Point-of-Care Kidney and Genitourinary Ultrasound in Adults: Image Acquisition.

A range of conditions involving the kidneys and urinary bladder can cause organ-threatening complications that are preventable if diagnosed promptly with diagnostic imaging. Common imaging modalities include either computed tomography or diagnostic ultrasound. Traditionally, ultrasound of the kidney-genitourinary system has required consultative teams consisting of a sonographer performing image acquisition and a radiologist performing image interpretation. However, diagnostic point-of-care ultrasound (POCUS) has recently emerged as a useful tool to troubleshoot acute kidney injury at the bedside. Studies have shown that non-radiologists can be trained to perform diagnostic POCUS of the kidneys and bladder with high accuracy for a set number of important conditions. Currently, diagnostic POCUS of the kidney-genitourinary system remains underused in actual clinical practice. This is likely because image acquisition for this organ system is unfamiliar to most clinicians in specialties that encounter acute kidney injury, including primary care, emergency medicine, intensive care, anesthesiology, nephrology, and urology. To address this multi-specialty educational gap, this narrative review was developed by a multi-disciplinary group to provide a specialty-agnostic framework for kidney-genitourinary POCUS image acquisition: indications/contraindications, patient positioning, transducer selection, acquisition sequence, and exam limitations. Finally, we describe foundational concepts in kidney-genitourinary ultrasound image interpretation, including key abnormal findings that every bedside clinician performing this modality should know.

Current Use, Training, and Barriers of Point-of-Care Ultrasound in Anesthesiology: A National Survey of Veterans Affairs Hospitals.

OBJECTIVES: The purpose of this study was to determine current use, training needs, and barriers to point-of-care ultrasound (POCUS) use among anesthesiologists in practice. DESIGN: Multicenter, prospective, observational study. SETTING: Anesthesiology departments in the Veterans Affairs Healthcare System in the United States. PARTICIPANTS: Chiefs of staff and chiefs of anesthesiology departments. INTERVENTIONS: A web-based survey was conducted between June 2019 and March 2020. Chiefs of staff answered questions about facility-level POCUS use, training, competency, and policies. Anesthesiology chiefs responded to a follow-up survey with specialty-specific POCUS questions. The results of the 2020 survey were compared with a similar survey conducted by the authors' group in 2015. MEASUREMENTS AND MAIN RESULTS: All chiefs of staff (n = 130) and 77% of anesthesiology chiefs (n = 96) completed the survey. The most common POCUS applications used were central and peripheral vascular access (69%-72%), peripheral nerve blocks (66%), and evaluation of cardiac function (29%-31%). Compared with 2015, there was a statistically significant increase in desire for training (p = 0.00015), but no significant change in POCUS use (p = 0.31). Training was most desired for volume-status assessment (52%), left ventricular function (47%), pneumothorax (47%), central line placement (40%), peripheral nerve blocks (40%), and pleural effusion (40%). The most common barriers to POCUS use were lack of funding for training (35%), trained providers (33%), and training opportunities (28%). CONCLUSIONS: A significant increase in desire for POCUS training was seen among anesthesiologists practicing in the Veterans Affairs healthcare system since 2015, and lack of training continues to be a top barrier for POCUS use among anesthesiologists.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis for characterization of lignin oligomers using cationization techniques and 2,5-dihydroxyacetophenone (DHAP) matrix.

RATIONALE: Effective analytical techniques are needed to characterize lignin products for the generation of renewable carbon sources. Application of matrix-assisted laser desorption/ionization (MALDI) in lignin analysis is limited because of poor ionization efficiency. In this study, we explored the potential of cationization along with a 2,5-dihydroxyacetophenone (DHAP) matrix to characterize model lignin oligomers. METHODS: Synthesized lignin oligomers were analyzed using the developed MALDI method. Two matrix systems, DHAP and α-cyano-4-hydroxycinnamic acid (CHCA), and three cations (lithium, sodium, silver) were evaluated using a Bruker UltraFlextreme time-of-flight mass spectrometer. Instrumental parameters, cation concentration, matrix, sample concentrations, and sample spotting protocols were optimized for improved results. RESULTS: The DHAP/Li+ combination was effective for dimer analysis as lithium adducts. Spectra from DHP and ferric chloride oligomers showed improved signal intensities up to decamers (m/z 1823 for the FeCl3 system) and provided insights into differences in the oligomerization mechanism. Spectra from a mixed DHP oligomer system containing H, G, and S units showed contributions from all monolignols within an oligomer level (e.g. tetramer level). CONCLUSIONS: The DHAP/Li+ method presented in this work shows promise to be an effective analytical tool for lignin analysis by MALDI and may provide a tool to assess lignin break-down efforts facilitating renewable products from lignin.

Phosphorylation of Src by phosphoinositide 3-kinase regulates beta-adrenergic receptor-mediated EGFR transactivation.

ß2-Adrenergic receptors (ß2AR) transactivate epidermal growth factor receptors (EGFR) through formation of a ß2AR-EGFR complex that requires activation of Src to mediate signaling. Here, we show that both lipid and protein kinase activities of the bifunctional phosphoinositide 3-kinase (PI3K) enzyme are required for ß2AR-stimulated EGFR transactivation. Mechanistically, the generation of phosphatidylinositol (3,4,5)-tris-phosphate (PIP3) by the lipid kinase function stabilizes ß2AR-EGFR complexes while the protein kinase activity of PI3K regulates Src activation by direct phosphorylation. The protein kinase activity of PI3K phosphorylates serine residue 70 on Src to enhance its activity and induce EGFR transactivation following ßAR stimulation. This newly identified function for PI3K, whereby Src is a substrate for the protein kinase activity of PI3K, is of importance since Src plays a key role in pathological and physiological signaling.

Muscle giants: molecular scaffolds in sarcomerogenesis.

Myofibrillogenesis in striated muscles is a highly complex process that depends on the coordinated assembly and integration of a large number of contractile, cytoskeletal, and signaling proteins into regular arrays, the sarcomeres. It is also associated with the stereotypical assembly of the sarcoplasmic reticulum and the transverse tubules around each sarcomere. Three giant, muscle-specific proteins, titin (3-4 MDa), nebulin (600-800 kDa), and obscurin (approximately 720-900 kDa), have been proposed to play important roles in the assembly and stabilization of sarcomeres. There is a large amount of data showing that each of these molecules interacts with several to many different protein ligands, regulating their activity and localizing them to particular sites within or surrounding sarcomeres. Consistent with this, mutations in each of these proteins have been linked to skeletal and cardiac myopathies or to muscular dystrophies. The evidence that any of them plays a role as a "molecular template," "molecular blueprint," or "molecular ruler" is less definitive, however. Here we review the structure and function of titin, nebulin, and obscurin, with the literature supporting a role for them as scaffolding molecules and the contradictory evidence regarding their roles as molecular guides in sarcomerogenesis.

The rho-guanine nucleotide exchange factor domain of obscurin activates rhoA signaling in skeletal muscle.

Obscurin is a large ( approximately 800-kDa), modular protein of striated muscle that concentrates around the M-bands and Z-disks of each sarcomere, where it is well positioned to sense contractile activity. Obscurin contains several signaling domains, including a rho-guanine nucleotide exchange factor (rhoGEF) domain and tandem pleckstrin homology domain, consistent with a role in rho signaling in muscle. We investigated the ability of obscurin's rhoGEF domain to interact with and activate small GTPases. Using a combination of in vitro and in vivo approaches, we found that the rhoGEF domain of obscurin binds selectively to rhoA, and that rhoA colocalizes with obscurin at the M-band in skeletal muscle. Other small GTPases, including rac1 and cdc42, neither associate with the rhoGEF domain of obscurin nor concentrate at the level of the M-bands. Furthermore, overexpression of the rhoGEF domain of obscurin in adult skeletal muscle selectively increases rhoA expression and activity in this tissue. Overexpression of obscurin's rhoGEF domain and its effects on rhoA alter the expression of rho kinase and citron kinase, both of which can be activated by rhoA in other tissues. Injuries to rodent hindlimb muscles caused by large-strain lengthening contractions increases rhoA activity and displaces it from the M-bands to Z-disks, similar to the effects of overexpression of obscurin's rhoGEF domain. Our results suggest that obscurin's rhoGEF domain signals at least in part by inducing rhoA expression and activation, and altering the expression of downstream kinases in vitro and in vivo.

Obscurin interacts with a novel isoform of MyBP-C slow at the periphery of the sarcomeric M-band and regulates thick filament assembly.

Obscurin is a multidomain protein composed of adhesion and signaling domains that plays key roles in the organization of contractile and membrane structures in striated muscles. Overexpression of the second immunoglobulin domain of obscurin (Ig2) in developing myotubes inhibits the assembly of A- and M-bands, but not Z-disks or I-bands. This effect is mediated by the direct interaction of the Ig2 domain of obscurin with a novel isoform of myosin binding protein-C slow (MyBP-C slow), corresponding to variant-1. Variant-1 contains all the structural motifs present in the known forms of MyBP-C slow, but it has a unique COOH terminus. Quantitative reverse transcription-polymerase chain reaction indicated that MyBP-C slow variant-1 is expressed in skeletal muscles both during development and at maturity. Immunolabeling of skeletal myofibers with antibodies to the unique COOH terminus of variant-1 demonstrated that, unlike other forms of MyBP-C slow that reside in the C-zones of A-bands, variant-1 preferentially concentrates around M-bands, where it codistributes with obscurin. Overexpression of the Ig2 domain of obscurin or reduction of expression of obscurin inhibited the integration of variant-1 into forming M-bands in skeletal myotubes. Collectively, our experiments identify a new ligand of obscurin at the M-band, MyBP-C slow variant-1 and suggest that their interaction contributes to the assembly of M- and A-bands.

The rho-guanine nucleotide exchange factor domain of obscurin regulates assembly of titin at the Z-disk through interactions with Ran binding protein 9.

Obscurin is an approximately 800-kDa protein composed of structural and signaling domains that organizes contractile structures in striated muscle. We have studied the Rho-GEF domain of obscurin to understand its roles in morphogenesis and signaling. We used adenoviral overexpression of this domain, together with ultrastructural and immunofluorescence methods, to examine its effect on maturing myofibrils. We report that overexpression of the Rho-GEF domain specifically inhibits the incorporation of titin into developing Z-disks and disrupts the structure of the Z-disk and Z/I junction, and alters features of the A/I junction. The organization of other sarcomeric markers, including alpha-actinin, was not affected. We identified Ran binding protein 9 (RanBP9) as a novel ligand of the Rho-GEF domain and showed that binding is specific, with an apparent binding affinity of 1.9 microM. Overexpression of the binding region of RanBP9 also disrupted the incorporation of titin into developing Z-disks. Immunofluorescence localization during myofibrillogenesis indicated that the Rho-GEF domain assembles into sarcomeres before RanBP9, which first occurs in myonuclei and later in development translocates to the myoplasm, where it colocalizes with obscurin. Both the Rho-GEF domain and its binding region on RanBP9 bind directly to the N-terminal Ig domains of titin, which flank the Z-disk. Our results suggest that the Rho-GEF domain interacts with RanBP9 and that both can interact with the N-terminal region of titin to influence the formation of the Z-disk and A/I junction.

Different obscurin isoforms localize to distinct sites at sarcomeres.

We used four antibodies to regions of obscurin isoforms A and B, encoded by the obscurin gene, to investigate the location of these proteins in skeletal myofibers at resting and stretched lengths. Obscurin A ( approximately 800 kDa) which was recognized by antibodies generated to the N-terminal, Rho-GEF, and the non-modular C-terminal domain that lacks the kinase-like domains, localizes at the level of the M-band. Obscurin B ( approximately 900 kDa) which has the N-terminal, Rho-GEF, and the C-terminal kinase-like domains, localizes at the level of the A/I junction. Additional isoforms, which lack one or more of these epitopes, are present at the Z-disk and Z/I junction.

Cloning and characterization of cytokeratins 8 and 19 in adult rat striated muscle. Interaction with the dystrophin glycoprotein complex.

We used degenerate primers for the amino- and carboxyl-terminal ends of the rod domains of intermediate filament proteins in reverse transcriptase-PCR experiments to identify and clone cytokeratins 8 and 19 (K8 and K19) from cardiac muscle of the adult rat. Northern blots showed that K8 has a 2.2-kb transcript and K19 has a 1.9-kb transcript in both adult cardiac and skeletal muscles. Immunolocalization of the cytokeratins in adult cardiac muscle with isoform-specific antibodies for K8 and K19 showed labeling at Z-lines within the muscle fibers and at Z-line and M-line domains at costameres at the sarcolemmal membrane. Dystrophin and K19 could be co-immunoprecipitated and co-purified from extracts of cardiac muscle, suggesting a link between the cytokeratins and the dystrophin-based cytoskeleton at the sarcolemma. Furthermore, transfection experiments indicate that K8 and K19 may associate with dystrophin through a specific interaction with its actin-binding domain. Consistent with this observation, the cytokeratins are disrupted at the sarcolemmal membrane of skeletal muscle of the mdx mouse that lacks dystrophin. Together these results indicate that at least two cytokeratins are expressed in adult striated muscle, where they may contribute to the organization of both the myoplasm and sarcolemma.