UPLC/Q-TOF-MS-based metabolomics and molecular docking analysis of Bifidobacterium adolescentis exposure to levofloxacin.

Antibiotic-associated diarrhea is a common adverse reaction caused by the widespread use of antibiotics. The decrease in probiotics is one of the reasons why antibiotics cause drug-induced diarrhea. However, few studies have addressed the intrinsic mechanism of antibiotics inhibiting probiotics. To investigate the underlying mechanism of levofloxacin against Bifidobacterium adolescentis, we used a metabolomics mass spectrometry-based approach and molecular docking analysis for a levofloxacin-induced B. adolescentis injury model. The results showed that levofloxacin reduced the survival rate of B. adolescentis and decreased the number of B. adolescentis. The untargeted metabolomics analysis identified 27 potential biomarkers, and many of these metabolites are involved in energy metabolism, amino acid metabolism and the lipid metabolism pathway. Molecular docking showed that levofloxacin can bind with aminoacyl-tRNA synthetase and lactic acid dehydrogenase. This result provides a novel insight into the mechanism of the adverse reactions of levofloxacin.

Adsorptive decontamination of antibiotics from livestock wastewater by using alkaline-modified biochar.

Efficient abatement of antibiotics from livestock wastewater is in urgent demand, but still challenging. In this study, alkaline-modified biochar with larger surface area (130.520 m2 g-1) and pore volume (0.128 cm3 g-1) was fabricated and explored for the adsorption of different types of antibiotics from livestock wastewater. Batch adsorption experiments demonstrated that the adsorption process was mainly determined by chemisorption and was heterogeneous, which could be moderately affected by the variations of solution pH (3-10). Furthermore, the computational analysis based on density functional theory (DFT) indicated that the -OH groups on biochar surface could serve as the dominant active sites for antibiotics adsorption due to the strongest adsorption energies between antibiotics and -OH groups. In addition, the antibiotics removal was also evaluated in multi-pollutants system, where biochar performed synergistic adsorption towards Zn2+/Cu2+ and antibiotics. Overall, these findings not only deepen our understandings on the adsorption mechanism between biochar and antibiotics, but also promote the application of biochar in the remediation of livestock wastewater.

Metabolomics and molecular docking analysis of antibiotic exposure in Bifidobacterium adolescentis.

Bifidobacterium adolescentis is a probiotic. This research aimed to investigate the mechanism of antibiotics led to decrease in the number of B. adolescentis. The metabolomics approach was employed to explore the effects of amoxicillin on metabolism of B.adolescentis, while MTT assay and scanning electron microscopy were applied to analyse changes in viability and morphology of bacteria. Molecular docking was used to illuminate the mechanism by which amoxicillin acts on a complex molecular network. The results showed that increasing the concentration of amoxicillin led to a gradual decrease in the number of live bacteria. Untargeted metabolomics analysis identified 11 metabolites that change as a result of amoxicillin exposure. Many of these metabolites are involved in arginine and proline metabolism, glutathione metabolism, arginine biosynthesis, cysteine, and methionine metabolism, and tyrosine and phenylalanine metabolism. Molecular docking revealed that amoxicillin had a good binding effect on the proteins AGR1, ODC1, GPX1, GSH, MAT2A, and CBS. Overall, this research provides potential targets for screening probiotic regulatory factors and lays a theoretical foundation for the elucidation of its mechanisms.

Discovery of a novel analogue of FR901533 and the corresponding biosynthetic gene cluster from Streptosporangium roseum No. 79089.

FR901533 (1, also known as WS79089B), WS79089A (2), and WS79089C (3) are polycyclic aromatic natural products with promising inhibitory activity to endothelin-converting enzymes. In this work, we isolated five tridecaketide products from Streptosporangium roseum No. 79089, including 1-3, benaphthamycin (4) and a novel FR901533 analogue (5). The structure of 5 was characterized based on spectroscopic data. Compared with the major product 2, the new compound 5 has an additional hydroxyl group at C-12 and an extra methyl group at the 13-OH. The configuration of C-19 of these compounds was determined to be R using Mosher's method. A putative biosynthetic gene cluster for compounds 1-5 was discovered by analyzing the genome of S. roseum No. 79089. This 38.6-kb gene cluster contains 38 open reading frames, including a minimal polyketide synthase (wsaA-C), an aromatase (wsaD), three cyclases (wsaE, F, and W), and a series of tailoring enzymes such as monooxygenases (wsaO1-O7) and methyltransferases (wsaM1 and M2). Disruption of the ketosynthase gene (wsaA) in this gene cluster abolished the production of 1-5, confirming that this gene cluster is indeed responsible for the biosynthesis of 1-5. A type II polyketide biosynthetic pathway was proposed for this group of natural endothelin-converting enzyme inhibitors. KEY POINTS: • Five aromatic tridecaketides were isolated from Streptosporangium roseum No. 79089. • A novel FR901533 analogue, 12-hydroxy-13-O-methyl-WS79089A, was characterized. • The absolute configuration of C-19 of FR901533 and analogues was determined. • The biosynthetic gene cluster of FR901533 and analogues was discovered.

Six new furostanol glycosides from Smilax glauco-china and their cytotoxic activity.

Six new steroidal saponins, namely glauco-chinaosides A-F, and one known compound were isolated from the tubers of Smilax glauco-china. Their structures were elucidated by a combination of spectroscopic analysis and hydrolysis followed by spectral and chromatographic analysis. Compounds 1-7 were tested in vitro for their cytotoxic activities against four human tumor cell lines (SH-SY5Y, SGC-7901, HCT-116, and Lovo). Compounds 1, 2, and 5 exhibited cytotoxic activity against SGC-7901, with IC50 values of 2.7, 11.5, and 6.8 µM, respectively.

[Discrimination of Chemical Constituents Changes of Citrus reticulata Pericarp Before and After Processing with Vinegar Based on Partial Least Squares-Discriminate Analysis].

Objective: To investigate the chemical constituents changes of Citrus reticulata pericarp before and after processing with vinegar,and to provide scientific basis for the study of active components in Citrus reticulata pericarp. Methods: Citrus reticulata pericarp was processed with vinegar according to the Zhongyao Paozhixue Cidian. The extracts of unprocessed and processed Citrus reticulata pericarp were detected by HPLC. Partial least squares-discriminate analysis( PLS-DA) was employed to reveal the discrimination between different samples. Results: 15 chemical constituents had significant changes in Citrus reticulata pericarp before and after processing with vinegar. Two components increased significantly after processing with vinegar were characterized as narirutin and hesperidin respectively. Conclusion: The chemical constituents in Citrus reticulata pericarp are found to be changed after processing with vinegar, two components with significant increasing indicate the basis of the variation of bioactivity in vinegar-processed Citrus reticulata pericarp, which provides evidence to interpret its clinical usage.

[¹H-NMR based metabonomic approach to evaluate anti-coagulant effect of Danggui Sini decoction].

To study the anti-coagulant effect and influence of danggui Sini decoction (DSD) on rat's plasma endogenous metabolites by animal experiment and ¹H-NMR based metabolomics method. After intragastric administration of Danggui Sini Decoction for 7 days, Plasma thrombin time (TT) was measured. Rat plasma metabolic fingerprint in two groups was analyzed using ¹H-NMR, based on which the principal component analysis( PCA) and orthogonal partial least-squares discriminant analysis(OPLS-DA) models for metabonomic analysis. Potential biomarkers were screened by using variable importance in the projection (VIP) and T test. DSD could prolong TT of the rat significantly (P < 0.05). Compared with control group, six kinds of endogenous metabolites in DSD group change significantly (P < 0.05), among which isobutyrate, carnitine and phenylalanine content had an upward trend (P < 0.01) and lysine, Histidine and cholesterol content had a downward trend (P < 0.05). It is likely that carnitine, phenylalanine, Histidine and cholesterol are the potential metabolic markers in the anti-coagulant process and DSD affects the platelet aggregation and the expression of tissue factor and fiber protease by regulating the energy, amino acid and lipid metabolism.

Comprehensive chemical analysis of the rhizomes of Drynaria fortunei by orthogonal pre-separation and liquid chromatography mass spectrometry.

The chemical composition of Drynaria fortunei, a traditional Chinese herbal medicine, is very complicated. In order to separate these chemicals to obtain their structural information, an orthogonal sample enrichment procedure was established. The ethyl acetate extract of D. fortunei was pre-separated by Sephadex LH-20 × polyamide columns to yield 15 fractions. These fractions were analyzed successively using a reversed-phase Agilent Zorbax SB-C18 column, coupled with diode array detection and electrospray ionization tandem mass spectrometry. The method reduced co-elution and enriched minor compounds on the basis of their chemical features. A total of 369 compounds were detected by LC/MSn, compared to less than 50 compounds without pre-separation. The pretreatment facilitated the analytical separation of flavonoids, proanthocyanidins, triterpenoids, phenolic acids, and lignans in D. fortunei, and allowed a comprehensive chemical profiling of these constituents. This method could be applied to other multicomponent herbal extracts.

Treatment of a non-typical hepatic pseudolesion complicated by greatly elevated alpha fetoprotein: case report and literature review.

BACKGROUND: Hepatic pseudolesions detected by helical computed tomography (CT) are not rare, but it is difficult to make a final diagnosis when the hepatic lesion is complicated by the presence of greatly elevated alpha fetoprotein (AFP). Clinical treatment of non-typical hepatic pseudolesions complicated by greatly elevated AFP should confirm the diagnosis and minimize trauma. CASE PRESENTATION: Non-invasive procedures including ultrasonography, CT, and micro-invasive digital subtraction angiography could not safely differentiate this lesion from a malignant focus when it was complicated by greatly elevated AFP. Laparoscopic hepatectomy was performed, and pathological analysis showed chronic hepatitis, nodular regenerative hyperplasia, focal nodular hyperplasia of the liver, and mild vascular malformation. The tissue was HbsAg(-), HbcAg(-), and AFP(+). CONCLUSION: Heightened awareness of hepatic pseudolesion complicated by primarily elevated AFP will help physicians avoid unnecessary invasive procedures. Hepatic biopsy is inevitable because of greatly elevated AFP. For suspected hepatic pseudolesion with elevated AFP, needle-core biopsy and follow-up surveillance instead of hepatectomy are recommended to find the source of AFP and make a final diagnosis of pseudolesion.

[Evolution of miRNA in plants].

Due to the important role of miRNA in regulation of plant gene expression, researchers have focused on the prediction, identification, functional and evolutionary analysis of miRNA. As genome sequencing has been completed in many species, the integration of genomic sequences with evolutionary data of miRNA provides more and more evidence and hypotheses for the origin and evolution of miRNA. However, the evolution of miRNA in plants has not been extensively reviewed. In this article, we reviewed several aspects of the evolution of miRNA in plants, including the origin hypothesis of miRNA, the birth and death of miRNA, and the functional evolution of miRNA, which provides a reference for future research.

Proactive dynamic vehicle routing problems considering cooperation services for the store-depot-integrated retailer.

Distribution costs remain consistently high in crowded city road networks, posing challenges for traditional distribution methods in efficiently handling dynamic online customer orders. To address this issue, this paper introduces the Proactive Dynamic Vehicle Routing Problem considering Cooperation Service (PDVRPCS) model. Based on proactive prediction and order-matching strategies, the model aims to develop a cost-effective and responsive distribution system. A novel solution framework is proposed, incorporating a proactive prediction method, a matching algorithm and a hybrid Genetic Algorithm-Simulated Annealing (GA-SA) algorithm. To validate the effectiveness of the proposed model and algorithm, a case study is conducted. The experimental results demonstrate that the dynamic scheme can significantly reduce the number of vehicles required for distribution, leading to cost reduction and increased efficiency.

Toosendanin induced hepatotoxicity via triggering PERK-eIF2α-ATF4 mediated ferroptosis.

Toosendanin (TSN) is the main active compound of Melia toosendan Sieb et Zucc with various bioactivities. In this study, we investigated the role of ferroptosis in TSN-induced hepatotoxicity. The characteristic indicators of ferroptosis were detected including reactive oxygen species (ROS), lipid-ROS, glutathione (GSH), ferrous ion and the expression of glutathione peroxidase 4 (GPX4), which showed that TSN caused ferroptosis in hepatocytes. The results of qPCR analysis and western blotting assay showed that TSN-induced activation of protein kinase R-like endoplasmic reticulum kinase (PERK)- eukaryotic initiation factor 2 α subunit (eIF2α)- activation transcription factor 4 (ATF4) signaling pathway resulted in increasing activation transcription factor 3 (ATF3) expression, which upregulated the expression of transferrin receptor 1 (TFRC). Furthermore, TFRC mediated iron accumulation leading to ferroptosis in hepatocytes. To clarify whether TSN triggered ferroptosis in vivo, male Balb/c mice were treated with the different doses of TSN. The results of hematoxylin-eosin (H&E) staining, 4-hydroxynonenal (4-HNE) staining, malondialdehyde (MDA) content and the protein expression of GPX4 showed that ferroptosis contributed to TSN-induced hepatotoxicity. Iron homeostasis relative protein and PERK- eIF2α- ATF4 signaling pathway also involved in hepatotoxicity of TSN in vivo.

Effects of different soil water holding capacities on vegetable residue return and its microbiological mechanism.

With the gradual expansion of the protected vegetable planting area, dense planting stubbles and increasing labor cost, the treatment of vegetable residues has become an urgent problem to be solved. Soil bacterial community structure plays an important role in vegetable residue return and is susceptible to environmental changes. Therefore, understanding the influences of different soil water holding capacities on plant residue decomposition and soil bacterial communities is important for biodegradation. During the whole incubation period, the weight loss ratio of plant residue with 100% water holding capacity was 69.60 to 75.27%, which was significantly higher than that with 60% water holding capacity in clay and sandy soil, indicating that high water holding capacity promoted the decomposition of plant residue. The degradation of lignin and cellulose was also promoted within 14 days. Furthermore, with the increase in soil water holding capacity, the contents of NH4+ increased to 5.36 and 4.54 times the initial value in the clay and sandy soil, respectively. The increase in napA and nrfA resulted in the conversion of NO3- into NH4+. The increase in water holding capacity made the bacterial network structure more compact and changed the keystone bacteria. The increase in water holding capacity also increased the relative abundance of Firmicutes at the phylum level and Symbiobacterium, Clostridium at the genus level, which are all involved in lignin and cellulose degradation and might promote their degradation. Overall, these findings provide new insight into the effects of different soil water holding capacities on the degradation of plant residues in situ and the corresponding bacterial mechanisms.

Integrated Gut Microbiota and Urine Metabolite Analyses of T2DM with NAFLD Rat Model.

Globally 80% type 2 diabetes mellitus (T2DM) patients suffer nonalcoholic fatty liver disease (NAFLD). The interplay of gut microbiota and endogenous metabolic networks has not yet been reported in the setting of T2DM with NAFLD. As such, this study utilized 16S rRNA gene sequencing to assess the changes in intestinal flora and nuclear magnetic resonance spectroscopy (1H NMR) to identify potential metabolites in a T2DM with NAFLD rat model. Spearman correlation analysis was performed to explore the relationship between gut microbiota and metabolites. Results revealed that among T2DM with NAFLD rats, diversity indexes of intestinal microbiota were distinctly decreased while levels of 18 bacterial genera within the intestinal tract were significantly altered. In addition, levels of eight metabolites mainly involved in the synthesis and degradation of ketone bodies, the TCA cycle, and butanoate metabolism were altered. Correlation analysis revealed that gut bacteria such as Blautia, Ruminococcus torques group, Allobaculum, and Lachnoclostridium strongly associate with 3-hydroxybutyrate, acetone, acetoacetate, 2-oxoglutarate, citrate, creatinine, hippurate, and allantoin. Our findings can provide a basis for future development of targeted treatments.

Integrating metabonomics and metagenomics sequencing to study the anti-liver fibrosis effects of palmatine in Corydalis saxicola Bunting.

ETHNOPHARMACOLOGICAL RELEVANCE: Corydalis saxicola Bunting (CS), a traditional Chinese folk medicine, has been effectively used for treating liver disease in Zhuang nationality in South China. However, the main anti-liver fibrosis ingredients in CS are incompletely understood. AIM OF THE STUDY: To elucidate the main anti-liver fibrosis ingredients in CS and its underlying mechanism. MATERIAL AND METHODS: Firstly, spectrum-effect relationship (SER) strategy was applied to identify the major ingredients against liver fibrosis in CS. Subsequently, 1H NMR metabonomics and metagenomics sequencing techniques were used to clarify the intervention of palmatine (PAL) on liver fibrosis. Furthermore, the expression of tight junction proteins and the levels of liver inflammation factors were examined, the effect of PAL on microbiota was verified by FMT. RESULTS: The SER model revealed that PAL was the most important active ingredient in CS. 1H NMR fecal metabonomics showed that PAL could reserve the abnormal levels of gut microbial-mediated metabolites of liver fibrosis, such as isoleucine, taurine, butyrate, propionate, lactate, glucose, which mainly involved in amino acid metabolism, intestinal flora metabolism and energy metabolism. Metagenomics sequencing found that PAL could callback the abundance of s__Lactobacillus_murinus, s__Lactobacillus_reuteri, s__Lactobacillus_johnsonii, s__Lactobacillus_acidophilus and s__Faecalibaculum_rodentium to varying degree. Furthermore, the intestinal barrier function and the levels of hepatic inflammation factors were significantly ameliorated by PAL. FMT demonstrated that the therapeutic efficiency of PAL was closely associated with gut microbiota. CONCLUSION: The effects of CS on liver fibrosis were attributed in part to PAL by alleviating metabolic disorders and rebalancing gut microbiota. The SER strategy may be a useful method for the discovery of active constituents in natural plants.

A spectrum-effect based method for screening antibacterial constituents in Niuhuang Shangqing Pill using comprehensive two-dimensional liquid chromatography.

To investigate and screen the active antibacterial constituents of Niuhuang Shangqing Pill (NSP), the current study developed a two-dimensional liquid chromatography (2DLC) method combining microcalorimetry technique. 60% ethanol extracts from 10 batches of different commercial NSP samples were analyzed and their chemical fingerprint were developed by the comprehensive 2DLC system of Shimadzu Nexera X2. Anti-streptococcus pneumoniae (SP) constituents were determined by microcalorimetry. Thermal kinetic parameters of the SP thermogram affected by 60% ethanol extracts from 10 NSP samples were analyzed by principal component analysis. Spectrum-effect correlation between comprehensive 2DLC fingerprint and the antibacterial activity were analyzed by orthogonal partial least squares (OPLS) and orthogonal partial least squares discriminant analysis (OPLS-DA). Findings showed that peak X1 (unknown), X9 (aloe-emodin), X10 (baicalein), X11 (unknown), X14 (wogonin), X15 (glycyrrhizic acid) and X17 (unknown) are the relevant components that are in positive correlation with inhibitory rate. Regarding inhibitory rate, X17 is the most powerful one, followed by X14, X15, X10, X11, X1 and X9, suggesting that compound X17, wogonin, glycyrrhizic acid and baicalein are the major active antibacterial components of NSP. The current method employing 2DLC with microcalorimetry technique proposes a new insight for screening and identifying antibacterial components in complex herbal formula.

The recognition of aristolochic acid I based on fluorescence quenching of bovine serum albumin-stabilized gold nanoclusters.

Aristolochic acid I (AAI) is one of the nephrotoxic derivatives present in genera Aristolochia and Asarum. Although some detection strategies for monitoring AAI have been reported, the application of these methods is limited because they involve tedious preparation and require professional operation. In this work, bovine serum albumin (BSA) has been introduced as a reducing agent and stabilizing agent to synthesize gold nanoclusters with strong red fluorescence for the rapid and effective detection of AAI. Under excitation at 328 nm, the fluorescence intensity at the maximum emission wavelength of the bovine serum albumin-stabilized gold nanoclusters (BSA-AuNCs) decreased with the addition of AAI, and the degree of quenching showed a linear relationship with the concentration of AAI from 0.1-12.8 µg mL-1. The obtained BSA-AuNCs were stable, and quenching in the presence of AAI could be achieved within 10 seconds. Here, we have focused on the application of these gold nanoclusters as an optical sensing material for AAI in rat urine samples, including a discussion on the detection mechanism. The detection result of the fluorescent probe was consistent with that of the HPLC method. In view of this reality, the reported protein-AuNCs sensing platform can serve as a convenient detection strategy in toxicological analyses.

Toosendanin Induces Hepatocyte Damage by Inhibiting Autophagic Flux via TFEB-Mediated Lysosomal Dysfunction.

Toosendanin (TSN) is a triterpenoid from the fruit or bark of Melia toosendan Sieb et Zucc, which has clear antitumor and insecticidal activities, but it possesses limiting hepatotoxicity in clinical application. Autophagy is a degradation and recycling mechanism to maintain cellular homeostasis, and it also plays an essential role in TSN-induced hepatotoxicity. Nevertheless, the specific mechanism of TSN on autophagy-related hepatotoxicity is still unknown. The hepatotoxicity of TSN in vivo and in vitro was explored in this study. It was found that TSN induced the upregulation of the autophagy-marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) and P62, the accumulation of autolysosomes, and the inhibition of autophagic flux. The middle and late stages of autophagy were mainly studied. The data showed that TSN did not affect the fusion of autophagosomes and lysosomes but significantly inhibited the acidity, the degradation capacity of lysosomes, and the expression of hydrolase cathepsin B (CTSB). The activation of autophagy could alleviate TSN-induced hepatocyte damage. TSN inhibited the expression of transcription factor EB (TFEB), which is a key transcription factor for many genes of autophagy and lysosomes, such as CTSB, and overexpression of TFEB alleviated the autophagic flux blockade caused by TSN. In summary, TSN caused hepatotoxicity by inhibiting TFEB-lysosome-mediated autophagic flux and activating autophagy by rapamycin (Rapa), which could effectively alleviate TSN-induced hepatotoxicity, indicating that targeting autophagy is a new strategy to intervene in the hepatotoxicity of TSN.

Influence of Sequential Liquid Ammonia and Caustic Mercerization Pre-Treatment on Dyeing Performance of Knit Cotton Fabric.

A two-stage sequential pretreatment including caustic mercerization (CM) and liquid ammonia (LA) treatment was applied to investigate the influence on dyeing performance and handle of knit cotton fabric, and the relationship between dye size and dyeing properties. Various techniques were applied to characterize all the treated fabrics. X-ray diffraction (XRD) and Fourier-transform infrared (FTIR) analyses of the treated fabrics confirmed that both sequential treatments decreased the crystallinity of cotton fabric more than only the CM or LA treatment. The pattern of cellulose I was transferred to a mixed configuration of cellulose II and cellulose III after the CM/LA or LA/CM treatment. Thermal performances measured by thermogravimetric analysis (TGA) and differential thermogravimetry (DTG) techniques showed that the thermal stability of the treated cotton only marginally decreased. The wicking height increased after the sequential CM/LA treatment, indicating that the hydrophilicity of the fabric increased. The dye absorption and color uniformity were better for the reactive dye with a smaller molecular weight (Reactive Red 2) compared with the one with a larger molecular weight (Reactive Red 195). The total dye fixation efficiency (T%) increased to 72.93% and 73.24% for Reactive Red 2 dyeings of CM/LA- and LA/CM-cotton fabric from 46.75% of the untreated fabric, respectively; the T% increased to 65.33% and 72.27% for Reactive Red 195 dyeings of CM/LA- and LA/CM-cotton fabric from 35.17% of the untreated fabric, respectively. The colorfastness and dye exhaustion and fixation percentages of the samples were enhanced after the treatments. Furthermore, compared to the single CM or LA treatment, the softness handle properties were further improved after the fabrics were sequentially treated by CM/LA. The developed pre-treatment of CM/LA can be used in the textile industry to promote the dyeability, handle, and mechanical properties of knit cotton fabrics.

Combination of Pre- and Post-Mercerization Processes for Cotton Fabric.

The dyeing process commonly deteriorates the luster of pre-mercerized cotton fabric, so post-mercerization processes are regularly applied to compensate for this. Herein, the influence of combining pre-mercerization with CS (caustic solution) or LA (liquid ammonia) and post-mercerization with CS or LA on the morphological structure, dyeing performance, tensile strength, and stiffness of woven cotton fabric was investigated. The crystallinity index values greatly decreased from 73.12 to 51.25, 58.73, 38.42, and 40.90% after the combined mercerization processes of LA-LA, CS-CS, LA-CS, and CS-LA, respectively. Additionally, the CS-LA- and LA-CS-treated samples exhibited a mixture of cellulose II and cellulose III allomorphs. The combined mercerization processing of cotton fabric resulted in slightly worse thermal stability. The LA and CS pre-mercerization processes increased the dye exhaustion, although the former decreased the dye fixation rate while the latter increased it by 4% for both dyes. The color strength of the dyed cotton fabric increased after both post-mercerization processes. Moreover, the fabric stiffness and mechanical properties showed an increasing trend due to the combined mercerization efforts.