The Lasso-Loop Technique: A Simple Way to Attach the Semitendinosus Tendon to an Endobutton Device.

During reconstruction of the anterior cruciate ligament, graft preparation is an essential step. The semitendinosus tendon is most frequently used, usually with a 4-strand graft and endobutton fixation. Our lasso-loop technique for tendon fixation is rapid and without sutures, providing a graft with a regular diameter, with no weak points and satisfactory primary stability.

Protein folds as synapomorphies of the tree of life.

Several studies showed that folds (topology of protein secondary structures) distribution in proteomes may be a global proxy to build phylogeny. Then, some folds should be synapomorphies (derived characters exclusively shared among taxa). However, previous studies used methods that did not allow synapomorphy identification, which requires congruence analysis of folds as individual characters. Here, we map SCOP folds onto a sample of 210 species across the tree of life (TOL). Congruence is assessed using retention index of each fold for the TOL, and principal component analysis for deeper branches. Using a bicluster mapping approach, we define synapomorphic blocks of folds (SBF) sharing similar presence/absence patterns. Among the 1232 folds, 20% are universally present in our TOL, whereas 54% are reliable synapomorphies. These results are similar with CATH and ECOD databases. Eukaryotes are characterized by a large number of them, and several SBFs clearly support nested eukaryotic clades (divergence times from 1100 to 380 mya). Although clearly separated, the three superkingdoms reveal a strong mosaic pattern. This pattern is consistent with the dual origin of eukaryotes and witness secondary endosymbiosis in their phothosynthetic clades. Our study unveils direct analysis of folds synapomorphies as key characters to unravel evolutionary history of species.

Percutaneous Medial Ligament Reconstruction for Valgus and Rotational Knee Instability.

The following surgical technique is intended for patients with chronic valgus laxity and rotational knee instability. It is a percutaneous 2-bundle ligament reconstruction method that uses the semitendinosus tendon, allowing it to remain pedicled to its distal tibial insertion. The aim is to correct the laxity without otherwise limiting the motion of the knee.

Percutaneous Medial Ligament Reconstruction for Valgus Knee Instability.

Injuries to stabilizing elements on the medial side of the knee are one of the most common knee ailments. Because of the good healing capacity of these structures, acute injuries are typically treated conservatively. However, valgus laxity near full extension can persist in some patients. This laxity may be the source of instability due to medial joint space opening, which then requires surgical treatment. Various procedures have been described that aim to reproduce the anatomy of the medial collateral ligament (MCL) and the posterior oblique ligament (POL), which work together to stabilize the medial aspect of the knee. However, these are complex open surgical procedures, technically demanding to achieve the favorable isometry, which prevent joint contracture or recurrence of laxity. The purpose of this study was to describe a short construct that minimizes the risk of secondary loss of tension and complies with the principle of favorable anisometry. The graft is positioned in the joint opening axis, between the deep bundle of the MCL and the POL.

Knee laxity modifications after ACL rupture and surgical intra- and extra-articular reconstructions: intra-operative measures in reconstructed and healthy knees.

PURPOSE: Quantifying the effects of anterior cruciate ligament (ACL) deficiency on knee joint laxity is fundamental for understanding the outcomes of its reconstruction techniques. The general aim of this study was to determine intra-operatively the main modifications in knee laxity before and after standard isolated intra-articular and additional extra-articular anterolateral reinforcement. Our main hypothesis was that laxity abnormalities, particularly axial rotation, can still result from these ACL reconstruction techniques. METHODS: Thirty-two patients with primary ACL deficiency were analysed by a navigation system immediately before and after each of the two reconstructions. Laxity measurements in terms of knee translations and rotations were taken during the anteroposterior drawer test, with internal-external rotation at 20° and 90° of flexion, and varus-valgus and pivot-shift tests. All these laxity measures were also taken originally from the contralateral healthy knee. RESULTS: With respect to the contralateral healthy knee, in the ACL-deficient knee significantly increased laxity (expressed in %) was found in the medial compared with that of the lateral compartment, respectively, 115 and 68 % in the drawer test at 20° flexion, and 55 and 46 % at 90° flexion. In the medial compartment, a significant 35 % increment was also observed for the coupled tibial anteroposterior translation during axial knee rotation at 20° of flexion. After isolated intra-articular reconstruction, normal values of anteroposterior laxity were found restored in the pivot-shift and drawer tests in the lateral compartment, but not fully in the medial compartment. After the reinforcement, laxity in the medial compartment was also found restored in the axial rotation test at 20° flexion. CONCLUSION: In ACL reconstruction, with respect to the contralateral knee, intra-articular plus additional anterolateral reinforcement procedures do not restore normal joint laxity. This combined procedure over-constrained the lateral compartment, while excessive laxity still persists at the medial one. LEVEL OF EVIDENCE: III.

High Throughput Random Mutagenesis and Single Molecule Real Time Sequencing of the Muscle Nicotinic Acetylcholine Receptor.

High throughput random mutagenesis is a powerful tool to identify which residues are important for the function of a protein, and gain insight into its structure-function relation. The human muscle nicotinic acetylcholine receptor was used to test whether this technique previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the α1 subunit of the channel was generated by error-prone PCR, and full length sequences of all 2816 mutants were retrieved using single molecule real time sequencing. Each α1 mutant was co-transfected with wildtype ß1, δ, and ε subunits, and the channel function characterized by an ion flux assay. To test whether the strategy could map the structure-function relation of this receptor, we attempted to identify mutations that conferred resistance to competitive antagonists. Mutant hits were defined as receptors that responded to the nicotinic agonist epibatidine, but were not inhibited by either α-bungarotoxin or tubocurarine. Eight α1 subunit mutant hits were identified, six of which contained mutations at position Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were studied further, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations made the channel resistant to the antagonists, not by impairing antagonist binding, but rather by producing a gain-of-function phenotype, e.g. increased agonist sensitivity. Our data show that random high throughput mutagenesis is applicable to multimeric proteins to discover novel functional mutants, and outlines the benefits of using single molecule real time sequencing with regards to quality control of the mutant library as well as downstream mutant data interpretation.

Pulmonary Artery Bullet Embolism following Cardiac Gunshot Wound.

Penetrating chest trauma is common but few need surgical treatment (10-20%). The mortality of gunshot wounds of the heart is 45%, among the wounded arriving at the hospital. The suspicion of wound heart with an inlet in the heart area (limited by costal awnings down, clavicles top, and mid-clavicular line outside) and pericardial effusion remains a surgical indication. Gunshot wounds of the heart with migration of the projectile in the pulmonary artery are rare. Migration of projectile into the pulmonary artery is described most often with lesions of peripheral veins without concomitant cardiac involvement. The indication of projectile extraction is not clearly defined in the literature. Conservative management of selected cases of pulmonary artery bullet emboli may be warranted in light of the risks of extraction.

Combined Anterior and Anterolateral Stabilization of the Knee With the Iliotibial Band.

Interest and knowledge on the anatomy, function, and biomechanical properties of the anterolateral ligament has led to the recognition of the importance of this structure in rotational control of the knee. This article describes a technique that allows for a combined anterior cruciate ligament (ACL) and anterolateral reconstruction, using an Iliotibial band (ITB) autograft. The graft is detached from the vastus lateralis from proximal to distal, at the center portion from ITB, preserving its distal insertion on the Gerdy tubercle. Its width is 1 cm for the distal part, used for the anterolateral ligament reconstruction, and 3 cm for the proximal part. An outside-in femoral tunnel is drilled respecting both the preferred favorable isometric femoral insertion site and the femoral ACL footprint. An ACL reconstruction combined with a lateral tenodesis with a continuous ITB graft respects the anatomical and isometric rules providing superior internal rotational control of the knee in comparison with a stand-alone ACL reconstruction.

Isometric Characteristics of the Anterolateral Ligament of the Knee: A Cadaveric Navigation Study.

PURPOSE: To measure the variations in length during flexion and internal tibial rotation of the 3 different femoral insertions of the anterolateral ligament (ALL) while maintaining a fixed tibia insertion. METHODS: Twelve fresh-frozen cadaver knees were analyzed using a navigation system. Maximal distance variations of the 3 different anatomic femoral insertions of the ALL were measured during knee flexion and internal tibial rotation at 20° (IR20°) and 90° (IR90°). The 3 different femoral attachments were, as published, at the center of the lateral epicondyle, distal and anterior from this position, and proximal and posterior. Each of these 3 femoral insertions was coupled to the same tibial insertion at the tibial margin, halfway between the tip of the fibular head and the prominence of the Gerdy tubercle. RESULTS: During IR20°, variation in the distance between paired points is not different between the proximal-posterior, epicondyle, and distal-anterior femoral insertions. These variations were statistically different during IR90° for the 3 different femoral locations. In increasing degrees of flexion, there was a length decrease between paired points observed with the proximal-posterior position. A length increase was observed for both the epicondyle location and the distal-anterior location. CONCLUSIONS: The ALL did not reveal an isometric behavior at any of the femoral insertion locations but had different length change patterns during knee flexion and internal tibial rotation at 90°. The proximal and posterior to epicondyle femoral position is the only position with a favorable isometry, as shown by being tight in extension and in internal rotation at 20° and then relaxed when the knee goes to flexion at 120° and during internal rotation at 90°. CLINICAL RELEVANCE: Clinical relevance is significant with respect to optimizing the femoral position of an ALL reconstruction.

The Involvement of the Anterolateral Ligament in Rotational Control of the Knee.

BACKGROUND: Rotational control of the knee is crucial for knee stability. The anterolateral ligament (ALL) has been identified as a potentially important structure involved in rotational control of the knee. PURPOSE/HYPOTHESIS: The purpose of this study was to determine, utilizing a navigation system, the involvement of the anterior cruciate ligament (ACL), the iliotibial band (ITB), and the ALL in tibial internal rotational control of the knee. The hypothesis was that the ALL would be involved in rotational control of the knee at varying degrees of knee flexion. STUDY DESIGN: Controlled laboratory study. METHODS: Twelve fresh-frozen cadaveric knees were tested in internal rotation at 20° and 90° of flexion and then subsequently tested using a simulated pivot-shift test consisting of coupled axial rotation at 30° of flexion. Serial sectioning of the ACL, ALL, and ITB was performed. On the contralateral knee, sectioning was performed in the reverse order. Measurements were collected using a surgical navigation system before and after each sectioning. RESULTS: After ACL sectioning, an incision of the ALL induced a significant increase in internal rotation (+19.2% [P = .0002] at 20°; +21.8% [P = .0029] at 90°) and in coupled axial rotation (+43.0%; P = .0035) compared with the intact knee as well as a significant increase in internal rotation at 90° (+13.4%; P = .009) and in coupled axial rotation (+30.8%; P = .0124) compared with the ACL-deficient knee. After ITB sectioning, an additional ALL section caused a significant increase in internal rotation (+39.0% [P = .002] at 20°; +63.0% [P = .0147] at 90°) and in coupled axial rotation (+59.7%; P = .0003) compared with the intact knee as well as a significant increase in internal rotation at 90° (+14.8%; P = .0067) in comparison to the ITB-deficient knee. CONCLUSION: The ALL is involved in rotational control of the knee at varying degrees of knee flexion and during a simulated pivot shift. Concomitant to an ACL or ITB transection, sectioning the ALL further increased rotational laxity. CLINICAL RELEVANCE: This laboratory study demonstrated that the ALL provides rotational control of the knee in combination with the ACL and/or ITB.

Femoral Origin of the Anterolateral Ligament: An Anatomic Analysis.

PURPOSE: To determine the location and variability of the anterolateral ligament (ALL) femoral origin. METHODS: The ALL was dissected and examined in 52 embalmed specimens, and the femoral origin was isolated. The presence of a bony or soft-tissue attachment, the relation to the lateral collateral ligament, the average diameter of the proximal origin, and the specific location of the origin relative to the lateral femoral epicondyle were recorded. RESULTS: The ALL was present in all 52 specimens, with a mean diameter of 11.85 mm, and was consistently attached to bone in all specimens. The ALL consistently overlapped the lateral collateral ligament near its attachment, with the location of the origin directly on the lateral epicondyle in 12 specimens (23%), with a shared lateral femoral condyle and with the origin slightly posterior and proximal to the lateral epicondyle in 30 specimens (58%), and with the origin completely posterior and proximal to the lateral epicondyle in 10 specimens (19%). CONCLUSIONS: The ALL showed a consistent bony origin overlapping the lateral collateral ligament in all specimens, with some variability in the femoral attachment, ranging from directly on the lateral epicondyle to posterior to the lateral epicondyle. CLINICAL RELEVANCE: The identification and description of the femoral origin of the ALL are crucial in understanding its role in the stability of the knee, as well as determining the appropriate position for the femoral origin placement in ALL reconstruction.

FR171456 is a specific inhibitor of mammalian NSDHL and yeast Erg26p.

FR171456 is a natural product with cholesterol-lowering properties in animal models, but its molecular target is unknown, which hinders further drug development. Here we show that FR171456 specifically targets the sterol-4-alpha-carboxylate-3-dehydrogenase (Saccharomyces cerevisiae--Erg26p, Homo sapiens--NSDHL (NAD(P) dependent steroid dehydrogenase-like)), an essential enzyme in the ergosterol/cholesterol biosynthesis pathway. FR171456 significantly alters the levels of cholesterol pathway intermediates in human and yeast cells. Genome-wide yeast haploinsufficiency profiling experiments highlight the erg26/ERG26 strain, and multiple mutations in ERG26 confer resistance to FR171456 in growth and enzyme assays. Some of these ERG26 mutations likely alter Erg26 binding to FR171456, based on a model of Erg26. Finally, we show that FR171456 inhibits an artificial Hepatitis C viral replicon, and has broad antifungal activity, suggesting potential additional utility as an anti-infective. The discovery of the target and binding site of FR171456 within the target will aid further development of this compound.

Human knee laxity in ACL-deficient and physiological contralateral joints: intra-operative measurements using a navigation system.

BACKGROUND: The comprehension of human knee laxity and of the failures of relevant surgical reconstructions of the anterior cruciate ligament (ACL) can be enhanced by the knowledge of the laximetric status of the contralateral healthy knee (CHK). Rarely this is available in patients, directly from the skeletal structures, and for a number of the standard clinical tests. The general aim of this study was to measure the extent to which laxity occurs immediately before surgery in the ACL deficient knee (ADK) with respect to CHK, in a number of standard clinical evaluation tests. METHOD: Thirty-two patients with ACL deficiency were analyzed at ADK and at CHK by a navigation system immediately before reconstructions. Knee laxity was assessed based on digitized anatomical references during the antero-posterior drawer, Lachman, internal-external rotation, varus-valgus, and pivot-shift tests. Antero-posterior laxity was normalized based on patient-specific length of the tibial plateau. RESULTS: In the drawer test, statistical significance (p<0.05) was found for the larger antero-posterior laxity in ADK than in CHK, on average, of 54' in the medial and 47' in the lateral compartments, when measured in normalized translations. In the Lachman test, these were about 106' and 68'. The pivot-shift test revealed a significant 70' larger antero-posterior central laxity and a 32' larger rotational laxity. No statistically relevant differences were observed in the other tests. CONCLUSION: The first conclusion is that it is important to measure also the antero-posterior and rotational laxity of the uninjured contralateral knee in assessing the laxity of the injured knee. A second is that the Lachman test shows knee laxity better than the AP drawer, and that the pivot-shift test was the only one able to reveal rotational instability. The present original measurements and analyses contribute to the knowledge of knee joint mechanics, with possible relevant applications in biomedical and clinical research.

Importance of correlation between gene expression levels: application to the type I interferon signature in rheumatoid arthritis.

BACKGROUND: The analysis of gene expression data shows that many genes display similarity in their expression profiles suggesting some co-regulation. Here, we investigated the co-expression patterns in gene expression data and proposed a correlation-based research method to stratify individuals. METHODOLOGY/PRINCIPAL FINDINGS: Using blood from rheumatoid arthritis (RA) patients, we investigated the gene expression profiles from whole blood using Affymetrix microarray technology. Co-expressed genes were analyzed by a biclustering method, followed by gene ontology analysis of the relevant biclusters. Taking the type I interferon (IFN) pathway as an example, a classification algorithm was developed from the 102 RA patients and extended to 10 systemic lupus erythematosus (SLE) patients and 100 healthy volunteers to further characterize individuals. We developed a correlation-based algorithm referred to as Classification Algorithm Based on a Biological Signature (CABS), an alternative to other approaches focused specifically on the expression levels. This algorithm applied to the expression of 35 IFN-related genes showed that the IFN signature presented a heterogeneous expression between RA, SLE and healthy controls which could reflect the level of global IFN signature activation. Moreover, the monitoring of the IFN-related genes during the anti-TNF treatment identified changes in type I IFN gene activity induced in RA patients. CONCLUSIONS: In conclusion, we have proposed an original method to analyze genes sharing an expression pattern and a biological function showing that the activation levels of a biological signature could be characterized by its overall state of correlation.

Minimally invasive extra-articular anterolateral reinforcement: a new technique.

Degenerative changes that occur after anterior cruciate ligament (ACL) reconstruction most often are due to poor rotary control with the use of usually 1-bundle intra-articular techniques. For this reason, double-bundle procedures were developed; however, they must be evaluated. The other solution designed to improve rotational stability, which was initiated by MacIntosh in the 1980s, involves extra-articular lateral reinforcement during intra-articular ACL reconstruction. This surgical combination is invasive because of the need for long grafts from the extensor apparatus or the fascia lata, and because anterolateral anatomic stabilizing structures must be dissected. In response to these criticisms, our technique uses the semitendinosus for the usual intra-articular reconstruction procedure and the gracilis in an effort to enhance the original minimally invasive procedure by performing an extra-articular lateral reinforcement procedure. The gracilis is folded to create a free 10-cm-long graft. Interference screws inside bone tunnels are used for femoral and tibial fixation of the graft. The site of fixation adheres to the best isometric principles of Krackow and Draganish. Drilling of the tunnels, insertion of the graft, and its fixation with interference screws are performed through two 1.5-cm-long incisions. Between them, the graft application is completed through Blount dissection under the fascia lata through the distal incision with a pincer.

Recommendations for the reduction of compound artifacts in time-resolved fluorescence resonance energy transfer assays.

Time-resolved (TR) fluorescence resonance energy transfer (FRET) is a widely accepted technology for high throughput screening (HTS), being able to detect and quantify the interactions of specific biomolecules in a homogeneous format. TR-FRET has several advantages for HTS applications that reduce assay artifacts such as compound interference. However, in some cases artifacts due to compound autofluorescence, color quenching, or signal stability are still observed. This report presents strategies addressing these issues by several means. One recommendation is the recording and visualization of differences in the donor/acceptor fluorescence, which allows the identification of compound artifacts. Another suggestion is to adjust the time delay, between excitation and recording of the fluorescence, in order to reduce compound interference. Furthermore, configuring the assay to allow the TR-FRET measurement to be taken at different time points, creating a reaction time course, allows background correction for each sample. Finally, the optimization of the FRET pair, to ensure assay signal stability under screening conditions, can improve the assay quality. This report presents examples of how these simple steps can be applied to enhance the quality of TR-FRET screening campaigns.