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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.

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 efect of press-it structural impacted bone allograft with locking plate ixation 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 inal 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-efect model was used for repeated measures over time of the clinical scores. ResultsThe NRS decreased by 1.5 at 1 month (p < 0.01) and 2.1 at 3 months (p < 0.01), while KOOS pain signiicantly 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. ConclusionThe 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).

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. Candida albicans is an opportunistic human fungal pathogen. This pathogen is able to cause relatively harmless superficial infections of the skin and mucosa or severe invasive bloodstream infections with a mortality rate of 46 to 75% 1. Because of the limited amount of antifungal drug classes available and the diverse set of resistance mechanisms against existing antifungals, research on this pathogenic fungus is necessary in order to discover novel targets for antifungal therapy 2. Molecular research on yeast species such as C. albicans relies on biochemical techniques and tools that were often first developed for use in the model organism Saccharomyces cerevisiae or mammalian cell systems. For example; bimolecular fluorescence complementation (BiFC) was first applied in COS-1 cells and has only recently been introduced in C. albicans by our group 3,4. Even simple plasmids from the closely related yeast S. cerevisiae have to be adapted because of the instability of episomal plasmids in C. albicans and the CUG codon, which is translated as serine instead of leucine in 97 out of 100 cases 5,6. Heterologous expression of genes in C. albicans requires additional manipulations, such as specific codon adaptations due to the previously described CUG codon, but also due to differing overall codon usage of C. albicans compared to other fungi 7. Even in Escherichia coli and S. cerevisiae, where significant enhancements in synthetic biology were already made, acquiring a functional and highly expressed heterologous protein often requires the construction and screening of large libraries of codon variants 8,9. Furthermore, the selection of a specific strain for overexpressing a protein can play a role in protein solubility and expression levels, since codon bias-adjusted strains do not always improve protein folding 10. There is a clear need for a better understanding of codon usage and its application in heterologous expression. Light microscopy is one...

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.

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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.

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.