Synthetic Biology-Driven Microbial Therapeutics for Disease Treatment.

The human microbiome, consisting of microorganisms that coexist symbiotically with the body, impacts health from birth. Alterations in gut microbiota driven by factors such as diet and medication can contribute to diseases beyond the gut. Synthetic biology has paved the way for engineered microbial therapeutics, presenting promising treatments for a variety of conditions. Using genetically encoded biosensors and dynamic regulatory tools, engineered microbes can produce and deliver therapeutic agents, detect biomarkers, and manage diseases. This review organizes engineered microbial therapeutics by disease type, emphasizing innovative strategies and recent advancements. The scope of diseases includes gastrointestinal disorders, cancers, metabolic diseases, infections, and other ailments. Synthetic biology facilitates precise targeting and regulation, improving the efficacy and safety of these therapies. With promising results in animal models, engineered microbial therapeutics provide a novel alternative to traditional treatments, heralding a transformative era in diagnostics and treatment for numerous diseases.

Functional pathogenicity of ESRRB variant of uncertain significance contributes to hearing loss (DFNB35).

Advances in next-generation sequencing technologies have led to elucidation of sensorineural hearing loss genetics and associated clinical impacts. However, studies on the functional pathogenicity of variants of uncertain significance (VUS), despite their close association with clinical phenotypes, are lacking. Here we identified compound heterozygous variants in ESRRB transcription factor gene linked to DFNB35, specifically a novel splicing variant (NM_004452.4(ESRRB): c.397 + 2T>G) in trans with a missense variant (NM_004452.4(ESRRB): c.1144C>T p.(Arg382Cys)) whose pathogenicity remains unclear. The splicing variant (c.397 + 2T>G) caused exon 4 skipping, leading to premature stop codon formation and nonsense-mediated decay. The p.(Arg382Cys) variant was classified as a VUS due to its particularly higher allele frequency among East Asian population despite disease-causing in-silico predictions. However, functional assays showed that p.(Arg382Cys) variant disrupted key intramolecular interactions, leading to protein instability. This variant also reduced transcriptional activity and altered expression of downstream target genes essential for inner ear function, suggesting genetic contribution to disease phenotype. This study expanded the phenotypic and genotypic spectrum of ESRRB in DFNB35 and revealed molecular mechanisms underlying ESRRB-associated DFNB35. These findings suggest that variants with high allele frequencies can also possess functional pathogenicity, providing a breakthrough for cases where VUS, previously unexplored, could be reinterpreted by elucidating their functional roles and disease-causing characteristics.

Apoptosis of Pancreatic Cancer Cells after Co-Treatment with Eugenol and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand.

Pancreatic cancer is a refractory cancer with limited treatment options. Various cancer types are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Eugenol, the main component of clove oil, exhibits anticancer, anti-inflammatory, and antioxidant effects. However, no studies have reported that eugenol increases TRAIL sensitivity by upregulating death receptor (DR) expression. Here, we aimed to investigate eugenol as a potent TRAIL sensitizer. Increased apoptosis and inhibition of cell proliferation was observed in pancreatic cancer cells treated with eugenol and TRAIL compared with those treated with eugenol alone. Eugenol upregulated the expression of DR5, inhibited the FLICE-inhibitory protein (FLIP), an anti-apoptotic protein, and increased p53, a tumor suppressor protein. In addition, eugenol induced the generation of reactive oxygen species (ROS) and caused endoplasmic reticulum (ER) stress. C/EBP-homologous protein (CHOP) knockdown using siRNA decreased the expression of DR5 and reduced the combined effects of eugenol and TRAIL. These results demonstrate that eugenol enhances TRAIL-induced apoptosis by upregulating DR5 through the ROS-mediated ER stress-CHOP pathway, which enhances ER stress by inducing p53 and downregulating FLIP expression. This suggests that eugenol has the potential to treat pancreatic cancer by increasing cell sensitivity to TRAIL.

Codium fragile extract prevents atopic dermatitis in DNCB-induced mice.

Atopic dermatitis (AD) is a chronic inflammatory-allergic skin disorder that causes pruritic and eczematous skin lesions. Effect of Codium fragile extract (CFE) on AD has not been reported yet. In this study, inhibitory effects of CFE against skin severity scores, skin lesions, AD characteristics, and histological features of BALB/c mice with AD caused by 2,4-dinitrochlorobenzene (DNCB) were investigated. Results indicated that AD effects of CFE reduced body, skin, ear, spleen, thymus, and lymph node weights. Histopathological changes in skin reactions on the back and ears showed that CFE inhibited thickening of the epidermis and ear. Moreover, CFE reduced epidermal swelling and ear thickness compared with the DNCB group. These results suggest that CFE might be effective in alleviating AD with potential as a promising candidate for therapeutic and cosmetic treatment of inflammatory dermatitis. CFE may be useful in alleviating AD and could be a potential treatment for inflammatory dermatitis.

Discovery of novel disease-causing mutation in SSBP1 and its correction using adenine base editor to improve mitochondrial function.

Mutations in nuclear genes regulating mitochondrial DNA (mtDNA) replication are associated with mtDNA depletion syndromes. Using whole-genome sequencing, we identified a heterozygous mutation (c.272G>A:p.Arg91Gln) in single-stranded DNA-binding protein 1 (SSBP1), a crucial protein involved in mtDNA replisome. The proband manifested symptoms including sensorineural deafness, congenital cataract, optic atrophy, macular dystrophy, and myopathy. This mutation impeded multimer formation and DNA-binding affinity, leading to reduced efficiency of mtDNA replication, altered mitochondria dynamics, and compromised mitochondrial function. To correct this mutation, we tested two adenine base editor (ABE) variants on patient-derived fibroblasts. One variant, NG-Cas9-based ABE8e (NG-ABE8e), showed higher editing efficacy (≤30%) and enhanced mitochondrial replication and function, despite off-target editing frequencies; however, risks from bystander editing were limited due to silent mutations and off-target sites in non-translated regions. The other variant, NG-Cas9-based ABE8eWQ (NG-ABE8eWQ), had a safer therapeutic profile with very few off-target effects, but this came at the cost of lower editing efficacy (≤10% editing). Despite this, NG-ABE8eWQ-edited cells still restored replication and improved mtDNA copy number, which in turn recovery of compromised mitochondrial function. Taken together, base editing-based gene therapies may be a promising treatment for mitochondrial diseases, including those associated with SSBP1 mutations.

Outcomes of Osteochondral Autologous Transplantation with Ipsilateral Lateral Talar Autograft for Medial Osteochondral Lesions of the Talus.

Background: Osteochondral autologous transplantation (OAT) has been widely used in the treatment of osteochondral lesion of the talus (OLT). Previous studies have reported successful outcomes following the use of osteochondral autogenous grafts from the intercondylar notch of the knee or a non-weight-bearing region of the femoral condyle. However, donor-site morbidity of the knee joint has been observed in several cases. This study aimed to investigate the outcomes and safety of OAT with autografts from the ipsilateral lateral talar articular facet as an alternative donor site for medial OLT. Methods: Among 40 patients who underwent OAT, 29 patients were excluded. Eleven patients who underwent OAT with an osteochondral graft harvested from the ipsilateral lateral talar articular facet from 2011 to 2022 were retrospectively analyzed. The size of OLT was measured on ankle magnetic resonance imaging, including coronal length, sagittal length, depth, and area. Clinical outcomes were evaluated using the American Orthopaedic Foot and Ankle Society (AOFAS) ankle-hindfoot scale and a visual analog scale (VAS). Weight-bearing ankle radiographs were obtained postoperatively and at 1 year after surgery. Results: The average follow-up time after surgery was 64.7 months (range, 14-137 months). The average diameter of lesions was 8.8 mm (range, 8-9.9 mm). The average size of lesions was 51.2 mm2 (range, 33.6-71.3 mm2) , and all lesions included subchondral cysts. The average depth of lesions was 7.3 mm (range, 6.2-9.1 mm). Graft sizes ranged from 8 to 10 mm in diameter (8 mm, n = 1; 10 mm, n = 10) All measured clinical outcomes improved postoperatively, including the AOFAS scores (preoperative, 55.4 ± 9.0; 1-year follow-up, 92.1 ± 7.6; p = 0.001) and VAS scores (preoperative, 5.5 ± 0.7; 1-year follow-up, 1.9 ± 0.8; p = 0.001). All weight-bearing ankle radiographs of the graft and donor sites did not reveal arthritic change in the ankle joint, lateral talar dome collapse, and graft-site delayed union or nonunion at 1 year after surgery. Conclusions: For a single medial OLT, harvesting autografts from the ipsilateral lateral talar articular facet without knee donor-site morbidities can be a good alternative in OAT for OLT.

The Osteotomy Gap Filling Rate of Multiple Time Points After Medial Opening Wedge High Tibial Osteotomy Without Bone Graft.

BACKGROUND: The purpose of the present study was to analyze osteotomy gap filling of multiple time points until 2 years post-medial opening wedge high tibial osteotomy (MOWHTO). The absolute value of gap filling and postoperative changes relative to the preoperative void osteotomy gap were evaluated using computed tomography (CT) at each time point. METHODS: Data of thirty patients who underwent MOWHTO between September 2019 and July 2021 were retrospectively analyzed. Surgical procedures without bone grafts were performed; a standardized rehabilitation protocol was implemented. The osteotomy gap filling rate was measured using CT scans at the immediate postoperative period and at 6, 12, and 24 months after surgery. Statistical analysis was performed to assess changes over time. RESULTS: The osteotomy gap filling rate showed a significant continuous increase after MOWHTO, reaching 45.2% at 6 months and 66.7 and 84.4% at 1 and 2 years postoperatively, respectively. The most substantial increase occurred within the initial 6 months, thus indicating a critical period for bone healing. CONCLUSION: The osteotomy gap filling rate showed a significant and gradual increase from immediately after surgery to 2 years after MOWTHO without bone grafting, the greatest of which was achieved in the initial 6-month period. Therefore, this study may be helpful for planning postoperative rehabilitation, including the extent of weight-bearing load and the period of crutch use.

Role of Corn Peptide Powder in Lipopolysaccharide-Induced Inflammatory Responses in 3T3-L1 Adipocytes.

Corn peptide (CP) is a short, naturally occurring, and physiologically active peptide generated from corn-protease-catalyzed hydrolysis. CP plays a role in preventing obesity-related disorders, but its impact on reducing inflammation is unknown. Hence, this study examined the possible protective effects of corn peptide powder (CPP) against the harmful effects of lipopolysaccharide (LPS), with a particular emphasis on reducing oxidative damage and inflammation in adipocytes. Hence, mature 3T3-L1 adipocytes underwent exposure to 10 ng/mL LPS, with or without CPP (10 and 20 µg/mL). LPS stimulation increased reactive oxygen species and superoxide anion generation. However, this effect was reduced in a dose-dependent manner by pretreatment with CPP. CPP treatment elevated the mRNA expressions of the antioxidant enzymes manganese superoxide dismutase (mnSOD) and glutathione peroxidase 1 (Gpx1) while reducing the mRNA expressions of the cytosolic reactive oxygen species indicators p40 and p67 (NADPH oxidase 2). In addition, CPP inhibited the monocyte chemoattractant protein-1, tumor necrosis factor-alpha, Toll-like receptor 4, and nuclear factor kappa B mRNA expressions induced by LPS. These findings demonstrate that CPP may ameliorate adipocyte dysfunction by suppressing oxidative damage and inflammatory responses through a new mechanism known as Toll-like receptor 4/nuclear factor kappa B-mediated signaling.

The secreted protein Amuc_1409 from Akkermansia muciniphila improves gut health through intestinal stem cell regulation.

Akkermansia muciniphila has received great attention because of its beneficial roles in gut health by regulating gut immunity, promoting intestinal epithelial development, and improving barrier integrity. However, A. muciniphila-derived functional molecules regulating gut health are not well understood. Microbiome-secreted proteins act as key arbitrators of host-microbiome crosstalk through interactions with host cells in the gut and are important for understanding host-microbiome relationships. Herein, we report the biological function of Amuc_1409, a previously uncharacterised A. muciniphila-secreted protein. Amuc_1409 increased intestinal stem cell (ISC) proliferation and regeneration in ex vivo intestinal organoids and in vivo models of radiation- or chemotherapeutic drug-induced intestinal injury and natural aging with male mice. Mechanistically, Amuc_1409 promoted E-cadherin/ß-catenin complex dissociation via interaction with E-cadherin, resulting in the activation of Wnt/ß-catenin signaling. Our results demonstrate that Amuc_1409 plays a crucial role in intestinal homeostasis by regulating ISC activity in an E-cadherin-dependent manner and is a promising biomolecule for improving and maintaining gut health.

Proximal row carpectomy with interposition arthroplasty using both capsular flap and acellular human dermal matrix.

BACKGROUND: In cases of wrist arthritis, proximal row carpectomy (PRC) has been widely utilized and shown favorable long-term outcomes. However, its applicability is limited in cases where arthritis extends to the lunate fossa or capitate. Recently, surgical approaches combining various methods of interposition arthroplasty have been introduced to overcome these drawbacks. The purpose of this study was to perform PRC and interposition arthroplasty with dorsal capsule and acellular dermal matrix(ADM),and analyze the clinical outcomes of these procedures. METHODS: Fourteen cases who underwent PRC and interposition arthroplasty using both dorsal capsular flap and ADM were retrospectively recruited. The researchers assessed the patients' Visual Analog Scale (VAS) pain score, Disabilities of the Arm, Shoulder and Hand (DASH) scores, range of motion (ROM), retear, and radiocarpal distance (RCD). RESULTS: One year post-surgery, both the VAS pain scores, DASH scores, and ROM showed statistically significant improvement compared to before the surgery. Upon reviewing the radiological results, the postoperative mean RCD was 4.8 ± 0.8 mm and one year follow up mean RCD was 3.6 ± 0.5 mm at one year post-surgery. Moreover, in the one year follow-up, there was no observed failure of the allodermis graft in any of the cases. CONCLUSION: The PRC and interposition arthroplasty with ADM demonstrated significantly improved clinical outcomes after surgery, showing a maintain of RCD without graft failure effectively.

Knee Joint Line Obliquity With Adaptational Hip and Ankle Joint Orientation After Medial Open Wedge High Tibial Osteotomy.

BACKGROUND: Time-dependent postoperative changes in knee joint line obliquity (KJLO) and subsequent adaptational changes in the hip and ankle joints have not been fully proven after medial open wedge high tibial osteotomy (MOWHTO). PURPOSE: To investigate the serial postoperative changes in KJLO and subsequent adaptational changes in the hip and ankle joints over time after MOWHTO. STUDY DESIGN: Case series, Level of evidence, 4. METHODS: A total of 92 patients who underwent MOWHTO between April 2015 and December 2020 were evaluated. Radiographic parameters, including KJLO, ankle joint line obliquity (ALO), hip abduction angle (HAA), joint line convergence angle, weightbearing line ratio, and hip-knee-ankle angle, were analyzed in time sequence (preoperatively and 3, 6, 12, and 24 months postoperatively). Repeated-measures analysis of variance and post hoc analysis were used to demonstrate alterations and the statistical significance of KJLO and other related radiographic parameters over time. RESULTS: The mean KJLO values were -1.9°, -2.1°, -2.7°, and -3.2° at 3, 6, 12, and 24 months postoperatively, respectively, indicating that there was consistent increase in valgus tilting of KJLO from 6 to 24 months (P < .001 for both 6-12 months and 12-24 months). ALO and HAA showed significant changes from 6 to 12 months (ALO, P < .001; HAA, P = .002), but not between 12 and 24 months (ALO: -3.0°, -2.7°, -1.9°, and -1.6°; HAA: -0.8°, -0.9°, -1.5°, and -1.8° at 3, 6, 12, and 24 months, respectively). The mean joint line convergence angle, weightbearing line ratio, and hip-knee-ankle angle did not change significantly from 3 months to 24 months postoperatively. CONCLUSION: There was a consistent increase in valgus tilting of the postoperative KJLO from 6 to 24 months after MOWHTO. The adaptive ALO and HAA significantly changed between 6 and 12 months and were maintained until 24 months after MOWHTO. It is necessary to consider the adaptive change when hip or ankle surgery is planned within this period.

Comparison of Clinical and Radiological Outcomes after Vertebroplasty and Balloon Kyphoplasty in the Treatment of Osteoporotic Vertebral Compression Fractures.

BACKGROUND: Vertebroplasty (VP) and balloon kyphoplasty (KP) are effective means with which to improve pain and function in osteoporotic vertebral compression fractures. However, the risk of complications after these procedures is poorly understood, with concerns regarding adjacent vertebral fractures. This study retrospectively investigated the clinical and radiological outcomes of these procedures. METHODS: A total of 115 patients who experienced their first vertebral fracture were treated with VP (N=63) or KP (N=52) at the Dankook University Hospital between January 2013 and December 2022. The clinical outcomes were evaluated using the visual analog scale (VAS) preoperative and at 1-year follow-up. Radiological comparisons were performed for kyphosis correction, vertebral height restoration, and postoperative cement leakage. RESULTS: KP was more effective than VP, especially for vertebral body height restoration and kyphotic angle reduction (P<0.05). However, the incidence of cement leakage, new adjacent vertebral fractures, and improvement in pain assessed by VAS did not differ statistically between the 2 groups (P>0.05). CONCLUSIONS: Considering that KP was performed on fractures with severe deformity, no differences were observed in the clinical outcomes and incidence of adjacent vertebral fractures compared Considering that KP was performed for fractures with severe deformity, there was no difference in clinical outcomes and incidence of adjacent vertebral fractures compared to VP. Improvements in radiological measurements were demonstrated. Therefore, KP may be a good treatment option for pain relief and long-term prognosis in patients with high-compressive-rate vertebral fractures.

Unlocking synergies: Harnessing the potential of biological methane sequestration through metabolic coupling between Methylomicrobium alcaliphilum 20Z and Chlorella sp. HS2.

A halotolerant consortium between microalgae and methanotrophic bacteria could effectively remediate in situ CH4 and CO2, particularly using saline wastewater sources. Herein, Methylomicrobium alcaliphilum 20Z was demonstrated to form a mutualistic association with Chlorella sp. HS2 at a salinity level above 3.0%. Co-culture significantly enhanced the growth of both microbes, independent of initial inoculum ratios. Additionally, increased methane provision in enclosed serum bottles led to saturated methane removal. Subsequent analyses suggested nearly an order of magnitude increase in the amount of carbon sequestered in biomass in methane-fed co-cultures, conditions that also maintained a suitable cultural pH suitable for methanotrophic growth. Collectively, these results suggest a robust metabolic coupling between the two microbes and the influence of the factors other than gaseous exchange on the assembled consortium. Therefore, multi-faceted investigations are needed to harness the significant methane removal potential of the identified halotolerant consortium under conditions relevant to real-world operation scenarios.

In Vitro Anti-Inflammatory and Skin Protective Effects of Codium fragile Extract on Macrophages and Human Keratinocytes in Atopic Dermatitis.

Codium fragile has been traditionally used in oriental medicine to treat enterobiasis, dropsy, and dysuria, and it has been shown to possess many biological properties. Atopic dermatitis (AD) is one of the types of skin inflammation and barrier disruption, which leads to chronic inflammatory skin diseases. In the current investigation, the protective effects of C. fragile extract (CFE) on anti-inflammation and skin barrier improvement were investigated. In LPS-stimulated RAW 264.7 cells, nitric oxide generation and the expression levels of interleukin (IL)-1ß, IL-4, IL-6, iNOS, COX-2, and tumor necrosis factor-alpha (TNF)-α were reduced by CFE. CFE also inhibited the phosphorylation of NF-κB-p65, ERK, p-38, and JNK. Additionally, CFE showed inhibitory activity on TSLP and IL-4 expression in HaCaT cells stimulated with TNF-α/interferon-gamma (IFN-γ). Enhanced expression of factors related to skin barrier function, FLG, IVL, and LOR, was confirmed. These findings implied that CFE may be used as a therapeutic agent against AD due to its skin barrier-strengthening and anti-inflammatory activities, which are derived from natural marine products.

Novel Signal Peptides and Episomal Plasmid System for Enhanced Protein Secretion in Engineered Bacteroides Species.

The genus Bacteroides, a predominant group in the human gut microbiome, presents significant potential for microbiome engineering and the development of live biotherapeutics aimed at treating gut diseases. Despite its promising capabilities, tools for effectively engineering Bacteroides species have been limited. In our study, we have made a breakthrough by identifying novel signal peptides in Bacteroides thetaiotaomicron and Akkermansia muciniphila. These peptides facilitate efficient protein transport across cellular membranes in Bacteroides, a critical step for therapeutic applications. Additionally, we have developed an advanced episomal plasmid system. This system demonstrates superior protein secretion capabilities compared to traditional chromosomal integration plasmids, making it a vital tool for enhancing the delivery of therapeutic proteins in Bacteroides species. Initially, the stability of this episomal plasmid posed a challenge; however, we have overcome this by incorporating an essential gene-based selection system. This novel strategy not only ensures plasmid stability but also aligns with the growing need for antibiotic-free selection methods in clinical settings. Our work, therefore, not only provides a more robust secretion system for Bacteroides but also sets a new standard for the development of live biotherapeutics.

Engineered Methylococcus capsulatus Bath for efficient methane conversion to isoprene.

Isoprene has numerous industrial applications, including rubber polymer and potential biofuel. Microbial methane-based isoprene production could be a cost-effective and environmentally benign process, owing to a reduced carbon footprint and economical utilization of methane. In this study, Methylococcus capsulatus Bath was engineered to produce isoprene from methane by introducing the exogenous mevalonate (MVA) pathway. Overexpression of MVA pathway enzymes and isoprene synthase from Populus trichocarpa under the control of a phenol-inducible promoter substantially improved isoprene production. M. capsulatus Bath was further engineered using a CRISPR-base editor to disrupt the expression of soluble methane monooxygenase (sMMO), which oxidizes isoprene to cause toxicity. Additionally, optimization of the metabolic flux in the MVA pathway and culture conditions increased isoprene production to 228.1 mg/L, the highest known titer for methanotroph-based isoprene production. The developed methanotroph could facilitate the efficient conversion of methane to isoprene, resulting in the sustainable production of value-added chemicals.

Development of in-vitro pulsatile flow generator for evaluating the performance of hemodialysis catheters.

Hemodialysis (HD) using an HD catheter is performed widely on renal failure patients. The catheter was evaluated using the recirculation ratio in pre-clinical status, which is a crucial index indicating its performance. However, pre-clinical in-vivo experiments have limitations: high cost, and ethical issues. Hence, computational and in-vitro methods have been developed as alternatives. However, computational methods require fluid dynamic knowledge, whereas in-vitro experiments are complicated and expensive. In this study, we developed a pulsatile flow generator to mimic blood flow achieving cost effectiveness and user convenience. The device used iterative learning control, achieving blood flow in the superior and inferior vena cava within a 3.3% error. Furthermore, the recirculation ratios were measured based on two insertion directions and two different external pipe materials to evaluate the catheter regarding patients' posture and blood vessel stiffness. The results provide a better understanding of cardiovascular device performance without complicated and costly pre-clinical tests.

Novel autosomal dominant TMC1 variants linked to hearing loss: insight into protein-lipid interactions.

BACKGROUND: TMC1, which encodes transmembrane channel-like protein 1, forms the mechanoelectrical transduction (MET) channel in auditory hair cells, necessary for auditory function. TMC1 variants are known to cause autosomal dominant (DFNA36) and autosomal recessive (DFNB7/11) non-syndromic hearing loss, but only a handful of TMC1 variants underlying DFNA36 have been reported, hampering analysis of genotype-phenotype correlations. METHODS: In this study, we retrospectively reviewed 338 probands in an in-house database of genetic hearing loss, evaluating the clinical phenotypes and genotypes of novel TMC1 variants associated with DFNA36. To analyze the structural impact of these variants, we generated two structural models of human TMC1, utilizing the Cryo-EM structure of C. elegans TMC1 as a template and AlphaFold protein structure database. Specifically, the lipid bilayer-embedded protein database was used to construct membrane-embedded models of TMC1. We then examined the effect of TMC1 variants on intramolecular interactions and predicted their potential pathogenicity. RESULTS: We identified two novel TMC1 variants related to DFNA36 (c.1256T > C:p.Phe419Ser and c.1444T > C:p.Trp482Arg). The affected subjects had bilateral, moderate, late-onset, progressive sensorineural hearing loss with a down-sloping configuration. The Phe419 residue located in the transmembrane domain 4 of TMC1 faces outward towards the channel pore and is in close proximity to the hydrophobic tail of the lipid bilayer. The non-polar-to-polar variant (p.Phe419Ser) alters the hydrophobicity in the membrane, compromising protein-lipid interactions. On the other hand, the Trp482 residue located in the extracellular linker region between transmembrane domains 5 and 6 is anchored to the membrane interfaces via its aromatic rings, mediating several molecular interactions that stabilize the structure of TMC1. This type of aromatic ring-based anchoring is also observed in homologous transmembrane proteins such as OSCA1.2. Conversely, the substitution of Trp with Arg (Trp482Arg) disrupts the cation-π interaction with phospholipids located in the outer leaflet of the phospholipid bilayer, destabilizing protein-lipid interactions. Additionally, Trp482Arg collapses the CH-π interaction between Trp482 and Pro511, possibly reducing the overall stability of the protein. In parallel with the molecular modeling, the two mutants degraded significantly faster compared to the wild-type protein, compromising protein stability. CONCLUSIONS: This results expand the genetic spectrum of disease-causing TMC1 variants related to DFNA36 and provide insight into TMC1 transmembrane protein-lipid interactions.

Changes in ankle and hip joints following medial opening-wedge high tibial osteotomy affect knee joint line obliquity.

PURPOSE: This study aimed to measure the change in knee joint line obliquity (KJLO) and the changes in radiologic parameters of the ankle and hip joints after medial opening-wedge high tibial osteotomy (MOWHTO), and to evaluate the correlation and causal relationship between these parameters. METHODS: This study evaluated 109 patients who underwent MOWHTO between April 2015 and December 2021. Radiologic parameters, including KJLO, medial proximal tibial angle (MPTA), ankle joint line obliquity (AJLO), and hip abduction angle (HAA), were analysed before and 1 year after MOWHTO. Multiple linear regression analysis was used to identify independent variables that significantly affected the change in KJLO after MOWHTO. Receiver operating characteristic (ROC) analysis was used to evaluate the cutoff value for a change in KJLO that exceeded 5° postoperatively, and the predicting values of radiologic parameters. RESULTS: Multiple linear regression analysis showed that changes in MPTA, AJLO, and HAA (ß = 0.440, P < 0.001; ß = - 0.310, P < 0.001; ß = 0.164, P = 0.035, respectively) were predictors of the change in KJLO after MOWHTO. ROC analysis showed that the threshold value for a change in KJLO which exceeded 5° postoperatively was 4.6° (66.7% sensitivity, 63.8% specificity, P = 0.025). Moreover, ROC curves for predicting a change in KJLO of > 4.6° showed that the AUC was significantly higher for the change in MPTA than that of the other two parameters (P = 0.011 for AJLO and P < 0.001 for HAA). CONCLUSION: MOWHTO increases the KJLO by valgization of the proximal tibia and causes hip adduction and ankle valgization. The postoperative ankle valgization after MOWHTO could reduce the increase in KJLO, counteracting the effects of proximal tibial valgization and hip adduction. Therefore, the effects of the hip and ankle joints should be considered to achieve an optimal KJLO and satisfactory clinical outcomes after MOWHTO. LEVEL OF STUDY: Cohort study, IV.

Serial change of femoral and tibial tunnel width after anterior cruciate ligament reconstruction with allograft.

PURPOSE: To investigate progressive tunnel widening and its correlation with postoperative outcomes after anterior cruciate ligament (ACL) reconstruction using allografts. METHODS: Sixty-five patients who underwent ACL reconstruction using a tibialis anterior allograft between 2015 and 2017 were enrolled. Femoral and tibial tunnel widths were measured on anteroposterior (AP) and lateral radiographs immediately and at 3, 6, 12, and 24 months postoperatively. Average femoral and tibial tunnel widths in AP and lateral views were calculated at three different measurement points. Tunnel widening was calculated as the difference in tunnel width immediately and 2 years postoperatively. The correlation between tunnel widening and the postoperative results was analysed. RESULTS: Tunnel width changes between immediate and 2 years postoperatively were as follows, in AP and lateral views, respectively: femur, 3.0 mm ± 1.5 mm and 2.4 mm ± 1.4 mm; and tibia, 2.8 mm ± 1.4 mm and 2.9 mm ± 1.5 mm. Femoral tunnel widths significantly increased until 1 year, but not from 1 to 2 years postoperatively. Tibial tunnel width significantly increased until 2 years postoperatively. In all tunnels, the increments in tunnel widening decreased over time. Increased knee laxity significantly correlated with greater femoral tunnel widening in AP (r = 0.346, P = 0.006) and lateral views (r = 0.261, P = 0.049). CONCLUSION: Femoral tunnel widths gradually increased until 1 year postoperatively, and tibial tunnel widths increased until 2 years after ACL reconstruction with allografts. The tunnel widening rate gradually decreased over time. Femoral tunnel widening of 3.7 mm and 3.2 mm on AP and lateral views, respectively, were the cut-off values for postoperative knee laxity. LEVEL OF EVIDENCE: Level III.