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.

Measurement of the Acetabular Cup Orientation after Total Hip Arthroplasty Based on Three-Dimensional Reconstruction from a Single X-ray Image Using Generative Adversarial Networks.

BACKGROUND: The purpose of this study was to reconstruct three-dimensional (3D) computed tomography (CT) images from single anteroposterior (AP) postoperative total hip arthroplasty (THA) X-ray images using a deep learning algorithm known as generative adversarial networks (GANs) and to validate the accuracy of cup angle measurement on GAN-generated CT. METHODS: We used two GAN-based models, CycleGAN and X2CT-GAN, to generate 3D CT images from X-ray images of 386 patients who underwent primary THAs using a cementless cup. The training dataset consisted of 522 CT images and 2,282 X-ray images. The image quality was validated using the peak signal-to-noise ratio (PSNR) and the structural similarity index measure (SSIM). The cup anteversion and inclination measurements on the GAN-generated CT images were compared with the actual CT measurements. Statistical analyses of absolute measurement errors were performed using Mann-Whitney U tests and nonlinear regression analyses. RESULTS: The study successfully achieved 3D reconstruction from single AP postoperative THA X-ray images using GANs, exhibiting excellent PSNR (37.40) and SSIM (0.74). The median absolute difference in radiographic anteversion (RA) was 3.45° and the median absolute difference in radiographic inclination (RI) was 3.25°, respectively. Absolute measurement errors tended to be larger in cases with cup malposition than in those with optimal cup orientation. CONCLUSION: This study demonstrates the potential of GANs for 3D reconstruction from single AP postoperative THA X-ray images to evaluate cup orientation. Further investigation and refinement of this model are required to improve its performance.

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.

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.

Non-Glycosylated SARS-CoV-2 Omicron BA.5 Receptor Binding Domain (RBD) with a Native-like Conformation Induces a Robust Immune Response with Potent Neutralization in a Mouse Model.

The Omicron BA.5 variant of SARS-CoV-2 is known for its high transmissibility and its capacity to evade immunity provided by vaccine protection against the (original) Wuhan strain. In our prior research, we successfully produced the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein in an E. coli expression system. Extensive biophysical characterization indicated that, even without glycosylation, the RBD maintained native-like conformational and biophysical properties. The current study explores the immunogenicity and neutralization capacity of the E. coli-expressed Omicron BA.5 RBD using a mouse model. Administration of three doses of the RBD without any adjuvant elicited high titer antisera of up to 7.3 × 105 and up to 1.6 × 106 after a booster shot. Immunization with RBD notably enhanced the population of CD44+CD62L+ T cells, indicating the generation of T cell memory. The in vitro assays demonstrated the antisera's protective efficacy through significant inhibition of the interaction between SARS-CoV-2 and its human receptor, ACE2, and through potent neutralization of a pseudovirus. These findings underscore the potential of our E. coli-expressed RBD as a viable vaccine candidate against the Omicron variant of SARS-CoV-2.

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.

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.

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.

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.

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.

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.

Development of a Clinical Prediction Rule to Identify Physical Activity After Total Hip Arthroplasty.

OBJECTIVE: To develop clinical prediction rule (CPR) of physical activity 1 year after total hip arthroplasty (THA). DESIGN: Retrospective cohort study. SETTING: University hospital with orthopedic surgery. PARTICIPANTS: The study group included 321 patients (56 men) who underwent primary THA (N=321). INTERVENTION: Not applicable. MAIN OUTCOMES MEASURES: The data collected included age, body mass index, clinical score from the questionnaires, hip pain, range of motion, muscle strength, and Physical functions (10-meter walk test [10MWT], timed Up and Go test, sit-to-stand test). Patients were classified into sufficient and insufficient activity groups based on their University of California, Los Angeles (UCLA) activity score 1 year after THA. Variables measured preoperatively and 3 weeks postoperatively were analyzed using univariate and multivariate methods to derive CPR for physical activity. RESULTS: A CPR was developed using the following 5 factors and cutoffs: age 70.5 years or younger, preoperative UCLA activity score ≥3.5, preoperative hip abduction strength ≥0.54 Nm/kg, preoperative knee extension strength ≥1.04 Nm/kg, and 10MWT ≤8.49 seconds 3 weeks after surgery. The presence of 4 of the 5 factors predicted a sufficient physical activity level at 1 year, with a positive likelihood ratio of 5.94 and probability of 85.4%. The presence of 5 predictor variables increased the probability of sufficient physical activity after THA to 94.7%. CONCLUSIONS: This study developed a CPR for physical activity 1 year after THA. Having 4 or more of the 5 measurements were useful indicators for predicting of physical activity 1 year postoperatively.

The superior accuracy of a novel method in total hip wear calculations following radiographic measurement.

BACKGROUND: Polyethylene wear is one of the major concerns of orthopedic surgeons. However, there is no standardized calculation method for the wear rate following radiographic measurement. The purpose of this study was to propose a novel method of wear calculation and to compare its accuracy with a representative conventional method. METHODS: Relative position of the center of the femoral head to that of the cup progresses in one direction following arthroplasty surgery because of bedding-in and wear. We predetermined the amount of bedding-in, wear rate, and random error in measuring the head center position in a 2-dimensional plane. We calculated the wear rate using the head center coordinates over a certain number of measurement periods using a representative conventional method and our novel method. The conventional method consisted of transforming vector data into scalars and conducting a least-squares method. The least-squares method was directly applied to each component of the vector in the novel method. We evaluated the accuracy of these methods by comparing the expected value for the wear rate with their predetermined true values. RESULTS: If the error were limited to being random, the novel method could provide the predetermined wear rate as the calculation result. However, the conventional method could not. CONCLUSION: We recommend using the novel method for the wear calculation rather than the conventional method because of its mathematical accuracy.

Preoperative Factors Associated With Low Back Pain Improvement After Total Hip Arthroplasty in a Japanese Population.

BACKGROUND: Spinal disorders and low back pain (LBP) have been associated with worse clinical outcomes of total hip arthroplasty (THA). It is essential to identify spinal factors associated with post-THA LBP improvement. Therefore, we aimed to determine the proportion of patients with improved LBP after THA and to identify the preoperative spinal factors associated with LBP improvement. METHODS: We included 151 patients who underwent primary THA between December 2015 and December 2019 and had a preoperative visual analog scale score for LBP of ≥2. The patients were classified into the LBP improved or LBP continued group based on a visual analog scale score for LBP at 1 year after THA. Preoperative spinal parameters were compared between the 2 groups. RESULTS: Ninety-five patients (62.9%) were classified into the LBP improved group. Among the coronal spinal parameters, the Cobb angle was significantly lower in the LBP improved group. Among the sagittal spinal parameters, the LBP continued group showed a significantly more posteriorly titled pelvis, lower lumbar lordosis, greater sagittal vertical axis, and greater pelvic incidence minus lumbar lordosis mismatch, indicating a sagittal spinal imbalance. Logistic regression analysis found that preoperative factors associated with LBP improvement after THA had a low Cobb angle and high anterior pelvic plane angle (anteriorly tilted pelvis). CONCLUSION: Among patients with LBP before THA, 62.9% had improved LBP. Sagittal spinal imbalance and high Cobb angle were the key spinal factors associated with persistent LBP. Our findings suggest that hip surgeons should evaluate spinal alignment before THA in patients with LBP.

Corticosteroid-associated osteonecrosis of the femoral head after orthotopic liver transplantation and the outcomes of subsequent total hip arthroplasty.

BACKGROUND: Corticosteroids are essential for immunosuppression after orthotopic liver transplantation (OLT), but also have many side effects. Osteonecrosis of the femoral head (ONFH) is one of the most serious complications requiring prostheses. However, few studies have investigated ONFH after OLT. The purpose of this study is to survey the incidence of corticosteroid-induced ONFH after OLT and the outcomes of total hip arthroplasty (THA). METHODS: Between January 2001 and December 2010, a series of 926 patients underwent OLT at our Hospital. A retrospective analysis was performed on a total of 738 patients who survived at least 2 years after OLT. The incidence of symptomatic ONFH, the interval from OLT to the initial diagnosis of ONFH, and the cumulative dose of corticosteroids were analyzed. The side effects related to OLT, such as other osteonecrosis lesions, osteoporotic fractures, and infection, were monitored. For patients who underwent THA, radiological findings and Japanese Orthopaedic Association (JOA) scores were evaluated. RESULTS: ONFH occurred in 10 patients (13 hips) (6 men [7 hips], 4 women [6 hips]), with an incidence of 1.36%. The average age at OLT was 51.4 years (range, 31-61 years). The average interval from OLT to ONFH was 86.7 months (range, 22-155 months). The average cumulative dose of corticosteroids was 7274 mg (range, 1342-29,514 mg). Twenty patients suffered from side effects related to OLT. Seven patients (8 hips) underwent THA. No adverse events including infection arose during the perioperative process. One hip dislocated, and one femoral stem displayed a radiolucent line. The average JOA score improved from 45.4 (range, 25-76) preoperatively to 86.9 (range, 73-99) at final follow-up. No patients required revision surgery. CONCLUSIONS: The incidence of symptomatic ONFH after OLT was 1.36%. Once the graft function becomes stable, THA can be a safe and effective treatment option for patients with ONFH after OLT.

Closed suction drainage is not beneficial in hybrid total hip arthroplasty with intra-articular administration of tranexamic acid: a propensity score-matched cohort study.

PURPOSE: This retrospective study aimed to evaluate the benefits of closed suction drainage (CSD) in hybrid total hip arthroplasty (THA) with intra-articular administration of tranexamic acid (TXA). METHODS: We included 369 hips that underwent primary hybrid THA between November 2015 and September 2020. We compared peri-operative blood test results, blood loss, and post-operative complications including transfusion, wound complications, and venous thromboembolism (VTE) with or without CSD. Propensity score matching was performed to balance baseline patient demographics. RESULTS: Transfusion, wound complications, and VTE were observed in 1.9% (seven), 2.4% (nine), and 2.2% (eight) of hips, respectively. There were no significant differences in transfusion, blood loss, wound complications, and deep venous thrombosis in both overall patients and propensity score-matched patients with or without CSD. The calculated total blood loss was approximately 600 ml and showed no significant difference between the two groups in the matched cohort (p = 0.59). CONCLUSION: CSD does not provide any benefits and is not needed in primary hybrid THA with intra-articular administration of TXA.

A Multi-Disulfide Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike Protein Expressed in E. coli Using a SEP-Tag Produces Antisera Interacting with the Mammalian Cell Expressed Spike (S1) Protein.

An Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 (isolate Wuhan-Hu-1) spike protein would significantly accelerate the search for anti-COVID-19 therapeutics because of its versatility and low cost. However, RBD contains four disulfide bonds and its expression in E. coli is limited by the formation of aberrant disulfide bonds resulting in inclusion bodies. Here, we show that a solubility-enhancing peptide (SEP) tag containing nine arginine residues (RBD-C9R) attached at the C-terminus can overcome this problem. The SEP-tag increased the expression in the soluble fraction and the final yield by five times (2 mg/L). The folding properties of the E. coli expressed RBD-C9R were demonstrated with biophysical characterization using RP-HPLC, circular dichroism, thermal denaturation, fluorescence, and light scattering. A quartz crystal microbalance (QCM) analysis confirmed the binding activity of RBD-C9R with ACE2, the host cell's receptor. In addition, RBD-C9R elicited a Th-2 immune response with a high IgG titer in Jcl: ICR mice. The RBD-C9R antisera interacted with both itself and the mammalian-cell expressed spike protein (S1), as demonstrated by ELISA, indicating that the E. coli expressed RBD-C9R harbors native-like epitopes. Overall, these results emphasize the potential of our SEP-tag for the E. coli production of active multi-disulfide-bonded RBD.

Direct Analysis of Mitochondrial Damage Caused by Misfolded/Destabilized Proteins.

Protein quality control is essential for cellular homeostasis. In this study, we examined the effect of improperly folded proteins that do not form amyloid fibrils on mitochondria, which play important roles in ATP production and cell death. First, we prepared domain 3 of the dengue envelope protein in wild type and four mutants with widely different biophysical properties in misfolded/aggregated or destabilized states. The effects of the different proteins were detected using fluorescence microscopy and Western blotting, which revealed that three of the five proteins disrupted both inner and outer membrane integrity, while the other two proteins, including the wild type, did not. Next, we examined the common characteristics of the proteins that displayed toxicity against mitochondria by measuring oligomer size, molten globule-like properties, and thermal stability. The common feature of all three toxic proteins was thermal instability. Therefore, our data strongly suggest that thermally unstable proteins generated in the cytosol can cause cellular damage by coming into direct contact with mitochondria. More importantly, we revealed that this damage is not amyloid-specific.

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.

Reverse Engineering Analysis of the High-Temperature Reversible Oligomerization and Amyloidogenicity of PSD95-PDZ3.

PSD95-PDZ3, the third PDZ domain of the post-synaptic density-95 protein (MW 11 kDa), undergoes a peculiar three-state thermal denaturation (N ↔ In ↔ D) and is amyloidogenic. PSD95-PDZ3 in the intermediate state (I) is reversibly oligomerized (RO: Reversible oligomerization). We previously reported a point mutation (F340A) that inhibits both ROs and amyloidogenesis and constructed the PDZ3-F340A variant. Here, we "reverse engineered" PDZ3-F340A for inducing high-temperature RO and amyloidogenesis. We produced three variants (R309L, E310L, and N326L), where we individually mutated hydrophilic residues exposed at the surface of the monomeric PDZ3-F340A but buried in the tetrameric crystal structure to a hydrophobic leucine. Differential scanning calorimetry indicated that two of the designed variants (PDZ3-F340A/R309L and E310L) denatured according to the two-state model. On the other hand, PDZ3-F340A/N326L denatured according to a three-state model and produced high-temperature ROs. The secondary structures of PDZ3-F340A/N326L and PDZ3-wt in the RO state were unfolded according to circular dichroism and differential scanning calorimetry. Furthermore, PDZ3-F340A/N326L was amyloidogenic as assessed by Thioflavin T fluorescence. Altogether, these results demonstrate that a single amino acid mutation can trigger the formation of high-temperature RO and concurrent amyloidogenesis.