Mechanical activation of TWIK-related potassium channel by nanoscopic movement and rapid second messenger signaling.

Rapid conversion of force into a biological signal enables living cells to respond to mechanical forces in their environment. The force is believed to initially affect the plasma membrane and then alter the behavior of membrane proteins. Phospholipase D2 (PLD2) is a mechanosensitive enzyme that is regulated by a structured membrane-lipid site comprised of cholesterol and saturated ganglioside (GM1). Here we show stretch activation of TWIK-related K+ channel (TREK-1) is mechanically evoked by PLD2 and spatial patterning involving ordered GM1 and 4,5-bisphosphate (PIP2) clusters in mammalian cells. First, mechanical force deforms the ordered lipids, which disrupts the interaction of PLD2 with the GM1 lipids and allows a complex of TREK-1 and PLD2 to associate with PIP2 clusters. The association with PIP2 activates the enzyme, which produces the second messenger phosphatidic acid (PA) that gates the channel. Co-expression of catalytically inactive PLD2 inhibits TREK-1 stretch currents in a biological membrane. Cellular uptake of cholesterol inhibits TREK-1 currents in culture and depletion of cholesterol from astrocytes releases TREK-1 from GM1 lipids in mouse brain. Depletion of the PLD2 ortholog in flies results in hypersensitivity to mechanical force. We conclude PLD2 mechanosensitivity combines with TREK-1 ion permeability to elicit a mechanically evoked response.

"Ouch!": you have just stabbed your little toe on the sharp corner of a coffee table. That painful sensation stems from nerve cells converting information about external forces into electric signals the brain can interpret. Increasingly, new evidence is suggesting that this process may be starting at fat-based structures within the membrane of these cells. The cell membrane is formed of two interconnected, flexible sheets of lipids in which embedded structures or molecules are free to move. This organisation allows the membrane to physically respond to external forces and, in turn, to set in motion chains of molecular events that help fine-tune how cells relay such information to the brain. For instance, an enzyme known as PLD2 is bound to lipid rafts ­ precisely arranged, rigid fatty 'clumps' in the membrane that are partly formed of cholesterol. PLD2 has also been shown to physically interact with and then activate the ion channel TREK-1, a membrane-based protein that helps to prevent nerve cells from relaying pain signals. However, the exact mechanism underpinning these interactions is difficult to study due to the nature and size of the molecules involved. To address this question, Petersen et al. combined a technology called super-resolution imaging with a new approach that allowed them to observe how membrane lipids respond to pressure and fluid shear. The experiments showed that mechanical forces disrupt the careful arrangement of lipid rafts, causing PLD2 and TREK-1 to be released. They can then move through the surrounding membrane where they reach a switch that turns on TREK-1. Further work revealed that the levels of cholesterol available to mouse cells directly influenced how the clumps could form and bind to PLD2, and in turn, dialled up and down the protective signal mediated by TREK-1. Overall, the study by Petersen et al. shows that the membrane of nerve cells can contain cholesterol-based 'fat sensors' that help to detect external forces and participate in pain regulation. By dissecting these processes, it may be possible to better understand and treat conditions such as diabetes and lupus, which are associated with both pain sensitivity and elevated levels of cholesterol in tissues.

Analysis of free cisplatin in microdialysates and plasma ultrafiltrate by liquid chromatography-tandem mass spectrometry.

Cisplatin is a potent cytotoxic agent used in the treatment of various malignancies and exerts its antitumor effect through malignant cell DNA damage and apoptosis induction. Evaluation of systemic delivery of cisplatin is important in optimization of cisplatin treatment. However, accurate quantification of systemic cisplatin is challenging due to its various forms in circulation. This study aimed to develop a sensitive (LOQ < 0.1 µg/mL) and precise Ultra Performance Liquid Chromatography (UPLC) - Tandem Mass Spectrometry (MS/MS) method for quantifying free cisplatin in microdialysates and plasma. Furthermore the aim was to compare free cisplatin concentrations measured in standard plasma samples with those obtained from intravenous microdialysis catheters in a porcine model. The method developed utilizes dichloro(ethylenediamine)platinum(II) as an internal standard that co-elutes with cisplatin, ensuring precise correction for ion suppression/enhancement effects. The method was validated, demonstrating linearity up to 100 µg/mL and good intermediate precision (CV% < 6 %) in the range of 1.0-100 µg/mL, with an LOQ of 0.03 µg/mL. The pharmacokinetic parameters (AUC0-last, Cmax, T1/2, and Tmax) showed no significant differences between the two sampling methods. This validated LC-MS/MS method provides a reliable tool for quantifying systemic free cisplatin concentrations, facilitating future systemic and local pharmacokinetic evaluations for optimization of cisplatin-based cancer treatments.

Metabolic flexibility in postmenopausal women: Hormone replacement therapy is associated with higher mitochondrial content, respiratory capacity, and lower total fat mass.

AIM: To investigate effects of hormone replacement therapy in postmenopausal women on factors associated with metabolic flexibility related to whole-body parameters including fat oxidation, resting energy expenditure, body composition and plasma concentrations of fatty acids, glucose, insulin, cortisol, and lipids, and for the mitochondrial level, including mitochondrial content, respiratory capacity, efficiency, and hydrogen peroxide emission. METHODS: 22 postmenopausal women were included. 11 were undergoing estradiol and progestin treatment (HT), and 11 were matched non-treated controls (CONT). Peak oxygen consumption, maximal fat oxidation, glycated hemoglobin, body composition, and resting energy expenditure were measured. Blood samples were collected at rest and during 45 min of ergometer exercise (65% VO2peak). Muscle biopsies were obtained at rest and immediately post-exercise. Mitochondrial respiratory capacity, efficiency, and hydrogen peroxide emission in permeabilized fibers and isolated mitochondria were measured, and citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HAD) activity were assessed. RESULTS: HT showed higher absolute mitochondrial respiratory capacity and post-exercise hydrogen peroxide emission in permeabilized fibers and higher CS and HAD activities. All respiration normalized to CS activity showed no significant group differences in permeabilized fibers or isolated mitochondria. There were no differences in resting energy expenditure, maximal, and resting fat oxidation or plasma markers. HT had significantly lower visceral and total fat mass compared to CONT. CONCLUSION: Use of hormone therapy is associated with higher mitochondrial content and respiratory capacity and a lower visceral and total fat mass. Resting energy expenditure and fat oxidation did not differ between HT and CONT.

Analysis and applications of respiratory surface EMG: report of a round table meeting.

Surface electromyography (sEMG) can be used to measure the electrical activity of the respiratory muscles. The possible applications of sEMG span from patients suffering from acute respiratory failure to patients receiving chronic home mechanical ventilation, to evaluate muscle function, titrate ventilatory support and guide treatment. However, sEMG is mainly used as a monitoring tool for research and its use in clinical practice is still limited-in part due to a lack of standardization and transparent reporting. During this round table meeting, recommendations on data acquisition, processing, interpretation, and potential clinical applications of respiratory sEMG were discussed. This paper informs the clinical researcher interested in respiratory muscle monitoring about the current state of the art on sEMG, knowledge gaps and potential future applications for patients with respiratory failure.

An experimental apparatus for the study of high-temperature degradation and solid-deposit formation of lubricants.

When exposed to high surface temperatures, engine lubricating oils degrade and may form solid deposits, which cause operational issues and increase shutdown time and maintenance costs. Despite its being a common issue in engine operation, the information available on the mechanics of this phenomenon is still lacking, and the experimental data and conditions must be updated to match the improvements in both lubricant stability and engine efficiency. To this end, an experimental apparatus has been developed to study the mechanisms that lead to the degradation and deposit formation of lubricants at high temperatures. The apparatus is designed to operate at pressures up to 69 bar, surface temperatures up to 650 °C, oil bulk temperatures up to 550 °C, and flow rates of <14 mL/min. In this apparatus, the oil is cycled through a heated test section, and deposits accumulate on the heated surface. The time required for deposits to start accumulating under the test conditions is determined based on the recorded temperature traces, and collected oil and deposit samples may be analyzed to determine changes in composition over time due to high-temperature exposure. The removable test section can be modified to accommodate different geometries, surface materials, and flow paths to adapt the instrument to a range of potential research directions. This paper presents the technical details of the new apparatus and the steps taken to characterize the experimental conditions. In addition, sample data are provided to show the unique capabilities of the new instrument, and an Arrhenius plot for Castrol Perfecto X 32 in the surface temperature range of 445-475 °C is presented as a demonstration of its use for quantifying the coking delay time. The new instrument detailed herein is the first such device to demonstrate a reliable, lab-scale technique for studying lube oil coke formation and deposition at temperatures and pressures of interest to power generation gas turbines.

Is loneliness associated with cancellation of medical appointments during the COVID-19 pandemic? Evidence from the Hamburg City Health Study (HCHS).

BACKGROUND: The COVID-19 pandemic engendered numerous societal and economic challenges in addition to health-related concerns. Maintenance of healthcare utilization assumed immense significance during this period. However, few studies have examined the association between loneliness and cancelled medical appointments during the COVID-19 pandemic. This study aimed to examine whether medical appointments are less likely to be cancelled with increased loneliness during a pandemic. We analyzed the association between loneliness and both patient- and provider-initiated appointment cancellations. METHODS: Cross-sectional data from the Hamburg City Health Study (HCHS) were collected during April 2020-November 2021. The analytical sample included 1,840 participants with an average age of 55.1 years (standard deviation: 6.5, range 45-76 years). Medical appointments cancelled by individuals-medical appointments in general, and GP, specialist, and dentist appointments-and appointments cancelled by healthcare providers served as outcome measures. Loneliness was quantified using a single item ranging from 0 to 10. Accordingly, we created empirical loneliness tertiles. Covariates were selected based on the Andersen model. Several penalized maximum likelihood logistic regressions were utilized to examine the association between loneliness and cancellation of medical appointments during the COVID-19 pandemic. RESULTS: The penalized maximum likelihood logistic regressions showed that, compared to individuals in the lowest loneliness tertiles, individuals in the other two tertiles reported a higher chance of medical appointments cancellation by individuals, particularly driven by cancelled GP appointments. Except for age and sex, none of the covariates were comparably associated with the outcomes. When appointments cancelled by healthcare providers served as outcomes, only a higher number of chronic conditions was significantly positively associated with it. CONCLUSIONS: Individuals scoring higher in loneliness had a greater chance of cancelling medical (particularly GP) appointments. This may contribute to a potential cascade of loneliness and skipped medical appointments in the future, resulting in adverse health outcomes over the medium-to-long term. Future research should examine whether lonely people are more likely to lack the social motivation to visit the doctor.

Orthogonal and complementary measurements of properties of drug products containing nanomaterials.

Quality control of pharmaceutical and biopharmaceutical products, and verification of their safety and efficacy, depends on reliable measurements of critical quality attributes (CQAs). The task becomes particularly challenging for drug products and vaccines containing nanomaterials, where multiple complex CQAs must be identified and monitored. To reduce (i) the risk of measurement bias and (ii) the uncertainty in decision-making during product development, the combination of orthogonal and complementary analytical techniques are generally recommended by regulators. However, despite frequent reference to "orthogonal" and "complementary" in guidance documents, neither term is clearly defined. How does one determine if two analytical methods are orthogonal or complementary to one another? Definitions are needed to design a robust characterization strategy aligned to regulatory needs. Definitions for "orthogonal" and "complementary" are proposed that are compatible with existing metrological terminology and are applicable to complex measurement problems. Orthogonal methods target the quantitative evaluation of the true value of a product attribute to address unknown bias or interference. Complementary measurements include a broader scope of methods that reinforce each other to support a common decision. Examples of the application of these terms are presented, with a focus on measurement of physical properties of nano-enabled drug products, including liposomes and polymeric nanoparticles for cancer treatment, lipid-based nanoparticles (LNPs) and virus-like particles for nucleic acid delivery. The proposed framework represents a first step in advancing the assessment of the orthogonality and complementarity of two measurements and it can potentially serve as the basis for a future international standard. This framework may help product developers to implement more efficient product characterization strategies, accelerate the introduction of novel medicines to the clinic and be applicable to other therapeutics beyond nanomaterial-containing pharmaceuticals.

Sociocultural influences on dietary behavior and meal timing among Native Hawaiian and Pacific Islander women at risk of endometrial cancer: a qualitative investigation.

PURPOSE: Determine sociocultural influences on dietary behavior, body image, weight loss, and perceptions of the cultural appropriateness of a meal-timing intervention design and menu among Native Hawaiian and Pacific Islander (NHPI) women at risk of endometrial cancer. METHODS: Six 90-min videoconference focus groups among NHPI women (n = 35) recruited by a community champion in Utah. Eligible women were aged ≥ 18 years at risk of endometrial cancer (i.e., BMI ≥ 25 kg/m2, history of non-insulin-dependent diabetes or complex atypical endometrial hyperplasia) had a working cell phone capable of downloading a phone app, could use their cell phone during the day, and were not night-shift workers. Twelve semi-structured questions were posed during the focus groups. Using inductive qualitative methods based on Hatch's 9-step approach, de-identified transcript data were analyzed. RESULTS: Overarching themes included economic factors, cultural influences, meal choice and timing, and perceptions of health. Subthemes included affordability, waste avoidance, inundated schedules, and cultural influences. Perceptions of body size and weight loss were influenced by family, community, and social media, whose messages could be conflicting. Important intervention components included satisfying, convenient pre-made meals, while barriers included the need to cook for family members. CONCLUSIONS: Dietary interventions targeting metabolic health among NHPI women should consider the multitude of sociocultural and economic factors that influence food choices and meal timing in this population, including affordability, hectic schedules, and immigrant adjustment. Promoting the link between physical and mental well-being as opposed to weight loss is a key approach to reaching this population.

Where does spinal cord stimulation fit into the international guidelines for refractory painful diabetic neuropathy? a consensus statement.

BACKGROUND: Although pharmacotherapy with anticonvulsants and/or antidepressants can be effective for many people with painful diabetic neuropathy (PDN), albeit with frequent side-effects, a critical juncture occurs when neuropathic pain no longer responds to standard first- and second-step mono- and dual therapy and becomes refractory. Subsequent to these pharmacotherapeutic approaches, third-line treatment options for PDN may include opioids (short-term), capsaicin 8% patches, and spinal cord stimulation (SCS). AIM: This document summarizes consensus recommendations regarding appropriate treatment for refractory peripheral diabetic neuropathy (PDN), based on outcomes from an expert panel convened on December 10, 2022, as part of the Worldwide Initiative for Diabetes Education Virtual Global Summit, "Advances in the Management of Painful Diabetic Neuropathy." PARTICIPANTS: Nine attendees, eminent physicians and academics, comprising six diabetes specialists, two pain specialists, and one health services expert. EVIDENCE: For individuals with refractory PDN, opioids are a high-risk option that do not provide a long-term solution and should not be used. For appropriately selected individuals, SCS is an effective, safe, and durable treatment option. In particular, high-frequency (HF) SCS (10 kHz) shows strong efficacy and improves quality of life. To ensure treatment success, strict screening criteria should be used to prioritize candidates for SCS. CONSENSUS PROCESS: Each participant voiced their opinion after reviewing available data, and a verbal consensus was reached during the meeting. CONCLUSION: Globally, the use of opioids should rarely be recommended for refractory, severe PDN. Based on increasing clinical evidence, SCS, especially HF-SCS, should be considered as a treatment for PDN that is not responsive to first- or second-line monotherapy/dual therapy.

Patients with knee osteoarthritis can be divided into subgroups based on tibiofemoral joint kinematics of gait - an exploratory and dynamic radiostereometric study.

OBJECTIVE: Patients with advanced knee osteoarthritis (KOA) frequently alter their gait patterns in an attempt to alleviate symptoms. Understanding the underlying pathomechanics and identifying KOA phenotypes are essential to improve treatments. We investigated kinematics in patients with KOA to identify subgroups of homogeneous knee joint kinematics. METHOD: A total of 66 patients with symptomatic KOA scheduled for total knee arthroplasty and 15 age-matched healthy volunteers with asymptomatic, non-arthritic knees were included. We used k-means clustering to divide patients into subgroups based on dynamic radiostereometry-assessed tibiofemoral joint kinematics. Clinical characteristics such as knee ligament lesions and KOA scores were graded by magnetic resonance imaging and radiographs, respectively. RESULTS: We identified four clusters that were supported by clinical characteristics. The flexion group (n = 20) consisted primarily of patients with medial KOA. The abduction group (n = 17) consisted primarily of patients with lateral KOA. The anterior draw group (n = 10) was composed of patients with medial KOA, some degree of anterior cruciate ligament lesion and the highest KOA score. The external rotation group (n = 19) primarily included patients with medial collateral and posterior cruciate ligament lesions. CONCLUSION: Based on tibiofemoral gait patterns, patients with advanced KOA can be divided into four subgroups with specific clinical characteristics and different KOA-affected compartments. The findings add to our understanding of how knee kinematics may affect the patient's development of different types of KOA. This may inspire improved and more patient-specific treatment strategies in the future.

The matrix-dependent 3D spheroid model of the migration of non-small cell lung cancer: a step towards a rapid automated screening.

In vitro 3D cell culture systems utilizing multicellular tumor spheroids (MCTS) are widely used in translational oncology, including for studying cell migration and in personalized therapy. However, early stages of cellular migration from MCTS and cross-talk between spheroids are overlooked, which was addressed in the current study. Here, we investigated cell migration from MCTS derived from human non-small cell lung cancer (NSCLC) cell line A549 cultured on different substrates, collagen gel or plastic, at different time points. We found that migration starts at 4-16 h time points after the seeding and its speed is substrate-dependent. We also demonstrated that co-culture of two NSCLC-derived MCTS on collagen gel, but not on plastic, facilitates cell migration compared with single MTCS. This finding should be considered when designing MCTS-based functional assays for personalized therapeutic approach and drug screenings. Overall, our work characterizes the in vitro 3D cell culture model resembling NSCLC cell migration from the clusters of CTCs into surgical wound, and describes microscopy-based tools and approaches for image data analysis with a potential for further automation. These tools and approaches also might be used to predict patterns of CTCs migration based on ex vivo analysis of patient biopsy in a 3D culture system.

Studies on the mechanism of general anesthesia.

Inhaled anesthetics are a chemically diverse collection of hydrophobic molecules that robustly activate TWIK-related K+ channels (TREK-1) and reversibly induce loss of consciousness. For 100 y, anesthetics were speculated to target cellular membranes, yet no plausible mechanism emerged to explain a membrane effect on ion channels. Here we show that inhaled anesthetics (chloroform and isoflurane) activate TREK-1 through disruption of phospholipase D2 (PLD2) localization to lipid rafts and subsequent production of signaling lipid phosphatidic acid (PA). Catalytically dead PLD2 robustly blocks anesthetic TREK-1 currents in whole-cell patch-clamp recordings. Localization of PLD2 renders the TRAAK channel sensitive, a channel that is otherwise anesthetic insensitive. General anesthetics, such as chloroform, isoflurane, diethyl ether, xenon, and propofol, disrupt lipid rafts and activate PLD2. In the whole brain of flies, anesthesia disrupts rafts and PLDnull flies resist anesthesia. Our results establish a membrane-mediated target of inhaled anesthesia and suggest PA helps set thresholds of anesthetic sensitivity in vivo.

Toxoplasma gondii-specific IgG avidity testing in pregnant women.

BACKGROUND: The parasite Toxoplasma gondii can cause congenital toxoplasmosis following primary infection in a pregnant woman. It is therefore important to distinguish between recent and past infection when both T. gondii-specific IgM and IgG are detected in a single serum in pregnant women. Toxoplasma gondii-specific IgG avidity testing is an essential tool to help to date the infection. However, interpretation of its results can be complex. OBJECTIVES: To review the benefits and limitations of T. gondii-specific avidity testing in pregnant women, to help practitioners to interpret the results and adapt the patient management. SOURCES: PubMed search with the keywords avidity, toxoplasmosis and Toxoplasma gondii for articles published from 1989 to 2019. CONTENT: Toxoplasma gondii-specific IgG avidity testing remains a key tool for dating a T. gondii infection in immunocompetent pregnant women. Several commercial assays are available and display comparable performances. A high avidity result obtained on a first-trimester serum sample is indicative of a past infection, which occurred before pregnancy. To date, a low avidity result must still be considered as non-informative to date the infection, although some authors suggest that very low avidity results are highly suggestive of recent infections depending on the assay. Interpretation of low or grey zone avidity results on a first-trimester serum sample, as well as any avidity result on a second-trimester or third-trimester serum sample, is more complex and requires recourse to expert toxoplasmosis laboratories. IMPLICATIONS: Although used for about 30 years, T. gondii-specific avidity testing has scarcely evolved. The same difficulties in interpretation have persisted over the years. Some authors have proposed additional thresholds to exclude an infection of <9 months, or in contrast to confirm a recent infection. Such thresholds would be of great interest to adapt management of pregnant women and avoid unnecessary treatment; however, they need confirmation and further studies.

COVID-19, SARS and MERS: are they closely related?

BACKGROUND: The 2019 novel coronavirus (SARS-CoV-2) is a new human coronavirus which is spreading with epidemic features in China and other Asian countries; cases have also been reported worldwide. This novel coronavirus disease (COVID-19) is associated with a respiratory illness that may lead to severe pneumonia and acute respiratory distress syndrome (ARDS). Although related to the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome (MERS), COVID-19 shows some peculiar pathogenetic, epidemiological and clinical features which to date are not completely understood. AIMS: To provide a review of the differences in pathogenesis, epidemiology and clinical features of COVID-19, SARS and MERS. SOURCES: The most recent literature in the English language regarding COVID-19 has been reviewed, and extracted data have been compared with the current scientific evidence about SARS and MERS epidemics. CONTENT: COVID-19 seems not to be very different from SARS regarding its clinical features. However, it has a fatality rate of 2.3%, lower than that of SARS (9.5%) and much lower than that of MERS (34.4%). The possibility cannot be excluded that because of the less severe clinical picture of COVID-19 it can spread in the community more easily than MERS and SARS. The actual basic reproductive number (R0) of COVID-19 (2.0-2.5) is still controversial. It is probably slightly higher than the R0 of SARS (1.7-1.9) and higher than that of MERS (<1). A gastrointestinal route of transmission for SARS-CoV-2, which has been assumed for SARS-CoV and MERS-CoV, cannot be ruled out and needs further investigation. IMPLICATIONS: There is still much more to know about COVID-19, especially as concerns mortality and its capacity to spread on a pandemic level. Nonetheless, all of the lessons we learned in the past from the SARS and MERS epidemics are the best cultural weapons with which to face this new global threat.

Disruption of palmitate-mediated localization; a shared pathway of force and anesthetic activation of TREK-1 channels.

TWIK related K+ channel (TREK-1) is a mechano- and anesthetic sensitive channel that when activated attenuates pain and causes anesthesia. Recently the enzyme phospholipase D2 (PLD2) was shown to bind to the channel and generate a local high concentration of phosphatidic acid (PA), an anionic signaling lipid that gates TREK-1. In a biological membrane, the cell harnesses lipid heterogeneity (lipid compartments) to control gating of TREK-1 using palmitate-mediated localization of PLD2. Here we discuss the ability of mechanical force and anesthetics to disrupt palmitate-mediated localization of PLD2 giving rise to TREK-1's mechano- and anesthetic-sensitive properties. The likely consequences of this indirect lipid-based mechanism of activation are discussed in terms of a putative model for excitatory and inhibitory mechano-effectors and anesthetic sensitive ion channels in a biological context. Lastly, we discuss the ability of locally generated PA to reach mM concentrations near TREK-1 and the biophysics of localized signaling. Palmitate-mediated localization of PLD2 emerges as a central control mechanism of TREK-1 responding to mechanical force and anesthetic action. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.