Physeal-sparing posteromedial portal approach reduced distance between guide pin and neurovascular structures.

PURPOSE: To compare a femoral physeal sparing anterior cruciate ligament (ACL) reconstruction technique utilizing a posteromedial portal to traditional transphyseal techniques with regards to anatomic tunnel positioning and proximity to important neurovascular structures. METHODS: Eight cadaveric knees were obtained for the study. Femoral tunnel guide pins were placed utilizing four different techniques: accessory anterior medial portal, posteromedial portal, trans-tibial, and figure-4 methods. The knees were then dissected and the following measurements taken: distance of each pin to the saphenous and peroneal nerve, popliteus tendon, neurovascular bundle, femoral ACL footprint and articular cartilage, and the angle of the guide pin entering the lateral femoral condyle. Fluoroscopic imaging was taken to determine the disruption of the physeal scar. RESULTS: Posteromedial portal guide pin was significantly closer to the neurovascular bundle, popliteal tendon and saphenous nerve when compared to the other 3 techniques, but was further from the peroneal nerve. It also had the smallest distance from the anatomic footprint of the ACL, and the largest angle to the lateral femoral condyle. The posteromedial portal guide pin had similar distance from the articular cartilage as the accessory anterior medial portal and figure-4 guide pin, with the trans-tibial guide pin being the farthest. The posteromedial portal guide pin failed to disrupt the physeal scar in all specimens, while the other three techniques consistently violated the physeal scar. CONCLUSION: The posteromedial portal technique offers an appropriate method for anatomic ACL reconstruction while protecting the distal femoral physis from injury. Care needs to be taken with this technique as it comes in closer proximity to some of the important neurovascular structures. This study indicates that posteromedial portal technique is a less technically challenging approach for physeal-sparing ACL repair with special attention required for the protection of neurovascular bundle from potential injury.

Acetaminophen, bupivacaine, Duramorph, and Toradol: A comparison of chondrocyte viability and gene expression changes in osteoarthritic human chondrocytes.

BACKGROUND: A multitude of chemical agents are currently used intra-articularly to decrease pain after orthopaedic procedures including total knee arthroplasty. However, the possible deleterious effects of these injectable chemicals on chondrocyte viability have not been weighed against their potential benefits. Using a human osteoarthritic chondrocyte model, the purpose of this study was to assess the potential for cartilage damage caused by bupivacaine, Toradol, Duramorph, and acetaminophen from surgical local anesthesia. METHODS: Human distal femur and proximal tibia cross sections were obtained during total knee arthroplasty and divided into control group and experimental groups treated by bupivacaine, Toradol, Duramorph, and acetaminophen respectively. Chondrocytes obtained from enzymatically digested cartilage were cultured using a 3D alginate bead culture method to ensure lower rates of dedifferentiation. Chondrocyte bead cultures were exposed to the study chemicals. The gene expression and chondrocyte viability were measured by RT-PCR and flow cytometry, respectively. RESULTS: Compared with untreated group bupivacaine treatment led to the greatest cellular apoptosis with 30.5 ± 11% dead cells (P = 0.000). Duramorph and acetaminophen did not result in a significant increase in cell death. Bupivacaine treatment led to an increase in Caspase 3 gene expression (P = 0.000) as well as the acetaminophen treatment (P = 0.001) when compared to control. CONCLUSION: Our data demonstrated that Duramorph and Toradol were not cytotoxic to human chondrocytes and may be better alternatives to the frequently used and more cytotoxic bupivacaine. Acetaminophen did not result in increased cell death; however, it did show increased caspase 3 gene expression and caution should be considered.

Patellofemoral Joint Contact Pressures: Current Concepts and Use in Patellar Instability Studies.

The patellofemoral joint is thought to be a common source for knee pain. Improper alignment and function of the patellofemoral joint can lead to abnormal contact pressures, which may explain patients' symptoms. In this review, the authors examine techniques for measuring patellofemoral joint contact pressures and summarize the relevant patellofemoral joint anatomy and contact pressures in normal knee kinematics. Finally, they discuss the results of studies investigating contact pressure changes in cases of patellar instability. This includes both reconstruction of the medial patellofemoral ligament and tibial tubercle osteotomy. [Orthopedics. 2019; 42(2):e172-e179.].

Articular cartilage surface roughness as an imaging-based morphological indicator of osteoarthritis: A preliminary investigation of osteoarthritis initiative subjects.

Current imaging-based morphometric indicators of osteoarthritis (OA) using whole-compartment mean cartilage thickness (MCT) and volume changes can be insensitive to mild degenerative changes of articular cartilage (AC) due to areas of adjacent thickening and thinning. The purpose of this preliminary study was to evaluate cartilage thickness-based surface roughness as a morphometric indicator of OA. 3D magnetic resonance imaging (MRI) datasets were collected from osteoarthritis initiative (OAI) subjects with Kellgren-Lawrence (KL) OA grades of 0, 2, and 4 (n = 10/group). Femoral and tibial AC volumes were converted to two-dimensional thickness maps, and MCT, arithmetic surface roughness (Sa ), and anatomically normalized Sa (normSa ) were calculated. Thickness maps enabled visualization of degenerative changes with increasing KL grade, including adjacent thinning and thickening on the femoral condyles. No significant differences were observed in MCT between KL grades. Sa was significantly higher in KL4 compared to KL0 and KL2 in the whole femur (KL0: 0.55 ± 0.10 mm, KL2: 0.53 ± 0.09 mm, KL4: 0.79 ± 0.18 mm), medial femoral condyle (KL0: 0.42 ± 0.07 mm, KL2: 0.48 ± 0.07 mm, KL4: 0.76 ± 0.22 mm), and medial tibial plateau (KL0: 0.42 ± 0.07 mm, KL2: 0.43 ± 0.09 mm, KL4: 0.68 ± 0.27 mm). normSa was significantly higher in KL4 compared to KL0 and KL2 in the whole femur (KL0: 0.22 ± 0.02, KL2: 0.22 ± 0.02, KL4: 0.30 ± 0.03), medial condyle (KL0: 0.17 ± 0.02, KL2: 0.20 ± 0.03, KL4: 0.29 ± 0.06), whole tibia (KL0: 0.34 ± 0.04, KL2: 0.33 ± 0.05, KL4: 0.48 ± 0.11) and medial plateau (KL0: 0.23 ± 0.03, KL2: 0.24 ± 0.04, KL4: 0.40 ± 0.10), and significantly higher in KL2 compared to KL0 in the medial femoral condyle. Surface roughness metrics were sensitive to degenerative morphologic changes, and may be useful in OA characterization and early diagnosis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2755-2764, 2017.

Rotator cuff rehabilitation: current theories and practice.

A fully functioning, painless shoulder joint is essential to maintain a healthy, normal quality of life. Disease of the rotator cuff tendons (RCTs) is a common issue that affects the population, increasing with age, and can lead to significant disability and social and health costs. RCT injuries can affect younger, healthy patients and the elderly alike, and may be the result of trauma or occur as a result of chronic degeneration. They can be acutely painful, limited to certain activities or completely asymptomatic and incidental findings. A wide variety of treatment options exists ranging from conservative local and systemic pain modalities, to surgical fixation. Regardless of management ultimately chosen, physiotherapy of the RCT, rotator cuff muscles and surrounding shoulder girdle plays an essential role in proper treatment. Length of treatment, types of therapy and timing may vary if therapy is definitive care or part of a postoperative protocol. Allowing time for adequate RCT healing must always be considered when implementing ROM and strengthening after surgery. With current rehabilitation methods, patients with all spectrums of RCT pathology can improve their function, pain and quality of life. This manuscript reviews current theories and practice involving rehabilitation for RCT injuries.

Diagnosis, costs, and utilization for psychogenic non-epileptic seizures in a US health care setting.

BACKGROUND: Psychogenic non-epileptic seizures (PNES) pose a substantial burden to patients and the health care system, due to long diagnostic and treatment delays. METHODS: This study used medical chart notes from 103 patients to shed light on the diagnostic process. Electronic medical records and cost data from a US health system were also used to investigate costs and utilization for the 12-months before and after PNES diagnosis. RESULTS: The results show that accurate diagnosis was typically achieved via the use of multiple medical tests, including vEEG, magnetic resonance imaging (MRI), and computed tomography (CT) scans, as well as historical diagnostic and clinical information. In the year following PNES diagnosis, a decline in average visits (-1.45) and costs (-$1784) were observed. The largest cost savings were seen in neurology care and inpatient stays. CONCLUSIONS: This study has implications for timely and accurate diagnosis of PNES, which may decrease the overall health care burden for individuals and the health care system.

Atrial fibrillation significantly increases total mortality and stroke risk beyond that conveyed by the CHADS2 risk factors.

BACKGROUND: Atrial fibrillation (AF) is associated with an increased risk of mortality and stroke. However, it is unclear if AF is independently associated with these poor outcomes or it is merely a risk marker of other processes that convey the risk. METHODS: Consecutive patients who underwent angiography for suspicion of coronary artery disease, but without a history of AF, were studied. Traditional CHADS2 (congestive heart failure, hypertension, age >75 years, diabetes, stroke/transient ischemic attack) risk factors for each patient were recorded. RESULTS: A total of 343 AF patients (age = 69 +/- 10 years, 215 [63%] male) and 2,945 non-AF patients (age = 63 +/- 12 years, 2,012 [67%] male) were studied. Among AF patients, 51 (15%) had a myocardial infarction (MI), 35 (10%) had a stroke, and 180 (52%) died. CHADS2 score incrementally increased risk of stroke (adjusted hazard ratio [HR] for 1:1.92, 2:2.30, 3:1.14, 4:3.83, 5:10.96; P-trend = 0.14), death (HR for 1:1.83, 2:2.34, 3:3.69, 4:2.27, 5:4.53; P-trend < 0.001), and major adverse cardiac event (MACE)(HR for 1:1.29, 2:1.54, 3:2.07, 4:2.41, 5:2.68; P-trend = 0.002). Among non-AF patients, CHADS2 score incrementally increased risk of stroke (HR for 1:1.18, 2:3.17, 3:5.08, 4:10.78, 5:7.50; P-trend < 0.001), MI (HR for 1:1.05, 2:1.46, 3:1.57, 4:0.53, 5:4.76; P-trend = 0.002), death (HR for 1:1.79, 2:3.22, 3:6.23, 4:9.09, 5:14.00; P-trend < 0.001), and MACE (HR for 1:1.47, 2:2.36, 3:4.16, 4:5.91, 5:7.56; P-trend < 0.001). Among all patients, both CHADS2 score (all P < or = 0.001) and AF were independent risk factors for stroke (AF: P = 0.002), MI (AF: P = 0.035), death (AF: P < 0.001), and MACE (AF: P < 0.001). CONCLUSION: The CHADS2 score is a powerful predictor of stroke and death. AF increases the risk of these outcomes in an independent manner. These data support the concept that AF is a risk factor of future cardiovascular disease.

Hydroxyatrazine N-ethylaminohydrolase (AtzB): an amidohydrolase superfamily enzyme catalyzing deamination and dechlorination.

Hydroxyatrazine [2-(N-ethylamino)-4-hydroxy-6-(N-isopropylamino)-1,3,5-triazine] N-ethylaminohydrolase (AtzB) is the sole enzyme known to catalyze the hydrolytic conversion of hydroxyatrazine to N-isopropylammelide. AtzB, therefore, serves as the point of intersection of multiple s-triazine biodegradative pathways and is completely essential for microbial growth on s-triazine herbicides. Here, atzB was cloned from Pseudomonas sp. strain ADP and its product was purified to homogeneity and characterized. AtzB was found to be dimeric, with subunit and holoenzyme molecular masses of 52 kDa and 105 kDa, respectively. The k(cat) and K(m) of AtzB with hydroxyatrazine as a substrate were 3 s(-1) and 20 microM, respectively. Purified AtzB had a 1:1 zinc-to-subunit stoichiometry. Sequence analysis revealed that AtzB contained the conserved mononuclear amidohydrolase superfamily active-site residues His74, His76, His245, Glu248, His280, and Asp331. An intensive in vitro investigation into the substrate specificity of AtzB revealed that 20 of the 51 compounds tested were substrates for AtzB; this allowed for the identification of specific substrate structural features required for catalysis. Substrates required a monohydroxylated s-triazine ring with a minimum of one primary or secondary amine substituent and either a chloride or amine leaving group. AtzB catalyzed both deamination and dechlorination reactions with rates within a range of one order of magnitude. This differs from AtzA and TrzN, which do not catalyze deamination reactions, and AtzC, which is not known to catalyze dechlorination reactions.

Glutamate 107 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli is protonated and near the heme d/heme b595 binuclear center.

Cytochrome bd is a quinol oxidase from Escherichia coli, which is optimally expressed under microaerophilic growth conditions. The enzyme catalyzes the two-electron oxidation of either ubiquinol or menaquinol in the membrane and scavenges O2 at low concentrations, reducing it to water. Previous work has shown that, although cytochrome bd does not pump protons, turnover is coupled to the generation of a proton motive force. The generation of a proton electrochemical gradient results from the release of protons from the oxidation of quinol to the periplasm and the uptake of protons used to form H2O from the cytoplasm. Because the active site has been shown to be located near the periplasmic side of the membrane, a proton channel must facilitate the delivery of protons from the cytoplasm to the site of water formation. Two conserved glutamic acid residues, E107 and E99, are located in transmembrane helix III in subunit I and have been proposed to form part of this putative proton channel. In the current work, it is shown that mutations in either of these residues results in the loss of quinol oxidase activity and can result in the loss of the two hemes at the active site, hemes d and b595. One mutant, E107Q, while being totally inactive, retains the hemes. Fourier transform infrared (FTIR) redox difference spectroscopy has identified absorption bands from the COOH group of E107. The data show that E107 is protonated at pH 7.6 and that it is perturbed by the reduction of the heme d/heme b595 binuclear center at the active site. In contrast, mutation of an acidic residue known to be at or near the quinol-binding site (E257A) also inactivates the enzyme but has no substantial influence on the FTIR redox difference spectrum. Mutagenesis shows that there are several acidic residues, including E99 and E107 as well as D29 (in CydB), which are important for the assembly or stability of the heme d/heme b595 active site.

Deinococcus radiodurans engineered for complete toluene degradation facilitates Cr(VI) reduction.

Toluene and other fuel hydrocarbons are commonly found in association with radionuclides at numerous US Department of Energy sites, frequently occurring together with Cr(VI) and other heavy metals. In this study, the extremely radiation-resistant bacterium Deinococcus radiodurans, which naturally reduces Cr(VI) to the less mobile and less toxic Cr(III), was engineered for complete toluene degradation by cloned expression of tod and xyl genes of Pseudomonas putida. The recombinant Tod/Xyl strain showed incorporation of carbon from 14C-labelled toluene into cellular macromolecules and carbon dioxide, in the absence or presence of chronic ionizing radiation. The engineered bacteria were able to oxidize toluene under both minimal and complex nutrient conditions, and recombinant cells reduced Cr(VI) in sediment microcosms. As such, the Tod/Xyl strain could provide a model for examining the reduction of metals coupled to organic contaminant oxidation in aerobic radionuclide-contaminated sediments.

Arginine 391 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli stabilizes the reduced form of the hemes and is essential for quinol oxidase activity.

The cytochrome bd quinol oxidase is one of two respiratory oxidases in Escherichia coli. It oxidizes dihydroubiquinol or dihydromenaquinol while reducing dioxygen to water. The bd-type oxidases have only been found in prokaryotes and have been implicated in the survival of some bacteria, including pathogens, under conditions of low aeration. With a high affinity for dioxygen, cytochrome bd not only couples respiration to the generation of a proton motive force but also scavenges O(2). In the current work, the role of a highly conserved arginine residue is explored by site-directed mutagenesis. Four mutations were made: R391A, R391K, R391M, and R391Q. All of the mutations except R391K result in enzyme lacking ubiquinol oxidase activity. Oxidase activity using the artificial reductant N,N,N',N'-tetramethyl-p-phenylenediamine in place of ubiquinol was, however, unimpaired by the mutations, indicating that the catalytic center where O(2) is reduced is intact. UV-visible spectra of each of the mutant oxidases show no perturbations to any of the three heme components (heme b(558), heme b(595), and heme d). However, spectroelectrochemical titrations of the R391A mutant reveal that the midpoint potentials of all of the heme components are substantially lower compared with the wild type enzyme. Since Arg(391) is close to Met(393), one of the axial ligands to heme b(558), it is to be expected that the R391A mutation might destabilize the reduced form of heme b(558). The fact that the midpoint potentials of heme d and heme b(595) are also significantly lowered in the R391A mutant is consistent with these hemes being physically close together on the periplasmic side of the membrane.

Proton NMR study of the heme environment in bacterial quinol oxidases.

The heme environment and ligand binding properties of two relatively large membrane proteins containing multiple paramagnetic metal centers, cytochrome bo3 and bd quinol oxidases, have been studied by high field proton nuclear magnetic resonance (NMR) spectroscopy. The oxidized bo3 enzyme displays well-resolved hyperfine-shifted 1H NMR resonance assignable to the low-spin heme b center. The observed spectral changes induced by addition of cyanide to the protein were attributed to the structural perturbations on the low-spin heme (heme b) center by cyanide ligation to the nearby high-spin heme (heme o) of the protein. The oxidized hd oxidase shows extremely broad signals in the spectral region where protons near high-spin heme centers resonate. Addition of cyanide to the oxidized bd enzyme induced no detectable perturbations on the observed hyperfine signals, indicating the insensitive nature of this heme center toward cyanide. The proton signals near the low-spin heme b558 center are only observed in the presence of 20% formamide, consistent with a critical role of viscosity in detecting NMR signals of large membrane proteins. The reduced bd protein also displays hyperfine-shifted 1H NMR signals, indicating that the high-spin heme centers (hemes b595 and d) remain high-spin upon chemical reduction. The results presented here demonstrate that structural changes of one metal center can significantly influence the structural properties of other nearby metal center(s) in large membrane paramagnetic metalloproteins.

Atrazine chlorohydrolase from Pseudomonas sp. strain ADP is a metalloenzyme.

Atrazine chlorohydrolase (AtzA) from Pseudomonas sp. ADP initiates the metabolism of the herbicide atrazine by catalyzing a hydrolytic dechlorination reaction to produce hydroxyatrazine. Sequence analysis revealed AtzA to be homologous to metalloenzymes within the amidohydrolase protein superfamily. AtzA activity was experimentally shown to depend on an enzyme-bound, divalent transition-metal ion. Loss of activity obtained by incubating AtzA with the chelator 1,10-phenanthroline or oxalic acid was reversible upon addition of Fe(II), Mn(II), or Co(II) salts. Experimental evidence suggests a 1:1 metal to subunit stoichiometry, with the native metal being Fe(II). Our data show that the inhibitory effects of metals such as Zn(II) and Cu(II) are not the result of displacing the active site metal. Taken together, these data indicate that AtzA is a functional metalloenzyme, making this the first report, to our knowledge, of a metal-dependent dechlorinating enzyme that proceeds via a hydrolytic mechanism.

Purification, substrate range, and metal center of AtzC: the N-isopropylammelide aminohydrolase involved in bacterial atrazine metabolism.

N-Isopropylammelide isopropylaminohydrolase, AtzC, the third enzyme in the atrazine degradation pathway in Pseudomonas sp. strain ADP, catalyzes the stoichiometric hydrolysis of N-isopropylammelide to cyanuric acid and isopropylamine. The atzC gene was cloned downstream of the tac promoter and expressed in Escherichia coli, where the expressed enzyme comprised 36% of the soluble protein. AtzC was purified to homogeneity by ammonium sulfate precipitation and phenyl column chromatography. It has a subunit size of 44,938 kDa and a holoenzyme molecular weight of 174,000. The K(m) and k(cat) values for AtzC with N-isopropylammelide were 406 micro M and 13.3 s(-1), respectively. AtzC hydrolyzed other N-substituted amino dihydroxy-s-triazines, and those with linear N-alkyl groups had higher k(cat) values than those with branched alkyl groups. Native AtzC contained 0.50 eq of Zn per subunit. The activity of metal-depleted AtzC was restored with Zn(II), Fe(II), Mn(II), Co(II), and Ni(II) salts. Cobalt-substituted AtzC had a visible absorbance band at 540 nm (Delta epsilon = 84 M(-1) cm(-1)) and exhibited an axial electron paramagnetic resonance (EPR) signal with the following effective values: g((x)) = 5.18, g((y)) = 3.93, and g((z)) = 2.24. Incubating cobalt-AtzC with the competitive inhibitor 5-azacytosine altered the effective EPR signal values to g((x)) = 5.11, g((y)) = 4.02, and g((z)) = 2.25 and increased the microwave power at half saturation at 10 K from 31 to 103 mW. Under the growth conditions examined, our data suggest that AtzC has a catalytically essential, five-coordinate Zn(II) metal center in the active site and specifically catalyzes the hydrolysis of intermediates generated during the metabolism of s-triazine herbicides.