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.

Role of ERK signaling protein in morphine preconditioning reducing global ischemia-reperfusion injury in isolated rat hearts / 中国药理学通报

Aim To observe the role of ERK signaling protein in morphine preconditioning reducing global ischemia-reperfusion injury in isolated rat hearts.Methods Adult male Sprague-Dawley rats were distributed into six groups (n =10 for each) using a random number table:control group (CON),ischemia-reperfusion group (I/R),ischemia preconditioning group (IPC),morphine preconditioning group at the concentration of 1 μmol · L-1 (MPC),ERK inhibitor PD98059 + MPC (MPD),and group of ERK inhibitor-PD98059 (PD).The isolated rat hearts were treated on a Langendorff perfusion apparatus system.The coronary effluent was collected at 15 min of equilibration (baseline),5 and 10 min of reperfusion for detection of the activity of LDH.Meanwhile,a water-filled balloon was inserted into the left ventricular for continuous LVDP measurement.The IS and AAR and IS/AAR ratios were observed by TTC.Western blot was used to examine the level of phosphorylated ERK in myocardium.Results As compared with the I/R group,MPC significantly decreased IS and IS/AAR ratio as well as LDH activities at 5 min and 10 min of reperfusion,but improved the LVDP at the end of reperfusion.Moreover,the phosphorylation level of ERK in myocardium was up-regulated by MPC.However,ERK inhibitor PD98059 could block the protective effects of MPC,as indicated by the increased IS and IS/AAR ratio,elevated LDH activity at the reperfusion of 5 and 10 min,and the suppressed LVDP at the end of reperfusion.Furthermore,the MPC-induced phosphorylation of ERK was also reversed by PD98059.Conclusion Morphine preconditioning may confer cardio-protection against the global ischemia-reperfusion injury in rat hearts through enhancing the phosphorylation of ERK.