Impact of Presarcopenic Dysphagia on 1-Year Mortality After Videofluoroscopic Swallowing Study in Patients with Cancer.

Sarcopenic dysphagia is the term for swallowing difficulty associated with loss of mass, strength, and physical performance, which leads to increased pharyngeal residues. Unlike sarcopenia, presarcopenia is characterized by low muscle mass without decreased muscle strength or physical performance and can develop into dysphagia due to low skeletal muscle mass. This retrospective study investigated the impact of presarcopenic dysphagia (PSD) on 1-year mortality in patients with cancer and dysphagia who underwent a videofluoroscopic swallowing study (VFSS). An operational definition of PSD based on presarcopenia and pharyngeal residues was adopted. The psoas muscle mass index (cm2/height [m2]), calculated by the psoas muscle area at the third lumber vertebra via abdominal computed tomography (CT) and related to height, was used to assess presarcopenia with cutoff values of 4.62 for men and 2.66 for women. Pharyngeal residues were assessed using a VFSS to evaluate dysphagia. Patients' medical charts were analyzed to investigate 1-year mortality after a VFSS. Out of 111 consecutive patients with cancer, 53 (47.7%) were defined as having PSD. In a forward-stepwise Cox proportional regression analysis, PSD (HR 2.599; 95% CI 1.158-5.834; p = 0.021) was significantly associated with 1-year mortality after a VFSS, even after adjusting for the factors of operation, Functional Oral Intake Scale (FOIS) scores at discharge, and modified Barthel Index (BI) scores at discharge. PSD, defined as CT-based presarcopenia and pharyngeal residues observed during a VFSS, is associated with increased 1-year mortality in patients with cancer and dysphagia.

Survey of patients with stroke in the Kitakyushu Area, Japan: a 12-year retrospective analysis of the critical pathway.

OBJECTIVES: We aimed to clarify the incidence, clinical profile, outcome, and activities of daily living of patients with stroke using the Kitakyushu clinical pathway database and to investigate the characteristics of patients with stroke in the Kitakyushu medical area in Japan. MATERIALS AND METHODS: The clinical data of patients with stroke registered in the Kitakyushu database between April 1, 2009 and December 31, 2021 were retrospectively examined. The National Institutes of Health Stroke Scale was used to classify stroke severity. A descriptive analysis of basic variables, including age, stroke type, length of hospital stay, and activities of daily living, according to stroke severity was conducted. RESULTS: There were 7,487 acute care hospital patients and 5,441 rehabilitation hospital patients. Compared with patients in similar cities in Japan, patients in the Kitakyushu area tended to be older at the time of stroke onset with a higher proportion of cases of hemorrhagic stroke. Length of hospital stay in both acute and rehabilitation hospitals increased with stroke severity. The Functional Independence Measure gain was highest in patients with moderate disability. CONCLUSIONS: Compared with patients in similar cities in Japan, in the Kitakyushu area, patients with stroke were older and the proportion of patients with hemorrhagic stroke was higher. Stroke rehabilitation therapy is effective for patients with moderately severe stroke.

Mizoribine Promotes Molecular Chaperone HSP60/HSP10 Complex Formation.

It has been reported that Mizoribine is an immunosuppressant used to suppress rejection in renal transplantation, nephrotic syndrome, lupus nephritis, and rheumatoid arthritis. The molecular chaperone HSP60 alone induces inflammatory cytokine IL-6 and the co-chaperone HSP10 alone inhibits IL-6 induction. HSP60 and HSP10 form a complex in the presence of ATP. We analyzed the effects of Mizoribine, which is structurally similar to ATP, on the structure and physiological functions of HSP60-HSP10 using Native/PAGE and transmission electron microscopy. At low concentrations of Mizoribine, no complex formation of HSP60-HSP10 was observed, nor was the expression of IL-6 affected. On the other hand, high concentrations of Mizoribine promoted HSP60-HSP10 complex formation and consequently suppressed IL-6 expression. Here, we propose a novel mechanism of immunosuppressive action of Mizoribine.

Preoperative decline in skeletal muscle strength of patients with cardiovascular disease affects postoperative pulmonary complication occurrence: a single-center retrospective study.

BACKGROUND: Dynapenia, defined as age-related skeletal muscle strength decline, has been reported as a poor prognostic factor in patients with cardiovascular disease. Decline in skeletal muscle strength (DS), the main symptom of dynapenia, may be an important clinical indicator in patients undergoing cardiac surgery. However, the relationship between DS and postoperative pulmonary complication occurrence is unclear. Herein, we investigated the relationship between preoperative DS and postoperative pulmonary complication occurrence in patients undergoing cardiac surgery. METHODS: We enrolled 125 patients who underwent cardiac surgery. DS was determined by low grip strength and quadriceps isometric strength. The patients were divided into DS and non-DS groups. The relationship between the clinical characteristics and preoperative physical function was compared, and factors associated with postoperative pulmonary complication occurrence were investigated using multivariate logistic regression analysis. RESULTS: There were 42 (33.6%) patients in the DS group and 83 (66.4%) patients in the non-DS group. Compared with the non-DS group, the DS group was significantly older and had a higher body mass index and Japan SCORE (operative mortality rate and major complication rate). The DS group also had a lower estimated glomerular filtration rate and preoperative Barthel index than the non-DS group. Furthermore the DS group had a significantly higher incidence of postoperative pulmonary complications and length of intensive care unit stay, and their postoperative rehabilitation was prolonged compared to the non-DS group. Multivariate logistic regression analysis revealed that DS was a determinant of postoperative pulmonary complications (odds ratio 4.26, 95% confidence interval 1.63‒11.14). CONCLUSIONS: We showed that preoperative DS was an independent risk factor for postoperative pulmonary complications in patients undergoing cardiac surgery. Skeletal muscle strength before cardiac surgery may be an important clinical indicator for predicting the prognosis of patients from post-surgery to discharge and for planning postoperative rehabilitation programs.

The Relationship Between Preoperative Frailty Risk as Assessed by the Hospital Frailty Risk Score and the Outcome at Discharge in Coronary Artery Bypass Grafting (CABG) Patients: A Retrospective Observational Study Using the Diagnosis Procedure Combination Database.

The relationship between the Hospital Frailty Risk Score (HFRS)-based frailty risk and outcomes after coronary artery bypass grafting (CABG) is yet unclear. The objective of this study was to investigate the relationship between preoperative frailty risk as assessed by the HFRS and postoperative outcomes in patients undergoing CABG. This observational study used the diagnosis procedure combination (DPC) system in Japan (2014-2017). In total, 35,015 adults aged ≥ 65 years and diagnosed with angina pectoris and acute myocardial infarction who had undergone CABG were enrolled. We investigated the association between the HFRS-based frailty risk and the home discharge rate, as well as the prevalence of complications. Multilevel logistic regression analysis revealed that having an HFRS ≥ 5 was a determinant of lower home discharge rate (odds ratio [OR] 0.60, 95% confidence interval [CI] 0.49-0.74, P <0.01), aspiration pneumonia (OR 2.25, 95%CI 1.27-3.96, P <0.01) and disuse syndrome (OR 1.90, 95%CI 1.23-2.94, P <0.01). Preoperative stratification of frailty risk using HFRS may help in predicting postoperative progress and in planning postoperative rehabilitation.

[Use of a Guidebook for Stroke Patients to Promote Health and Employment Support].

When stroke patients consider a return to work, several difficulties are encountered regarding the promotion of support for both their health and employment due to the different perspectives of the patients, their families, and medical institutions. The Japanese Ministry of Health, Labour and Welfare has published a guideline for medical institutions and offices, but not for stroke patients or their families. As a result, patients and their families do not know how to process both medical treatments and their work after the occurrence of a stroke. We have therefore published the "Guidebook for Stroke Patients to Promote Health and Employment Support" based on the concept of the previously published "Guidebook for Cancer Patients for Promoting Health and Employment Support". This publication offers an overview of stroke and the flow from stroke onset to return to work. Patients can confirm how they should be handled at different phases of stroke. A stroke affects the patient, their family, and the individuals providing support, so this publication offers an information tool to facilitate the participation of stroke patients in society. The guide can thus be expected to contribute to an increased rate of return to work by stroke patients.

Pre-operative Physical Performance Is Associated With Early Return to Work in Peri-operative Lung Cancer Patients.

Little is known about the factors related to return to work (RTW) in patients with peri-operative lung cancer (LC). This study aimed to investigate whether pre-operative physical performance is associated with early RTW in patients with peri-operative LC. A total of 59 patients who wished to resume work after lung resection surgery were included and were divided into three groups: early RTW (within 14 days after discharge), delayed RTW (within 15-90 days), and non-RTW (failure of RTW within 90 days). The early RTW group had significantly lower scores on the modified Medical Research Council dyspnea scale (mMRC) and significantly higher scores on the Euro Quality of Life 5-Dimension 3-Level (EQ-5D-3L) than the non-RTW group. Multivariate logistic regression analysis showed that EQ-5D-3L scores were significantly associated with early RTW, and mMRC scores and knee extensor strength tended to be associated with early RTW. Better pre-operative quality of life, mild dyspnea, and greater lower limb muscle strength tended to be associated with early RTW in patients with peri-operative LC.

Rosmarinic Acid and Sodium Citrate Have a Synergistic Bacteriostatic Effect against Vibrio Species by Inhibiting Iron Uptake.

BACKGROUND: New strategies are needed to combat multidrug-resistant bacteria. The restriction of iron uptake by bacteria is a promising way to inhibit their growth. We aimed to suppress the growth of Vibrio bacterial species by inhibiting their ferric ion-binding protein (FbpA) using food components. METHODS: Twenty spices were selected for the screening of FbpA inhibitors. The candidate was applied to antibacterial tests, and the mechanism was further studied. RESULTS: An active compound, rosmarinic acid (RA), was screened out. RA binds competitively and more tightly than Fe3+ to VmFbpA, the FbpA from V. metschnikovii, with apparent KD values of 8 µM vs. 17 µM. Moreover, RA can inhibit the growth of V. metschnikovii to one-third of the control at 1000 µM. Interestingly, sodium citrate (SC) enhances the growth inhibition effect of RA, although SC only does not inhibit the growth. The combination of RA/SC completely inhibits the growth of not only V. metschnikovii at 100/100 µM but also the vibriosis-causative pathogens V. vulnificus and V. parahaemolyticus, at 100/100 and 1000/100 µM, respectively. However, RA/SC does not affect the growth of Escherichia coli. CONCLUSIONS: RA/SC is a potential bacteriostatic agent against Vibrio species while causing little damage to indigenous gastrointestinal bacteria.

Insights into the mechanism of paradoxical low-flow, low-pressure gradient severe aortic stenosis: association with reduced O2 consumption by the whole body.

The mechanism of reduced stroke volume index (SVi) in paradoxical low-flow, low-pressure gradient (PLFLPG) aortic stenosis (AS) remains unclarified. Guyton et al. ( 21 ) demonstrated that SVi is determined by whole body O2 consumption (V̇o2) in many subjects, including patients with heart disease. We hypothesized that reduced SVi in PLFLPG AS is associated with reduced V̇o2 by the whole body. This study investigated the relationship between V̇o2, SVi, and AS severity in patients with AS to examine the association between reduced V̇o2 and PLFLPG AS. In 59 patients (24 men and 35 women, mean age: 78 ± 7 yr old) with severe AS, SVi, AS severity, and type were evaluated by echocardiography, and V̇o2 was measured by the fraction of O2 in expired gases. SVi and V̇o2 were significantly decreased in 20 patients with PLFLPG AS compared with 39 patients with non-PLFLPG AS (30 ± 4 vs. 41 ± 7 ml/m2 and 2.4 ± 0.5 vs. 3.0 ± 0.5 ml·min-1·kg-1, respectively, P < 0.01). The SVi-to-V̇o2 ratio was not different between the two groups (13.1 ± 2.6 vs. 13.6 ± 2.1, not significant). SVi was independently correlated with V̇o2 ( r = 0.74, P < 0.01) but not with the aortic valve area index. Categorized PLFLPG AS was also significantly associated with reduced V̇o2 ( P < 0.001). PLFLPG AS is associated with reduced V̇o2 by the whole body, which may offer insights into the mechanism of PLFLPG AS. NEW & NOTEWORTHY Paradoxical low-flow, low-pressure gradient severe aortic stenosis (PLFLPG AS) is an important and problematic subtype, and its central pathophysiology with reduced stroke volume is yet to be clarified. We hypothesized and subsequently clarified that reduced stroke volume in PLFLPG AS is associated with reduced O2 consumption by the whole body. This study suggests important insights into the mechanism of PLFLPG AS and may further promote studies to investigate further mechanisms and novel treatment.

Physicochemical Properties of the Mammalian Molecular Chaperone HSP60.

The E. coli GroEL/GroES chaperonin complex acts as a folding cage by producing a bullet-like asymmetric complex, and GroEL exists as double rings regardless of the presence of adenosine triphosphate (ATP). Its mammalian chaperonin homolog, heat shock protein, HSP60, and co-chaperonin, HSP10, play an essential role in protein folding by capturing unfolded proteins in the HSP60/HSP10 complex. However, the structural transition in ATPase-dependent reaction cycle has remained unclear. We found nucleotide-dependent association and dissociation of the HSP60/HSP10 complex using various analytical techniques under near physiological conditions. Our results showed that HSP60 exist as a significant number of double-ring complexes (football- and bullet-type complexes) and a small number of single-ring complexes in the presence of ATP and HSP10. HSP10 binds to HSP60 in the presence of ATP, which increased the HSP60 double-ring formation. After ATP is hydrolyzed to Adenosine diphosphate (ADP), HSP60 released the HSP10 and the dissociation of the double-ring to single-rings occurred. These results indicated that HSP60/HSP10 undergoes an ATP-dependent transition between the single- and double-rings in their system that is highly distinctive from the GroEL/GroES system particularly in the manner of complex formation and the roles of ATP binding and hydrolysis in the reaction cycle.

NIP-SNAP-1 and -2 mitochondrial proteins are maintained by heat shock protein 60.

NIP-SNAP-1 and -2 are ubiquitous proteins thought to be associated with maintenance of mitochondrial function, neuronal transmission, and autophagy. However, their physiological functions remain largely unknown. To elucidate their functional importance, we screened for proteins that interact with NIP-SNAP-1 and -2, resulting in identification of HSP60 and P62/SQSTM1 as binding proteins. NIP-SNAP-1 and -2 localized in the mitochondrial inner membrane space, whereas HSP60 localized in the matrix. Native gel electrophoresis and filter trap assays revealed that human HSP60 prevented aggregation of newly synthesized NIP-SNAP-2 in an in vitro translation system. Moreover, expression levels of NIP-SNAP-1 and -2 in cells were decreased by knockdown of HSP60, but not HSP10. These findings indicate that HSP60 promotes folding and maintains the stability of NIP-SNAP-1 and -2.

Comparison of the Time Course of Return to Work After Stroke Between Two Cohort Studies in Japan.

The conditions for stroke rehabilitation such as individual therapeutic procedure and medical treatment system in Japan have drastically changed over the past decade: increasing incidence of ischemic stroke, the use of intravenous recombinant tissue plasminogen activator, hospital specialization, introduction of convalescent rehabilitation wards, and public long-term care insurance. However, it is not known whether these changes have influenced the time course of return to work (RTW) after stroke. In this study we compared the time course of RTW after stroke in Japan that was reported in two cohort studies performed 20 years apart. The cumulative rate of RTW after first stroke was similar in the two studies, even though they were separated by an interval of two decades. This shows that advances in stroke rehabilitation have not impacted RTW, and we suggest that the social security system, particularly sickness benefit, has a strong influence on RTW.

ATPase activity and ATP-dependent conformational change in the co-chaperone HSP70/HSP90-organizing protein (HOP).

Co-chaperones help to maintain cellular homeostasis by modulating the activities of molecular chaperones involved in protein quality control. The HSP70/HSP90-organizing protein (HOP) is a co-chaperone that cooperates with HSP70 and HSP90 in catalysis of protein folding and maturation in the cytosol. We show here that HOP has ATP-binding activity comparable to that of HSP70/HSP90, and that HOP slowly hydrolyzes ATP. Analysis of deletion mutants revealed that the ATPase domain of HOP is in the N-terminal TPR1-DP1-TPR2A segment. In addition, HOP changes its conformation in the presence of ATP. These results indicate that HOP is a unique co-chaperone that undergoes an ATP-dependent conformational change.

Functional structure and physiological functions of mammalian wild-type HSP60.

The Chaperonins comprise a family of molecular chaperones having a double-ring structure and similar sequence homology. These proteins play an essential role in biological reactions that mediate the folding of newly synthesized polypeptides and partially denatured proteins. In the prokaryotic group I chaperonins, structural and reaction cycle analyses of GroEL and its co-chaperone GroES have been performed in detail. While in eukaryotes, there have been limited reports analyzing the group I chaperonin HSP60 and its co-chaperone HSP10. In the present study, we purified the wild type HSP60 from porcine liver and investigated the interaction between HSP60 and HSP10, including conformation and physiological relationships. Based on the results of transmission electron microscopy, native PAGE, and gel filtration column chromatography, the wild type HSP60 displayed a heptameric single-ring structure in the absence of ATP. In contrast, HSP60 formed mainly a "football-type" complex with HSP10 in the presence of ATP and mediated the refolding of denatured substrate protein. The functional conformation cycle of the purified mammalian HSP60 is distinct from the cycle of the prokaryotic GroEL/GroES chaperonin.

Aminoglycosides suppress the protein folding activity of the molecular chaperone HSC70: implication of a structure-activity relationship.

BACKGROUND: The mechanism of aminoglycoside (AG)-induced nephrotoxicity has not been fully elucidated. We previously reported that gentamicin suppresses the functions of HSC70, a heat shock cognate protein, which is a cytosolic molecular chaperone in mammalian cells. However, the relationship between HSC70 dysfunction and nephrotoxicity has not been clarified yet. METHODS: The effects of 11 AGs, including gentamicin, on the function, protein refolding and substrate binding of HSC70 were examined in vitro. The protein refolding activity of HSC70 was examined by enzymatic activity restoration of the denatured firefly luciferase. Substrate binding activity of HSC70 was examined by the binding of fluorescent-dye-labeled reduced carboxymethlylated α-lactalbumin determined by HSC70/substrate complex formation by native polyacrylamide gel electrophoresis. RESULTS: Strong inhibitory activity of AGs on the protein refolding activity of HSC70 was observed for arbekacin, gentamicin, neomycin and sisomicin, but not spectinomycin, streptomycin, ribostamycin and paromomycin, and inhibition was weak for tobramycin, amikacin and kanamycin. Neomycin strongly suppressed the formation of the HSC70/substrate complex and had the lowest concentration that resulted in 50% inhibition (IC50; in the order of 10(-4) M). Arbekacin, gentamicin, sisomicin and tobramycin had IC50 values in the order of 10(-3) M. Amikacin, ribostamycin and paromomycin had no effective potencies. CONCLUSIONS: The inhibition efficacies of AGs for protein refolding of HSC70 were as follows: neomycin >> gentamicin, arbekacin, and sisomicin > tobramycin, amikacin and kanamycin > spectinomycin, streptomycin, ribostamycin and paromomycin. Amino groups and/or hydroxyl groups located at 2', 3', 4', and 6' of amino sugar residue II, as well as the number of amino sugar rings were closely associated with the inhibition efficacies of AGs. Inhibition efficacies of AGs towards the function of HSC70 correlated well with the nephrotoxicity of AGs deduced from the in vivo studies previously reported. Amino groups/hydroxyl groups and the number of amino sugar rings of AGs were shown to be important for inhibition efficacies. The results strongly suggest that the AG-induced dysfunction of HSC70 is involved in the nephrotoxicity.

Low phase angle is associated with swallowing outcomes in patients with acute stroke.

BACKGROUND AND PURPOSE: Stroke-induced dysphagia is a critical complication that can lead to severe secondary worldwide health issues. This retrospective cohort study investigated the association between phase angle (PhA) and swallowing function in the acute phase of stroke. We aim to establish whether nutritional and muscular interventions associated with PhA could enhance swallowing recovery and reduce related risks post-stroke. METHODS: Stroke patients requiring rehabilitation were assessed for the association between low PhA-with cutoff values for low PhA defined as less than 5.28 for men and 4.32 for women-and swallowing function using a functional oral intake scale (FOIS) on Day 7 after admission and after the completion of acute stroke treatment. RESULTS: In this study of 140 acute stroke patients (median age 74[69-81], and 85 men and 55 women), 76 patients with low PhA significantly exhibited older age, lower body mass index, more decline in skeletal muscle mass index, and lower premorbid modified Rankin Scale scores compared to 64 patients with high PhA. Multivariate linear regression revealed that low PhA was independently associated with FOIS scores on Day 7 after admission (ß=-0.143 and p=0.036) and after the completion of acute stroke treatment (ß=-0.513 and p=0.024), even when adjusting for confounding factors. CONCLUSIONS: Low PhA is associated with swallowing function in patients with acute stroke. Nutritional and physical interventions improving PhA may lead to a reduction of the risk associated with stroke sequelae.

Correlation between phase angle and the number of medications in older inpatients: A cross-sectional study.

Background: Muscle weakness in older adults elevates mortality risk and impairs quality of life, with the phase angle (PhA) indicating cellular health. Polypharmacy, common in geriatric care, could influence PhA. This investigates whether the number of medications and polypharmacy with PhA as a biomarker of muscle quality in older inpatients aged ≧ 65 and determines the extent to which multiple medications contribute to the risk of reduced muscle quality. Methods: This retrospective cross-sectional study analyzed data from older inpatients requiring rehabilitation. PhA was measured using bioelectrical impedance analysis. The number of medications taken by each patient was recorded at admission. Polypharmacy was defined as the concurrent use of five or more medications at admission. Results: In this study of 517 hospitalized older adults (median age: 75 years; 47.4% men), 178 patients (34.4%) were diagnosed with sarcopenia. Polypharmacy was present in 66% of patients. The median PhA was 4.9° in men and 4.3° in women. Multivariate linear regression analysis was performed separately for men and women. In men, PhA was negatively correlated with the number of medications (ß = -0.104, p=0.041) and polypharmacy (ß = -0.045, p=0.383). In women, PhA was negatively correlated with the number of medications (ß = -0.119, p=0.026) and polypharmacy (ß = -0.098, p=0.063). Analyses were adjusted for age, BMI, sarcopenia, CRP, and hemoglobin levels. Conclusions: The number of medications at admission negatively impacted PhA in older inpatients, highlighting the importance of reviewing prescribed drugs and their interactions.

Probable Respiratory Sarcopenia Decreases Activities of Daily Living in Older Patients Hospitalized with Respiratory Diseases: A Cross-sectional Study.

Objectives: Respiratory sarcopenia is characterized by low respiratory muscle mass and respiratory muscle strength, but its impact on activities of daily living (ADL) remains unknown. We aimed to investigate the association between respiratory sarcopenia and decreased ADL. Methods: This retrospective cross-sectional study included older inpatients (≥65 years old) with respiratory diseases who underwent rehabilitation. Because the evaluation of respiratory muscle mass is challenging, probable respiratory sarcopenia was defined according to low appendicular skeletal muscle index (<7 kg/m2 for men, <5.7 kg/m2 for women) and peak expiratory flow rate (<4.4 L/s for men, <3.21 L/s for women). ADL was assessed on the first day of rehabilitation using the baseline Barthel Index (BI). Results: Of 111 inpatients (median age 75 years; 57 women), 13 (11.7%) had probable respiratory sarcopenia. Forty-five patients (40.5%) had sarcopenia and 12 of these had probable respiratory sarcopenia. Pulmonary functions (Forced Vital Capacity and expiratory volume in 1 s) were significantly lower in patients with probable respiratory sarcopenia than those without. Spearman's rank coefficient analysis showed probable respiratory sarcopenia did not significantly correlate with age, phase angle, Charlson Comorbidity Index (CCI), or hemoglobin (Hb). Multivariate linear regression analysis with baseline BI revealed probable respiratory sarcopenia (ß -0.279 and P=0.004) was the significant factor after adjusting for age, sex, body mass index, chronic obstructive pulmonary disease, CCI, and Hb. Conclusions: Probable respiratory sarcopenia was independently associated with decreased ADL in patients aged 65 years and older who were hospitalized with respiratory diseases.

Molecular mechanism of Fe3+ binding inhibition to Vibrio metschnikovii ferric ion-binding protein, FbpA, by rosmarinic acid and its hydrolysate, danshensu.

Global warming has increased the growth of pathogenic Vibrio bacteria, which can cause foodborne illnesses and death. Vibrio bacteria require iron for growth and survival. They utilize a ferric ion-binding protein (FbpA) to bind and transport Fe3+ into the cell. FbpA from Vibrio metschnikovii (Vm) is a potential target for inhibiting its growth. Rosmarinic acid (RA) can block the binding of VmFbpA to Fe3+ ; however, the molecular mechanism of Fe3+ binding and RA inhibition to VmFbpA is unclear. In this study, we used x-ray crystallography to determine the Fe3+ -binding mode of VmFbpA and the mechanism of RA inhibition. The structures revealed that in the Fe3+ bound form, Fe3+ was coordinated to VmFbpA by two Tyr residues, two HCO3 - ions, and two water molecules in a six-coordinated geometry. In contrast, in the inhibitor bound form, RA was initially bound to VmFbpA following gel filtration purification, but it was hydrolyzed to danshensu (DSS), which occupied the binding site as shown in an electron density map and reverse phase chromatography (RPC) analysis. Both RA and DSS exhibited a stronger binding affinity to VmFbpA, higher Fe3+ reduction capacity, and more potent bacteriostatic effect on V. metschnikovii compared with caffeic acid (CA), another hydrolysis product of RA. These results provide insight into the mechanism of iron acquisition by V. metschnikovii and inhibition by RA and DSS. Our findings offer clues on the development of effective strategies to prevent Vibrio infections.

Refolding, Crystallization, and Crystal Structure Analysis of a Scavenger Receptor Cysteine-Rich Domain of Human Salivary Agglutinin Expressed in Escherichia coli.

Scavenger receptors are a protein superfamily that typically consists of one or more repeats of the scavenger receptor cysteine-rich structural domain (SRCRD), which is an ancient and highly conserved protein module. The expression and purification of eukaryotic proteins containing multiple disulfide bonds has always been challenging. The expression systems that are commonly used to express SRCRD proteins mainly consist of eukaryotic protein expression systems. Herein, we established a high-level expression strategy of a Type B SRCRD unit from human salivary agglutinin using the Escherichia coli expression system, followed by a refolding and purification process. The untagged recombinant SRCRD was expressed in E. coli using the pET-32a vector, which was followed by a refolding process using the GSH/GSSG redox system. The SRCRD expressed in E. coli SHuffle T7 showed better solubility after refolding than that expressed in E. coli BL21(DE3), suggesting the importance of the disulfide bond content prior to refolding. The quality of the refolded protein was finally assessed using crystallization and crystal structure analysis. As proteins refolded from inclusion bodies exhibit a high crystal quality and reproducibility, this method is considered a reliable strategy for SRCRD protein expression and purification. To further confirm the structural integrity of the refolded SRCRD protein, the purified protein was subjected to crystallization using sitting-drop vapor diffusion method. The obtained crystals of SRCRD diffracted X-rays to a resolution of 1.47 Å. The solved crystal structure appeared to be highly conserved, with four disulfide bonds appropriately formed. The surface charge distribution of homologous SRCRD proteins indicates that the negatively charged region at the surface is associated with their calcium-dependent ligand recognition. These results suggest that a high-quality SRCRD protein expressed by E. coli SHuffle T7 can be successfully folded and purified, providing new options for the expression of members of the scavenger receptor superfamily.