Getting to the Outer Leaflet: Physiology of Phosphatidylserine Exposure at the Plasma Membrane.

Phosphatidylserine (PS) is a major component of membrane bilayers whose change in distribution between inner and outer leaflets is an important physiological signal. Normally, members of the type IV P-type ATPases spend metabolic energy to create an asymmetric distribution of phospholipids between the two leaflets, with PS confined to the cytoplasmic membrane leaflet. On occasion, membrane enzymes, known as scramblases, are activated to facilitate transbilayer migration of lipids, including PS. Recently, two proteins required for such randomization have been identified: TMEM16F, a scramblase regulated by elevated intracellular Ca(2+), and XKR8, a caspase-sensitive protein required for PS exposure in apoptotic cells. Once exposed at the cell surface, PS regulates biochemical reactions involved in blood coagulation, and bone mineralization, and also regulates a variety of cell-cell interactions. Exposed on the surface of apoptotic cells, PS controls their recognition and engulfment by other cells. This process is exploited by parasites to invade their host, and in specialized form is used to maintain photoreceptors in the eye and modify synaptic connections in the brain. This review discusses what is known about the mechanism of PS exposure at the surface of the plasma membrane of cells, how actors in the extracellular milieu sense surface exposed PS, and how this recognition is translated to downstream consequences of PS exposure.

Effect of intraarticular pressure on glenohumeral kinematics during a simulated abduction motion: a cadaveric study.

BACKGROUND: The current understanding of glenohumeral joint stability is defined by active restrictions and passive stabilizers including naturally-occurring negative intraarticular pressure. Cadaveric specimens have been used to evaluate the role of intraarticular pressure on joint stability, although, while the shoulder's negative intraarticular pressure is universally acknowledged, it has been inconsistently accounted for. HYPOTHESIS: During continuous, passive humeral abduction, releasing the native intraarticular pressure increases joint translation, and restoring this pressure decreases joint translations. STUDY DESIGN: Descriptive Laboratory Study. METHODS: A validated shoulder testing system was used to passively abduct the humerus in the scapular plane and measure joint translations for seven (n = 7) cadaveric specimens. The pressure within the glenohumeral joint was measured via a 25-gauge needle during passive abduction of the arm, which was released and subsequently restored. During motion, the rotator cuff muscles were loaded using stepper motors in a force feedback loop and electromagnetic sensors were used to continuously measure the position of the humerus and scapula. Joint translation was defined according to the instant center of rotation of the glenohumeral head according to the recommendations by the International Society of Biomechanics. RESULTS: Area under the translation versus abduction angle curve suggests that releasing the pressure within the capsule results in significantly less posterior translation of the glenohumeral head as compared to intact (85-90˚, p < 0.05). Posterior and superior translations were reduced after 70˚ of abduction when the pressure within the joint was restored. CONCLUSION: With our testing system employing a smooth continuous passive motion, we were able to show that releasing intraarticular pressure does not have a major effect on the path of humeral head motion during glenohumeral abduction. However, both violating the capsule and restoring intraarticular pressure after releasing alter glenohumeral translations. Future studies should study the effect of simultaneous external rotation and abduction on the relationship between joint motion and IAP, especially in higher degrees of abduction. CLINICAL RELEVANCE: Thoroughly simulating the glenohumeral joint environment in the cadaveric setting may strengthen the conclusions that can be translated from this setting to the clinic.

Intraarticular injection of relaxin-2 alleviates shoulder arthrofibrosis.

Arthrofibrosis is a prevalent condition affecting greater than 5% of the general population and leads to a painful decrease in joint range of motion (ROM) and loss of independence due to pathologic accumulation of periarticular scar tissue. Current treatment options are limited in effectiveness and do not address the underlying cause of the condition: accumulation of fibrotic collagenous tissue. Herein, the naturally occurring peptide hormone relaxin-2 is administered for the treatment of adhesive capsulitis (frozen shoulder) and to restore glenohumeral ROM in shoulder arthrofibrosis. Recombinant human relaxin-2 down-regulates type I collagen and α smooth muscle actin production and increases intracellular cAMP concentration in human fibroblast-like synoviocytes, consistent with a mechanism of extracellular matrix degradation and remodeling. Pharmacokinetic profiling of a bolus administration into the glenohumeral joint space reveals the brief systemic and intraarticular (IA) half-lives of relaxin-2: 0.96 h and 0.62 h, respectively. Furthermore, using an established, immobilization murine model of shoulder arthrofibrosis, multiple IA injections of human relaxin-2 significantly improve ROM, returning it to baseline measurements collected before limb immobilization. This is in contrast to single IA (sIA) or multiple i.v. (mIV) injections of relaxin-2 with which the ROM remains constrained. The histological hallmarks of contracture (e.g., fibrotic adhesions and reduced joint space) are absent in the animals treated with multiple IA injections of relaxin-2 compared with the untreated control and the sIA- and mIV-treated animals. As these findings show, local delivery of relaxin-2 is an innovative treatment of shoulder arthrofibrosis.

Efficiency Analysis of an Interoperable Healthcare Operations Platform.

(1) Develop an enterprise platform to unify isolated information, software applications and team members. (2) Assess the efficiency of one benefit of the platform through comparative testing of employee document retrieval times. (3) Evaluate the level of satisfaction among our target audience. We developed an infrastructure to integrate information throughout our practice and make it available on a unified, secure, and remotely accessible platform. We solicited our practice for volunteers to test the new system. All interested volunteers participated. Thirteen employees searched for the same four items in both the new system and our legacy systems. Testing was performed in the pre-deployment stage. In our evaluation, we introduced an innovative method to precisely and objectively obtain data through the use of a widely available tool which could be leveraged for a variety of other studies. On average, it took our participants 7 min and 48 s to find four assigned items in our legacy systems. It only took our volunteers 1 min and 1 s to find the same items with the new platform (p-value 0.002). On a scale of 10 being the highest level of satisfaction, participants ranked the new system to be 8.7 while the traditional system was ranked at 6.3. An overarching enterprise platform is critical due to the ability to unify otherwise isolated applications, people and documents. Because navigating a new system would be expected to take longer than a familiar one, we were surprised by the dramatically improved efficiency and satisfaction of our new interoperable platform compared to the status quo. Since this platform was evaluated in the pre-deployment stage, we expect results to improve with employee experience as well as ongoing enhancements.

Isothiourea-catalysed enantioselective pyrrolizine synthesis: synthetic and computational studies.

The catalytic enantioselective synthesis of a range of cis-pyrrolizine carboxylate derivatives with outstanding stereocontrol (14 examples, >95 : 5 dr, >98 : 2 er) through an isothiourea-catalyzed intramolecular Michael addition-lactonisation and ring-opening approach from the corresponding enone acid is reported. An optimised and straightforward three-step synthetic route to the enone acid starting materials from readily available pyrrole-2-carboxaldehydes is delineated, with benzotetramisole (5 mol%) proving the optimal catalyst for the enantioselective process. Ring-opening of the pyrrolizine dihydropyranone products with either MeOH or a range of amines leads to the desired products in excellent yield and enantioselectivity. Computation has been used to probe the factors leading to high stereocontrol, with the formation of the observed cis-steroisomer predicted to be kinetically and thermodynamically favoured.

Substrate trajectory through phospholipid-transporting P4-ATPases.

A difference in the lipid composition between the two leaflets of the same membrane is a relatively simple instance of lipid compositional heterogeneity. The large activation energy barrier for transbilayer movement for some (but not all) membrane lipids creates a regime governed by active transport processes. An early step in eukaryote evolution was the development of a capacity for generating transbilayer compositional heterogeneity far from equilibrium by directly tapping energy from the ATP pool. The mechanism of the P-type ATPases that create lipid asymmetry is well understood in terms of ATP hydrolysis, but the trajectory taken by the phospholipid substrate through the enzyme is a matter of current active research. There are currently three different models for this trajectory, all with support by mutation/activity measurements and analogies with known atomic structures.

Mixed-metal MIL-100(Sc,M) (M=Al, Cr, Fe) for Lewis acid catalysis and tandem C-C bond formation and alcohol oxidation.

The trivalent metal cations Al(3+) , Cr(3+) , and Fe(3+) were each introduced, together with Sc(3+) , into MIL-100(Sc,M) solid solutions (M=Al, Cr, Fe) by direct synthesis. The substitution has been confirmed by powder X-ray diffraction (PXRD) and solid-state NMR, UV/Vis, and X-ray absorption (XAS) spectroscopy. Mixed Sc/Fe MIL-100 samples were prepared in which part of the Fe is present as α-Fe2 O3 nanoparticles within the mesoporous cages of the MOF, as shown by XAS, TGA, and PXRD. The catalytic activity of the mixed-metal catalysts in Lewis acid catalysed Friedel-Crafts additions increases with the amount of Sc present, with the attenuating effect of the second metal decreasing in the order Al>Fe>Cr. Mixed-metal Sc,Fe materials give acceptable activity: 40 % Fe incorporation only results in a 20 % decrease in activity over the same reaction time and pure product can still be obtained and filtered off after extended reaction times. Supported α-Fe2 O3 nanoparticles were also active Lewis acid species, although less active than Sc(3+) in trimer sites. The incorporation of Fe(3+) into MIL-100(Sc) imparts activity for oxidation catalysis and tandem catalytic processes (Lewis acid+oxidation) that make use of both catalytically active framework Sc(3+) and Fe(3+) . A procedure for using these mixed-metal heterogeneous catalysts has been developed for making ketones from (hetero)aromatics and a hemiacetal.

A Passive Ankle Dorsiflexion Testing System for an In Vivo Model of Overuse-induced Tendinopathy.

Tendinopathy is a chronic tendon condition that results in pain and loss of function and is caused by repeated overload of the tendon and limited recovery time. This protocol describes a testing system that cyclically applies mechanical loads via passive dorsiflexion to the rat Achilles tendon. The custom-written code consists of pre- and post-cyclic loading measurements to assess the effects of the loading protocol along with the feedback control-based cyclic fatigue loading regimen. We used 25 Sprague-Dawley rats for this study, with 5 rats per group receiving either 500, 1,000, 2,000, 3,600, or 7,200 cycles of fatigue loads. The percentage differences between the pre- and post-cyclic loading measurements of the hysteresis, peak stress, and loading and unloading moduli were calculated. The results demonstrate that the system can induce varying degrees of damage to the Achilles tendon based on the number of loads applied. This system offers an innovative approach to apply quantified and physiological varying degrees of cyclic loads to the Achilles tendon for an in vivo model of fatigue-induced overuse tendon injury.

Mapping functional interactions in a heterodimeric phospholipid pump.

Type 4 P-type ATPases (P(4)-ATPases) catalyze phospholipid transport to generate phospholipid asymmetry across membranes of late secretory and endocytic compartments, but their kinship to cation-transporting P-type transporters raised doubts about whether P(4)-ATPases alone are sufficient to mediate flippase activity. P(4)-ATPases form heteromeric complexes with Cdc50 proteins. Studies of the enzymatic properties of purified P(4)-ATPase·Cdc50 complexes showed that catalytic activity depends on direct and specific interactions between Cdc50 subunit and transporter, whereas in vivo interaction assays suggested that the binding affinity for each other fluctuates during the transport reaction cycle. The structural determinants that govern this dynamic association remain to be established. Using domain swapping, site-directed, and random mutagenesis approaches, we here show that residues throughout the subunit contribute to forming the heterodimer. Moreover, we find that a precise conformation of the large ectodomain of Cdc50 proteins is crucial for the specificity and functionality to transporter/subunit interactions. We also identified two highly conserved disulfide bridges in the Cdc50 ectodomain. Functional analysis of cysteine mutants that disrupt these disulfide bridges revealed an inverse relationship between subunit binding and P(4)-ATPase-catalyzed phospholipid transport. Collectively, our data indicate that a dynamic association between subunit and transporter is crucial for the transport reaction cycle of the heterodimer.

Biochemical characterization of P4-ATPase mutations identified in patients with progressive familial intrahepatic cholestasis.

Mutations in the P4-ATPase ATP8B1 cause the inherited liver disease progressive familial intrahepatic cholestasis. Several of these mutations are located in conserved regions of the transmembrane domain associated with substrate binding and transport. Assays for P4-ATPase-mediated transport in living yeast cells were developed and used to characterize the specificity and kinetic parameters of this transport. Progressive familial intrahepatic cholestasis mutations were introduced into the yeast plasma membrane P4-ATPase Dnf2p, and the effect of these mutations on its catalysis of phospholipid transport were determined. The results of these measurements have implications for the basis of the disease and for the mechanism of phospholipid transit through the enzyme during the reaction cycle.

A Validated Three-Dimensional, Heterogenous Finite Element Model of the Rotator Cuff and The Effects of Collagen Orientation.

Continuum mechanics-based finite element models of the shoulder aim to quantify the mechanical environment of the joint to aid in clinical decision-making for rotator cuff injury and disease. These models allow for the evaluation of the internal loading of the shoulder, which cannot be measured in-vivo. This study uses human cadaveric rotator cuff samples with surface tendon strain estimates, to validate a heterogeneous finite element model of the supraspinatus-infraspinatus complex during various load configurations. The computational model was considered validated when the absolute difference in average maximum principal strain for the articular and bursal sides for each load condition estimated by the model was no greater than 3% compared to that measured in the biomechanical study. The model can predict the strains for varying infraspinatus loads allowing for the study of load sharing between these two tightly coordinated tendons. The future goal is to use the modularity of this validated model to study the initiation and propagation of rotator cuff tear and other rotator cuff pathologies to ultimately improve care for rotator cuff tear patients.

A passive ankle dorsiflexion testing system to assess mechanobiological and structural response to cyclic loading in rat Achilles tendon.

Tendinopathy is thought to be caused by repeated overload of the tendon with insufficient recovery time, leading to an inadequate healing response and incomplete recovery of preinjury material strength and function. The etiology of tendinopathy induced by mechanical load is being explored with a variety of mechanical load scenarios in small animals. This study establishes a testing system that applies passive ankle dorsiflexion to a rat hindlimb, estimates the force applied to the tendon during cyclic loading and enables the assessment of subsequent structural and biological changes. We demonstrated that the system had no drift in the applied angle, and the registered maximum angle and torque inputs and outputs were consistent between tests. We showed that cyclic loading decreased hysteresis and loading and unloading moduli with increasing cycles applied to the tendon. Histology showed gross changes to tendon structure. This work establishes a system for passively loading the rat Achilles tendon in-vivo in a physiological manner, facilitating future studies that will explore how mechanics, structure, and biology are altered by mechanical repetitive loading.

Plaster of Paris: Squeeze, But Not Too Hard!

Plaster of Paris (PoP) has been the predominant treatment option for most acute and chronic orthopedic conditions. Water immersion significantly decreases the PoP bandage strength. Moreover, concerns have been raised about the possibility of breaks in PoP splints and cast failures once solid. The current study was designed to account for the increase in weight associated with increased PoP layers. The authors hypothesized that by controlling for weight variation as layers increased, they could determine the number of layers of PoP bandage that truly results in optimal mechanical properties. They assessed whether adequate plaster weight control while increasing layers could improve the mechanical properties of the splint. [Orthopedics. 2022;45(1):e57-e61.].

Minimally invasive, sustained-release relaxin-2 microparticles reverse arthrofibrosis.

Substantial advances in biotherapeutics are distinctly lacking for musculoskeletal diseases. Musculoskeletal diseases are biomechanically complex and localized, highlighting the need for novel therapies capable of addressing these issues. All frontline treatment options for arthrofibrosis, a debilitating musculoskeletal disease, fail to treat the disease etiology-the accumulation of fibrotic tissue within the joint space. For millions of patients each year, the lack of modern and effective treatment options necessitates surgery in an attempt to regain joint range of motion (ROM) and escape prolonged pain. Human relaxin-2 (RLX), an endogenous peptide hormone with antifibrotic and antifibrogenic activity, is a promising biotherapeutic candidate for musculoskeletal fibrosis. However, RLX has previously faltered through multiple clinical programs because of pharmacokinetic barriers. Here, we describe the design and in vitro characterization of a tailored drug delivery system for the sustained release of RLX. Drug-loaded, polymeric microparticles released RLX over a multiweek time frame without altering peptide structure or bioactivity. In vivo, intraarticular administration of microparticles in rats resulted in prolonged, localized concentrations of RLX with reduced systemic drug exposure. Furthermore, a single injection of RLX-loaded microparticles restored joint ROM and architecture in an atraumatic rat model of arthrofibrosis with clinically derived end points. Finally, confirmation of RLX receptor expression, RXFP1, in multiple human tissues relevant to arthrofibrosis suggests the clinical translational potential of RLX when administered in a sustained and targeted manner.

Tendinopathy and tendon material response to load: What we can learn from small animal studies.

Tendinopathy is a debilitating disease that causes as much as 30% of all musculoskeletal consultations. Existing treatments for tendinopathy have variable efficacy, possibly due to incomplete characterization of the underlying pathophysiology. Mechanical load can have both beneficial and detrimental effects on tendon, as the overall tendon response depends on the degree, frequency, timing, and magnitude of the load. The clinical continuum model of tendinopathy offers insight into the late stages of tendinopathy, but it does not capture the subclinical tendinopathic changes that begin before pain or loss of function. Small animal models that use high tendon loading to mimic human tendinopathy may be able to fill this knowledge gap. The goal of this review is to summarize the insights from in-vivo animal studies of mechanically-induced tendinopathy and higher loading regimens into the mechanical, microstructural, and biological features that help characterize the continuum between normal tendon and tendinopathy. STATEMENT OF SIGNIFICANCE: This review summarizes the insights gained from in-vivo animal studies of mechanically-induced tendinopathy by evaluating the effect high loading regimens have on the mechanical, structural, and biological features of tendinopathy. A better understanding of the interplay between these realms could lead to improved patient management, especially in the presence of painful tendon.

A P4-ATPase protein interaction network reveals a link between aminophospholipid transport and phosphoinositide metabolism.

High-throughput analysis of protein-protein interactions can provide unprecedented insight into how cellular processes are integrated at the molecular level. Yet membrane proteins are often overlooked in these studies owing to their hydrophobic nature and low abundance. Here we used a proteomics-based strategy with the specific intention of identifying membrane-associated protein complexes. One important aspect of our approach is the use of chemical cross-linking to capture transient and low-affinity protein interactions that occur in living cells prior to cell lysis. We applied this method to identify binding partners of the yeast Golgi P(4)-ATPase Drs2p, a member of a conserved family of putative aminophospholipid transporters. Drs2p was endogeneously tagged with both a polyhistidine and a biotinylation peptide, allowing tandem-affinity purification of Drs2p-containing protein complexes under highly stringent conditions. Mass-spectrometric analysis of isolated complexes yielded one known and nine novel Drs2p binding partners. Binding specificity was verified by an orthogonal in vivo membrane protein interaction assay, confirming the efficacy of our method. Strikingly, three of the novel Drs2p interactors are involved in phosphoinositide metabolism. One of these, the phosphatidylinositol-4-phosphatase Sac1p, also displays genetic interactions with Drs2p. Together, these findings suggest that aminophospholipid transport and phosphoinositide metabolism are interconnected at the Golgi.

Cdc50p plays a vital role in the ATPase reaction cycle of the putative aminophospholipid transporter Drs2p.

Members of the P(4) subfamily of P-type ATPases are believed to catalyze transport of phospholipids across cellular bilayers. However, most P-type ATPases pump small cations or metal ions, and atomic structures revealed a transport mechanism that is conserved throughout the family. Hence, a challenging problem is to understand how this mechanism is adapted in P(4)-ATPases to flip phospholipids. P(4)-ATPases form heteromeric complexes with Cdc50 proteins. The primary role of these additional polypeptides is unknown. Here, we show that the affinity of yeast P(4)-ATPase Drs2p for its Cdc50-binding partner fluctuates during the transport cycle, with the strongest interaction occurring at a point where the enzyme is loaded with phospholipid ligand. We also find that specific interactions with Cdc50p are required to render the ATPase competent for phosphorylation at the catalytically important aspartate residue. Our data indicate that Cdc50 proteins are integral components of the P(4)-ATPase transport machinery. Thus, acquisition of these subunits may have been a crucial step in the evolution of flippases from a family of cation pumps.

Effect of rotator cuff muscle activation on glenohumeral kinematics: A cadaveric study.

Healthy shoulder function requires the coordination of the rotator cuff muscles to maintain the humeral head's position in the glenoid. While glenohumeral stability has been studied in various settings, few studies have characterized the effect of dynamic rotator cuff muscle loading on glenohumeral translation during shoulder motion. We hypothesize that dynamic rotator cuff muscle activation decreases joint translation during continuous passive abduction of the humerus in a cadaveric model of scapular plane glenohumeral abduction. The effect of different rotator cuff muscle activity on glenohumeral translation was assessed using a validated shoulder testing system. The Dynamic Load profile is a novel approach, based on musculoskeletal modeling of human subject motion. Passive humeral elevation in the scapular plane was applied via the testing system arm, while the rotator cuff muscles were activated according to the specified force profiles using stepper motors and a proportional control feedback loop. Glenohumeral translation was defined according to the International Society of Biomechanics. The Dynamic load profile minimized superior translation of the humeral head relative to the conventional loading profiles. The total magnitude of translation was not significantly different (0.805) among the loading profiles suggesting that the compressive forces from the rotator cuff primarily alter the direction of humeral head translation, not the magnitude. Rotator cuff muscle loading is an important element of cadaveric shoulder studies that must be considered to accurately simulate glenohumeral motion. A rotator cuff muscle activity profile based on human subject muscle activity reduces superior glenohumeral translation when compared to previous RC loading profiles.

Tendon lengthening after achilles tendon rupture-passive effects on the ankle joint in a cadaveric pilot study simulating weight bearing.

BACKGROUND: In recent years, primary Achilles tendon ruptures have increased due to the aging population's participation in physically demanding activities. These injuries commonly occur during recreational sports and frequently lead to a long-term reduction in activity despite treatment. Non-operative methods of treatment for Achilles tendon ruptures may result in the Achilles healing in a lengthened position compared to the pre-injury state. This study uses a cadaveric model that simulates static weight bearing to explore the effect of a lengthened Achilles tendon on ankle joint load distribution. METHODS: Five lower limb cadaveric specimens were placed on a custom jig, where a 334 N (75 lb) load was applied at the femoral head, and the foot was supported against a plate to simulate static double-leg stance. A pressure mapping sensor was inserted into the ankle joint. A percutaneous triple hemiresection tendo-Achilles lengthening procedure (Hoke procedure) was performed on each specimen to simulate tendon lengthening after conservative treatment. Contact pressure, peak pressure, and center-of-pressure were measured for native and tendon-lengthened conditions. RESULTS: Tendon rupture did not significantly alter average contact pressure, peak contact pressures, or center-of-pressure in the ankle joint compared with native tendon. CONCLUSION: Achilles lengthening does not significantly change contact pressures of the ankle joint in this model . This result suggests that the passive restraint on ankle joint translation imposed by the Achilles tendon is minimal without muscle activation.