Design and Escherichia coli Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD).

Refolding multi-disulfide bonded proteins expressed in E. coli into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and versatility, the E. coli expression of viral envelope proteins, such as the RBD (Receptor-Binding Domain) of the influenza Hemagglutinin protein, could significantly advance research on viral infections. Here, we show that H1N1-PR8-RBD (27 kDa, containing four cysteines forming two disulfide bonds) expressed in E. coli and was purified with nickel affinity chromatography, and reversed-phase HPLC was successfully refolded into its native structure, as assessed with several biophysical and biochemical techniques. Analytical ultracentrifugation indicated that H1N1-PR8-RBD was monomeric with a hydrodynamic radius of 2.5 nm. Thermal denaturation, monitored with DSC and CD at a wavelength of 222 nm, was cooperative with a midpoint temperature around 55 °C, strongly indicating a natively folded protein. In addition, the 15N-HSQC NMR spectrum exhibited several 1H-15N resonances indicative of a beta-sheeted protein. Our results indicate that a significant amount (40 mg/L) of pure and native H1N1-PR8-RBD can be produced using an E. coli expression system with our refolding procedure, offering potential insights into the molecular characterization of influenza virus infection.

Effect of high disease activity on spinal sagittal malalignment in patients with rheumatoid arthritis.

OBJECTIVE: Atlantoaxial subluxation is a well-known serious complication encountered in patients with rheumatoid arthritis (RA). However, it is unknown whether RA affects global spinal alignment. The aim of this study was to investigate whether high disease activity in patients with RA exacerbates spinal sagittal malalignment. METHODS: The authors included 197 patients with RA who were followed up for > 2 years; standing spinal radiographs were obtained in all patients. Patients were divided into persistent moderate disease activity/high disease activity (pMDA/HDA; n = 64) and non-pMDA/HDA (n = 133) groups based on the disease activity at follow-up visits. Radiographic parameters assessed included pelvic incidence, pelvic tilt (PT), lumbar lordosis (LL), thoracic kyphosis (TK), and C7 sagittal vertical axis (SVA). RESULTS: Over an average 5-year follow-up, increases in SVA, PT, and TK and a decrease in LL were observed. The pMDA/HDA group had a larger increase in PT and a higher incidence of vertebral fractures than the non-pMDA/HDA group. After adjusting variables using propensity score matching, the authors still found a higher rate of increase in PT (0.79°/year vs 0.01°/year, p = 0.001) in the pMDA/HDA group than in the non-pMDA/HDA group. This trend remained consistent even when patients who developed vertebral fractures were excluded. CONCLUSIONS: Spinal sagittal alignment deteriorates over time in patients with RA. High disease activity in RA exacerbates spinal deformity.

Investigational regenerative medicine for non-traumatic osteonecrosis of the femoral head: a survey of registered clinical trials.

INTRODUCTION: Osteonecrosis of the femoral head (ONFH) is a refractory disease requiring joint replacement in young patients. Regenerative therapies have been developed. AREAS COVERED: This study surveyed clinical trials on regenerative medicine for ONFH. We extracted clinical trials on non-traumatic ONFH from the websites of five publicly available major registries (EuropeanUnion Clinical Trials Register ([EU-CTR],ClinicalTrials.gov, Chinese ClinicalTrial Registry [ChiCTR], University Hospital Medical InformationNetwork - Clinical Trial Registry [UMIN-CTR] and Australian New Zealand Clinical Trials Registry [ANZCTR]).The trials were classified into six categories based on purpose: surgical treatment, non-drug conservative treatment, conservative drug treatment, therapeutic strategy, diagnosis and pathogenesis, and regenerative therapy.) We extracted 169 clinical trials on ONFH. Of these, 37 were on regenerative medicine, including 29 on cell therapy. Surgical treatment was the most common treatment, followed by regenerative therapy.There were 9 clinical trials registered in the EU-CTR, with 5 on regenerative medicine; 79 trials registered on ClinicalTrials.gov, with 24 on regenerativemedicine; 54 trials registered in the ChiCTR, with 6 on regenerative medicine. EXPERT OPINION: The focus of the joint-preserving surgery has shifted to regenerative therapy based on using cell therapy in early-stage ONFH. The global standardisation of regenerative therapy is still ongoing.

What are the differences in the recovery of physical function and clinical score between patients with steroid-related osteonecrosis of the femoral head and hip osteoarthritis undergoing total hip arthroplasty? A propensity score-matched study.

PURPOSE: Steroid-related osteonecrosis of the femoral head (ONFH), arising from steroid administration for underlying diseases, represents a unique pathology for total hip arthroplasty (THA) and typically affects a younger demographic compared to osteoarthritis (OA). Given the significant age-related differences, this study aims to employ propensity score matching to align patient backgrounds between these two diseases and compare physical function. Additionally, our objective is to scrutinize the patterns of clinical score recovery over the course of one year following THA. METHODS: Using propensity score matching, 29 patients each with steroid-related ONFH and OA were selected. Muscle strength (hip abductor and knee extensor) were assessed before and after THA. Additionally, recovery of the Harris Hip Score (HHS) and Oxford Hip Score (OHS) up to one year postoperatively was analyzed. RESULTS: The steroid-related ONFH group exhibited gender bias and significantly younger age compared to the OA group. Propensity score matching achieved balanced patient backgrounds. Physical function showed trends of lower hip abduction and knee extensor strength on the operative side in the steroid-related ONFH group. Notably, nonoperative knee extensor strength decreased significantly after matching. HHS and OHS were poor in steroid-related ONFH up to three months postoperatively but recovered after six months. CONCLUSIONS: Patients with steroid-related ONFH experience positive outcomes in clinical score following THA. Propensity score matching effectively identified muscle weakness on both operative and nonoperative sides, highlighting its utility in comparative analyses.

Association of physical activity with fatty infiltration of muscles after total hip arthroplasty.

OBJECTIVE: This study aimed to investigate the association between muscle density as an indicator of fatty infiltration of lower extremity muscles and physical activity (PA) after total hip arthroplasty (THA) and identify the patient characteristics with high postoperative PA. METHODS: This study included 62 female patients who underwent THA for unilateral hip osteoarthritis. Muscle density of the gluteus maximus, gluteus medius, iliopsoas, and quadriceps muscles was measured using computed tomography (CT). PA was assessed using University of California, Los Angeles (UCLA) activity scores. CT and UCLA activity score were obtained before and 1 year after THA. The patients were divided into two groups, sufficient (score ≥ 6) and insufficient (score < 6) activity groups, based on their level of PA as determined by their UCLA activity score 1 year after THA. The association of PA with the amount of changes in muscle density was examined with Spearman's rank correlation coefficient. Logistic regression analysis was performed to identify postoperative factors determining PA at 1 year after THA. RESULTS: Spearman's rank correlation coefficient showed a significantly positive association between recovery in PA and an increase in muscle density of the gluteus maximus, gluteus medius, iliopsoas, and quadriceps muscles. Additionally, logistic regression analysis confirmed that postoperative muscle densities of the gluteus maximus and quadriceps muscles were variables determining the PA 1 year after THA. CONCLUSION: The findings of this study indicated that the improvement of fatty infiltration in lower limb muscles, especially in the gluteus maximus and quadriceps, is likely to promote the increase in postoperative PA.

Investigating sagittal spinopelvic alignment and equivalent stress on the femoral head in patients with rapidly destructive coxarthrosis.

PURPOSE: Rapidly destructive coxarthrosis (RDC) is a rare syndrome of unknown etiology. This study evaluated sagittal spinopelvic alignment (SSPA) in patients with RDC and compared it with that in patients with hip osteoarthritis (HOA). In addition, finite element analysis (FEA) was performed to investigate the distribution of stress on the femoral head in RDC versus HOA. METHODS: This retrospective study included patients who had undergone primary total hip arthroplasty for RDC (n = 33) and HOA (n = 99; age- and sex-matched to patients with RDC) at three hospitals from June 2014 to September 2020. Preoperative SSPA parameters and inflammatory blood markers were compared between the two groups. FEA on the computed tomography data was performed for four patients from each group with similar pelvic tilt (PT) and lateral center-edge angle (LCEA). The distribution of Drucker-Prager equivalent stress was assessed at the loaded area of the femoral head. RESULTS: Patients with RDC had significantly higher PT, lower sacral slope, decreased lumbar lordosis (LL), higher sagittal vertical axis, and higher pelvic incidence minus LL than patients with HOA, indicating sagittal spinal imbalance. Blood test revealed patients with RDC had higher levels of inflammation markers than patients with HOA. FEA revealed no statistically significant difference in the degree of stress concentration or the maximum equivalent stress between the two groups when PT and LCEA were comparable. CONCLUSION: Patients with RDC tend to have sagittally imbalanced spine. Decreased acetabular coverage of the femoral head may heighten mechanical load of the hip joint in patients with RDC.

Antisera Produced Using an E. coli-Expressed SARS-CoV-2 RBD and Complemented with a Minimal Dose of Mammalian-Cell-Expressed S1 Subunit of the Spike Protein Exhibits Improved Neutralization.

E. coli-expressed proteins could provide a rapid, cost-effective, and safe antigen for subunit vaccines, provided we can produce them in a properly folded form inducing neutralizing antibodies. Here, we use an E. coli-expressed SARS-CoV-2 receptor-binding domain (RBD) of the spike protein as a model to examine whether it yields neutralizing antisera with effects comparable to those generated by the S1 subunit of the spike protein (S1 or S1 subunit, thereafter) expressed in mammalian cells. We immunized 5-week-old Jcl-ICR female mice by injecting RBD (30 µg) and S1 subunit (5 µg) according to four schemes: two injections 8 weeks apart with RBD (RBD/RBD), two injections with S1 (S1/S1), one injection with RBD, and the second one with S1 (RBD/S1), and vice versa (S1/RBD). Ten weeks after the first injection (two weeks after the second injection), all combinations induced a strong immune response with IgG titer > 105 (S1/RBD < S1/S1 < RBD/S1 < RBD/RBD). In addition, the neutralization effect of the antisera ranked as S1/RBD~RBD/S1 (80%) > S1/S1 (56%) > RBD/RBD (42%). These results indicate that two injections with E. coli-expressed RBD, or mammalian-cell-produced spike S1 subunit alone, can provide some protection against SARS-CoV-2, but a mixed injection scheme yields significantly higher protection.

Closed Suction Drainage May Not be Beneficial in Revision Total Hip Arthroplasty: A Propensity Score-Matched Cohort Study.

Background: Many studies on closed suction drainage (CSD) in primary total hip arthroplasty (THA) have demonstrated that it has no definite benefit. However, evidence of the clinical benefits of CSD in revision THA has not yet been established. Therefore, this retrospective study aimed to investigate the benefits of CSD in revision THA. Materials and Methods: We reviewed 107 hips of patients who underwent revision THA between June 2014 and May 2022, excluding cases of fracture and infection. We compared perioperative blood test results, calculated total blood loss (TBL), and postoperative complications, including allogenic blood transfusion (ABT), wound complications, and deep venous thrombosis (DVT), between the groups with and without CSD. Propensity score matching was conducted to balance patients' demographics and surgical factors. Results: ABT, wound complications, and DVT were observed in 10.3% (n = 11), 5.6% (six), and 5.6% (six) of patients, respectively. There were no significant differences in ABT, calculated TBL, wound complications, and DVT between all patients and propensity score-matched patients with or without CSD. The calculated TBL was approximately 1200 mL and showed no significant difference between the two groups in the matched cohort (p = 0.40) but tended to have a greater volume in the drain group than in the non-drain group. Conclusion: The routine use of CSD in revision THA for aseptic loosening may not be useful in clinical practice.

Biophysical and biochemical nature of amorphous protein oligomers determines the strength of immune response and the generation of T-cell memory.

Here, we used domain 3 of dengue virus serotype 3 envelope protein (D3ED3), a natively folded globular low-immunogenicity protein, to ask whether the biophysical nature of amorphous oligomers can affect immunogenicity. We prepared nearly identical 30 ~ 50 nm-sized amorphous oligomers in five distinct ways and looked at any correlation between their biophysical properties and immunogenicity. One oligomer type was produced using our SCP tag (solubility controlling peptide) made of 5 isoleucines (C5I). The others were prepared by miss-shuffling the SS bonds (Ms), heating (Ht), stirring (St) and freeze-thaw (FT). Dynamic light scattering showed that all five formulations contained oligomers of approximately identical sizes with hydrodynamic radii (Rh) between 30 and 55 nm. Circular dichroism (cd) indicated that the secondary structure content of oligomers formed by stirring and freeze-thaw was essentially identical to that of the native monomeric D3ED3. The secondary structure content of the Ms showed moderate changes, whereas the C5I and heat-induced (Ht) oligomers exhibited a significant change. The Ms contained D3ED3 with intermolecular SS bonds as assessed by nonreducing size exclusion chromatography (SEC). Immunization in JcL:ICR mice showed that both C5I and Ms significantly increased the anti-D3ED3 IgG titre. Ht, St and FT were only mildly immunogenic, similar to the monomeric D3ED3. Cell surface CD marker analysis by flow cytometry confirmed that immunization with Ms generated a strong central and effector T-cell memory. Our observations indeed suggest that controlled oligomerization can provide a new, adjuvant-free method for increasing a protein's immunogenicity, yielding a potentially powerful platform for protein-based (subunit) vaccines.

Total hip arthroplasty in patients with vertebral compression fracture is associated with poor clinical outcomes - retrospective analysis on 453 cases.

PURPOSE: Total hip arthroplasty (THA) is increasingly performed in older adults, and the prevalence of vertebral compression fracture (VCF) increases with age. We aimed to investigate the clinical outcomes of THA in patients with VCF. METHODS: We reviewed the records of 453 patients who underwent THA at our institution between 2015 and 2021. We classified patients into those with and without VCF. VCF was identified using preoperative upright whole-spine radiographs. Spinal parameters, preoperative and 1-year postoperative clinical outcomes of the Harris hip score (HHS), Oxford hip score (OHS), and visual analog scale (VAS) for low back pain (LBP) were assessed. Furthermore, propensity score-matched cohorts for age, sex, body mass index, and spinal parameters were created, and the clinical outcomes were compared between the two groups. RESULTS: Among the 453 patients, 51 (11.3%) with VCF and 402 without VCF were identified. Before matching, patients with VCF were older (p < 0.01), had sagittal spinal imbalance (p < 0.01), and had worse clinical outcomes pre- and postoperatively. After matching 47 patients in both groups, patients with VCF had worse HHS (p < 0.05), especially regarding support and distance walked, and worse VAS scores for LBP (p < 0.05) pre- and postoperatively. However, the improvements in scores were not significantly different between the groups. CONCLUSIONS: HHS, especially regarding support and distance walked, and VAS scores for LBP were poorer in patients with VCF preoperatively and 1-year postoperatively. Our findings suggest that hip surgeons should evaluate not only spinal alignment but also the presence of VCF before performing THA. LEVEL OF EVIDENCE: Level III, Retrospective cohort study.

Identifying Muscle Function-based Phenotypes Associated With Radiographic Progression of Secondary Hip Osteoarthritis.

OBJECTIVE: The purposes of our study were to (1) identify muscle function-based clinical phenotypes in patients with hip osteoarthritis (OA) and (2) determine the association between those phenotypes and radiographic progression of hip OA. DESIGN: Prospective cohort study. SETTING: Clinical biomechanics laboratory of a university. PARTICIPANTS: Fifty women patients with mild-to-moderate secondary hip OA (N=50) were recruited from the orthopedic department of a single institution. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Two-step cluster analyses were performed to classify the patients, using hip flexion, extension, abduction, and external/internal rotation muscle strength (cluster analysis 1); relative hip muscle strength to total hip strength (ie, hip muscle strength balance; cluster analysis 2); and both hip muscle strength and muscle strength balance (cluster analysis 3) as variables. The association between the phenotype and hip OA progression over 12 months (indicated by joint space width [JSW] >0.5 mm) was investigated by logistic regression analyses. Hip joint morphology, hip pain, gait speed, physical activity, Harris hip score, and SF-36 scores were compared between the phenotypes. RESULTS: Radiographic progression of hip OA was observed in 42% of the patients. The patients were classified into 2 phenotypes in each of the 3 cluster analyses. The solution in cluster analyses 1 and 3 was similar, and high-function and low-function phenotypes were identified; however, no association was found between the phenotypes and hip OA progression. The phenotype 2-1 (high-risk phenotype) extracted in cluster analysis 2, which had relative muscle weakness in hip flexion and internal rotation, was associated with subsequent hip OA progression, even after adjusting for age and minimum JSW at baseline (adjusted odds ratio [95% confidence interval], 3.60 [1.07-12.05]; P=.039). CONCLUSION: As preliminary findings, the phenotype based on hip muscle strength balance, rather than hip muscle strength, may be associated with hip OA progression.

Biophysical analysis of Gaussia luciferase bioluminescence mechanisms using a non-oxidizable coelenterazine.

Gaussia luciferase (GLuc 18.2kDa; 168 residues) is a marine copepod luciferase that emits a bright blue light when oxidizing coelenterazine (CTZ). It is a helical protein where two homologous sequential repeats form two anti-parallel bundles, each made of four helices. We previously identified a hydrophobic cavity as a prime candidate for the catalytic site, but GLuc's fast bioluminescence reaction hampered a detailed analysis. Here, we used azacoelenterazine (Aza-CTZ), a non-oxidizable coelenterazine (CTZ) analog, as a probe to investigate its binding mode to GLuc. While analysing GLuc's activity, we unexpectedly found that salt and monovalent anions are absolutely required for Gluc's bioluminescence, which retrospectively appears reasonable for a sea-dwelling organism. The NMR-based investigation, using chemical shift perturbations monitored by 15N-1H HSQC, suggested that Aza-CTZ (and thus unoxidized CTZ) binds to residues in or near the hydrophobic cavity. These NMR data are in line with a recent structural prediction of GLuc, hypothesizing that large structural changes occur in regions remote from the hydrophobic cavity upon the addition of CTZ. Interestingly, these results point toward a unique mode of catalysis to achieve CTZ oxidative decarboxylation.

Evaluating the contribution of fat infiltration in anterior gluteus minimus muscle to walking ability in female with unilateral hip osteoarthritis and candidates for total hip arthroplasty.

BACKGROUND: The purpose of this study is to investigate the relationship between gait and fat infiltration in anterior and posterior gluteus minimus in the patients with hip osteoarthritis. METHODS: Ninety-one female patients who were diagnosed as the unilateral hip osteoarthritis, classified into Kellgren-Lawrence global scoring system grades 3 or 4, and candidate for total hip arthroplasty were retrospectively reviewed. The horizontally cross-sectional regions of interest for the gluteus medius and anterior and posterior gluteus minimus were manually circumscribed in a single transaxial computed tomography image and muscle density of those regions were obtained. The gait was assessed as the step and speed with the 10-Meter Walk Test. The multiple regression analysis was used to compare the step and speed with age, height, range of motion in flexion, the muscle density of anterior gluteus minimus in the affected side, and that of gluteus medius muscle in both affected and unaffected sides. FINDINGS: Multiple regression analysis for step revealed that the muscle density of anterior gluteus minimus in the affected side and height were the independent predictors for step (R2 = 0.389, p < 0.001). That for speed identified the muscle density of anterior gluteus minimus in the affected side as the only factor determining speed (R2 = 0.287, p < 0.001). INTERPRETATION: The fatty infiltration of anterior gluteus minimus muscle in affected side can be a predictor for the gait in in female with unilateral hip osteoarthritis and candidates for total hip arthroplasty.

Assessment of the Damage to Hip Abductor Muscles in Primary Total Hip Arthroplasty With a Minimally Invasive Anterolateral Approach With or Without Trochanteric Flip Osteotomy.

BACKGROUND: Although introduction of minimally invasive techniques via anterolateral approaches considerably decreased prolonged limp after total hip arthroplasty (THA), potential damage to the abductor muscles is still a concern. This study aimed to evaluate the residual damage after primary THA with two types of anterolateral approach by assessing fatty infiltration and atrophy of the gluteus medius (GMed) and gluteus minimus (GMin) muscles. METHODS: We retrospectively analyzed 100 primary THAs using computed tomography; surgeries were performed by detaching the anterior part of the abductor muscles with a bone fragment (anterolateral approach with trochanteric flip osteotomy) or without it (anterolateral approach without trochanteric flip osteotomy). The change in radiodensities (RDs), cross-sectional areas (CSAs), and clinical scores preoperatively and at 1 year after operation were evaluated. RESULTS: The RD and CSA of GMed increased 1 year postoperatively in 86 and 81% of patients, respectively, while RD and CSA of GMin decreased in 71 and 94%, respectively. The improvement of RD of GMed was more frequently seen in the posterior rather than the anterior part, while the GMin decreased in both parts. The RD decrease of GMin was significantly lower in the anterolateral approach with trochanteric flip osteotomy group than the anterolateral approach without trochanteric flip osteotomy group (P = .0250). However, there was no difference in the clinical scores between the two groups. The change in the RD of GMed was the only factor that correlated with clinical scores. CONCLUSION: The two anterolateral approaches both improved the RD of GMed, recovery of which was significantly associated with postoperative clinical scores. Although the two approaches showed different recovery patterns in GMin until 1 year after THA, both showed similar improvements in clinical scores.

The Immunogenicity of DENV1-4 ED3s Strongly Differ despite Their Almost Identical Three-Dimensional Structures and High Sequence Similarities.

The development of a dengue (DENV) vaccine remains challenging due to the heteroserotypic infection, which can result in a potentially deadly hemorrhagic fever or dengue shock syndrome, and only a tetravalent vaccine can overcome this issue. Here, we report the immunogenicity of DENV envelope protein domain 3 (ED3) from all four DENV serotypes (DENV1-4) in Swiss albino and BALB/c mice models. Firstly, we observed that despite having very similar sequences and structures, both the humoral and cellular immunogenicity of ED3s varied significantly, with strength ranging from DENV2 ED3 (2ED3)~3ED3 > 1ED3 > 4ED3, which was assessed through anti-ED3 IgG titers, and DENV1 ED3 (1ED3) > 2ED3~3ED3 > 4ED3 as determined by monitoring T-cell memory (CD44+CD62L+ T cells with IL-4 and IFN-γ expression). Secondly, anti-1ED3 sera cross-reacted with 2ED3 and 3ED3; anti-2ED3 and anti-3ED3 sera cross-reacted with each other, but anti-4ED3 was completely serotype-specific. The lack of reciprocity of anti-1ED3's cross-reaction was unanticipated. Such disparity in the ED3 responses and cross-reaction might underlie the appearance of hemorrhagic fever and dengue shock syndrome. Hence, the development of an ED3-based tetravalent subunit vaccine would require understanding the aforementioned disparities.

Long-term outcome of primary total hip arthroplasty with cementless bioactive glass ceramic bottom-coated implants and highly cross-linked polyethylene: A minimum 10-year analysis.

BACKGROUND: Cementless glass ceramics containing apatite and wollastonite (AW-GC) bottom-coated titanium hip implants were developed; early excellent clinical and radiographic results have been reported previously. This study aimed to investigate the long-term clinical and radiographic outcomes in detail, and the wear rate of HXLPE. METHODS: We retrospectively evaluated 99 patients (117 hips) between November 2001 and December 2007. The survival rate was estimated using the Kaplan-Meier method. Hip joint function was evaluated using the Japanese Orthopaedic Association (JOA) score. The extent of radiographic signs was determined from the radiographs performed at the last follow-up. Polyethylene wear was measured using Martell's Hip Analysis Suite. We assessed the possible factors affecting the steady-state linear wear rate. RESULTS: The mean follow-up period was 14.8 ± 2.1 (10-18.6) years. The mean JOA score improved to 88.7 ± 9.4 (59-100) at the final follow-up from 47.8 ± 12.5 (17-76) before surgery. The overall survival rate with the end point of all-cause revision and wear-related revision was 99% and 100% respectively. There was no osteolysis or loosening of either the acetabular or femoral component. All hips were classified as having bone ingrowth fixation. The mean steady-state wear rate was 0.008 ± 0.025 mm/year. We found no significant correlation between the wear rate and age, body weight, body mass index, cup inclination and femoral head size. CONCLUSION: The combination of AW-GC bottom-coated implants and HXLPE showed excellent implant survival and wear resistance for 15 years.

Reproducibility, criterion-related validity, and minimal clinically important difference of the stair negotiation test after total Hip arthroplasty.

OBJECTIVE: To determine the reproducibility, criterion-related validity, and minimal clinically important difference (MCID) of the stair negotiation test (SNT) after total hip arthroplasty (THA). METHODS: Sixty patients who underwent THA were included in this study. They performed the SNT and rated their difficulty in stair negotiation (question 7 of the Oxford Hip Score [OHSQ7]) before and 6 months after surgery. The SNT determined the time taken by a patient to ascend, turn around, and descend the stairs (15 cm × 4 steps) and was measured twice each time. As a measure of reproducibility, the intraclass correlation coefficient (ICC1,1) was calculated using the preoperative SNT. As an index of criterion-related validity, Spearman's rank correlation coefficient was used to evaluate the relationship between the better score of two trials in the preoperative SNT and the OHSQ7. The MCID of the SNT was calculated using the distribution-based method and the anchor-based method. The change in the OHSQ7 between before and after surgery was used as an anchor in the latter method. RESULTS: The ICC1,1 of the SNT was 0.97. The SNT was significantly correlated with the OHSQ7 (r = 0.40, p < .05). Moreover, the anchor-based MCID of the SNT was 1.98 seconds. CONCLUSION: The SNT is an objective assessable test of stair negotiation ability in post-THA patients that has good reproducibility and moderate criterion-related validity. Changes in the SNT beyond the MCID (1.98 seconds) represent clinically important changes in stair negotiation ability.

E. coli production of a multi-disulfide bonded SARS-CoV-2 Omicron BA.5 RBD exhibiting native-like biochemical and biophysical properties.

Low-cost bacterial production of the receptor binding domain (RBD) of the SARS-CoV-2 Omicron spike protein holds significant potential in expediting the development of therapeutics against COVID-19. However, RBD contains eight cysteines forming four disulfide bonds, and expression in E. coli using standard protocols produces insoluble RBD forming non-native disulfide bonds. Here, we expressed RBD in E. coli T7 SHuffle with high aeration, which enhanced disulfide formation in the cytoplasm and reshuffling of non-native disulfide bonds, and at a low temperature of 16°C, which stabilized the native conformation and thus the formation of the native disulfide bonds. The yield of RBD was as high as 3 mg per 200 mL culture. We analyzed the conformational and biophysical properties of our E. coli-expressed RBD. First, the RP-HPLC elution profile indicated a single peak, suggesting that RBD was folded with a single disulfide bond pairing pattern. Next, circular dichroism analysis indicated a secondary structure content very close to that computed from the crystal structure. RBD's thermal denaturation monitored by CD was cooperative, strongly indicating a well-folded protein structure. Moreover, limited proteolysis showed that RBD was nearly as stable as RNase A, and the formation of native disulfide bonds was confirmed by LC-MS analysis. Furthermore, BLI analysis indicated a strong binding of RBD with the hACE2 with a dissociation constant of 0.83 nM, confirming the folded nature of RBD. Altogether, these results demonstrate that our E. coli-expression system can provide a large amount of highly purified RBD with correct disulfide bonds and native-like biochemical and biophysical properties.

Biophysical studies of amorphous protein aggregation and in vivo immunogenicity.

Amorphous protein aggregates are oligomers that lack specific, high-order structures. Soluble amorphous aggregates are smaller than ~1 µm. Despite their lack of high-order structure, amorphous protein aggregates exhibit specific biophysical properties such as reversibility of formation, density, conformation, and biochemical stability. Our mutational analysis using a Solubility Controlling Peptide (SCP) tag strongly suggests that amorphous aggregation of small globular proteins can significantly increase in vivo immune response and that the magnitude of enhanced immune responses depends on the aggregates' biophysical and biochemical properties. We propose that SCP tags might help develop subunit (protein) adjuvant-free (immunostimulant-free) vaccines by controlling the aggregation propensity of target proteins.

An Escherichia coli Expressed Multi-Disulfide Bonded SARS-CoV-2 RBD Shows Native-like Biophysical Properties and Elicits Neutralizing Antisera in a Mouse Model.

A large-scale Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 could yield a versatile and low-cost antigen for a subunit vaccine. Appropriately folded antigens can potentially elicit the production of neutralizing antisera providing immune protection against the virus. However, E. coli expression using a standard protocol produces RBDs with aberrant disulfide bonds among the RBD's eight cysteines resulting in the expression of insoluble and non-native RBDs. Here, we evaluate whether E. coli expressing RBD can be used as an antigen candidate for a subunit vaccine. The expressed RBD exhibited native-like structural and biophysical properties as demonstrated by analytical RP-HPLC, circular dichroism, fluorescence, and light scattering. In addition, our E. coli expressed RBD binds to hACE2, the host cell's receptor, with a binding constant of 7.9 × 10-9 M, as indicated by biolayer interferometry analysis. Our E. coli-produced RBD elicited a high IgG titer in Jcl:ICR mice, and the RBD antisera inhibited viral growth, as demonstrated by a pseudovirus-based neutralization assay. Moreover, the increased antibody level was sustained for over 15 weeks after immunization, and a high percentage of effector and central memory T cells were generated. Overall, these results show that E. coli-expressed RBDs can elicit the production of neutralizing antisera and could potentially serve as an antigen for developing an anti-SARS-CoV-2 subunit vaccine.