeled plant proteins such as whole mung bean (n = 6) and dehulled mung bean (n = 6), chickpea (n = 5), and yellow pea (n = 5), and protein from animal source foods such as egg white (n = 6), whole egg (n = 6), chicken meat (n = 6), and goat milk (n = 7) was determined against the known digestibility of U-13C spirulina whole cell protein as reference, except for goat milk protein that was measured against free crystalline 13C-Trp as reference. Banked samples from earlier studies conducted to determine true IAA digestibility of different protein sources were used for the analysis. DIAAS was calculated for each test protein using digestibility corrected IAA scores (mg IAA/g of protein) in comparison with the IAA requirement score for adults. ResultsThe true Trp digestibility of whole mung bean, dehulled mung bean, chickpea, yellow pea, egg white, whole egg, chicken meat, and goat milk were 67.6 ± 3.7%, 74.5 ± 4.4%, 72.6 ± 2.3%, 72.5 ± 2.2%, 89.7 ± 2.5%, 91.4 ± 2.6%, 95.9 ± 2.2%, and 92.8 ± 2.9%, respectively. The true Trp digestibility of plant protein sources was significantly lower than that of animal protein sources (P < 0.05). Trp was not a limiting IAA in all the tested proteins. ConclusionThe true Trp digestibility determined in this study ranged from 67.6 ± 3.7% to 95.9 ± 2.2% for whole mung bean and chicken meat, respectively, and adds to the database of individual true IAA digestibility of different protein sources. Trial registration numberThis study was registered in Clinical Trials Registry of India (CTRI) with registration numbers CTRI/2017/11/010468, CTRI/2020/04/024512, and CTRI/2018/03/012265.

Effects of Different Protein Sources on Amino Acid Absorption and Plasma Appearance of Tryptophan, Large Neutral Amino Acids, and Tryptophan Metabolites in Pigs

BackgroundAbsorption of tryptophan (TRP) across the gut epithelium is potentially modulated by competing large neutral amino acids (LNAAs), which could affect the appearance of TRP and its metabolites in the bloodstream. ObjectivesThis study aimed to determine, in a growing pig model of an adult human, the absorption of TRP and other LNAAs from the gastrointestinal tract, and plasma appearance of TRP, LNAAs, and TRP metabolites, in response to dietary proteins varying in TRP content. MethodsPigs were adapted for 7 d to each of 4 diets that differed in their protein source and TRP content: 1) alpha-lactalbumin (AL; 9.95 mg TRP/g diet DM), 2) whey protein (6.59 mg TRP/g), 3) casein (3.73 mg TRP/g), or 4) zein (0.14 mg TRP/g). On day 8, pigs were euthanised after a 12-h fast (baseline), or 1, 2, 3, 4, or 6 h after they received a test meal consisting of 45 g protein, or a protein-free meal (n = 6 pigs at each time in each meal group). Tryptophan and LNAA absorption from the small intestine, and appearance of TRP, LNAAs, and TRP metabolites (melatonin, serotonin, kynurenine pathway metabolites), in the portal vein and systemic circulation, were determined. ResultsAL intake resulted in sustained elevated plasma TRP concentrations after an overnight fast. The amount of TRP absorbed was dose-dependently related to protein TRP content (P = 0.028), with fastest rates for pigs fed AL (371 mg/h). Portal and systemic plasma TRP, TRP/LNAA, and the TRP metabolites were highest (P ≤ 0.05) after AL intake, and remained above baseline levels for ∼4 h postprandially. Absorption rates of TRP correlated with postprandial plasma TRP and TRP metabolites (P ≤ 0.05). ConclusionsIn adult humans, postprandial plasma TRP and TRP metabolite concentrations can likely be modulated by the TRP content of the meal.

A structural role for tryptophan in proteins, and the ubiquitous Trp Cδ1-H...O=C (backbone) hydrogen bond.

Tryptophan is the most prominent amino acid found in proteins, with multiple functional roles. Its side chain is made up of the hydrophobic indole moiety, with two groups that act as donors in hydrogen bonds: the Nϵ-H group, which is a potent donor in canonical hydrogen bonds, and a polarized Cδ1-H group, which is capable of forming weaker, noncanonical hydrogen bonds. Due to adjacent electron-withdrawing moieties, C-H...O hydrogen bonds are ubiquitous in macromolecules, albeit contingent on the polarization of the donor C-H group. Consequently, Cα-H groups (adjacent to the carbonyl and amino groups of flanking peptide bonds), as well as the Cϵ1-H and Cδ2-H groups of histidines (adjacent to imidazole N atoms), are known to serve as donors in hydrogen bonds, for example stabilizing parallel and antiparallel β-sheets. However, the nature and the functional role of interactions involving the Cδ1-H group of the indole ring of tryptophan are not well characterized. Here, data mining of high-resolution (r ≤ 1.5 Å) crystal structures from the Protein Data Bank was performed and ubiquitous close contacts between the Cδ1-H groups of tryptophan and a range of electronegative acceptors were identified, specifically main-chain carbonyl O atoms immediately upstream and downstream in the polypeptide chain. The stereochemical analysis shows that most of the interactions bear all of the hallmarks of proper hydrogen bonds. At the same time, their cohesive nature is confirmed by quantum-chemical calculations, which reveal interaction energies of 1.5-3.0 kcal mol-1, depending on the specific stereochemistry.

Metabolism mechanisms of biogenic methane production by synergistic biodegradation of lignite and guar gum

Coalbed methane (CBM) presents a promising energy source for addressing global energy shortages. Nonetheless, challenges such as low gas production from individual wells and difficulties in breaking gels at low temperatures during extraction hinder its efficient utilization. Addressing this, we explored native microorganisms within coal seams to degrade guar gum, thereby enhancing CBM production. However, the underlying mechanisms of biogenic methane production by synergistic biodegradation of lignite and guar gum remain unclear. Research results showed that the combined effect of lignite and guar gum enhanced the production, yield rate and concentration of biomethane. When the added guar gum content was 0.8 % (w/w), methane production of lignite and guar gum reached its maximum at 561.9 mL, which was 11.8 times that of single lignite (47.3 mL). Additionally, guar gum addition provided aromatic and tryptophan proteins and promoted the effective utilization of CC/CH and OCO groups on the coal surface. Moreover, the cooperation of lignite and guar gum accelerated the transformation of volatile fatty acids into methane and mitigated volatile fatty acid inhibition. Dominant bacteria such as Sphaerochaeta, Macellibacteroides and Petrimonas improved the efficiency of hydrolysis and acidification. Electroactive microorganisms such as Sphaerochaeta and Methanobacterium have been selectively enriched, enabling the establishment of direct interspecies electron transfer pathways. This study offers valuable insights for increasing the production of biogenic CBM and advancing the engineering application of microbial degradation of guar gum fracturing fluid. Future research will focus on exploring the methanogenic capabilities of lignite and guar gum in in-situ environments, as well as elucidating the specific metabolic pathways involved in their co-degradation.

Electrochemical Bioconjugation of Tryptophan Residues: A Strategy for Peptide Modification.

Interest in electrocatalytic bioconjugation reactions has surged, particularly for modifying tryptophan and tyrosine residues in proteins. We used a cost-effective graphite felt electrode and low-current methodology to achieve selective bioconjugation of tryptophan with thiophenols, yielding up to 92%. This method exclusively labeled tryptophan residues and incorporated fluorinated tryptophan for NMR analysis. Eight polypeptides, including lanreotide and leuprorelin, were effectively coupled, demonstrating the method's versatility and potential for novel diagnostic and therapeutic agents.

DTT protein equalization and Tryptophan protein quantification as a powerful tool in analytical proteomics.

Assessing total protein levels in biological samples is a common procedure in biochemistry and molecular biology. In this study, we compare tryptophan fluorescence (WF) with Bradford and BCA assays to determine total protein in serum samples. Our results indicate that tryptophan fluorescence spectrometry is an efficient, sensitive, and straightforward technique for quantifying proteins in serum. We observed minimal variation between the three methods: BCA de one with the lowers LOD and LOQ. The tryptophan method offers the possibility of reusing the intact sample that does not need colourimetric reagents for quantification. Consequently, free tryptophan serves as a reliable universal standard. This assay can be performed using a conventional fluorescence spectrometer with cuvettes or in a 96-well plate format with a plate reader. The method was successfully used as proof of concept, using serum from patients diagnosed with myeloma and serum from healthy donors.

Magnetic nanoparticle loading and application of weak magnetic field to reconstruct the cake layer of coagulation-ultrafiltration process to achieve efficient antifouling: Performance and mechanism analysis

Abandoning the costly development of new membrane materials and instead directly remodeling the naturally occurring cake layer constitutes a dynamic, low-cost, long-lasting, and proactive strategy to "fight fouling with fouling". Several optimization strategies, including coagulation/modified magnetic seed loading and applying a weak magnetic force (0.01T) at the ultrafiltration end, improved the anti-fouling, retention, and sieving performances of conventional ultrafiltration process during the treatment of source water having complex natural organic matter (NOMs) and small molecule micropollutants. Two modified magnetic seeds we prepared were composite nano-seed particles (Fe3O4@SiO2-NH2 (FS) and Fe3O4@SiO2@PAMAM-NH2 (FSP)). Aim of the study was to regulate the formation of cake layer via comprehensive testing of the antifouling properties of optimized processes and related mechanistic studies. It was found to be essential to enhance the interception of xanthate and tryptophan proteins in the cake layer for improving the anti-fouling performance based on the correlation and redundancy analyses, while the use of modified magnetic seeds and magnetic field showed a significant positive impact on water production. Blockage modeling demonstrated the ability to form a mature cake layer during the initial filtration stage swiftly. This mitigated the risk of irreversible fouling caused by pore blockage during the early stage of coagulation-ultrafiltration. Morphologically, the reconstructed cake layer exhibited elevated surface porosity, an internal cavity channel structure, and enhanced roughness that can promote increased water flux and retention of water impurities. These optimized the maturity of the cake layer in both time and space. Density Functional Theory (DFT), Extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory, and Modified Extended Derjaguin-Landau-Verwey-Overbeek (MDLVO) calculations indicated aggregation behavior of matter on the cake layer to be enhanced effectively due to magnetic seed loading. This is mainly due to the strengthening of polar interactions, including hydrogen bonding, π-π* conjugation, etc., which can happen between the cake layer loaded with FSP and the organic matter. Under the influence of a magnetic field, magnetic force energy (VMF) significantly impacts the system by eliminating energy barriers. This research will provide innovative strategies for effectively purifying intricate source water through ultrafiltration while controlling membrane fouling.

Insight into the Effect of Submicellar Concentrations of Sodium Deoxycholate on the Structure, Stability, and Activity of Bovine and Human Serum Albumin: An Interesting Comparison between Single and Double Tryptophan Proteins.

The progressive escalation in the applications of bile salts in diverse fields has triggered research on their interaction with various biological macromolecules, especially with proteins. A proper understanding of the interaction process of bile salts, particularly in the lower concentrations range, with the serum albumin seems important since the normal serum concentration of bile salts is approximately in the micromolar range. The current study deals with a comprehensive and comparative analysis of the interaction of submicellar concentrations of sodium deoxycholate (NaDC) with two homologous transport proteins: bovine serum albumin (BSA) and human serum albumin (HSA). HSA and BSA with one and two tryptophans, respectively, provide the opportunity for an interesting comparison of tryptophan fluorescence behavior on interaction with NaDC. The study suggests a sequential interaction of NaDC in three discrete stages with the two proteins. A detailed study using warfarin and ibuprofen as site markers provides information about the sites of interaction, which is further confirmed by inclusive molecular dynamics simulation analysis. Moreover, the comparison of the thermodynamics and stability of the NaDC-serum albumin complexes confirms the stronger interaction of NaDC with BSA as compared to that with HSA. The differential interaction between the bile salt and the two serum albumins is further established from the difference in the extent of decrease in the esterase-like activity assay of the proteins in the presence of NaDC. Therefore, the present study provides important insight into the effect of submicellar concentrations of NaDC on the structure, stability, and activity of the two homologous serum albumins and thus can contribute not only to the general understanding of the complex nature of serum albumin-bile salt interactions but also to the design of more effective pharmaceutical formulations in the field of drug delivery and biomedical research.

Ecotoxicity of three typical tire wear particles to periphytic biofilms: The potentiating role after natural water-incubation-aging

Tire wear particles (TWPs), abundant in the aquatic environment, pose potential ecological risks, yet their implications have not been extensively studied. Rolling friction TWPs, sliding friction TWPs (S-TWPs) and cryogenically milled tire treads were used as research objects to study the ecotoxicity and difference of the above materials before and after aging in natural water (AS-TWPs) to the periphytic biofilm. The results showed that there were significant differences in the microstructure, surface elements, size, functional groups and environmentally persistent free radicals (EPFRs) of the three TWPs. After aging in natural water, the properties of the three TWPs mentioned above showed homogenization, but the EPFRs and reactive oxygen species (ROS) yield were different. After exposure to TWPs (10 mg L−1), total organic carbon and adenosine triphosphate decreased significantly (p < 0.05), and the production of extracellular polymeric substances (EPS) in the periphytic biofilm increased, in which the content of humic-like substance and proteins (tryptophan protein and humic acid-like substances) increased obviously. The increment of TB-EPS was higher than that of LB-EPS, and S-TWPs and AS-TWPs had the strongest promoting effect on EPS secretion. In addition, 10 mg L−1 TWPs caused massive cell death in the periphytic biofilm, which was more obvious in the S-TWPs and AS-TWPs exposure group. The toxic mechanism of TWPs promotes intracellular ROS accumulation and leads to the release of lactate dehydrogenase, which was attributed to the formation of EPFRs on the surface of TWPs and an increase in EPFRs intensity after aging in natural water. TWPs at environmentally relevant concentrations (0.1 mg L−1) had no biological toxicity to periphytic biofilms. This study fills the gap in the study of the surface structure characteristics of TWPs on the toxicity of periphytic biofilms, and is of great significance to the study of the aquatic toxicity mechanism of TWPs.

Short-time aerobic digestion treatment of waste activated sludge to enhance EPS production and sludge dewatering performance by changing microbial communities: The impact of temperature

Short-time aerobic digestion (STAD) of sludge requires low investment and has the advantages of short sludge retention time and rapid degradation of organic matter in waste activated sludge (WAS). This research conducted STAD at three temperature gradients (15 °C, 25 °C, and 35 °C). Characteristics of EPS and microbial community structure of raw and fermented sludge were analyzed, and environmental application potential of STAD at different temperatures was explored. STAD treatment improved both polysaccharide (PS) and protein (PN) contents in EPS, with increases of 5.7–18.35 % and 20.98–28.57 %, respectively. Aromatic protein-like substances (Peak E) and tryptophan protein substances (Peak F) were main components of PN in EPS. The microbial species richness decreased as the STAD temperature increased. At the class level, STAD temperature significantly affected Alphaproteobacteria, Gemm-1, Chloracidobacteria, Acidimicrobiia, Ellin6529, Betaproteobacteria, and SJA-28. From an application perspective, the specific resistance to filtration (SRF) value of WAS after STAD treatment at 35 °C was decreased to 7.01 × 107 m/kg with a 17.34 % reduction, indicating improvement in the dewaterability of the sludge. The Pb2+ adsorption efficiency per unit volume of EPS increased by 10.0–16.2 % after STAD treatment of WAS at different temperatures for 4 h. This research elucidated the impact of temperature on WAS in STAD systems, finding that microbial abundance correlated with EPS characteristics. The research results provide a basis for optimizing the system temperatures in STAD systems and a deeper understanding of the benefits of STAD in practical applications for sludge resource utilization.

Sediment Microbial Fuel Cells with Algae-Assisted Cathodes for Electricity Generation and Bio-Treatment of Sewage Sludge

In this study, an algal–bacterial symbiotic consortium was integrated with the sediment microbial fuel cell (SMFC) to construct an algal–bacterial cathode SMFC (AC-SMFC) for excess sewage sludge treatment and electricity generation. A bacterial cathode SMFC (BC-SMFC) and a static settling system (SS-system) were used as controls. Electrochemical analysis confirmed that the algal–bacterial biofilm on the cathode improved electricity production. The maximum power density of AC-SMFC was 75.21 mW/m2, which was 65.70% higher than that of the BC-SMFC (45.39 mW/m2). After 60 days of treatment, AC-SMFC achieved much higher removal efficiencies of the total chemical oxygen demand (TCOD) (59.60%), suspended solids (SS) (62.42%), and volatile suspended solids (VSS) (71.44%) in the sediment, compared to BC-SMFC and the SS-system, exhibiting an effective degradation of the organic matter in the sediment sludge. Moreover, the lower concentration of total nitrogen (TN) and total phosphorus (TP) in the overlying water of AC-SMFC demonstrated that the algae on the cathode could inhibit the accumulation of nitrogen and phosphorus released from the sediments. The three-dimensional excitation–emission matrix (EEM) fluorescence spectroscopy revealed that the tryptophan protein and aromatic protein in the loosely bound extracellular polymeric substances (LB-EPS) of the sediment sludge in the AC-SMFC were significantly decreased. Additionally, the abundance of functional microbiota in the AC-SMFC increased, such as Trichococcus, Alphaproteobacteria, and Clostridia, which contributed to electricity generation and sludge degradation. The combined application of microalgae and the SMFC provided a promising approach for excess sludge reduction and energy recovery.

Extracellular proteins enhance Cupriavidus pauculus nickel tolerance and cell aggregate formation

Heavy metal-resistant bacteria secrete extracellular proteins (e-PNs). However, the role of e-PNs in heavy metal resistance remains elusive. Here Fourier Transform Infrared Spectroscopy implied that N-H, C = O and NH2-R played a crucial role in the adsorption and resistance of Ni2+ in the model organism Cuprividus pauculus 1490 (C. pauculus). Proteinase K treatment reduced Ni2+ resistance of C. pauculus underlining the essential role of e-PNs. Further three-dimension excitation-emission matrix fluorescence spectroscopy analysis demonstrated that tryptophan proteins as part of the e-PNs increased significantly with Ni2+ treatment. Proteomic and quantitative real-time polymerase chain reaction data indicated that major changes were induced in the metabolism of C. pauculus in response to Ni2+. Among those lipopolysaccharide biosynthesis, general secretion pathways, Ni2+-affiliated transporters and multidrug efflux play an essential role in Ni2+ resistance. Altogether the results provide a conceptual model for comprehending how e-PNs contribute to bacterial resistance and adsorption of Ni2+.

Effects of Non-Allelic Interactions of O2 and SU2 Mutant Genes on Grain Biochemical Composition in Various Corn Inbreds

The use of combinations of non-allelic mutant genes of the maize endosperm structure creates opportunities for improving the quality of corn grain in comparison not only with forms of the common type but also with monogenic endospermic mutants. In this study, the effect of a combination of mutant genes O2 (Opaque-2) and SU2 (Sugary-2) according to the biochemical composition of the grain was studied. For the research, a series of inbreds - carriers of a combination of mutant genes O2SU2, inbreds - carriers of monogenic mutations O2 and SU2, as well as maize inbreds of the common type of two-year reproduction were used. In the experiments, the content of protein, starch, and oil and the main characteristics of their quality were studied. It was found that the inbred carriers of the O2SU2 combination are superior to the inbred carriers of monogenic mutations O2 and SU2 in terms of complex biochemical characteristics. In comparison with mutants O2 they were distinguished by an increased content of protein (by 12.3% on average), amylose in starch (by 38.9% on average), starch digestibility (by 24.4% on average), oil content (by 18.4% on average) and oleate content in oil (by 29.9% on average). In comparison with the carriers of SU2 mutation, they had a higher content of lysine and tryptophan in the total grain protein (on average, by 19.4% &amp; 14.3%, respectively). The main characteristics of grain quality in carriers of a combination of mutant genes O2SU2 were characterized by quantitative variability, which can modify the effect of non–allelic interaction of mutant genes O2 and SU2. The obtained results indicate the effectiveness of using non-allelic interactions between the O2 and SU2 mutant genes to improve the quality of corn grain.

Novel strategy for treating high salinity oilfield produced water: Pyrite-activated peroxymonosulfate coupled with heterotrophic ammonia assimilation

Existing conventional biological treatment techniques face numerous limitations in effectively removing total petroleum hydrocarbons (TPHs) and ammonia (NH4+-N) from oilfield-produced water (OPW), highlighting the pressing need for innovative pre-oxidation and biological treatment processes. In this study, a pyrite-activated peroxymonosulfate (PMS)-coupled heterotrophic ammonia assimilation (HAA) system was established to achieve satisfactory system performance for OPW treatment. Pyrite sustained-release Fe2+-activated PMS was used to produce SO4•− and •OH, and 71.0 % of TPHs were effectively removed from the oil wastewater. The average TPHs and NH4+-N removal efficiencies in the test group with pre-oxidation were 96.9 and 98.3 %, compared to 46.5 and 77.1 % in the control group, respectively. The maximum fluorescence intensities of tryptophan protein and aromatic protein in the test group declined by 83.7 %. Fourier transform ion cyclotron resonance mass spectrometry revealed that pre-oxidation degraded more long-chain hydrocarbons and aromatic family compound, whereas the HAA process produced more proteins and carbohydrates. Pyrite-PMS promoted the enrichment of ammonia-assimilating bacteria, alleviating the explosive increase in extracellular polymeric substances and reducing sludge settleability. The low cost, efficiency, green chemistry principles, and synergies of this approach make it a powerful solution for practical OPW treatment to reduce environmental impacts and promote sustainable wastewater treatment.

Human γS-Crystallin Mutation F10_Y11delinsLN in the First Greek Key Pair Destabilizes and Impairs Tight Packing Causing Cortical Lamellar Cataract.

Aromatic residues forming tyrosine corners within Greek key motifs are critical for the folding, stability, and order of βγ-crystallins and thus lens transparency. To delineate how a double amino acid substitution in an N-terminal-domain tyrosine corner of the CRYGS mutant p.F10_Y11delinsLN causes juvenile autosomal dominant cortical lamellar cataracts, human γS-crystallin c-DNA was cloned into pET-20b (+) and a p.F10_Y11delinsLN mutant was generated via site-directed mutagenesis, overexpressed, and purified using ion-exchange and size-exclusion chromatography. Structure, stability, and aggregation properties in solution under thermal and chemical stress were determined using spectrofluorimetry and circular dichroism. In benign conditions, the p.F10_Y11delinsLN mutation does not affect the protein backbone but alters its tryptophan microenvironment slightly. The mutant is less stable to thermal and GuHCl-induced stress, undergoing a two-state transition with a midpoint of 60.4 °C (wild type 73.1 °C) under thermal stress and exhibiting a three-state transition with midpoints of 1.25 and 2.59 M GuHCl (wild type: two-state transition with Cm = 2.72 M GuHCl). The mutant self-aggregates upon heating at 60 °C, which is inhibited by α-crystallin and reducing agents. Thus, the F10_Y11delinsLN mutation in human γS-crystallin impairs the protein's tryptophan microenvironment, weakening its stability under thermal and chemical stress, resulting in self-aggregation, lens opacification, and cataract.

Avoidance of the use of tryptophan in buried chromosomal proteins as a mechanism for reducing photo/oxidative damage to genomes.

In cells that are exposed to terrestrial sunlight, the indole moiety in the side chain of tryptophan (Trp) can suffer photo/oxidative damage (POD) by reactive oxygen species (ROS) and/or ultraviolet light (UV-B). Trp is oxidized to produce N-formylkynurenine (NFK), a UV-A-responsive photosensitizer that further degenerates into photosensitizers capable of generating ROS through exposure to visible light. Thus, Trp-containing proteins function as both victims, and perpetrators, of POD if they are not rapidly replaced through protein turnover. The literature indicates that protein turnover and DNA repair occur poorly in chromosomal interiors. We contend, therefore, that basic chromosomal proteins (BCPs) that are enveloped by DNA should have evolved to lack Trp residues in their amino acid sequences, since these could otherwise function as 'Trojan horse-type' DNA-damaging agents. Our global analyses of protein sequences demonstrates that BCPs consistently lack Trp residues, although DNA-binding proteins in general do not display such a lack. We employ HU-B (a wild-type, Trp-lacking bacterial BCP) and HU-B F47W (a mutant, Trp-containing form of the same bacterial BCP) to demonstrate that the possession of Trp is deleterious to BCPs and associated chromosomal DNA. Basically, we show that UV-B and UV-A (a) cause no POD in HU-B, but cause extensive POD in HU-B F47W (in vitro), as well as (b) only nominal DNA damage in bacteria expressing HU-B, but extensive DNA damage in bacteria expressing F47W HU-B (in vivo). Our results suggest that Trp-lacking BCPs could have evolved to reduce scope for protein-facilitated, sunlight-mediated damage of DNA by UV-A and visible light, within chromosomal interiors that are poorly serviced by protein turnover and DNA repair machinery.

Anaerobic digestion of hydrothermally pretreated dewatered sewage sludge: effects of process conditions on methane production and the fate of phosphorus.

The hydrothermal pretreatment (HTP) characteristics and the fate of phosphorus (P) and anaerobic digestion (AD) performance of dewatered sewage sludge (DSS) were investigated at different hydrothermal conditions. The maximum methane yield reached 241mL CH4/g COD when the hydrothermal conditions were 200°C-2h-10% (A4), and the yield was 78.28% higher than that without pretreatment (A0) and 29.62% higher than that of the initial hydrothermal conditions (A1, 140°C-1h-5%). Proteins, polysaccharides, and volatile fatty acids (VFAs) were the main hydrothermal products of DSS. 3D-EEM analysis revealed that tyrosine, tryptophan proteins, and fulvic acids decreased after HTP, but the content of humic acid-like substances increased, and this phenomenon was more noticeable after AD. Solid-organic P was converted into liquid-P during the hydrothermal process, and nonapatite inorganic P was converted into organic P during AD. All samples achieved positive energy balance, and the energy balance of A4 was 10.50kJ/g VS. Microbial analysis showed that the composition of the anaerobic microbial degradation community changed as the sludge organic composition was altered. Results showed that the HTP improved the anaerobic digestion of DSS.

Decoding the Role of Extracellular Polymeric Substances in Enhancing Nitrogen Removal from High-Ammonia and Low-C/N Wastewater in a Sequencing Batch Packed-Bed Biofilm Reactor

Although the role of extracellular polymeric substances (EPSs) as a viscous high-molecular polymer in biological wastewater treatment has been recognized, in-depth knowledge of how EPSs affect nitrogen removal remains limited in biofilm-based reactors. Herein, we explored EPS characteristics associated with nitrogen removal from high-ammonia (NH4+-N: 300 mg/L) and low carbon-to-nitrogen ratio (C/N: 2-3) wastewater in a sequencing batch packed-bed biofilm reactor (SBPBBR) under four different operating scenarios for a total of 112 cycles. Scanning electron microscopy (SEM), atomic force microscopy (AFM), and Fourier-transform infrared (FTIR) analysis revealed that the distinct physicochemical properties, interface microstructure, and chemical composition of the bio-carrier were conducive to biofilm formation and microbial immobilization and enrichment. Under the optimal conditions (C/N: 3, dissolved oxygen: 1.3 mg/L, and cycle time: 12 h), 88.9% ammonia removal efficiency (ARE) and 81.9% nitrogen removal efficiency (NRE) could be achieved in the SBPBBR. Based on visual and SEM observations of the bio-carriers, biofilm development, biomass concentration, and microbial morphology were closely linked with nitrogen removal performance. Moreover, FTIR and three-dimensional excitation-emission matrix (3D-EEM) spectroscopy demonstrated that tightly bound EPSs (TB-EPSs) play a more important role in maintaining the stability of the biofilm. Significant shifts in the number, intensity, and position of fluorescence peaks of EPSs determined different nitrogen removal. More importantly, the high presence of tryptophan proteins and humic acids might promote advanced nitrogen removal. These findings uncover intrinsic correlations between EPSs and nitrogen removal for better controlling and optimizing biofilm reactors.