University of Virginia Health System

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

OBJECTIVE: To develop clinical practice guidelines for the management of patients with primary aldosteronism. PARTICIPANTS: The Task Force included a chair, selected by the Clinical Guidelines Subcommittee of the Endocrine Society, six additional experts, a methodologist, and a medical writer. The guideline was cosponsored by American Heart Association, American Association of Endocrine Surgeons, European Society of Endocrinology, European Society of Hypertension, International Association of Endocrine Surgeons, International Society of Endocrinology, International Society of Hypertension, Japan Endocrine Society, and The Japanese Society of Hypertension. The Task Force received no corporate funding or remuneration. EVIDENCE: We searched for systematic reviews and primary studies to formulate the key treatment and prevention recommendations. We used the Grading of Recommendations, Assessment, Development, and Evaluation group criteria to describe both the quality of evidence and the strength of recommendations. We used "recommend" for strong recommendations and "suggest" for weak recommendations. CONSENSUS PROCESS: We achieved consensus by collecting the best available evidence and conducting one group meeting, several conference calls, and multiple e-mail communications. With the help of a medical writer, the Endocrine Society's Clinical Guidelines Subcommittee, Clinical Affairs Core Committee, and Council successfully reviewed the drafts prepared by the Task Force. We placed the version approved by the Clinical Guidelines Subcommittee and Clinical Affairs Core Committee on the Endocrine Society's website for comments by members. At each stage of review, the Task Force received written comments and incorporated necessary changes. CONCLUSIONS: For high-risk groups of hypertensive patients and those with hypokalemia, we recommend case detection of primary aldosteronism by determining the aldosterone-renin ratio under standard conditions and recommend that a commonly used confirmatory test should confirm/exclude the condition. We recommend that all patients with primary aldosteronism undergo adrenal computed tomography as the initial study in subtype testing and to exclude adrenocortical carcinoma. We recommend that an experienced radiologist should establish/exclude unilateral primary aldosteronism using bilateral adrenal venous sampling, and if confirmed, this should optimally be treated by laparoscopic adrenalectomy. We recommend that patients with bilateral adrenal hyperplasia or those unsuitable for surgery should be treated primarily with a mineralocorticoid receptor antagonist.

These guidelines were developed jointly by the American Society of Health-System Pharmacists (ASHP), the Infectious Diseases Society of America (IDSA), the Surgical Infection Society (SIS), and the Society for Healthcare Epidemiology of America (SHEA). This work represents an update to the previously published ASHP Therapeutic Guidelines on Antimicrobial Prophylaxis in Surgery,1 as well as guidelines from IDSA and SIS.2,3 The guidelines are intended to provide practitioners with a standardized approach to the rational, safe, and effective use of antimicrobial agents for the prevention of surgical-site infections (SSIs) based on currently available clinical evidence and emerging issues. Prophylaxis refers to the prevention of an infection and can be characterized as primary prophylaxis, secondary prophylaxis, or eradication. Primary prophylaxis refers to the prevention of an initial infection. Secondary prophylaxis refers to the prevention of recurrence or reactivation of a preexisting infection. Eradication refers to the elimination of a...

Task Group 101 of the AAPM has prepared this report for medical physicists, clinicians, and therapists in order to outline the best practice guidelines for the external-beam radiation therapy technique referred to as stereotactic body radiation therapy (SBRT). The task group report includes a review of the literature to identify reported clinical findings and expected outcomes for this treatment modality. Information is provided for establishing a SBRT program, including protocols, equipment, resources, and QA procedures. Additionally, suggestions for developing consistent documentation for prescribing, reporting, and recording SBRT treatment delivery is provided.

OBJECTIVE: This update of a 2004 guideline codeveloped by the American Academy of Otolaryngology-Head and Neck Surgery Foundation, the American Academy of Pediatrics, and the American Academy of Family Physicians, provides evidence-based recommendations to manage otitis media with effusion (OME), defined as the presence of fluid in the middle ear without signs or symptoms of acute ear infection. Changes from the prior guideline include consumer advocates added to the update group, evidence from 4 new clinical practice guidelines, 20 new systematic reviews, and 49 randomized control trials, enhanced emphasis on patient education and shared decision making, a new algorithm to clarify action statement relationships, and new and expanded recommendations for the diagnosis and management of OME. PURPOSE: The purpose of this multidisciplinary guideline is to identify quality improvement opportunities in managing OME and to create explicit and actionable recommendations to implement these opportunities in clinical practice. Specifically, the goals are to improve diagnostic accuracy, identify children who are most susceptible to developmental sequelae from OME, and educate clinicians and patients regarding the favorable natural history of most OME and the clinical benefits for medical therapy (eg, steroids, antihistamines, decongestants). Additional goals relate to OME surveillance, hearing and language evaluation, and management of OME detected by newborn screening. The target patient for the guideline is a child aged 2 months through 12 years with OME, with or without developmental disabilities or underlying conditions that predispose to OME and its sequelae. The guideline is intended for all clinicians who are likely to diagnose and manage children with OME, and it applies to any setting in which OME would be identified, monitored, or managed. This guideline, however, does not apply to patients <2 months or >12 years old. ACTION STATEMENTS: The update group made strong recommendations that clinicians (1) should document the presence of middle ear effusion with pneumatic otoscopy when diagnosing OME in a child; (2) should perform pneumatic otoscopy to assess for OME in a child with otalgia, hearing loss, or both; (3) should obtain tympanometry in children with suspected OME for whom the diagnosis is uncertain after performing (or attempting) pneumatic otoscopy; (4) should manage the child with OME who is not at risk with watchful waiting for 3 months from the date of effusion onset (if known) or 3 months from the date of diagnosis (if onset is unknown); (5) should recommend against using intranasal or systemic steroids for treating OME; (6) should recommend against using systemic antibiotics for treating OME; and (7) should recommend against using antihistamines, decongestants, or both for treating OME.The update group made recommendations that clinicians (1) should document in the medical record counseling of parents of infants with OME who fail a newborn screening regarding the importance of follow-up to ensure that hearing is normal when OME resolves and to exclude an underlying sensorineural hearing loss; (2) should determine if a child with OME is at increased risk for speech, language, or learning problems from middle ear effusion because of baseline sensory, physical, cognitive, or behavioral factors; (3) should evaluate at-risk children for OME at the time of diagnosis of an at-risk condition and at 12 to 18 months of age (if diagnosed as being at risk prior to this time); (4) should not routinely screen children for OME who are not at risk and do not have symptoms that may be attributable to OME, such as hearing difficulties, balance (vestibular) problems, poor school performance, behavioral problems, or ear discomfort; (5) should educate children with OME and their families regarding the natural history of OME, need for follow-up, and the possible sequelae; (6) should obtain an age-appropriate hearing test if OME persists for 3 months or longer OR for OME of any duration in an at-risk child; (7) should counsel families of children with bilateral OME and documented hearing loss about the potential impact on speech and language development; (8) should reevaluate, at 3- to 6-month intervals, children with chronic OME until the effusion is no longer present, significant hearing loss is identified, or structural abnormalities of the eardrum or middle ear are suspected; (9) should recommend tympanostomy tubes when surgery is performed for OME in a child <4 years old; adenoidectomy should not be performed unless a distinct indication exists (nasal obstruction, chronic adenoiditis); (10) should recommend tympanostomy tubes, adenoidectomy, or both when surgery is performed for OME in a child ≥4 years old; and (11) should document resolution of OME, improved hearing, or improved quality of life when managing a child with OME.

Recently it was demonstrated that exposure of the developing brain during the period of synaptogenesis to drugs that block NMDA glutamate receptors or drugs that potentiate GABA(A) receptors can trigger widespread apoptotic neurodegeneration. All currently used general anesthetic agents have either NMDA receptor-blocking or GABA(A) receptor-enhancing properties. To induce or maintain a surgical plane of anesthesia, it is common practice in pediatric or obstetrical medicine to use agents from these two classes in combination. Therefore, the question arises whether this practice entails significant risk of inducing apoptotic neurodegeneration in the developing human brain. To begin to address this problem, we have administered to 7-d-old infant rats a combination of drugs commonly used in pediatric anesthesia (midazolam, nitrous oxide, and isoflurane) in doses sufficient to maintain a surgical plane of anesthesia for 6 hr, and have observed that this causes widespread apoptotic neurodegeneration in the developing brain, deficits in hippocampal synaptic function, and persistent memory/learning impairments.

Parametric mapping techniques provide a non-invasive tool for quantifying tissue alterations in myocardial disease in those eligible for cardiovascular magnetic resonance (CMR). Parametric mapping with CMR now permits the routine spatial visualization and quantification of changes in myocardial composition based on changes in T1, T2, and T2*(star) relaxation times and extracellular volume (ECV). These changes include specific disease pathways related to mainly intracellular disturbances of the cardiomyocyte (e.g., iron overload, or glycosphingolipid accumulation in Anderson-Fabry disease); extracellular disturbances in the myocardial interstitium (e.g., myocardial fibrosis or cardiac amyloidosis from accumulation of collagen or amyloid proteins, respectively); or both (myocardial edema with increased intracellular and/or extracellular water). Parametric mapping promises improvements in patient care through advances in quantitative diagnostics, inter- and intra-patient comparability, and relatedly improvements in treatment. There is a multitude of technical approaches and potential applications. This document provides a summary of the existing evidence for the clinical value of parametric mapping in the heart as of mid 2017, and gives recommendations for practical use in different clinical scenarios for scientists, clinicians, and CMR manufacturers.

The ongoing explosion of antibiotic-resistant infections continues to plague global and US health care. Meanwhile, an equally alarming decline has occurred in the research and development of new antibiotics to deal with the threat. In response to this microbial "perfect storm," in 2001, the federal Interagency Task Force on Antimicrobial Resistance released the "Action Plan to Combat Antimicrobial Resistance; Part 1: Domestic" to strengthen the response in the United States. The Infectious Diseases Society of America (IDSA) followed in 2004 with its own report, "Bad Bugs, No Drugs: As Antibiotic Discovery Stagnates, A Public Health Crisis Brews," which proposed incentives to reinvigorate pharmaceutical investment in antibiotic research and development. The IDSA's subsequent lobbying efforts led to the introduction of promising legislation in the 109 th US Congress (January 2005-December 2006). Unfortunately, the legislation was not enacted. During the 110 th Congress, the IDSA has continued to work with congressional leaders on promising legislation to address antibiotic-resistant infection. Nevertheless, despite intensive public relations and lobbying efforts, it remains unclear whether sufficiently robust legislation will be enacted. In the meantime, microbes continue to become more resistant, the antibiotic pipeline continues to diminish, and the majority of the public remains unaware of this critical situation. The result of insufficient federal funding; insufficient surveillance, prevention, and control; insufficient research and development activities; misguided regulation of antibiotics in agriculture and, in particular, for food animals; and insufficient overall coordination of US (and international) efforts could mean a literal return to the preantibiotic era for many types of infections. If we are to address the antimicrobial resistance crisis, a concerted, grassroots effort led by the medical community will be required.

OBJECTIVE: Our objective was to develop clinical practice guidelines for the diagnosis and treatment of patients with primary aldosteronism. PARTICIPANTS: The Task Force comprised a chair, selected by the Clinical Guidelines Subcommittee (CGS) of The Endocrine Society, six additional experts, one methodologist, and a medical writer. The Task Force received no corporate funding or remuneration. EVIDENCE: Systematic reviews of available evidence were used to formulate the key treatment and prevention recommendations. We used the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) group criteria to describe both the quality of evidence and the strength of recommendations. We used "recommend" for strong recommendations and "suggest" for weak recommendations. CONSENSUS PROCESS: Consensus was guided by systematic reviews of evidence and discussions during one group meeting, several conference calls, and multiple e-mail communications. The drafts prepared by the task force with the help of a medical writer were reviewed successively by The Endocrine Society's CGS, Clinical Affairs Core Committee (CACC), and Council. The version approved by the CGS and CACC was placed on The Endocrine Society's Web site for comments by members. At each stage of review, the Task Force received written comments and incorporated needed changes. CONCLUSIONS: We recommend case detection of primary aldosteronism be sought in higher risk groups of hypertensive patients and those with hypokalemia by determining the aldosterone-renin ratio under standard conditions and that the condition be confirmed/excluded by one of four commonly used confirmatory tests. We recommend that all patients with primary aldosteronism undergo adrenal computed tomography as the initial study in subtype testing and to exclude adrenocortical carcinoma. We recommend the presence of a unilateral form of primary aldosteronism should be established/excluded by bilateral adrenal venous sampling by an experienced radiologist and, where present, optimally treated by laparoscopic adrenalectomy. We recommend that patients with bilateral adrenal hyperplasia, or those unsuitable for surgery, optimally be treated medically by mineralocorticoid receptor antagonists.

With mounting data on its accuracy and prognostic value, cardiovascular magnetic resonance (CMR) is becoming an increasingly important diagnostic tool with growing utility in clinical routine. Given its versatility and wide range of quantitative parameters, however, agreement on specific standards for the interpretation and post-processing of CMR studies is required to ensure consistent quality and reproducibility of CMR reports. This document addresses this need by providing consensus recommendations developed by the Task Force for Post-Processing of the Society for Cardiovascular Magnetic Resonance (SCMR). The aim of the Task Force is to recommend requirements and standards for image interpretation and post-processing enabling qualitative and quantitative evaluation of CMR images. Furthermore, pitfalls of CMR image analysis are discussed where appropriate. It is an update of the original recommendations published 2013.

The actual incidence of neurologic dysfunction resulting from hemorrhagic complications associated with neuraxial blockade is unknown. Although the incidence cited in the literature is estimated to be less than 1 in 150,000 epidural and less than 1 in 220,000 spinal anesthetics, recent epidemiologic surveys suggest that the frequency is increasing and may be as high as 1 in 3000 in some patient populations.Overall, the risk of clinically significant bleeding increase with age,associated abnormalities of the spinal cord or vertebral column, the presence of an underlying coagulopathy, difficulty during needle placement,and an indwelling neuraxial catheter during sustained anticoagulation( particularly with standard heparin or low-molecular weight heparin). The need for prompt diagnosis and intervention to optimize neurologic outcome is also consistently reported. In response to these patient safety issues, the American Society of Regional Anesthesia and Pain Medicine (ASRA) convened its Third Consensus Conference on Regional Anesthesia and Anticoagulation. Practice guidelines or recommendations summarize evidence-based reviews. However, the rarity of spinal hematoma defies a prospective randomized study, and there is no current laboratory model. As a result,the ASRA consensus statements represent the collective experience of recognized experts in the field of neuraxial anesthesia and anticoagulation. These are based on case reports, clinical series, pharmacology,hematology, and risk factors for surgical bleeding. An understanding of the complexity of this issue is essential to patient management.

BAX, a heterodimeric partner of BCL2, counters BCL2 and promotes apoptosis in gain-of-function experiments. A Bax knockout mouse was generated that proved viable but displayed lineage-specific aberrations in cell death. Thymocytes and B cells in this mouse displayed hyperplasia, and Bax-deficient ovaries contained unusual atretic follicles with excess granulosa cells. In contrast, Bax-deficient males were infertile as a result of disordered seminiferous tubules with an accumulation of atypical premeiotic germ cells, but no mature haploid sperm. Multinucleated giant cells and dysplastic cells accompanied massive cell death. Thus, the loss of Bax results in hyperplasia or hypoplasia, depending on the cellular context.

These guidelines from the Infectious Diseases Society of America (IDSA), the American College of Critical Care Medicine (for the Society of Critical Care Medicine), and the Society for Healthcare Epidemiology of America contain recommendations for the management of adults and children with, and diagnosis of infections related to, peripheral and nontunneled central venous catheters (CVCs), pulmonary artery catheters, tunneled central catheters, and implantable devices. The guidelines, written for clinicians, contain IDSA evidence-based recommendations for assessment of the quality and strength of the data. Recommendations are presented according to the type of catheter, the infecting organism, and the associated complications. Intravascular catheter-related infections are a major cause of morbidity and mortality in the United States. Coagulase-negative staphylococci, Staphylococcus aureus, aerobic gram-negative bacilli, and Candida albicans most commonly cause catheter-related bloodstream infection. Management of catheter-related infection varies according to the type of catheter involved. After appropriate cultures of blood and catheter samples are done, empirical i.v. antimicrobial therapy should be initiated on the basis of clinical clues, the severity of the patient's acute illness, underlying disease, and the potential pathogen(s) involved. In most cases of nontunneled CVC-related bacteremia and fungemia, the CVC should be removed. For management of bacteremia and fungemia from a tunneled catheter or implantable device, such as a port, the decision to remove the catheter or device should be based on the severity of the patient's illness, documentation that the vascular-access device is infected, assessment of the specific pathogen involved, and presence of complications, such as endocarditis, septic thrombosis, tunnel infection, or metastatic seeding. When a catheter-related infection is documented and a specific pathogen is identified, systemic antimicrobial therapy should be narrowed and consideration given for antibiotic lock therapy, if the CVC or implantable device is not removed. These guidelines address the issues related to the management of catheter-related bacteremia and associated complications. Separate guidelines will address specific issues related to the prevention of catheter-related infections. Performance indicators for the management of catheter-related infection are included at the end of the document. Because the pathogenesis of catheter-related infections is complicated, the virulence of the pathogens is variable, and the host factors have not been well defined, there is a notable absence of compelling clinical data to make firm recommendations for an individual patient. Therefore, the recommendations in these guidelines are intended to support, and not replace, good clinical judgment. Also, a section on selected, unresolved clinical issues that require further study and research has been included. There is an urgent need for large, well-designed clinical studies to delineate management strategies more effectively, which will improve clinical outcomes and save precious health care resources.

An update to the American Cancer Society (ACS) guideline regarding screening for the early detection of cervical precancerous lesions and cancer is presented. The guidelines are based on a systematic evidence review, contributions from 6 working groups, and a recent symposium cosponsored by the ACS, the American Society for Colposcopy and Cervical Pathology, and the American Society for Clinical Pathology, which was attended by 25 organizations. The new screening recommendations address age-appropriate screening strategies, including the use of cytology and high-risk human papillomavirus (HPV) testing, follow-up (eg, the management of screen positives and screening intervals for screen negatives) of women after screening, the age at which to exit screening, future considerations regarding HPV testing alone as a primary screening approach, and screening strategies for women vaccinated against HPV16 and HPV18 infections.

The protein tyrosine kinase focal adhesion kinase (FAK) plays a prominent role in integrin signaling. FAK activation, demonstrated by an increase in phosphorylation of Tyr397 as well as other sites in the protein, is best understood in the context of the engagement of integrins at the cell surface. Activation of FAK results in recruitment of a number of SH2-domain- and SH3-domain-containing proteins, which mediate signaling to several downstream pathways. FAK-dependent activation of these pathways has been implicated in a diverse array of cellular processes, including cell migration, growth factor signaling, cell cycle progression and cell survival.

In October 2002, a workshop was held in Ancona, Italy, to reach a Consensus on the management of Cushing's syndrome. The workshop was organized by the University of Ancona and sponsored by the Pituitary Society, the European Neuroendocrine Association, and the Italian Society of Endocrinology. Invited international participants included almost 50 leading endocrinologists with specific expertise in the management of Cushing's syndrome. The consensus statement on diagnostic criteria and the diagnosis and treatment of complications of this syndrome reached at the workshop is hereby summarized.

This study was undertaken to update and revise the estimate of the economic impact of obesity in the United States. A prevalence-based approach to the cost of illness was used to estimate the economic costs in 1995 dollars attributable to obesity for type 2 diabetes mellitus, coronary heart disease (CHD), hypertension, gallbladder disease, breast, endometrial and colon cancer, and osteoarthritis. Additionally and independently, excess physician visits, work-lost days, restricted activity, and bed-days attributable to obesity were analyzed cross-sectionally using the 1988 and 1994 National Health Interview Survey (NHIS). Direct (personal health care, hospital care, physician services, allied health services, and medications) and indirect costs (lost output as a result of a reduction or cessation of productivity due to morbidity or mortality) are from published reports and inflated to 1995 dollars using the medical component of the consumer price index (CPI) for direct cost and the all-items CPI for indirect cost. Population-attributable risk percents (PAR%) are estimated from large prospective studies. Excess work-lost days, restricted activity, bed-days, and physician visits are estimated from 88,262 U.S. citizens who participated in the 1988 NHIS and 80,261 who participated in the 1994 NHIS. Sample weights have been incorporated into the NHIS analyses, making these data generalizable to the U.S. population. The total cost attributable to obesity amounted to $99.2 billion dollars in 1995. Approximately $51.64 billion of those dollars were direct medical costs. Using the 1994 NHIS data, cost of lost productivity attributed to obesity (BMI> or =30) was $3.9 billion and reflected 39.2 million days of lost work. In addition, 239 million restricted-activity days, 89.5 million bed-days, and 62.6 million physician visits were attributable to obesity in 1994. Compared with 1988 NHIS data, in 1994 the number of restricted-activity days (36%), bed-days (28%), and work-lost days (50%) increased substantially. The number of physician visits attributed to obesity increased 88% from 1988 to 1994. The economic and personal health costs of overweight and obesity are enormous and compromise the health of the United States. The direct costs associated with obesity represent 5.7% of our National Health Expenditure in the United States.

In recent years, there has been an exponential growth in the number of complex documents and texts that require a deeper understanding of machine learning methods to be able to accurately classify texts in many applications. Many machine learning approaches have achieved surpassing results in natural language processing. The success of these learning algorithms relies on their capacity to understand complex models and non-linear relationships within data. However, finding suitable structures, architectures, and techniques for text classification is a challenge for researchers. In this paper, a brief overview of text classification algorithms is discussed. This overview covers different text feature extractions, dimensionality reduction methods, existing algorithms and techniques, and evaluations methods. Finally, the limitations of each technique and their application in real-world problems are discussed.

We here review mechanisms that can regulate the activity of myosin II, in smooth muscle and non-muscle cells, by modulating the Ca2+ sensitivity of myosin regulatory light chain (RLC) phosphorylation. The major mechanism of Ca2+ sensitization of smooth muscle contraction and non-muscle cell motility is through inhibition of the smooth muscle myosin phosphatase (MLCP) that dephosphorylates the RLC in smooth muscle and non-muscle. The active, GTP-bound form of the small GTPase RhoA activates a serine/threonine kinase, Rho-kinase, that phosphorylates the regulatory subunit of MLCP and inhibits phosphatase activity. G-protein-coupled release of arachidonic acid may also contribute to inhibition of MLCP acting, at least in part, through the Rho/Rho-kinase pathway. Protein kinase C(s) activated by phorbol esters and diacylglycerol can also inhibit MLCP by phosphorylating and thereby activating CPI-17, an inhibitor of its catalytic subunit; this mechanism is independent of the Rho/Rho-kinase pathway and plays only a minor, transient role in the G-protein-coupled mechanism of Ca2+ sensitization. Ca2+ sensitization by the Rho/Rho-kinase pathway contributes to the tonic phase of agonist-induced contraction in smooth muscle, and abnormally increased activation of myosin II by this mechanism is thought to play a role in diseases such as high blood pressure and cancer cell metastasis.