A multi-colour fluorogenic tag and its application in Candida albicans.

Fluorescent proteins (FPs) have always been a crucial part of molecular research in life sciences, including the research into the human fungal pathogen Candida albicans, but have obvious shortcomings such as their relatively large size and long maturation time. However, the next generation of FPs overcome these issues and rely on the binding of a fluorogen for the protein to become fluorescently active. This generation of FPs includes the improved version of Fluorescence activating and Absorption Shifting Tag (iFAST). The binding between the fluorogen and the iFAST protein is reversible, thus resulting in reversible fluorescence. The fluorogens of iFAST are analogues of 4-hydroxylbenzylidene-rhodanine (HBR). These HBR analogues differ in spectral properties depending on functional group substitutions, which gives the iFAST system flexibility in terms of absorbance and emission maxima. In this work we describe and illustrate the application of iFAST as a protein tag and its reversible multi-colour characteristics in C. albicans.

No clinical outcome difference between varus phenotypes after medial opening-wedge high tibial osteotomy at 2 years follow-up.

PURPOSE: Clinical studies regarding medial open-wedge high tibial osteotomy (MOWHTO) often analyse a large group of mechanical varus knees rather than differentiating for its primary varus-inducing component. This study aims to compare the radiological and clinical outcomes of the most prevalent varus malalignment phenotypes using the coronal plane alignment of the knee (CPAK) classification. METHODS: MOWHTO cases with minimal 2-year clinical follow-up were retrospectively selected from a knee osteotomy database (2016-2020). Based on the medial proximal tibial angle (MPTA) and lateral distal femoral angle (LDFA), subjects were allocated to the correct CPAK phenotype pre- and postoperatively. Clinical outcomes were the numeric rating scale (NRS), the knee injury and osteoarthritis outcome score (KOOS) and the therapeutic response rate (TRR) at 2-year follow-up. Inter-observer correlation coefficient (ICC) and unpaired student t test were performed for cross-phenotype comparison. RESULTS: One hundred thirty-five (135) subjects were found eligible (53.0 years old ±9.6 [19-77], 72% male, 53% left-sided). The most prevalent preoperative phenotype was CPAK 1 (n = 70 (52%)) and the postoperative phenotype was CPAK 6 (n = 66 (49%)). All CPAK phenotypes improved significantly relative to baseline but cross-phenotype comparison yielded no significant differences in clinical outcome. The TRR at 2 years was 67% for CPAK 1, 69% for CPAK 2 and 87% for CPAK 4. The TRR for CPAK 6 was 64% compared with 80% for CPAK 9, which was not significantly different. CONCLUSION: At 2-year follow-up, no clinically significant differences are observed between different CPAK phenotypes. Accurate MOWHTO corrections provide significant clinical improvement even in the femoral-driven varus knee and the constitutional varus knee dominated by intra-articular wear. The clinical indication for MOWHTO performance should not be reduced to the medial arthritic varus knee with underlying tibial varus alone. LEVEL OF EVIDENCE: Level IV, retrospective comparative study.

The position of the lateral tibial spine and the implications for high tibial osteotomy planning.

The lateral tibial spine (LTS) is frequently proposed as a correction target in high tibial osteotomy (HTO), although little is known about its exact radiographic position. This study primarily aims to define the position and variance of the LTS. Secondly, this study wants to investigate the relevance of the LTS position on the mechanical tibiofemoral angle (mTFA°) while planning and postoperatively landing the weight-bearing line (WBL) on this landmark. The LTS position was studied on preoperative full-leg standing radiographs (FLSR) and computed tomography (CT) scans in 70 cases. 3D models of the tibia were created in Mimics 23.0 and measurements were conducted in 3-matic 15.0 (Materialise, Leuven®). Next, 100 HTO cases were retrospectively planned with the WBL through the LTS according to Dugdale's method on FLSR. Finally, 55 postoperative FLSR which had the WBL on the LTS (±2%) were assessed for mTFA° outcome. Statistics were conducted in GraphPad 8.0. The LTS was located at 58.3%±1.9 [55-63%] in 2D and 57.3%±2.2 [53-63%] in 3D showing a high correlation (r=0.77 [0.65 to 0.85]). The planned mTFA on the LTS was 181.8°±0.3 (181.3°-182.5°). On postoperative FLSR, the mTFA was 182.2°±0.6 (180.9°-183.1°). The lateral tibial spine is located at 57-58% on the tibial plateau with a 10% maximal variation range. Good agreement was found between 2D and 3D imaging modalities while evaluating its position in the coronal plane. When aiming the WBL through the LTS during valgus-producing HTO, a consistent realignment of 181-183° mTFA can be expected when performing accurate surgery.

The intricate link between iron, mitochondria and azoles in Candida species.

Invasive fungal infections are rapidly increasing, and the opportunistic pathogenic Candida species are the fourth most common cause of nosocomial systemic infections. The current antifungal classes, of which azoles are the most widely used, all have shortcomings. Azoles are generally considered fungistatic rather than fungicidal, they do not actively kill fungal cells and therefore resistance against azoles can be rapidly acquired. Combination therapies with azoles provide an interesting therapeutic outlook and agents limiting iron are excellent candidates. We summarize how iron is acquired by the host and transported towards both storage and iron-utilizing organelles. We indicate whether these pathways alter azole susceptibility and/or tolerance, to finally link these transport mechanisms to mitochondrial iron availability. In this review, we highlight putative novel intracellular iron shuffling mechanisms and indicate that mitochondrial iron dynamics in relation to azole treatment and iron limitation is a significant knowledge gap.

Oteseconazole: a long-awaited diversification of the antifungal arsenal to manage recurrent vulvovaginal candidiasis (RVVC).

INTRODUCTION: Recurrent vulvovaginal candidiasis (RVVC) affects women worldwide and has far-reaching implications for a patient's quality of life. For decades, maintenance treatment using the azole antifungal fluconazole was the preferred treatment. Although efficient in controlling the symptoms, the development of azole resistance and high rates of recurrence after therapy cessation have emerged as significant limitations. Nevertheless, persistent efforts have delivered novel treatment options. Oteseconazole (VT-1161), marketed as VIVJOA, is an oral, tetrazole antifungal with unprecedented specificity toward the fungal lanosterol 14α-demethylase. AREAS COVERED: We reviewed literature data on oteseconazole with a focus on the management of RVVC. EXPERT OPINION: Therapeutic options for RVVC are limited, and novel, innovative approaches are needed to treat this debilitating condition. These therapies need to be well-tolerated and prevent RVVC recurrence. The available clinical data show excellent safety and efficacy, with an unprecedentedly low recurrence rate. However, we believe health-care providers should be mindful to monitor for the development of resistance, as this may result in treatment failure. Further, the availability and cost may, like for most novel drugs, affect the widespread clinical implementation of VIVJOA. Altogether, we are convinced that VIVJOA is a significant advance in RVVC management.

A novel patient-specific 3D printed guide for accurate femoral derotation osteotomies: a case report.

Background: Femoral malrotation errors are prevalent after intramedullary (IM) nailing for femur fractures. Supposing fracture consolidation has occurred, only a derotation osteotomy can offer a solution to this complication, despite in situ nail interference. The authors present a novel case-based surgical technique using 3D technology to obtain an accurate derotation correction and desired clinical outcome while facilitating surgery technics. Case Description: A 32-year-old woman was referred to our clinic with ongoing right anterior groin pain. Three months earlier, she sustained a high energy fall resulting in a subtrochanteric femur fracture. This was treated with a long IM femoral nail (PFNA™, Synthes, Solothurn, Switzerland) elsewhere. Postoperatively, she developed a limping gait pattern with 'toeing-in' and persistent hip pain during walking. In supine position, a notable axial malalignment towards endo-rotation was visible as compared to the contralateral side. CT-scan showed a side to side difference of 36° anteversion. It was decided to perform a subtrochanteric femoral derotation osteotomy. Outcomes concerning restoration of painless gait and bony healing were successfully obtained at 4 months. Conclusions: The application of a 3D guide with uni-cortical K-wire placement for derotation osteotomies provides additional correction control during surgery and accurate outcome, while facilitating the flow of this technically demanding procedure.

Impacted bone allograft personalised by a novel 3D printed customization kit produces high surgical accuracy in medial opening wedge high tibial osteotomy: a pilot study.

PURPOSE: Contemporary medial opening wedge high tibial osteotomy (MOWHTO) still seems to struggle with inconsistent accuracy outcomes. Our objective was to assess surgical accuracy and short-term clinical outcomes when using 3D planning and a patient-specific instrumentation (PSI) kit to prepare customized bone allografts. METHODS: Thirty subjects (age 48y ± 13) were included in a double-center prospective case series. A low-dose CT-scan was performed to generate 3D bone models, a MOWHTO was simulated, and PSI was designed and 3D printed based on the complementary negative of the planned osteotomy gap. Clinical outcome was assessed at two, four, 12 weeks and one year using NRS, KOOS, UCLA activity score, EQ-5D and anchor questions. A linear-mixed model approach was implemented for data analysis. RESULTS: Preoperative 3D values were 175.0° ± 2.2 mechanical tibiofemoral angle (mTFA), 85.0° ± 3.0 medial proximal tibial angle (MPTA), and 94.1° ± 3.4 medial posterior tibial slope (MPTS). Target planning ranged from slight varus to the lateral tibial spine (slight valgus). Postoperative 3D analysis showed an accuracy of 1.1° ± 0.7 ΔMPTA (p = 0.04) and 1.2° ± 1.2 ΔMPTS (p = 0.11). NRS decreased from baseline 6.1 ± 1.9 to 2.7 ± 1.9 at four weeks (p < 0.001) and 1.7 ± 1.9 at one year (p < 0.001). KOOS increased from 31.4 ± 17.6 to 50.6 ± 20.6 at 12 weeks (p < 0.001) and to 71.8 ± 15.6 at one year (p < 0.001). CONCLUSION: The study suggests that 3D printed instrumentation to personalize structural bone allograft is a viable alternative method in MOWHTO that has the benefit of optimizing surgical accuracy while providing early and consistent pain relief after surgery.

Assessment of cAMP-PKA Signaling in Candida glabrata by FRET-Based Biosensors.

Fluorescence or Förster resonance energy transfer (FRET)-based biosensors are used to monitor activity through molecular pathways inside the cell. Binding of secondary metabolites or enzyme-guided modification of protein targets can be assessed by quantifying the rate of energy transfer between two adequate fluorophores. The AKAR3 sensor contains a protein kinase A (PKA) phosphorylation site, which upon phosphorylation interacts with a ligand domain, bringing together FRET donor and acceptor fluorophores and allowing FRET. The EPAC2 sensor contains a cyclic adenosine monophosphate (cAMP)-binding domain. Upon binding of cAMP, donor and acceptor molecules are separated, thereby lowering energy transfer. Since the cAMP-PKA pathway is of great importance for regulation of growth, survival, and virulence in Candida species, monitoring the activity of this pathway in a time- and space-resolved manner allows for detailed investigation of potential drug targets. In this chapter, we describe how these FRET-based biosensors can be used in a practical setup.

Human meniscus allograft augmentation by allogeneic mesenchymal stromal/stem cell injections.

Meniscus allograft transplantations (MATs) represent established surgical procedures with proven outcomes. Yet, storage as frozen specimens and limited cellular repopulation may impair graft viability. This proof-of-concept study tests the feasibility of injecting allogeneic mesenchymal stromal/stem cells (MSCs) in meniscus allograft tissue. We investigated the injectable cell quantity, survival rate, migration, and proliferation ability of MSCs up to 28 days of incubation. In this controlled laboratory study, seven fresh-frozen human allografts were injected with human allogeneic MSCs. Cells were labeled and histological characteristics were microscopically imaged up to 28 days. Mock-injected menisci were included as negative controls in each experiment. Toluidine blue staining demonstrated that a 100-µl volume can be injected while retracting and rotating the inserted needle. Immediately after injection, labeled MSCs were distributed throughout the injection channel and eventually migrated into the surrounding tissues. Histological assessment revealed that MSCs cluster in disc-like shapes, parallel to the intrinsic lamination of the meniscus and around the vascular network. Quantification showed that more than 60% of cells were present in horizontally injected grafts and more than 30% were observed in vertically injected samples. On Day 14, cells adopted a spindle-shaped morphology and exhibited proliferative and migratory behaviors. On Day 28, live/dead ratio assessment revealed an approximately 80% cell survival. The study demonstrated the feasibility of injecting doses of MSCs (>0.1 million) in meniscus allograft tissue with active cell proliferation, migration, and robust cell survival.

The Sound of Cartilage Repair: The Importance of Using Pitch and Volume Cues in Cartilage Restoration Surgery.

Articular cartilage defects are common and can result in substantial pain and disability, prompting operative intervention, which commonly includes chondral debridement. Controlled defect preparation up to but not beyond the calcified cartilage layer is key to clinical success, but this remains technically challenging. We present a technique highlighting the substantial decrease in curette stroke volume and associated shift to a lower pitch when achieving satisfactory open cartilage defect debridement. These audiologic cues correlate well with histologic accuracy of debridement. Therefore, quantifiable pitch and volume changes serve as valuable technical cues for precise defect preparation at the time of joint preservation surgery. CLASSIFICATIONS: I: knee, II: cartilage.

Autologous Micro-Fragmented Adipose Tissue (MFAT) to Treat Symptomatic Knee Osteoarthritis: Early Outcomes of a Consecutive Case Series.

The study aimed to evaluate the short-term clinical effect, therapeutic response rate (TRR%), and therapy safety of a single intra-articular autologous MFAT injection for symptomatic knee OA. Secondly, patient- and pathology-related parameters were investigated to tighten patient selection for MFAT therapy. Sixty-four subjects with symptomatic mild-severe knee OA were enrolled in a single-center trial and received a unilateral (n = 37) or bilateral (n = 27) MFAT injection. After liposuction, the adipose tissue was mechanically processed with the Lipogem® device, which eventually produced 8-10 cc of MFAT. Subjects were clinically assessed by means of the KOOS, NRS, UCLA, and EQ-5D at baseline and 1, 3, 6, and 12 months after injection. Adverse events were recorded at each follow-up timepoint. The TRR was defined according to the OMERACT-OARSI criteria and baseline MRI was scored following the MOAKS classification. The TRR of the index knee was 64% at 3 months and 45% at 12 months after injection. Therapy responders at 12 months improved with 28.3 ± 11.4 on KOOS pain, while non-responders lost -2.1 ± 11.2 points. All clinical scores, except the UCLA, improved significantly at follow-up compared to baseline (p < 0.05). In the bilateral cohort, no difference in baseline scores or TRR was found between the index knee and contralateral knee (n.s.). An inflammatory reaction was reported in 79% of knees and resolved spontaneously within 16.6 ± 13.5 days after MFAT administration. Numerous bone marrow lesions (BML) were negatively correlated with the TRR at 12 months (p = 0.003). The study demonstrated an early clinical improvement but a mediocre response rate of 45% at 12 months after a single intra-articular injection with autologous MFAT. Assessment of bone marrow lesions on MRI can be helpful to increase the therapeutic responsiveness of MFAT up to 70% at 12 months. In comparison to repetitive injection therapies such as cortisone, hyaluronic acid, and PRP, administration of MFAT might become a relevant alternative in well-selected patients with symptomatic knee OA.

Photochromic Fluorophores Enable Imaging of Lowly Expressed Proteins in the Autofluorescent Fungus Candida albicans.

Fluorescence microscopy is a standard research tool in many fields, although collecting reliable images can be difficult in systems characterized by low expression levels and/or high background fluorescence. We present the combination of a photochromic fluorescent protein and stochastic optical fluctuation imaging (SOFI) to deliver suppression of the background fluorescence. This strategy makes it possible to resolve lowly or endogenously expressed proteins, as we demonstrate for Gcn5, a histone acetyltransferase required for complete virulence, and Erg11, the target of the azole antifungal agents in the fungal pathogen Candida albicans We expect that our method can be readily used for sensitive fluorescence measurements in systems characterized by high background fluorescence.IMPORTANCE Understanding the spatial and temporal organization of proteins of interest is key to unraveling cellular processes and identifying novel possible antifungal targets. Only a few therapeutic targets have been discovered in Candida albicans, and resistance mechanisms against these therapeutic agents are rapidly acquired. Fluorescence microscopy is a valuable tool to investigate molecular processes and assess the localization of possible antifungal targets. Unfortunately, fluorescence microscopy of C. albicans suffers from extensive autofluorescence. In this work, we present the use of a photochromic fluorescent protein and stochastic optical fluctuation imaging to enable the imaging of lowly expressed proteins in C. albicans through the suppression of autofluorescence. This method can be applied in C. albicans research or adapted for other fungal systems, allowing the visualization of intricate processes.

Fluorescent toys 'n' tools lighting the way in fungal research.

Although largely overlooked compared to bacterial infections, fungal infections pose a significant threat to the health of humans and other organisms. Many pathogenic fungi, especially Candida species, are extremely versatile and flexible in adapting to various host niches and stressful situations. This leads to high pathogenicity and increasing resistance to existing drugs. Due to the high level of conservation between fungi and mammalian cells, it is hard to find fungus-specific drug targets for novel therapy development. In this respect, it is vital to understand how these fungi function on a molecular, cellular as well as organismal level. Fluorescence imaging allows for detailed analysis of molecular mechanisms, cellular structures and interactions on different levels. In this manuscript, we provide researchers with an elaborate and contemporary overview of fluorescence techniques that can be used to study fungal pathogens. We focus on the available fluorescent labelling techniques and guide our readers through the different relevant applications of fluorescent imaging, from subcellular events to multispecies interactions and diagnostics. As well as cautioning researchers for potential challenges and obstacles, we offer hands-on tips and tricks for efficient experimentation and share our expert-view on future developments and possible improvements.

Joint Surface Lesions in the Knee Treated with an Acellular Aragonite-Based Scaffold: A 3-Year Follow-Up Case Series.

OBJECTIVE: The study aimed to evaluate the clinical outcome and repair capacity of a cell-free aragonite-based scaffold in patients with an isolated symptomatic joint surface lesion (JSL) of the knee. DESIGN: Thirteen patients (age 33.5 ± 8.9; female 23%; body mass index 25.3 ± 3.4, K/L [Kellgren-Lawrence] 1.8) with a JSL (2.6 ± 1.7 cm2 [1.0-7.5 cm2]) of the distal femur were enrolled in a single-center prospective case series. Safety and clinical outcome was assessed by the KOOS (Knee Injury and Osteoarthritis Outcome Score), IKDC (International Knee Documentation Committee), Lysholm, and Tegner activity scale at baseline and 6, 12, 18, 24, and 36 months follow-up. The MOCART 2.0 and scaffold integration were evaluated on magnetic resonance imaging at 12, 24, and 36 months postoperatively. RESULTS: Primary outcome (KOOS pain) improved with 36.5 ± 14.7 points at 12 months (P = 0.002) and 41.2 ± 14.7 points at 36 months (P = 0.002) follow-up. Similar increasing trends were observed for the other KOOS subscales, IKDC, and Lysholm score, which were significantly better at each follow-up time point relative to baseline (P < 0.05). Activity level increased from 2.75 ± 1.6 to 4.6 ± 2.2 points at final follow-up (P = 0.07). The MOCART was 61.7 ± 12.6 at 12 months and 72.9 ± 13.0 at 36 months postoperatively. Sixty-six to 100% implant integration and remodeling was observed in 73.3% cases at 36 months. No serious adverse events were reported. CONCLUSION: The study demonstrated that the biphasic aragonite-based scaffold is a safe and clinically effective implant for treating small-medium sized JSLs of the distal femur in a young and active patient cohort. The implant showed satisfying osteointegration and restoration of the osteochondral unit up to 3 years postimplantation.

Feasibility and 3D Planning of a Novel Patient-Specific Instrumentation Technique in Medial Opening-Wedge High Tibial Osteotomy.

A novel approach for opening-wedge high tibial osteotomy (OWHTO) with patient-specific instrumentation (PSI) was evaluated for its safety, feasibility, and accuracy. Next, the mechanical medial proximal tibial angle (mMPTA) was assessed as a potential planning angle by investigating the relation with the mechanical femorotibial angle (mFTA). Ten OWHTO cases were 3D planned using the mMPTA and operated with a customized 3D-printed wedge and cast which resembled the intended osteotomy opening. Patients were closely monitored for intraoperative and postoperative complications up to 1 year after surgery. Radiological assessment was conducted on full leg standing radiographs and supine lower limb computed tomography-scans preoperatively and 3 months after surgery. No intraoperative complications or logistical issues during PSI processing were observed. Absolute accuracy outcomes showed a correction error of 1.3° ± 1.1 mMPTA and 0.9° ± 0.6 mFTA with all osteotomies falling in (-2°; + 2°) mFTA around the target. The mMPTA and mFTA were found to have a strong correlation in both 3D (r = 0.842, p = 0.002) and 2D (r = 0.766, p = 0.01) imaging for effective correction. The study confirmed the development of a safe and feasible PSI technique in OWHTO with excellent accuracy outcomes. The strong correlation between the mMPTA and mFTA indicated that soft tissue changes after OWHTO are of minor significance to the final alignment in ligament-stable patients. Finally, the mMPTA was found to be a reliable planning angle in 3D software for obtaining the intended lower limb realignment and its use can therefore be recommended in modern OWHTO planning.

Autologous protein solution as selective treatment for advanced patellofemoral osteoarthritis in the middle-aged female patient: 54% response rate at 1 year follow-up.

PURPOSE: The study wanted to investigate the benefit, durability and safety of autologous protein solution (APS) injection(s) in a middle-aged female-only cohort suffering predominantly from patellofemoral osteoarthritis. METHODS: Fifty females (aged 50.4 ± 6.5) with mainly moderate-severe (86%) patellofemoral cartilage wear (PFCW) were treated with a unilateral intra-articular APS injection. The KOOS, NRS, Kujala, UCLA and EQ-5D were assessed at baseline and 1, 3, 6, and 12 months post-injection. Therapeutic response rate (TRR) was based on KOOS pain improvement > 10 points. Absolute improvement for, respectively, therapy responders and non-responders was determined. Second APS injection was administered if improvement was deemed insufficient by the patient after 3 months. RESULTS: The TRR remained stable averaging to 53.7% at final follow-up with subjects improving overall from 40.3 ± 18.7 to 57.3 ± 24.8 points on KOOS pain (p = 0.0002) and from 48.4 ± 13.0 to 56.3 ± 18.1 points on Kujala (p = 0.0203) at 12 months. Significant improvement was observed for the other KOOS subscales and NRS at each follow-up. In absolute values, APS responders improved with 30.5 ± 11.4 points on KOOS pain at 12 months. In contrast, non-responders deteriorated with 5.9 ± 8.9 points relative to baseline. A second APS injection was administered in 28 subjects. Patients with definite synovitis improved more on KOOS symptoms (p = 0.017) and KOOS ADL (p = 0.037) at 12 months compared to non-synovitis subjects. Mild-moderate arthralgia (46%) and effusion (29%) were commonly observed during the first month post-injection. CONCLUSION: This study evidenced a 54% response rate at 12 months to a single or second APS injection in a middle-aged female population with advanced patellofemoral cartilage wear. Moderate temporary flares can be expected without affecting clinical outcomes. Second APS injection has low efficacy in initially poor responding patients after 3 months. Major synovitis on baseline MRI appeared to be a beneficial prognosticator for pain relief and functional improvement after APS. LEVEL OF EVIDENCE: IV.

Presenting a codon-optimized palette of fluorescent proteins for use in Candida albicans.

Fluorescent proteins with varying colors are indispensable tools for the life sciences research community. These fluorophores are often developed for use in mammalian systems, with incremental enhancements or new versions published frequently. However, the successful application of these labels in other organisms in the tree of life, such as the fungus Candida albicans, can be difficult to achieve due to the difficulty in engineering constructs for good expression in these organisms. In this contribution, we present a palette of Candida-optimized fluorescent proteins ranging from cyan to red and assess their application potential. We also compare a range of reported expression optimization techniques, and find that none of these strategies is generally applicable, and that even very closely related proteins require the application of different strategies to achieve good expression. In addition to reporting new fluorescent protein variants for applications in Candida albicans, our work highlights the ongoing challenges in optimizing protein expression in heterologous systems.

Structural allograft impaction enables fast rehabilitation in opening-wedge high tibial osteotomy: a consecutive case series with one year follow-up.

PURPOSE: Painful and slow recovery are the presumed disadvantages after opening-wedge high tibial osteotomy (HTO) and play a role in favouring arthroplasty as treatment for moderate isolated medial knee arthritis. The primary study objective was to investigate the effect of press-fit structural impacted bone allograft with locking plate fixation on early ambulation, postoperative pain levels, and resumption of daily-life activities in opening-wedge HTO. METHODS: A prospective consecutive opening-wedge HTO case series was conducted, including 103 patients with final follow-up at 1 year. Weight-bearing was allowed from the day after surgery "as tolerated" by the patient. Clinical assessment included the Numeric Rating Scale (NRS), Knee injury and Osteoarthritis Outcome Score (KOOS), and Lysholm score. Additionally, the Knee Society Score (KSS) was assessed during consultation at 1, 3, and 12 months postoperatively with special attention for clinical anchor questions. Required sample size was calculated and a linear mixed-effect model was used for repeated measures over time of the clinical scores. RESULTS: The NRS decreased by 1.5 at 1 month (p < 0.01) and 2.1 at 3 months (p < 0.01), while KOOS pain significantly improved with 19.2 (p < 0.01) by this time compared to baseline. Under reduced pain levels, 98% were able to walk > 500 m without support, while all patients were able to climb up and down the stairs 3 months postoperatively. CONCLUSION: The study strongly supports the initial hypothesis that applying structural triangular bone allograft in HTO leads to low postoperative pain levels, early ambulation, and excellent short-term clinical outcomes. Study results have the potential to alter the general perception about HTO being a painful procedure with painstakingly slow recovery and consequently encourage the consideration of HTO as a highly valuable joint-preserving option, while treating unicompartmental knee arthritis. LEVEL OF EVIDENCE: IV (case series).

A Versatile Protocol for Studying Anterior Cruciate Ligament Reconstruction in a Rabbit Model.

Anterior cruciate ligament (ACL) injuries are frequent, as >200,000 injuries occur in the United States alone each year. Owing to the risks for associated meniscus and cartilage damage, ACL injuries are a significant source of both orthopedic care and research. Given the extended recovery course after ACL injury, which often lasts 1-2 years, and is associated with limited participation in sports and activities of daily living for patients, there is a critical need for the evolution of new and improved methods for ACL repair. Subsequently, animal models of ACL reconstruction (ACLR) play a key role in the development and initial trialing of novel ACL interventions. This article provides a clear operative description and associated illustrations for a validated, institutional animal care and use committee, and veterinarian approved and facile model of ACLR to serve researchers investigating ACLR.

Introducing fluorescence resonance energy transfer-based biosensors for the analysis of cAMP-PKA signalling in the fungal pathogen Candida glabrata.

The cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway is central to signal transduction in many organisms. In pathogenic fungi such as Candida albicans, this signalling cascade has proven to be involved in several processes, such as virulence, indicating its potential importance in antifungal drug discovery. Candida glabrata is an upcoming pathogen of the same species, yet information regarding the role of cAMP-PKA signalling in virulence is largely lacking. To enable efficient monitoring of cAMP-PKA activity in this pathogen, we here present the usage of two FRET-based biosensors. Both variations in the activity of PKA and the quantity of cAMP can be detected in a time-resolved manner, as we exemplify by glucose-induced activation of the pathway. We also present information on how to adequately process and analyse the data in a mathematically correct and physiologically relevant manner. These sensors will be of great benefit for scientists interested in linking the cAMP-PKA signalling cascade to downstream processes, such as virulence, possibly in a host environment.