Phage-driven coevolution reveals trade-off between antibiotic and phage resistance in Salmonella anatum.

Phage therapy faces challenges against multidrug-resistant (MDR) Salmonella due to rapid phage-resistant mutant emergence. Understanding the intricate interplay between antibiotics and phages is essential for shaping Salmonella evolution and advancing phage therapy. In this study, MDR Salmonella anatum (S. anatum) 2089b coevolved with phage JNwz02 for 30 passages (60 days), then the effect of coevolution on the trade-off between phage and antibiotic resistance in bacteria was investigated. Our results demonstrated antagonistic coevolution between bacteria and phages, transitioning from arms race dynamics (ARD) to fluctuating selection dynamics (FSD). The fitness cost of phage resistance, manifested as reduced competitiveness, was observed. Bacteria evolved phage resistance while simultaneously regaining sensitivity to amoxicillin, ampicillin, and gentamicin, influenced by phage selection pressure and bacterial competitiveness. Moreover, the impact of phage selection pressure on the trade-off between antibiotic and phage resistance was more pronounced in the ARD stage than in the FSD stage. Whole genome analysis revealed mutations in the btuB gene in evolved S. anatum strains, with a notably higher mutation frequency in the ARD stage compared to the FSD stage. Subsequent knockout experiments confirmed BtuB as a receptor for phage JNwz02, and the deletion of btuB resulted in reduced bacterial competitiveness. Additionally, the mutations identified in the phage-resistant strains were linked to multiple single nucleotide polymorphisms (SNPs) associated with membrane components. This correlation implies a potential role of these SNPs in reinstating antibiotic susceptibility. These findings significantly advance our understanding of phage-host interactions and the impact of bacterial adaptations on antibiotic resistance.

Paramyosin from field snail (Bellamya quadrata): Structural characteristics and its contribution to enhanced the gel properties of myofibrillar protein.

To assess the blending effect of field snails with grass carp muscle, the effects of paramyosin (PM) and actomyosin (AM) with different mixture ratios on the gel properties of the binary blend system were investigated in our work. The purified PM from field snail muscle was about 95 kDa on SDS-PAGE. Its main secondary structure was α-helix, which reached to 97.97 %. When the amount of PM increased in the binary blend system, their rheological indices and gel strength were improved. The water holding capacity (WHC) increased to 86.30 % at a mixture ratio of 2:8. However, the WHC and the area of immobile water (P22) dramatically decreased, and the area of free water (P23) increased when the mixture ratio exceeded 4:6. The low level of PM in binary blend system promoted the formation of a homogenous and dense gel network through non-covalent interactions as observed results of SEM and FTIR. When there were redundant PM molecules, the development of heterostructure via hydrophobic interaction of tail-tail contributed to the reduced gel properties of the binary blend system. These findings provided new insight into the binary blend system of PM and AM with different ratios to change the gel properties of myofibrillar protein.

Characterization of a broad-spectrum endolysin rLysJNwz and its utility against Salmonella in foods.

Salmonella is a common foodborne pathogen worldwide. The use of bacteriophage-encoded endolysins as antimicrobial agents is a promising approach for controlling pathogenic contamination. In this context, a recombinant endolysin named rLysJNwz, consisting of a single domain falling with the L-alanogyl-D-glutamate peptidase-like family, was cloned, expressed, and characterized. The yield of rLysJNwz was about 25 mg/L. Synergy between 7.5 µg/mL rLysJNwz and 0.5 mmol/L EDTA could decrease the viable counts of Salmonella NCTC 8271 by 93.28%. A synergistic effect between rLysJNwz and polymyxin B was demonstrated, exhibiting the MIC of polymyxin B decreased by twofold. Specifically, rlysJNwz had strong thermostability at temperatures (4-95 °C) and maintained high activity at pHs from 5.0 to 11.0. rlysJNwz was a metal ion-dependent peptidase, which activated by divalent metal ions such as Zn2+, Mn2+, or Ca2+. Moreover, it was also found that the synergism of rlysJNwz and EDTA had bactericidal activities against a broad range of Gram-negative bacteria, including several multidrug-resistant bacteria. The application of rLysJNwz combined with EDTA was evaluated on contaminated eggs and lettuce for 60 min, displaying more than 86.7% and 86.5% reduction of viable Salmonella, respectively. Hence, these results suggest that rLysJNwz is a potential antibacterial agent to control Salmonella, especially antibiotic-resistant pathogen contamination in the field of food safety. KEY POINTS: • rLysJNwz shows lytic activities against a broad range of Gram-negative bacteria. • Endolysin rLysJNwz is a stable metalloenzyme and has high thermostability. • rLysJNwz and 0.5 mmol/L EDTA synergistically inactivate Salmonella on eggs and lettuce.

Effects of partial substitution of NaCl on myofibrillar protein properties from pearl mussel Hyriopsis cumingii muscle: Structural characteristics and aggregation behaviors.

The effects of NaCl and its partial substitutes (KCl, MgCl2 and CaCl2) on solubility, structural characteristics and aggregation behaviors of myofibrillar protein (MP) from pearl mussel muscle were investigated and compared. MP at 0.6 M NaCl was beneficial to protein unfolding and showed excellent potential functional properties. When NaCl was substituted in low level, MPs also showed good solubility and ordered microstructure as well as NaCl, especially MgCl2 and CaCl2, due to the unfolding of α-helical structures and subsequently exposed tyrosine residues and hydrophobic groups. However, the obviously increased disulfide bonds and hydrophobic interactions in high substitution level indicated the excessive non-sodium salts had negative effects on molecular rearrangement, leading to irregular and overly tight of microstructure. Thus, NaCl partially substituted by KCl, MgCl2 and CaCl2 in low substitution level is promising to improve functional properties of MP in low-sodium meat products.

Femoral artery cannulation as a safe alternative for aortic dissection arch repair in the era of axillary artery cannulation.

BACKGROUND: To evaluate the safety and efficacy of femoral artery cannulation as an alternative to axillary artery cannulation, we retrospectively compared outcomes between patients with axillary or femoral artery cannulation during open aortic arch repair for type A aortic dissection (TAAD). METHODS: Between January 2014 and January 2019, 646 patients underwent open aortic arch repair with circulatory arrest for TAAD using antegrade selective cerebral perfusion (SACP) and were divided into two groups according to the site of arterial cannulation: an axillary artery group (axillary group, n=558) or a femoral artery group (femoral group, n=88). The axillary artery was considered as the primary cannulation site, and the femoral artery was used as an alternative when axillary artery cannulation was deemed unsuitable or had failed. Propensity score matching was performed to correct baseline differences. RESULTS: After propensity score matching, the patients' characteristics were comparable between groups (n=85 in each). The incidence of in-hospital mortality (10.6% vs. 14.1%; P=0.642) and stroke (3.5% vs. 5.9%; P=0.720) were comparable between the axillary and femoral groups. The incidence of newly required dialysis was lower in the femoral group, but the difference was not statistically significant (34.1% vs. 20.0%; P=0.050). Other outcomes and major adverse events were comparable. CONCLUSIONS: Femoral artery cannulation produced similar perioperative outcomes to axillary cannulation after open arch repair for TAAD. The femoral artery can be used as a safe and effective alternative to the axillary artery for arterial cannulation in TAAD patients undergoing open arch repair.

Tissue inhibitor of metalloproteinase-2 (TIMP-2) from red seabream (Pagrus major): Molecular cloning and biochemical characterization of highly expressed recombinant protein.

The tissue inhibitor of metalloproteinase-2 (TIMP-2) is originally characterized as an endogenous inhibitor of matrix metalloproteinases (MMPs) to response collagenolysis associated with immune challenge. In this study, the cDNA encoding TIMP-2a gene from red seabream (Pagrus major) muscle was cloned. It was 585 bp encoding a putative protein of 194 amino acids, which comprised all recognized functional domains and showed the high identity to TIMP-2as from other teleost fishes, revealing it belongs to TIMP-2a family. Soluble rTIMP-2a was efficiently expressed using a new constructed pPIC9K-rTIMP-2a vector with high inhibitory activity against to MMP-2 and MMP-9. The recombinant TIMP-2a tagged with 6 histidine residues showed the molecular mass of 23 kDa and isoelectric point of 6.50. Furthermore, the 6 disulfide bonds formed by 12 conserved cysteine residues were identified as functional motifs for its structural stability. In addition, rTIMP-2a possessed the high inhibitory activity against gelatinolytic hydrolysis and degradation of type I collagen which induced by endogenous MMPs in muscle. The results revealed the properties and inhibitory function of rTIMP-2a, which may be a pivotal role in regulation gelatinolytic MMPs metabolization during defense mechanism.

Production, optimisation and characterisation of angiotensin converting enzyme inhibitory peptides from sea cucumber (Stichopus japonicus) gonad.

In this study, production of bioactive peptides with angiotensin converting enzyme (ACE) inhibitory activity from sea cucumber (Stichopus japonicus) gonad using commercial protamex was optimised by response surface methodology (RSM). As a result, the optimal condition to achieve the highest ACE inhibitory activity in sea cucumber gonad hydrolysate (SCGH) was hydrolysis for 1.95 h and E/S of 0.75%. For further characterisation, three individual peptides (EIYR, LF and NAPHMR) were purified and identified. The peptide NAPHMR showed the highest ACE inhibitory activity with IC50 of 260.22 ± 3.71 µM. NAPHMR was stable against simulated gastrointestinal digestion and revealed no significant cytotoxicity toward Caco-2 cells. Molecular docking study suggested that Arg, His and Asn residues in NAPHMR interact with the S2 pocket or Zn2+ binding motifs of ACE via hydrogen or π-bonds, potentially contributing to ACE inhibitory effect. Sea cucumber gonad is thus a potential resource to produce ACE inhibitory peptides for preparation of functional foods.

Mast cell activation is involved in stress-induced epithelial barrier dysfunction in the esophagus.

OBJECTIVE: We aimed to investigate the role of mast cell in stress-induced barrier dysfunction in the esophagus and its possible pathway involved using mast cell-deficient (Ws/Ws) rats. METHODS: Ws/Ws rats and normal (+/+) rats were submitted to chronic restraint stress (CRS) 2 h/day for 7 days. Tissues were obtained from distal esophagus. Mast cells were counted under Alcian blue-safranin O stain. Activation of mast cells was assessed using transmission electron microscope. Esophageal epithelial barrier dysfunction was evaluated by measuring intercellular spaces (ICS) and by quantifying tight junction (TJ) proteins. The localization and expression of mast cell-derived tryptase and proteinase activated receptor 2 (PAR-2) were assessed. RESULTS: A higher number of mast cells and higher proportion of activated mast cells were observed in CRS +/+ rats compared with non-stress controls. Increased ICS and decreased expression of some TJ proteins were observed in the CRS +/+ rats but not in the CRS Ws/Ws rats. Tryptase and its receptor PAR-2 were found elevated concomitantly by nearly 100% in CRS +/+ rats, but not in CRS Ws/Ws rats. CONCLUSIONS: Mast cells play an important role in stress-induced epithelial barrier dysfunction in esophagus. The mechanism may involve the activation of PAR-2 by mast cell-derived tryptase, causing proinflammatory responses and the subsequent disruption of the epithelial TJ proteins and a disturbed cytoskeleton function, resulting in dilated intercellular spaces.

Mechanism study of high browning degree of mantle muscle meat from Japanese common squid Todarodes pacificus during air-drying.

Mantle meat from the Japanese common squid (Todarodes pacificus) browns more than other squid meats during air-drying. The factors contributing to the browning of Japanese common squid, long-finned squid (Photololigo edulis) and bigfin reef squid (Sepioteuthis lessoniana) were studied in boiled and raw meat both before and after air-drying. Dried raw meat from the Japanese common squid browned more than dried boiled meat (b(∗) value, from 4.7 to 28.5). The results from SDS-PAGE showed significant degradation of myosin heavy chain (MHC) suggesting that protease activity in raw Japanese common squid meat was higher than in the other two species. The concentration of arginine (1932.0mg/100g) and ribose (28.8µmol/g) in Japanese common squid meat was higher than in the other two species. These results suggest that high protease activity and high concentrations of arginine and ribose increase the browning discoloration of Japanese common squid during air-drying.

[Reconstitution of mast cell population in gastrointestinal tract by bone marrow transplantation in mast cell deficient rats].

OBJECTIVE: To explore whether bone marrow transplantation (BMT) can restore gastrointestinal mast cells in mast cell deficient (Ws/Ws) rats and understand the features of these reconstituted mast cells. METHODS: Thirty-six Ws/Ws rats were subjected to Co(60) radiation at 6 gradient doses (6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 Gy, n = 6) to confirm the appropriate dosage. And another 6 rats served as non-radiated controls. Sixteen Ws/Ws rats were exposed to a 7.5 Gy radiation and sacrificed at Day 1, 5, 8 and 12 (n = 4) to obtain hemogram and myelogram. And 4 non-radiated Ws/Ws rats served as control. Ws/Ws rats received an intravenous injection of bone marrow cells harvested from healthy congenic Brown Norway (BN) rats after a dosage of 7.5 Gy radiation. Y-chromosome fluorescence in situ hybridization (Y-FISH) was used to identify the survival and differentiation of bone marrow cells of male BN rats in female BMT-Ws/Ws rats (n = 4). Another set of 24 male BMT-Ws/Ws rats were sacrificed Weeks 5, 8, 13 and 23 post-BMT (n = 6). Tissues from esophagus, stomach, ileum and colon were harvested to perform mast cell quantification by Alcian blue staining. And mast cell derived proteinases (tryptase and chymase) were quantified by Western blotting. RESULTS: Y-FISH showed bone marrow cells from male BN rats survived in female BMT-Ws/Ws rats and mast cells were restored in gastrointestinal tract. Compared with control, the highest level of mast cell number (103 ± 6) vs (35 ± 4)/mm(2) for esophagus, (271 ± 23) vs (124 ± 13)/mm(2) for stomach, (200.1 ± 13.3) vs (103.2 ± 6.6)/mm(2), all P < 0.05) and mast cell proteinases (1.3 ± 0.3 vs 0.6 ± 0.2 for esophagus, 3.6 ± 0.8 vs 1.9 ± 0.4 for ileum, both P < 0.05) in BMT-Ws/Ws rats were observed at Week 8 post-BMT. The number of mast cell and proteinases decreased at Week 13 post-BMT. CONCLUSIONS: BMT may restore mast cells in Ws/Ws rats. The best time period for using BMT-Ws/Ws rats in the study of mast cell function is between Weeks 8-13 post-BMT.

Essential role of mast cells in the visceral hyperalgesia induced by T. spiralis infection and stress in rats.

Mast cells (MCs) deficient rats (Ws/Ws) were used to investigate the roles of MCs in visceral hyperalgesia. Ws/Ws and wild control (+/+) rats were exposed to T. spiralis or submitted to acute cold restraint stress (ACRS). Levels of proteinase-activated receptor 2 (PAR2) and nerve growth factor (NGF) were determined by immunoblots and RT-PCR analysis, and the putative signal pathways including phosphorylated extracellular-regulated kinase (pERK1/2) and transient receptor potential vanilloid receptor 1 (TRPV1) were further identified. Visceral hyperalgesia triggered by ACRS was observed only in +/+ rats. The increased expression of PAR2 and NGF was observed only in +/+ rats induced by T. spiralis and ACRS. The activation of pERK1/2 induced by ACRS occurred only in +/+ rats. However, a significant increase of TRPV1 induced by T. spiralis and ACRS was observed only in +/+ rats. The activation of PAR2 and NGF via both TRPV1 and pERK1/2 signal pathway is dependent on MCs in ACRS-induced visceral hyperalgesia rats.

2,2'-[4-Acetyl-1,3-phenyl-enebis(-oxy)]diacetic acid.

In the title compound, C12H12O7, the dihedral angles between the benzene ring and the mean planes of the 3-carb-oxy-meth-oxy, 1-carb-oxy-meth-oxy and acetyl substituents are 8.67 (7), 7.81 (6) and 10.3 (18)°, respectively. In the crystal, mol-ecules are linked by typical carb-oxy-lic acid O-H⋯O hydrogen bonds, forming a zigzag chain. C-H⋯O inter-actions also occur.

[Phenotype and genotype analysis of hemoglobin E].

OBJECTIVE: To analyze the genotype and phenotype correlation in the hemoglobin E (HbE) carriers, and to investigate the effect of HbE on hematological parameters. METHODS: The capillary electrophoresis was used to screen total 14 141 samples and blood cell analysis was further processed to the HbE carrying samples. Gap-PCR and reverse dot blot hybridization method were used for the detection of Chinese common mutation of α and ß thalassemia. RESULTS: There is a statistical difference in hematological phenotype index (HGB, MCV, MCH, HbE, HbA(2)) between samples of HbE heterozygous (53 samples), HbE homozygous (2 samples), HbE composite α thalassemia (α-thal, 7 samples) and HbE composite ß thalassemia (ß-thal, 8 samples). Among the four groups, HbE heterozygous \[HGB (122.7 ± 19.99) g/L, MCV (78.65 ± 5.03) fl\] and HbE composite α-thal \[HGB (113.6 ± 22.68) g/L, MCV (73.50 ± 7.73) fl\] had slight effect on hematological parameters, but HbE composite ß-thal \[HGB (76.4 ± 12.30) g/L\], MCV (59.23 ± 5.28) fl\] had the heaviest effect on hematological parameters. CONCLUSION: Co-existence of HbE heterozygous and other type thalassemias showed variation in their hematological phenotype, so patients should be informed of genetics in prenatal diagnosis.

Vesicular glutamate transporter-3 contributes to visceral hyperalgesia induced by Trichinella spiralis infection in rats.

BACKGROUND: According to a recent study, vesicular glutamate transporter-3 (VGLUT3) contributes to injury-induced mechanical hyperalgesia in mice. AIMS: The aims of the study were to investigate whether VGLUT3 is involved in visceral pain, and whether transient intestinal infection or acute cold restraint stress (ACRS) affects VGLUT3 expression levels in rats. METHODS: Changes in VGLUT3 and c-Fos proteins were evaluated in rats which received noxious colorectal distension (CRD) stimulation. Transient intestinal infection was effected by oral administration of Trichinella spiralis (T. spiralis) larvae in Brown Norway rats. On the 100th day post-infection (PI), half of the PI-rats and non infected controls were subjected to an ACRS procedure. The visceromotor response to CRD was measured using the abdominal withdrawal reflex (AWR) score. Immunofluorescence and western blot analysis were used to estimate the expression of VGLUT3 in both peripheral and central neurons. RESULTS: Noxious stimulation induced a significant increase in the expression of VGLUT3 in the L6S1 spinal dorsal horn. Compared with the control group, the pain threshold was significantly decreased in the ACRS, PI, and PI + ACRS groups. VGLUT3 expression in the L6S1 dorsal root ganglion (DRG) and spinal neurons were significantly increased in PI and PI + ACRS groups as compared with the control group. CONCLUSIONS: VGLUT3 is involved in conduction of visceral pain sensation and in visceral hyperalgesia induced by Trichinella spiralis infection in rats.

(E)-1-(2,4-Dihy-droxy-phen-yl)-3-(4-hydroxy-phen-yl)prop-2-en-1-one monohydrate.

In the title compound, C(15)H(12)O(4)·H(2)O, the two benzene rings are not coplanar, making a dihedral angle of 7.24 (16)°. An intra-molecular hy-droxy-carbonyl O-H⋯O hydrogen bond occurs. In the crystal, four inter-molecular O-H⋯O hydrogen bonds involving the hy-droxy residues, the carbonyl group and the water mol-ecule lead to the formation of a three-dimensional network. The supra-molecular structure is further stabilized by weak C-H⋯O inter-actions.