The use of remote monitoring and virtual visits has accelerated to support socially-distanced patient care during the COVID-19 pandemic. Despite the necessity of this expansion, ambiguity in coding is hindering adoption and patient access, most notably for remote physiologic monitoring due to a lack of definition of the term “physiologic”. In this analysis, we describe the history of remote monitoring code development, present several examples in respiratory disease and other chronic conditions in which gaps and confusion remain and suggest ways to clarify and broaden coverage to ensure equitable access to remote monitoring. The use of remote monitoring to evaluate and manage patients with chronic illness has become even more important in the wake of COVID-19. Remote monitoring, which uses digital health technologies to monitor patient data from anywhere, allows patients and providers to manage the chronic disease without relying on in-person appointments. With remote monitoring, patients can self-manage their condition from home, and providers can leverage data on the patient’s condition to inform their clinical decision-making.1, and reimbursement for these services became a focal point for the Centers for Medicare and Medicaid Services (CMS). Remote monitoring is related but distinct from telemedicine by definition and modality in that it involves the interpretation of medical information without a direct, synchronous interaction between the practitioner and beneficiary.Prior to the COVID-19 pandemic, remote monitoring was gaining traction across several disease states, most notably heart disease, mental health, lung disease, and diabetes. COVID-19 accelerated the adoption of remote monitoring dramatically. With patients unable to see their providers in person, remote monitoring, telemedicine, telehealth, and virtual care saw a 20-fold increase in use2 around reimbursement is still creating barriers to adoption3 and preventing the delivery of effective, socially distanced care for multiple conditions, which could exacerbate inequitable access to digital tools.Despite growing interest in the use of remote monitoring, ambiguity®) codes for remote monitoring services. Four CPT® codes now exist for the provision of “remote physiologic monitoring” services, but CMS has failed to define what it means by “physiologic”, causing providers and payers to question which technologies qualify for reimbursement.In the past several years, CMS has created multiple Current Procedural Terminology  in the USAIn this analysis, we explore the history and use of remote monitoring codes for reimbursement, present several examples highlighting existing gaps in coding and reimbursement both in respiratory disease and other chronic conditions, and make recommendations for further clarifying and broadening coverage to enhance equitable access to remote monitoring.® code set. CPT® codes are descriptive terms used in reporting medical, surgical, and diagnostic procedures and services that are performed by physicians and other qualified healthcare professionals in the USA. CPT® codes form the basis for reimbursement, research, and tracking medical utilization. Although CPT® codes have existed for numerous remote medical services, until recently, codes to describe general remote monitoring of physiologic data did not exist.The American Medical Association (AMA) maintains and develops the CPTIn the 2018 Physician Fee Schedule final rule, CMS concluded that “remote patient monitoring” services could be a significant part of ongoing medical care and that recognition and separate payment for “these services” was to be done “as soon as practicable”.® Code 99091—a code predicated on collection and interpretation of physiologic data, which CMS referred to as remote patient monitoring. Around the same time, CPT® approved three “remote physiologic monitoring” codes , which went into effect on January 1, 2019 (Table ® created a fourth “remote physiologic monitoring” code (CPT® 99458) that went into effect on January 1, 2020. It is important to note that these codes represent remote physiological monitoring, but also (and arguably more importantly) treatment management services that go well beyond the review of medical device data. However, CMS still had not defined “physiologic”, as reflected in Table CMS chose to activate, cover, and pay for CPTThe lack of definition of “physiologic” has created confusion within the reimbursement community, causing providers and payers to question which technologies and physiologic markers qualify for remote “physiologic” monitoring reimbursement, therefore limiting adoption.® 2020 Professional Edition codebook. The CPT® Editorial Panel publishes the annual codebook to report the rules and guidelines surrounding CPT® codes and descriptors. Despite urgings by stakeholders for clarity, “physiologic” is not formally defined in the CPT® manual, the CPT® Coding Assistant, a coding “Resource and Coverage Guide”, AMA’s Digital Health Implementation Playbook, nor on any associated website.The term “physiologic” appears nearly 80 times throughout the AMA CPT® remote physiologic monitoring codes 99453, 99454, and remote physiologic monitoring treatment management services code 99457. In November 2019, CMS announced that it would adopt, cover, and pay CPT® code 99458 for each “additional 20 min”, beginning January 1, 2020. In the Final Rule commentary11, CMS acknowledged the need for clarification of the term physiologic and indicated they would consider these issues in future rulemaking. However, as of September 2020, CMS has not issued regulatory, sub-regulatory, or clarifying policy specific to the entire suite of remote physiologic (9945X) monitoring codes. More broadly, the term physiologic does not have a formal definition by CMS within rule, policy, or even in its listing of chronic diseases and associated terms.In November 2018, CMS extended coverage and payment for CPTThere exist a multitude of examples where a narrow definition of physiologic limits the application of remote monitoring, which we describe briefly below. Here, we describe examples in respiratory disease that demonstrate the need for a broadened, clarified definition of physiologic. This respiratory exploration is proposed due to the prevalence and social and economic burden of COPD and asthma, prior published research in this area, provider confusion about coverage, and the relevance of the topic given COVID-19.13. Sensors passively monitor the use of inhaled medications, capturing the date and time of each usage, and approximate geographic location (when paired with a smartphone). These signals provide an assessment of daily adherence to controller medication therapy and changes in the use of rescue medications, or short-acting beta-agonists (SABA), which are used for acute relief of asthma and COPD symptoms. Patients have access to these data through web dashboards and smartphone applications. Clinical teams can also access sensor-collected data through a web dashboard to inform clinical decisions such as medication adjustments and receive notifications about patients whose status may be declining.Propeller is a Food and Drug Administration-cleared, digital therapeutic that combines inhaler sensors, companion mobile apps, predictive analytics, and monitoring and feedback to support self-management and clinical care for asthma and COPDDetermination of a patient’s respiratory status is made with a combination of clinical input, including physical examination focused on the work of breathing and chest auscultation, patient-reported outcomes and measures including spirometry, heart rate, respiratory rate, oxygen saturation, and exercise testing. We propose that the signals collected by the inhaler sensor provide an objective assessment of SABA use, providing physiological indicators of stable, worsening, or improving lung health.15. Studies have shown that exacerbations—events characterized by a sudden worsening in symptoms requiring an oral corticosteroid, emergency department (ED) visit or hospitalization—may also be identified through increased use of SABA medication. In COPD, SABA overuse is associated with greater disease burden16, and upward trends in use have been associated with an increased risk of moderate-to-severe exacerbations17. Similar outcomes have been observed in asthma18, where increased SABA use has been associated with worsening symptoms and acute exacerbations. Studies have shown that clinically validated asthma and COPD self-reported instruments, such as the Asthma Control Test (ACT)19, Asthma Control Questionnaire (ACQ)20, and COPD Assessment Test (CAT)21, are significantly correlated with SABA use. Lastly, SABA has also been incorporated into the Asthma Medication Ratio22, which is a specific HEDIS criteria measure23, and is a commonly tracked outcome reported in 68% of studies of exacerbations24.SABA use is an important marker of respiratory disease status, specifically level of control, disease burden, and riskPreviously, SABA use could only be captured based on medication pharmacy fill records, patient-reported history, or patients remembering to bring in their medications with a dose counter, making this component of respiratory status require record-keeping and/or presentation to a clinician for assessment. Passive monitoring of SABA with sensors provides a more real-time and objective component of evaluating respiratory status remotely, enabling early intervention to avoid unnecessary exacerbations.15, reduction in SABA use25, increase in the number of SABA-free days13, improvement in asthma control as measured by the ACT27, and improvement in adherence to controller medications31. These benefits have been observed in diverse participants, including underserved patients with both asthma and COPD26. Significant reduction in acute healthcare utilization, including ED visits and hospitalization events, has been demonstrated for both asthma and COPD32.Respiratory digital therapeutics have been studied in multiple randomized controlled trials and observational studies. Across these studies, significant improvements in asthma and COPD outcomes have been demonstrated in association with their use, including reduction in daytime and night-time self-reported symptoms33 are being developed; however, they do not necessarily cover other emerging signals, such as monitoring of smartphone usage or activity patterns, which may provide early indicators of depressive, anxious, or other pathologic behaviors34. For Parkinson’s disease, wearable sensors offer promise in remotely monitoring motor fluctuations, immobility, and tremors35, which can identify treatable symptoms such as dyskinesia and bradykinesia, but may not be covered by traditional physiologic monitoring codes.In other disease states, a similar promise yet ambiguity exists. In mental health, codes to support remote cognitive behavioral therapyThese signals from multiple disease states demonstrate that digital health deployed for remote monitoring can serve as an important tool for medical practitioners, specialists, and clinicians to assist patients living with a multitude of conditions. It is therefore in the interest of those medical professionals who employ the use of remote monitoring technologies to accurately understand medical diagnostic and therapeutic services, particularly in the context of coding, coverage, and payment.Without any definition or clarification by AMA/CPT, CMS, or a medical society that formally excludes digital health technologies as a valid mechanism by which to capture physiologic markers for purposes of remote physiologic monitoring, there is no conclusive exclusion to the broad use of the term “physiologic”.® code reimbursements are therefore vulnerable given the lack of clear definitions.CMS may at any given time produce regulatory or sub-regulatory policy guidance clarifying their position. In the absence of authorities limiting their power, such as national coverage determinations, the contractors in any one of the 12 Medicare Parts A and B and four Durable Medical Equipment Medicare Administrative Contractors regions, acting under their specific geographic jurisdiction, may at their discretion deny claims based on their particular opinion or other arbitrary factors. CPT® should continue to explore code gaps and create additional remote monitoring codes that describe other ways to evaluate patient status. As announced publicly by CPT® in anticipation of the CPT® Editorial Panel Meeting in October 202036, several applications to create new codes have been submitted for consideration, including one submitted by Propeller Health for Remote Respiratory Status Monitoring Services.As digital health innovation proliferates, CPT® codes for “Remote Physiologic Monitoring” should encompass these services. Inhaler sensors, used in conjunction with medicines and provider services, satisfy the professional and technical requirements11 for remote patient monitoring and remote physiologic monitoring CPT® codes.Based on all of the foregoing, including medical practice and evidence, “physiologic”, as used by CPTAs the US progresses through the COVID-19 pandemic, remote monitoring and other forms of virtual care will become increasingly critical to delivering effective healthcare while reducing infection risk and patient access to these tools should be equitable. An important step in addressing equity is to ensure that systemic reimbursement barriers, like the ambiguity of the definition of “physiologic”, do not disproportionately affect beneficiaries of public insurance more than those with commercial insurance or who are paying cash. In addition, temporary waivers, such as lifting geographic care site restrictions or administrative sanctions for providers who reduce or waive cost-sharing obligations, could temporarily incentivize providers.Increased utilization of virtual care in healthcare, which has been implemented on an emergency basis during the COVID-19 pandemic, now needs to be integrated permanently. Chronic respiratory disease care is just one example of the many conditions that could benefit from expanded access to remote monitoring.


Ursolic acid (UA), found widely in nature, exerts effective anti-tumoral activity against various malignant tumors. However, the low water solubility and poor bioavailability of UA have greatly hindered its translation to the clinic. To overcome these drawbacks, a simple redox-sensitive UA polymeric prodrug was synthesized by conjugating UA to polyethylene glycol using a disulfide bond. This formulation can self-assemble into micelles (U-SS-M) in aqueous solutions to produce small size micelles (∼62.5 nm in diameter) with high drug loading efficiency (∼16.7%) that exhibit pH and reduction dual-sensitivity. The cell and animal studies performed using the osteosarcoma MG-63 cell line and MG-63 cancer xenograft mice as the model systems consistently confirmed that the U-SS-M formulation could significantly prolong the circulation in blood and favor accumulation in tumor tissue. Targeted accumulation allows the U-SS-M to be effectively internalized by cancer cells, where the rapid release of UA is favored by a glutathione-rich and acidic intracellular environment, and ultimately achieves potent antitumor efficacy. PEG-SS-UA can self-assemble to form a micelle (U-SS-M) in an aqueous solution . U-SS-M 2.2.1.2, molecular weight [MW]: 2000 Da) and methoxy PEG succinimidyl carboxymethyl ester  were obtained from Jenkem Technology Co., Ltd. .Ursolic acid (UA), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), succinic anhydride, 3,3′-dithiodipropionic acid (DTPA), and 4-dimethylamino pyridine (DMAP) were bought from Aldrich . Methoxypolyethylene glycol amine , 100 μg/mL streptomycin, and 100 IU/mL penicillin as the medium. NIH-3T3 cells were maintained in a humidified incubator at 37 °C with 5% CO2 using DMEM containing 10% fetal bovine serum , 100 μg/mL streptomycin, and 100 IU/mL penicillin as the medium.Human osteosarcoma MG-63 cells and mouse normal fibroblasts NIH-3T3 cells were brought from the Chinese Academy of Science Cell Bank for Type Culture Collection . MG-63 cells were maintained in a humidified incubator at 37 °C with 5% COSprague Dawley (SD) rat  and BALB/c mice  were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd and used under the approval of the Animal Care and Use Committee of the University of Science and Technology of China.2.2.The 3,3′-dithiodipropionic anhydride (DTDPA) was synthesized as a previous report  was stirred for 10 h at 25 °C under an N2 environment. At the end of the reaction, the mixture was placed into a dialysis bag  and dialyzed against DMF and water to remove any reacted molecules. Then, PEG-DTPA was obtained after lyophilization.A solution consists of 1.0 g of PEG-NH1H nuclear magnetic resonance (NMR) spectroscopy on a spectrometer operating at 300 MHz .The structure of PEG-DTPA was assessed by 2.3.2 environment for 24 h at 25 °C. After the reaction, the mixture was dialyzed against DMF and water, and then lyophilized to obtain the PEG-SS-UA product.To synthesize PEG-SS-UA, 80 mL of anhydrous DMF contained 27.3 mg of DMAP, 42.1 mg of EDC, 114.3 mg of UA, and 400.0 mg of PEG-DTPA were stirred under an N2 environment for 24 h at 25 °C. After the reaction, the mixture was dialyzed against DMF and water, and then lyophilized to obtain the PEG-UA.For PEG-UA synthesis, PEG-NHS (400.0 mg) and UA (114.3 mg) were dissolved in 20 mL anhydrous DMF and stirred under an N1H NMR and high-performance liquid chromatography (HPLC) on a Shimadzu HPLC system  with the detector set at 282 nm using methanol: water: trifluoroacetate , as the mobile phase, using a C18 column  .All products were confirmed by 2.4.Micelles assembled by PEG-SS-UA (denoted UA-SS-M) and by PEG-UA (denoted UA-M) were synthesized via the simple solvent-evaporation method. Briefly, the preparation of U-SS-M will be described to illustrate the method. Typically, 30.0 mg of PEG-SS-UA was dissolved in 1.0 mL of ethanol under sonication. Subsequently, the solution was slowly added to 10.0 mL distilled water dropwise under continuous stirring. The ethanol was removed by stirring the micellar solution overnight. Finally, the mixture was filtered through a 0.4 μm filter to obtain the U-SS-M micelles.The drug loading efficiency (DLE) of the prodrug micelles was measured by HPLC method as mention above and calculated using the following equation:Coumarin-6 loaded U-SS-M and U-M micelles were also prepared using the same method. Briefly, 10.0 mg of PEG-SS-UA  and 0.5 mg coumarin-6 were dissolved in 0.5 mL ethanol under sonication. Subsequently, the solution was slowly added to 3.0 mL of distilled water dropwise under continuous stirring. The ethanol was removed by stirring the micellar solution overnight. Finally, the mixture was filtered through a 0.4-μm filter to obtain the coumarin-6 loaded U-SS-M (or U-M) micelles.The zeta potential, size, and polydispersity index (PDI) in an aqueous solution of these micelles were detected by dynamic light scatting (DLS) on a Malvern Zetasizer Nano-S90 (UK); and the structure and morphology of each micelle were recorded on a transmission electron microscope .2.5.in vivo looping environment, micelles were incubated in PBS or PBS containing 10% FBS at 37 °C. At predetermined time intervals, the size of the micelles was recorded.To explore the ability of the micelle to maintain stability under storage conditions and 2.6.in vitro drug release properties under various redox-conditions were measured. To investigate the size changes of micelles, U-SS-M and U-M were incubated in PBS at pH 7.4, PBS at pH 5.0, and PBS at 7.4 containing 10 mM GSH for 2, 4, or 8 h at 37 °C, respectively, and the size and PDI of the micelles were detected by DLS.To study the redox-sensitivity of the prepared micelles, size changes of the micelles and they are in vitro release experiment was performed in PBS at various conditions: pH 7.4, pH 7.4 with 20 μM GSH, pH 7.4 with 10 mM GSH, pH 5.0, and pH 5.0 with 10 mM GSH, respectively. Briefly, freshly prepared U-SS-M or U-M micelles (containing about 2.0 mg of UA) were suspended in 4.5 mL of release medium and then were placed into a dialysis bag (MWCO: 1000 Da). Subsequently, the dialysis bag was immersed in 45.5 mL of release medium and gently shaken in a thermotank at 37 °C. At the predetermined time point, 1.0 mL of the sample was removed from the incubation medium and replaced with an equal volume of fresh release medium. The amount of UA was determined HPLC method as described above.The 2.7.5 cells per well. After culturing overnight, the cells were treated with coumarin-6-loaded U-SS-M or U-M micelles for 1 or 3 h. After treatment, the cells were rinsed with PBS three times, fixed with formaldehyde (4% in PBS) for 10 min, and then stained with DAPI for 10 min, washed with PBS, and then observed under a CLSM . All steps were performed at room temperature.Cell internalization of coumarin-6-loaded U-SS-M or U-M micelles were observed using a confocal laser scanning microscope (CLSM). MG-63 cells were seeded into a six-well plate with coverslip at a density of 1.0 × 102.8.tretment/Acontrol) × 100, was used to calculated relative cell viabilities, where Atreatment and Acontrol represented the absorbances of the treatment well and control well, respectively.MG-63 cells and NIH-3T3 cells were seeded in 96-well plates at a density of 5000 cells/well. After culturing overnight, the cells were treated with UA, U-SS-M, or U-M at various concentrations. After treatment for 48 h, 20.0 μL of MTT solution was added to each well and then the cells were cultured for a further 3 h. After incubation, the solution was replaced with 150.0 μL DMSO followed by shaking for 15 min, then the adsorption of the solution was measured using a microplate reader  at 490 nm. The equation:  via the tail vein. After a 6-h or 12-h treatment, six mice per treatment were sacrificed and tumors and organs  were collected, weighed, and pulverized. The subsequent steps were the same as those used for processing the pharmacokinetic samples.The biodistribution of UA micelles was investigated using MG-63 tumor-bearing mice as the animal model. MG-63 cancer cells xenograft mice were prepared by the injection of six million cells into the left flank of BALB/c mice. Twenty days following implantation, mice were treated intravenously with UA, U-SS-M, or U-M  2.10.3, mice were intravenously injected with saline and 11.0 mg/kg UA equivalents of UA, U-SS-M, and U-M every three days for a total of five times. Mouse body weight and the tumor length and width were detected every three days after the first administration. The equation: tumor volume = length × (width)2/2, was used to calculate the tumor volume.MG-63 tumor-bearing mice were allocated to five groups randomly. Until the tumor volume reached about 80 mmOn day 21, all mice were sacrificed and collected from the tumor tissues. Subsequently, the tumor tissues were weighed and imaged. The degree of tumor growth inhibition (TGI) was calculated according the following equation:3.3.1.via esterification, as illustrated in 1H NMR was employed to assess the configuration of all products. As shown in 1H NMR spectra of PEG-UA, confirming that PEG-UA was also successfully produced. Moreover, we further evaluated the purity of PEG-SS-UA and PEG-UA using HPCL  and the redox-insensitive micelles formed by PEG-UA (U-M) were subjected to TEM and DLS to assess morphology and size distribution. As shown in The CMC values of U-SS-M and U-M were 2.7 and 3.1 μg/mL , respect3.3.According to our hypothesis, the disulfide linkage between UA and PEG will be cleaved once the U-SS-M are internalized by cancer cells. To investigate their redox-responsive capacity, the size changes of U-SS-M and U-M under various redox conditions were measured. In addition, many previous reports showed that the β-thiopropionate linkage was not only cleaved by reductive stimuli but it could also be degraded by acidic conditions . The superior tumor suppression of U-SS-M was mainly attributed to its efficient accumulation in the tumor tissue based on the EPR effect, which allowed internalization by cancer cells and rapid and complete drug release. Moreover, because the cellular uptake process, pharmacokinetics, and tumor tissue accumulation of U-M showed no differences when compared with U-SS-M, the superior tumor suppression effect of U-SS-M might be attributed solely to the more efficient intracellular sufficient drug release.Encouraged by the prolonged circulation in the blood and good tumor accumulation of UA polymeric prodrug micelles, the In addition, changes in body weight were monitored to assess the systemic toxicity of UA formulations. As indicated in 4.in vitro and in vivo when compared with control groups. Thus, U-SS-M preparation has great potential to achieve better therapeutic effects in OS patients.In summary, a simple redox-sensitive UA polymeric prodrug was synthesized, able to self-assemble into micelles (U-SS-M) in an aqueous solution. The U-SS-M preparation notably increased the water solubility of UA, significantly prolonged the blood circulation time, and efficiently accumulated in tumor tissue, where it was internalized by tumor cells to allow rapid release of UA in cancer cells in high redox conditions and a weakly acidic environment. Given these advantageous properties, the U-SS-M preparation effectively inhibited OS tumor properties


Unpaired fins, which are the most ancient form of locomotory appendages in chordates, had emerged at least 500 million years ago. While it has been suggested that unpaired fins and paired fins share structural similarities, cellular and molecular mechanisms that regulate the outgrowth of the former have not been fully elucidated yet. Using the ventral fin fold in zebrafish as a model, here, we investigate how the outgrowth of the unpaired fin is modulated. We show that Bone Morphogenetic Protein (BMP) signaling restricts extension of the ventral fin fold along the proximodistal axis by modulating diverse aspects of cellular behaviors. We find that lack of BMP signaling, either caused by genetic or chemical manipulation, prolongs the proliferative capacity of epithelial cells and substantially increases the number of cells within the ventral fin fold. In addition, inhibition of BMP signaling attenuates the innate propensity of cell division along the anteroposterior axis and shifts the orientation of cell division toward the proximodistal axis. Moreover, abrogating BMP signaling appears to induce excessive distal migration of cells within the ventral fin fold, and therefore precipitates extension along the proximodistal axis. Taken together, our data suggest that BMP signaling restricts the outgrowth of the ventral fin fold during zebrafish development. Teleosts are a paraphyletic group composed of diverse species, with characteristic anatomical features that enable living in aquatic environments. Fins, which are the major form of appendages, are arguably the most anatomically distinct features of teleosts. Based on their morphology and number, fins could be divided into two types, unpaired fins and paired fins . Recent hand2 and tbx5 similarly regulates development of the pectoral fins . Subsequently, the dorsal fin fold generates the dorsal fin, while the ventral fin fold is further divided into the anal and caudal fin . Based oIn this report, we investigated the role of Bmp signaling on the morphogenesis of unpaired fin, using ventral fin fold in zebrafish embryo as a model. We find that Bmp signaling negatively modulates the outgrowth of the ventral fin fold. We show that inhibition of Bmp signaling at late somitogenesis stages significantly extends the ventral fin fold along the proximodistal axis without affecting other organs. We demonstrate that Bmp signaling appears to restrict the proximodistal growth of unpaired fin by limiting proliferative capacity and thereby promotes quiescence of epithelial cells. In addition, we show that Bmp signaling modulates the orientation of cell division in the ventral fin fold, and thereby regulates innate anisotropic growth of unpaired fin. Taken together, our data suggest that Bmp signaling could negatively modulate the morphogenesis of unpaired fins in zebrafish.Danio rerio) were maintained under standard conditions in accordance with institutional and national guidelines, approved by the Institutional Animal Care and Use Committee. Fish stocks were maintained in an animal facility at 28.5°C on a 14 h/10 h light/dark cycle. The wild-type strain used was AB. Published strains used in this study include Tg(fli1a:EGFP) (Tg(-3.5ubb:Cerulean-gmnn-2A-mCherry-cdt1) (All zebrafish (1a:EGFP) . Tg(-3.5ry-cdt1) .P values were calculated using unpaired t-test with Welch’s correction (GraphPad Prism7).DMH1  used in experiments was reconstituted at stock concentrations (10 mM) in DMSO solvent. Embryos were dechorionated by hand using forceps and dechorionated embryos were transferred to 6-well plates. Embryo media with appropriate drug concentration or vehicle control was added to each well. For DMH1 treatments, embryos were treated from 28 hpf to 76 hpf. Embryos were incubated at 28.5°C for the duration of the drug treatment. For phenotypic analysis, embryos were mounted in 2% methylcellulose and documented using Leica M165FC microscope and Leica MC 170 HD camera. alk3a, alk3b, alk6a, and alk6b morpholinos were used as previously described (The sequence of escribed  (Supplemescribed . Each moin situ hybridization was performed as follows. Hybridized probes were detected using anti-digoxigenin (DIG) antibodies tagged with alkaline-phosphatase (AP) (Roche) using NBT/BCIP (Roche) solution per manufacturer’s instructions. Stained embryos were mounted in 90% glycerol/PBST and imaged using a Leica M165FC microscope and Leica MC 170 HD camera.RNA probes were synthesized using DIG RNA Labeling Kit (Roche) per manufacturer’s instruction. Fragments for synthesizing probes were amplified from wild type cDNA and cloned into pGEM-T Easy by TA cloning (Promega). The sequence of primers for PCR amplification are the shown in the P values were determined using unpaired t-test with Welch’s correction (GraphPad Prism7).Embryos were dechorionated at the desired stage and treated with the appropriate concentration of drug. At 48 hpf stage, EdU   solution was injected into the yolk of each embryo. Injected embryos were incubated at 28.5°C for 5 h. Embryos were washed thoroughly in fresh embryo media and fixed in 4% PFA at 4°C for 24 h. Following fixation, embryos were permeabilized by washing with 1% Tween-20 in PBS for 30 min. The EdU reaction mix was prepared per manufacturer’s instructions. Embryos were then transferred to the reaction mix and incubated in the dark for one hour at room temperature. For imaging, embryos were washed in PBST and mounted in 1% low melt agarose and imaged using a Zeiss LSM 700 confocal microscope. Embryos were fixed after drug treatment in 4% PFA for 24 h at 4°C. Embryos were permeabilized by washing with 1% Tween-20 in PBS for 30 min. Whole mount immunofluorescence was performed as previously described . pSMAD-1Tg(-3.5ubb:Cerulean-gmnn-2A-mCherry-cdt1) embryos were treated with 2 μM DMH1. Fish were anesthetized in tricaine in embryo media and mounted in 1% low melt agarose in embryo media with tricaine for imaging. To prevent shrinkage, confocal dish was filled with embryo media with DMH1 and tricaine. Fluorescent images were captured every 15 min using a Zeiss LSM 700 confocal microscope from 48 hpf to 60 hpf. Images were processed using Zen and IMARIS software. Dividing cells were counted and analyses were performed using IMARIS and GraphPad Prism 7 and Origin.To assess the behavioral changes of cells within the ventral fin fold in response to BMP signaling, 48 hpf To count the number of dividing cells, nuclei undergoing karyokinesis were counted. The coordinates of nuclei of original cells were measured and relative position was calculated. The underneath of caudal vein was assigned as relative position 0 and the distal end line of ventral fin fold was assigned as relative position 100. Distribution and density was defined by Kernel density smoothing. Significance were determined using Mann-Whitney test (Origin). To quantify the orientation of cell division, the coordinates of nuclei of daughter cells were measured and degree of orientation of dividing cells were calculated using Microsoft Excel. The value of calculated degree was presented in polar contour plot in both directions.To investigate migratory behavior of individual cells in ventral fin fold, 2D-projected time-lapse images from 50 hpf to 60 hpf were analyzed using IMARIS. Center of individual cells in ROI (region of interest) were automatically spotted and subsequently manually corrected. We filtered out low-quality cells following the criteria such as estimated XY diameter and spot intensity quality. Cells which are not considered epithelial cells such as clodronate-sensitive immune cells and PTU-sensitive melanophores were excluded. The cells which were tracked across the border line of ROI were excluded. Spots were automatically tracked using Autoregressive motion tracking algorithm. As a threshold to sort reliable tracks, we used track duration above 21 frames and max distance less than 2 μm between each frame. Tracks were presented in scatter plot by gathering the origin of tracks. The coordinates of spots per each frame from 1 to 40 were presented in contour plot.Previously, Bmp signaling has been shown to modulate the outgrowth of paired fins . HoweverTo further define the critical period when Bmp signaling limits growth of the ventral fin fold, embryos were treated with DMH1 for 6 h, starting from 28 hpf, and the effects of Bmp inhibition on the outgrowth of the ventral fin fold was assessed at 76 hpf. We found that the ventral fin fold is susceptible to inhibition of Bmp signaling between 28 hpf and 52 hpf. In particular, the extension of the ventral fin fold along the proximodistal axis was most pronounced in embryos treated with DMH1 between 28 hpf and 34 hpf . The effalk3a, alk3b, alk6a, and alk6b appear to be highly expressed within the ventral fin fold at 28 hpf  signaling pathways in a number of cell types , we exam+ cells compared to DMSO-treated embryos, suggesting that increased cell proliferation may promote the extension of the ventral fin fold along the proximodistal axis in the absence of BMP signaling activity  embryos   , which l embryos . While ty+ cells . ConsideTo better assess the dynamics of cell proliferation in the ventral fin fold in the absence of Bmp signaling, we examined the outgrowth of the ventral fin fold in detail by performing time-lapse imaging on the DMH1- or DMSO-treated Dual FUCCI transgenic embryos between 48 hpf and 60 hpf , 3. ConsSince it is possible that Bmp signaling can selectively enhance the proliferative capacity of cells within a specific sub-region within the ventral fin fold to modulate the outgrowth, we examined the distribution of proliferating cells along the proximodistal axis. Within the ventral fin fold, we identified a number of cell types with distinct morphology. While the majority of the cells appear to be have stereotypic morphology of epithelial cells, there were two types of cells with distinct morphology; highly mobile cells and highly arborized cells. Since clodronate liposome injection, which abrogates macrophages , abrogatSince the orientation of cell division could modulate the direction of outgrowth, we examined whether the orientation of cell division was different between DMSO- and DMH1-treated embryos (schematic diagram is shown in To assess whether directed cell migration also contributes to the proximodistal extension of the ventral fin fold in DMH1-treated embryos, we analyzed migratory behaviors of individual cells within the ventral find fold, in particular, those localized within the mid-section of the ventral fin fold in respect to the proximodistal axis . To quanTo better determine how Bmp signaling affects the direction of migration, contour maps based on the migratory tract of individual cells were constructed in DMSO- or DMH1-treated embryos . We founIn this report, we demonstrate that Bmp signaling provides indispensable inputs to modulate diverse aspects of ventral fin fold morphogenesis. We show that Bmp signaling promotes quiescence by limiting the proliferative capacity of cells within the ventral fin fold. In addition, Bmp signaling maintains the innate tendency of the ventral fin fold to grow along the anteroposterior axis by dictating the orientation of cell division. Taken together, our data show that Bmp signaling negatively regulates outgrowth of the ventral fin fold by limiting the extension of developing ventral fin fold along the proximodistal axis.While Bmp signaling promotes outgrowth of the pectoral fins , it appePreviously, it has been proposed that localized cell proliferation within the distal region, particularly in response to the signals emanating from the distal tip, serves as a major driving force for the proximodistal extension of the tetrapod appendages . For insDespite the distinct modes of regulation on cell proliferation, the ventral fin fold in zebrafish appears to undergo stereotypic extension based on anisotropic growth, as previously reported in paired fins . We founIn tetrapod limb buds, BMP7, which binds to ALK3 and ALK6, appears to be the most abundant BMP ligand . SimilarWhile our data collectively suggest that Bmp signaling modulates outgrowth of unpaired fin, there are a number of limitations which would warrant further investigation on the role of Bmp signaling on ventral fin fold; our analyses were primarily based on the loss-of-function analyses using chemical antagonists at embryonic stages, therefore, it is possible that our analyses might have not been able to capture dynamic changes in the effects of Bmp signaling on the ventral fin fold and its descendants during post-embryonic stages. It would be interesting to examine whether Bmp signaling exerts similar effects on the maintenance and regeneration of the adult fins. However, manipulating Bmp signaling to examine the effects on fin regeneration and/or maintenance may not be technically feasible given that inhibition of Bmp signaling during regeneration could interfere with angiogenesis, which is essential for regeneration and maintenance. In addition, it is not clear whether ectopic activation of Bmp signaling could attenuate the outgrowth of the ventral fin fold. Further analyses using transgenic lines which allow a tissue specific over-expression of Bmp signaling would be needed to precisely delineate the spatiotemporal requirement of Bmp signaling for the outgrowth of the fins. Furthermore, it is unclear how Bmp signaling exerts its effects on outgrowth of the ventral fin fold at molecular level, while our preliminary analyses alluded that Bmp signaling may attenuate Egf signaling by negatively regulating the transcription of key receptors. Therefore, further analyses would help us to fully appreciate the complexity of cellular and molecular mechanisms whereby Bmp signaling modulates the outgrowth of unpaired fins.Drosophila anterior and posterior midgut (Our data suggest that Bmp signaling negatively modulates cell proliferation in the ventral fin fold. While Bmp signaling is generally considered to promote cell proliferation during development, it has been shown to adversely affect cell proliferation in certain scenarios. For instance, it has been shown that Bmp signaling negatively modulates the size of hair follicles by inhibiting cell cycle-related genes . In addir midgut , and humr midgut . Similarr midgut . ConsideThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by Gwangju Institute of Science and Technology.JK, J-DK, BP, WC, OH, and HK performed the experiments. JK, J-DK, BP, WC, and HK analyzed the data. J-DK and S-WJ guided the experiments. S-WJ wrote and all authors edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This conclusion has recently been challenged for England by new empirical results and has also been widely rejected by theorists of long-run economic growth, where pre-industrial fertility control is integral to most models. In this study, we use the accident of twin births to show that for three Western European–derived pre-industrial populations—namely, England (1730–1879), France (1670–1788), and Québec (1621–1835)—we find no evidence for parity-dependent control of marital fertility. If a twin was born in any of these populations, family size increased by 1 compared with families with a singleton birth at the same parity and mother age, with no reduction of subsequent fertility. Numbers of children surviving to age 14 also increased. Twin births also show no differential effect on fertility when they occurred at high parities; this finding is in contrast to populations where fertility is known to have been controlled by at least some families, such as in England, 1900–1949, where a twin birth increased average births per family by significantly less than 1.A conclusion of the European Fertility Project in 1986 was that pretransition populations mostly displayed The online version of this article (10.1007/s13524-020-00898-0) contains supplementary material, which is available to authorized users. We propose a new test, based on parents’ response to the accident of a twin birth, of whether pretransitional populations were practicing parity-specific fertility control. We apply this quasi-experimental test to four micro-demographic data sets: the well-known family reconstitution studies of Enquête Louis Henry and CAMPOP, a large genealogical database from Québec, and a novel genealogical database from England.  In sum, we analyze the effects of 16,580 twins on 709,262 births and confirm that for English marriages pre-1880, French marriages pre-1789, and Québec marriages pre-1830, there was no sign of significant parity-specific fertility control.Why is such a test needed? After all, historical demographers, by the 1980s, concluded that parity-specific fertility control was absent from most pretransition populations Coale .11This test is needed for two reasons. First, in contradiction to this prior research, a literature has emerged claiming to establish that even in the pretransition era, there is strong empirical evidence of parity-dependent birth control. These researchers have argued that with populations that passed the traditional tests of natural fertility—for example, England pre-1850—substantial parity-dependent control does exist , there was no conscious attempt to control fertility. Families experiencing a twin birth ended up on average with 1 additional childbirth compared with those with only singleton births. In contrast in modern populations with fertility control, twins result in an increase in births within families that is significantly less than 1. However, given that families in the pre-industrial world with children had average numbers of births of 6 or more, if they had target numbers of births, they could adjust more easily to the biological accident of a twin birth than in a modern world, where the average number of births per family is only 2-–3.Our approach, using the random occurrence of twin births, has the advantage of being agnostic about the exact means that couples were employing to limit births: stopping, spacing, or some combination. If twinning induces earlier stopping, we will detect the effect. If twinning induces greater spacing between births, we will also detect the effect. We can test simply whether the accidental occurrence of an additional birth through twinning creates any behavioral response in families toward limiting fertility either through earlier stopping or increased spacing.The twins test, however, will not detect deliberate pre-industrial fertility control in response to adverse economic conditions. Nevertheless, it would be surprising if families had the ability and inclination to reduce fertility but used that capability only in response to external economic shocks and not to the equally significant shock of having many surviving children to provide for.Henry :81 definThis raises an immediate logical issue about how we know whether even in the reference group, there is an absence of any parity-specific control. The natural fertility literature of the 1960s to 1980s did not specifically test whether fertility truly was uncontrolled in such populations. Fertility levels at any age, however, varied substantially across the 13 reference populations. These level variations were not seen as evidence of parity-specific fertility control. Control was evidenced only by deviations from the age pattern of natural fertility. The reference populations were assumed, without any formal tests, to practice no fertility control. The decline in fertility with maternal age was asserted to be completely a product of declining fecundity.M and m as a way of formally characterizing fertility regimes and testing for the presence of parity-dependent birth control. M represents the average ratio of observed fertility relative to natural fertility at a given age, where natural fertility was initially represented by the Hutterite population, an early–twentieth century Anabaptist religious group that married early and prohibited contraception. m is the deviation of the observed age pattern of fertility from that of a natural fertility population, again represented by the Hutterites. m, alone, was the measure of parity-specific control. Thus,m = 0 the resultant schedule is simply a constant multiple at every age of “natural” fertility (represented by the Hutterite schedule); if m = 1 the schedule deviates from natural fertility to an extent that is the average degree of deviation of 43 schedules in the early 1960’s; if m is very large the schedule has very rapidly diminishing ratios of fertility relative to the Hutterite schedule as age increases. Only the second of the parameters (m) affects the age structure of fertility; the other (M) only helps determine the level of fertility.  and m = 0.4 (quite moderate control of fertility)” . Similarly, the Cambridge Group for the History of Population and Social Structure concluded that for England before 1838, “small groups may have been practicing family limitation, but the reconstitution evidence suggests that such behaviour was restricted to a small minority of the population, if present at all”  yet be undetectable from the Coale-Trussell m parameter. This was a possibility noted even by Knodel  in simulated populations in which as much as 40% of the population practices effective, parity-dependent control. In particular, values of m < 0.2 cannot justifiably be cited as evidence of the absence of significant fertility control .Consider a population with no fertility control within marriage. In this case, whenever and however the marriage terminates, the expected number of births will be increased by 1 with a twin birth, assuming that the twin birth has no effect on the length of the subsequent birth interval. Also, the increase in the final number of births will be the same whatever the parity at the time of the twin birth.t – θs, where θt is the twin survival rate to age 14, and θs is the single child survival rate.4Figure i, Ni. Assume also the marriage lasts to the end of planned births. Then with only singleton births, the number of births needed to achieve this target will average Ni / θs. With a twin birth, the number of births required to achieve the target becomesIn contrast, in populations where families control fertility and have a target number of children entering marriage, twin births will induce a more muted increase in completed family size. Suppose, as seems reasonable, the target is defined in terms of children reaching age 14. Call this number for family t> 0.5θs, the number of additional births required to achieve the target will be less than 1. If θt = θs, then there will be no additional births.As long as θt> 0.5θs). Because all marriages end with a last birth, the fraction of twin births that occur as the last planned birth will just be the inverse of average family size. In England, for example, for men whose first marriages took place 1730–1879, average family size for those with at least one child was 5.9. Thus, if each family had a planned target size, a twin birth would occur as the last planned birth 17% of the time. Suppose in general that the fraction of births that are the last planned birth is ϕ. For a marriage reaching completion of planned births, a twin birth will on average induce an increase in births ofHowever, if the twin birth is the last planned birth, it will add 1 additional birth (assuming θt − θs) with controlled fertility.The number of additional children reaching age 14 induced by the twin birth will, however, be just ϕ that has value 1 at parity p and mother age k, then the estimating equations will beFor pre-industrial populations with no fertility control, αPARITY is the parity of the twin birth. With no fertility control, λb and λc should be 0.With natural fertility, a twin birth will add one child to the family regardless of the parity at which the twinning occurs. If all families in a population are controllers with target family sizes, the average effect of a twin birth on total births and on completed family size will be greater the higher the parity at which a birth occurs. Pison and Couvert  found thAnother way to check for a behavior response to the accident of twinning is by looking at the length of the birth interval following a twin birth compared with a singleton at a given parity and mother age. If twin births add exactly one birth to the total number of births in a family, then this interval should be the same following a twin birth as following a singleton.l’Infrastructure intégrée des microdonnées historiques de la population québécoise) database. Table The data for analysis are multiple, independently constructed family history databases: the Families of England (FOE) database for England, the Henry data for France, the CAMPOP data for England, and the Québec IMPQ  is a set of family reconstitutions of the Catholic population of Québec using baptisms, burials, and marriages 1621–1849 . If we instead use fathers, there is no issue of twinning-induced parent mortality. If a mother died in childbirth, the father would often remarry. Some fathers will have died before their wives reach the end of their reproductive careers, but this effect will be found equally among twinning and nontwinning families.RSS is the same sex ratio, and Rt is the observed twinning rate per 100 births. If we miss many true twining events and instead misattribute singleton births, then the gender ratio will be closer to 0.5 than predicted by this formula when ϕ = 1. In general, as Table We can test the accuracy of our twin attributions by looking at the gender composition of twins. Two children who are not twins have a roughly 50% chance of being of the same gender.entclass1pt{minimaentclass1pt{minimaDTWIN toward 0. Thus, mistakes here in the data will bias us toward finding evidence of fertility control.The effects of attributing twins when the children in a family are actually singletons will be to bias the estimated coefficients on Table Figure Twinning is mostly uncorrelated with the observable social characteristics of families. Table A more important issue is whether twinning correlates with fecundity. As shown in Table Pison and Couvert :770 alsob the effect of a twin birth on total births for the six population samples we have for Eq. . Similab nonparametrically from Eq.   b and λcc in Eq. . Yet there was no sign that parents were limiting births within marriage to control such expenses.Clark and Cummins  argued tThe findings with the Henry data for France pre-1789 also cast doubt on the earlier claim of David and Mroz , b to haWith enough data, the nonresponse to twinning implies that twins can be used as an instrument for family size, as an exogenous source of variation in family size. However, the variance in size induced by twinning is a small component of the overall variance, so there would have to be enormous amounts of data to estimate with any precision the coefficients linking child outcomes to family size, especially if the relevant measure is completed family size as opposed to births. More promisingly, the response to twins—by showing that families were not choosing family size—suggests that in these populations, we can consider all the variation in family size as exogenous. In particular, in England, for marriages in the years 1780–1879, fertility was uncorrelated with family social status. Average completed family size was the same on average for the poorest as for the richest families. Thus in this period in England, we can get very simple estimates of the effect of family size on child outcomes given that the variation in family size is exogenous to the social status of the family and not a choice made by parents. In another paper, Clark and Cummins  estimateThe lack of any fertility response to twinning by families in England throughout the years 1780–1879 is also interesting in light of recent theories of the Industrial Revolution. By 1780, the rate of technological advance had clearly increased significantly from that of the previous six centuries in England. Technological advance has been attributed to a democratization in England of the ideas of the Enlightenment, an intellectual movement that emphasized rationality, experiment, and embrace of novel theories of both science and society


Sensitivity analyses and additional analyses also showed similar pooled results. In conclusion, no significant causality between serum UA levels and cancer risk was evidenced.The relationship between serum uric acid (UA) levels and cancer risk remains controversial. Here, a two-sample Mendelian randomization analysis was performed to identify a causal effect of serum UA levels on cancer risk. Twenty-six single nucleotide polymorphisms strongly associated with serum UA levels were screened as genetic variants from large-scale meta-analysis data of a genome-wide association study of 110,347 European individuals. Genetic associations with eight common site-specific cancers were subsequently explored. A total of six Mendelian randomization methods were used to estimate the potential effect of serum UA levels on cancer risk, including random effects inverse variance weighting, fix effects inverse variance weighting, MR-Egger, median weighting, mode weighting, and simple mode analysis. Our primary random effects inverse variance weighted analysis revealed that no significant associations with cancers was found (all  Serum Uxcretion , the latxcretion . Purine-xcretion . Hyperurxcretion .Previous studies have reported UA levels to be associated with the incidence of diabetes, cardiovascular disease, kidney disease, and malignancies . The preThe randomized controlled trial is the gold standard for demonstrating epidemiological causality between exposures and outcomes . HoweverThe role of UA in the pathogenesis of malignancies remains unclear. Here, we designed a two-sample MR study to analyze summary genetic data for the purposes of investigating any potential causal associations of genetically-proxied UA levels and the incidence of eight distinct malignancies.via UA concentrations and could not have a direct correlation  for serum UA levels were selected as instrumental variables from previously published genome-wide association study (GWAS) analyses. Three key assumptions were to be satisfied: first, the SNPs should have been associated with serum UA levels; second, the chosen SNPs should have been independent of confounders; and third, the SNPs should have affected cancer only relation .p-value threshold of 5 × 10−8, detailing a 7.0% phenotypic variance in serum UA levels. These genetic variants were pruned for linkage disequilibrium using LD-link (https://ldlink.nci.nih.gov/) with an r2 threshold of 0.01 . The average age of the participants was 52.12 years. The GWAS data were obtained from the Global Urate Genetics Consortium GUGC; . A total of 0.01 . After L of 0.01 .Data from eight, large-scale meta-analyses of GWASs studying eight common cancers were used to explore the association of genetically-proxied serum UA levels with risk of malignancy incidence rates; namely bladder, breast, colorectal, lung, prostate, renal cell, skin, and thyroid cancers. The breast cancer outcome dataset was composed of summary genetic data obtained from the Breast Cancer Association Consortium (BCAC) and consisted of a meta-analysis of 11 GWASs  in addition to 41 studies  genotyped on the iCOGs custom array . The proIn this study, we used five different methods of MR analysis to evaluate the causal effect of serum UA levels on cancer risk. Here, the random-effects inverse variance weighted (IVW) method was used as the primary analysis. The Wald estimator was used to calculate the ratio of the SNP-outcome estimate over the SNP-exposure estimate, while the Delta method was employed to calculate the standard errors . The ovevia leave-one-out sensitivity analysis. The remaining estimate effect was shown when one SNP was excluded  with the “TwoSampleMR” package (version 0.5.4). Results were considered to show strong evidence of an association between serum UA levels and cancer incidence if they surpassed a stringent Bonferroni-corrected p-value threshold of 6.25 × 10−3 (0.05/8 cancer outcomes).All statistical analyses in this paper were performed using R software  was observed. The minimum F-statistic value of these 26 SNPs was 30.05, suggesting that they were sufficiently effective in this study. All SNPs could thus be used to identify the potential effect of serum UA levels on cancer risk. Scatter plots were shown in the supplementary materials .The main MR results detailing the influence of UA levels on cancers were obtained using random-effects IVW methodology . Our priThe MR estimates of UA levels on cancer risk obtained using other sensitivity MR approaches are shown in the supplementary materials . Consistp = 0.03). In addition, some evidence of heterogeneity was also found using Cochran’s Q statistics for some cancers, including breast, colorectal and prostate cancer . One MR study also revealed causal relationships between serum UA levels and cancer risks. The study was based on the Copenhagen General Population Study and selected rs7442295  as the instrument for serum UA level representation. Results revealed causal relationships between high serum UA levels, high cancer incidence and high all-cause mortality, contrary to our findings. Possible explanations of such a paradoxical finding were that this study only used rs7442295 as the instrument which could only explain 2% of the variation in serum UA levels, and SLC2A9 was expressed differently in various organs, thus there might some biases in that study  for the publication of any potentially identifiable images or data included in this article.MJ and GL designed the study. MJ and LR assembled and analyzed the data. LR and SC visualized the data. MJ, LR, and SC wrote the paper. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Antidepressants are considered driving-impairing medicines (DIM). This is a population-based registry study that shows the trend in the use of antidepressants in Castile and León, Spain, from 2015 to 2018. Data on antidepressant dispensations at pharmacies and the adjusted use of these medicines by the driver population are presented. For the purposes of analysis, population distribution by age and gender has been taken into account, as well as the three Driving Under the Influence of Drugs, alcohol, and medicines (DRUID) categories. Antidepressants were used by 8.56% of the general population and 5.66% of drivers. Antidepressants were used more commonly by females than by males , and users increased as the age increased, even if women who drive used less antidepressants after turning 60 years of age. Chronic use of antidepressants was relevant (8.28%) in the same way as daily use (3.15%). Most of the consumption included SSRIs (4.99%), which are also known as “other antidepressants” (3.71%). Regardless of antidepressants consumed, users took 2.75 ± 1.19 DIMs, which are mainly anxiolytics (58.80%) and opioids (26.43%). Lastly, regarding consumption of antidepressants according to the DRUID classification, category I predominated over categories II and III. Our findings should serve as a starting point for health and traffic authorities to raise awareness of the risk for traffic accidents, especially involving SSRIs. Depression causes severe disabilities in the occupational and social life of patients  and it iDepression is characterized by loss of energy and cognitive impairment of the patient , and it Antidepressants that hold the Anatomical Therapeutic Chemical (ATC) code N06 are classified in four subgroups : (1) nonCurrently, SSRIs have practically replaced TCAs due to their greater tolerability and safety . StudiesImportantly, antidepressants are present in the three Driving Under the Influence of Drugs, alcohol and medicines (DRUID) categories: SSRIs and monoamine oxidase A inhibitors belong to category I (minor influence on fitness to drive), TCAs belong to categories II (moderate influence on fitness to drive), and III (severe influence on fitness to drive), and the so-called “other antidepressants” (ATC subgroup N06AX) may be encountered into category III , or categories I and II   20]..20].Trends in antidepressants use by the population is a topic of great interest. Since commercialization of SSRIs, an increase in their use for two to three times may be observed in western countries ,22. For This study presents the data on antidepressants consumption from 2015 to 2018 in Castile and León, Spain, based on dispensation of such DIMs at pharmacies. The adjusted use of antidepressants by the driver population is also presented in order to assess differences in patterns of use with respect to the general population ,25,26,27This manuscript presents findings from a population-based registry study carried out according to the Reporting of studies Conducted using Observational Routinely-collected Data (RECORD) recommendations in order to adequately provide real-world evidence into the topic addressed . Our finhttp://www.saludcastillayleon.es/portalmedicamento/es/indicadores-informes/concylia), where the entire information on pharmaceuticals care in Castile and León is routinely collected. The drivers’ license census data for the years 2015 to 2018 were also accessible  to calculate the adjusted use of antidepressants by drivers  yearly frequency of antidepressants consumption, (2) acute (1–7 days), sub-acute (8–29 days), and chronic use (≥30 days) of antidepressants each year, (3) yearly frequency of daily use of antidepressants, (4) yearly frequency of antidepressants consumption by DRUID categories, and (5) concomitant use of antidepressants with other DIMs during each year. All analyses were done considering age and gender distributions. This study was approved by our local ethics committee on 17 March 2016 (reference number PI 16-387).Results are expressed as frequencies (percentage) with their corresponding 95% confidence interval (95% CI) or as means accompanied by their standard deviations (SD). Differences between continuous variables were calculated using Student’s t-test, and those between categorical variables were calculated using Pearson’s chi-squared test. The Cochran-Armitage trend test was used to evaluate the consumption trend by years. The level of significance was set at p ≤ 0.05. All statistical analyses were performed by using the Statistical Package for the Social Sciences . Lastly, Microsoft Word and Excel  were used for preparing this manuscript.From 2015 to 2018, 8.56% of the general population took at least one antidepressant. Their use was three times greater in women than in men , which is followed by those in categories II (18.84%) and I (13.44%) .The results obtained for drivers were similar: 5.66% used antidepressants and 1.93% took these medicines every day . As for Our results show that the increase in antidepressants consumption between 2015 and 2018 in Castile and León, Spain was relevant. In each year of the study period, women users predominated over men users in both the general population and the driver population. With respect to the type of antidepressant, SSRIs were the most commonly used. In addition, antidepressants were mostly consumed chronically, and acute and subacute use was practically insignificant. More than half of individuals who used antidepressants during the study period also used other DIMs concomitantly, which can seriously influence fitness to drive.The increase in the prevalence of antidepressant use is consistent with other national data and comparable with those obtained in other European countries: SSRIs and the “other antidepressants” (ATC subgroup N06AX) were the most consumed ,22,23. IAntidepressants are the second most prescribed psychotropic medication for the elderly, with the first being benzodiazepines . AccordiNotwithstanding, the increase in antidepressants consumption may be due to other factors different from the prevalence of depression itself. An increase in detection by physicians, and an increase in clinical indications for antidepressants, e.g., neuropathic pain (TCAs) or for smoking cessation (bupropion) , should Antidepressants are mainly used chronically (≥30 days). A total of 40% of antidepressants were prescribed for more than 180 days, which is, according to the average use of these medications, 251 to 525 days for SSRIs that are frequently switched to SNRIs . FurtherThe effect on fitness to drive is not only due to medication. The depressive disorder also influences this factor . DepressRemarkably, the evidence on how antidepressants affect fitness to drive is contradictory, and there are studies that did not support a direct relation . For exaOn the other hand, the conclusions obtained by Ravera et al.  and BrunIt is considered important to remember that the psychomotor impairment of the patient with depression may have a greater impact on the increased risk of a traffic crash than the antidepressant treatment itself ,35. FurtRegardless of the need for more research, health professionals should be aware of the possible risks and have to inform their patients individually on adverse effects of antidepressants and concomitant use with other medications .Lastly, this study has some limitations. First, antidepressants dispensation cannot be considered as an equivalent to antidepressants consumption, as beneficiaries of our health system could have not consumed such medicines. Second, consumption in hospitals and by private prescriptions were not taken into account. However, in this case, it can be assumed that the biases produced when using dispensing data is not relevant, as antidepressants are financed medicines and their use require mandatory medical prescription. On the other hand, CONCYLIA does not include driver’s license information, so weighting was made on the basis of age and gender with the driver’s license census information, as performed in previous studies by our team ,25,26,27In conclusion, the use of antidepressants was frequent especially with chronic and daily use. Between 2015 and 2018, the use of these medicines increased, especially the new N06AX antidepressants. In addition, concomitant use of antidepressants with other DIMs was relevant, which involved benzodiazepines. Importantly, SSRIs were consumed by a considerable proportion of the population, even if risk of using these medicines may be greater into the firsts days of use .Lastly, it is considered necessary to carry out new actions to provide more accurate information to healthcare professionals, patients, and drivers. It would be appropriate to improve prescription and dispensing systems for medications including alerts related to antidepressants. It is necessary to propose actions to avoid the unsafe driving of vehicles by patients under treatment with antidepressants. These dissuasive actions could be the inclusion of antidepressants into the battery for road detection tests, adapt traffic regulations to the use of these medicines, information campaigns, etc. .


Mir140 locus in mice results in growth retardation and early-onset osteoarthritis-like pathology; however, the relative contribution of miR-140-5p or miR-140-3p to the phenotype remains to be determined. An unbiased small RNA sequencing approach identified miR-140-3p as significantly more abundant (>10-fold) than miR-140-5p in human cartilage. Analysis of these data identified multiple miR-140-3p isomiRs differing from the miRBase annotation at both the 5′ and 3′ end, with >99% having one of two seed sequences (5′ bases 2–8). Canonical (miR-140-3p.2) and shifted (miR-140-3p.1) seed isomiRs were overexpressed in chondrocytes and transcriptomics performed to identify targets. miR-140-3p.1 and miR-140-3p.2 significantly down-regulated 694 and 238 genes, respectively, of which only 162 genes were commonly down-regulated. IsomiR targets were validated using 3′UTR luciferase assays. miR-140-3p.1 targets were enriched within up-regulated genes in rib chondrocytes of Mir140-null mice and within down-regulated genes during human chondrogenesis. Finally, through imputing the expression of miR-140 from the expression of the host gene WWP2 in 124 previously published data sets, an inverse correlation with miR-140-3p.1 predicted targets was identified. Together these data suggest the novel seed containing isomiR miR-140-3p.1 is more functional than original consensus miR-140-3p seed containing isomiR.miR-140 is selectively expressed in cartilage. Deletion of the entire  MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression. Mature miRNAs are processed from primary transcripts containing stem loops by cleavage mediated by Drosha and then Dicer proteins . The 5′ In recent years, small RNA (sRNA) sequencing (sRNA-seq) has identified additional miRNAs that do not perfectly align to the annotated mature miRNA, known as isomiRs . IsomiRsMir140 in mice leads to a skeletal phenotype including an osteoarthritis (OA)-like disease. Although the contribution of the lack of miR-140-5p or miR-140-3p to the phenotype remains largely undetermined, the majority of studies have focused on miR-140-5p rather than miR-140-3p  have largely discrete target repertoires. We validated a number of common and discrete targets for each isomiR using a luciferase reporter system. We present evidence that miR-140-3p.1, which is not currently annotated in miRBase, may play roles in human chondrogenesis, mouse cartilage and in multiple other skeletal tissues.Supplemental Table S1). As 5′ isomiRs are most likely to alter function, we focused further sRNA-seq analysis on miRNAs with 5′ isomiRs. Using thresholds of >100 reads and >5% of total reads for that miRNA, we identified 5′ isomiR sequences for 29 miRNAs, 26 of which have a single addition (includes templated and nontemplated additions) or deletion at the 5′ nt, while miR-3074-5p, miR-455-3p, and let-7b-3p all have two additional 5′ nt . Although the isomiR for miR-1246 is just under our read threshold (read count of 99), it appears to have four additional 5′ nt compared to the miRBase annotation .We performed sRNA-seq of human cartilage RNA and identified novel cartilage specific miRNAs . Our carmiR-140-3p and its isomiRs account for more than half of all sequencing reads in our cartilage sRNA-seq A. As miRSupplemental Fig. S2; Supplemental Fig. S2). Furthermore analysis of CLEAR-CLIP, a modified version of high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP), from mouse brain . Together these data indicate miR-140-3p.1 and miR-140-3p.2 can be loaded into RISC and can target transcripts.Analysis of other published sRNA-seq data identified the presence of miR-140-3p.1 and miR-140-3p.2 in human tissue types, although expressed at a lower level than in cartilage . The majority of these targets also differ from predicted miR-140-5p targets .miRNAs generally target mRNAs through interaction of their seed sequence (nucleotides 2–7); the seed sequences for miR-140-3p.1 and miR-140-3p.2 differ  down-regulated by miR-140-3p.1 and miR-140-3p.2, respectively; however, only 161 genes were commonly down-regulated by both isomiRs . DAVID pathway analysis of repressed genes identified enrichment terms associated with “phosphoprotein” for miR-140-3p.1 . Direct comparison of miR-140-3p.1 transfected with miR-140-3p.2 transfected cells revealed 237 genes were significantly different between the two isomiRs . DAVID pathway analysis of the 237 genes differentially expressed between the two isomiRs identified an enrichment of triple helical collagen genes . Similarly, g-Profiler analysis of genes differentially expressed between miR-140-3p.1 and miR-140-3p.2 transfected chondrocytes, revealed an enrichment of genes associated with ECM structure including “connective tissue development” . The fraction of these genes that are decreased by, and are predicted to be a target of each isomiR, is greater for miR-140-3p.1 than for miR-140-3p.2, suggesting miR-140-3p.1 directly regulates genes belonging to these terms. However, the majority of genes contributing to the enrichment are not predicted direct targets of either miR-140-3p.1 or miR-140-3p.2, suggesting miR-140-3p isomiRs, in the most part, regulate these processes through indirect mechanisms. Together these data indicate that the sequence differences between the two isomiRs may be functionally important.Next, we used overexpression of miR-140-3p.1 and miR-140-3p.2 in primary human chondrocytes followed by genome-wide transcriptomics to experimentally identify similarities and differences in their target repertoire. Overexpression of miR-140-3p.1 and miR-140-3p.2 was validated using isomiR-specific qRT-PCR A. PrinciSylamer, an unbiased motif analysis tool, was used to investigate miRNA binding site enrichment within down-regulated 3′UTRs . TransfeSupplemental Table S3), unique and common targets were then selected for 3′UTR luciferase validation . Transcriptome RNA-seq data of rib cartilage from these mice identified 1894 and 1179 genes that were significantly up- and down-regulated, respectively . Pathway analysis of up-regulated genes following knock out (KO) of Mir140 identified terms associated with “phosphoprotein” , which is consistent with pathway analysis of genes decreased following miR-140-3p.1 overexpression in human chondrocytes . miR-140-5p, miR-140-3p.1, and miR-140-3p.2 predicted targets (identified using TargetScan 7.2) were enriched within up-regulated genes , may contribute to target repression. This analysis indicates miR-140-5p followed by miR-140-3p.1 have the largest contribution to target repression in mir140 KO mice , and that there are no lowly expressed Mir140-derived miRNAs that have a major contribution to target repression in mice.More than 99% of miRNAs produced from the murine pression . We usedP = 0.07)  chondrogenesis, with miR-140-5p predicted targets highly enriched within the down-regulated genes . Here we = 0.07) B. Sylame = 0.07) C. Consisogenesis C.WWP2, with expression of the miR and an abundant isoform of WWP2 (transcript variant 2) controlled by a common promoter  in WWP2 expression. The change in WWP2 expression (as a surrogate for miR-140 expression) was plotted against the average fold change of predicted miRNA isomiR targets for each study. Using regression analysis we identified a significant inverse relationship between WWP2 and miR-140-5p  and miR-140-3p.1, predicted targets . There was a minimal inverse relationship between WWP2 and miR-140-3p.2 predicted targets  or randomly selected genes . To determine if miR-140-5p, miR-140-3p.1, and miR-140-3p.2 targets are regulated in a similar way in each study, we correlated log2FC for miR-140-5p, miR-140-3p.1, and miR-140-3p.2 targets for all studies . Average log2FC for miR-140-5p targets correlated most strongly with average log2FC for miR-140-3p.1 targets . The direction of target FC tends to be in the opposite direction to WWP2 FC . Thus, where WWP2 expression changes the expression of predicted miR-140 targets have a tendency to change in the opposite direction.Of the 779 skeletal gene expression responses within SkeletalVis, 124 contained a significant alteration , where WWP2 expression was not altered, the percentage of targets within the up-regulated genes is greater than the percentage of targets within down-regulated genes . Similar to the log2FC analysis, the enrichment for miR-140-5p targets correlated most strongly with enrichment for miR-140-3p.1 targets .Next we calculated “Target Enrichment (up/down)” by dividing the percentage of targets within up-regulated genes by those within the down-regulated genes, for each of the studies . Target enrichment for miR-140-5p and miR-140-3p.1 was significantly higher in studies where WWP2 expression increases, both miR-140-5p and miR-140-3p.1 predicted targets are repressed. Furthermore, they also indicate that miR-140-3p.2, which contains the miRBase annotated seed sequence, has a less significant biological effect on gene expression than either miR-140-5p or miR-140-3p.1.These data indicate that when Mir140 locus, and genes up-regulated during MSC chondrogenesis. There were 3, 3, and 0 genes matching all of these criteria for miR-140-5p, miR-140-3p.1, and miR-140-3p.2, respectively . ABCA1 was common to both miR-140-5p and miR-140-3p.1. Furthermore, ABCA1 was inversely correlated with WWP2 across multiple skeletal data sets, with the inverse correlation being strongest in cartilage experiments , where miR-140 expression is highest.To further refine prediction of putative direct miR-140 targets, we investigated the overlap between four criteria; TargetScan predicted targets, genes down-regulated following overexpression in human chondrocytes, genes up-regulated in mice lacking the RNU2), which explains their similar sequence and expression pattern during chondrogenesis  facilitated by DGCR8, and subsequently cleaved in the cytoplasm by DICER to generate functionally mature miRNAs molecules. IsomiRs are generated because both DICER and DROSHA process miRNA precursors imprecisely generating miRNA variants with several plus/minus nucleotides at the 5′ and/or 3′ . In addiMIR140-null mice show some differences, which could perhaps be accounted for by the latter animals lacking miR-140-3p and the isomiRs described here.A mutation within miR-140-5p has recently been identified as causing a human skeletal dysplasia . Mice wiLIPA expression decreases during chondrocyte development in humans  , or by miR-140-3p.2 as a 7m8-BS or 8mer-BS if followed by U or UA, respectively (CUGUGGUGUGGUA) A. IndeedUGUGGUA)  may be sWWP2 host gene as a surrogate for miRNA expression, along with directly overexpressing the miRNAs and observing changes in gene expression, suggests these types of analyses can improve miRNA target prediction within skeletal tissues. Caution must however be taken as WWP2 and miR-140 can display tissue specific expression . Normalization of the quantified signals (background corrected) was performed using the global Lowess regression algorithm. Expression analysis was performed in R/bioconductor using the limma package . PCA andSylamer analysis was performed on ordered gene lists from most down-regulated to most up-regulated (including genes whose expression did not significantly change) . Target Custom miR-140-3p.1 and miR-140-3p.2 assays  were used to detect expression of miR-140-3p.1 and miR-140-3p.2. Assays were validated using spike-in of miR-140-3p.1 and miR-140-3p.2 mimic (Dharmacon).Supplemental Table S10; In-Fusion cloning  of selected 3′UTRs into the pmirGLO vector (Promega) was used to generate 3′UTR luciferase reporters essentially as previously described  and a 5′ PCR primer that included a T7 sequence and the crRNA. This was converted to RNA using the MEGAshortscript T7 kit (Fisher Scientific). sgRNA (50 ng/mL each) were mixed with recombinant Cas9  and injected into the cytoplasm of donor mouse zygotes and transferred into recipient foster mothers, all essentially as previously described .All animal experiments were performed under licenses granted from the Home Office (United Kingdom) in accordance with the guidelines and regulations for the care and use of laboratory animals outlined by the Animals (Scientific Procedures) Act 1986 according to Directive 2010/63/EU of the European Parliament, and conducted according to protocols approved by the Animal Ethics Committee of Newcastle University and the Home Office, United Kingdom. Postgeneration breeding and subsequent phenotyping were performed under licenses PPL60/4525 and P8A8B649A. CRISPR/Cas9 guide RNAs (crRNA) were designed using CHOPCHOP. crRNA linked with TRACR (sgRNA) were amplified by PCR with a pLKO vector (Addgene_52628) as template, using the primer T7 TRACR R (escribed . The mix−/−Mir140 and wild-type (WT) mice using collagenase digestion, essentially as previously described , leaving 124 experimental comparisons. The change in WWP2 expression was plotted against the average fold change of predicted (TargetScan7.2) miRNA targets for each experimental comparison. Correlation (R2) and regression analysis for all 124 experimental comparisons was calculated using the data analysis add-in in Microsoft Excel. The percentage (% of miRNA targets) was calculated by dividing the total number of predicted targets (TargetScan 7.2) that significantly increased or decreased by the total number of genes that significantly increased or decreased, multiplied by 100, for each of the 124 experimental comparisons. The percentage of miR-140-5p, miR-140-3p.1, and miR-140-3p.2predicted targets within up-, no change or down-regulated genes was then plotted against log2FC for WWP2; the trend lines represent the cumulative mean starting from studies where WWP2 is most up- or down-regulated to studies where WWP2 changes least, to demonstrate trend. A list of randomly generated genes was analyzed alongside miR-140-5p, miR-140-3p.1, and miR-140-3p.2. “Targets Enrichment (up/down)” was calculated by dividing the “% of miRNA targets within up-regulated genes” by the “% of miRNA targets within down-regulated genes,” this was performed for each miRNA in each study. The mean “Target enrichment (up/down)” for studies where WWP2 either decreased or increased was plotted. “Targets Enrichment (up/down)” for miR-140-5p, miR-140-3p.1, and miR-140-3p.2 predicted targets was also plotted against each other. Experimental comparisons where WWP2 decreased or increased were shown in different colors as described.Gene expression responses within SkeletalVis were filtered for human and mouse data sets and where Microarray data and RNA-seq data are available at NCBI GEO data sets with the accession numbers GSE144374.Supplemental material is available for this article.


S-glutathionylation and S-nitrosylation are reversible post-translational modifications on the cysteine thiol groups of proteins, which occur in cells under physiological conditions and oxidative/nitrosative stress both spontaneously and enzymatically. They are important for the regulation of the functional activity of proteins and intracellular processes. Connecting link and “switch” functions between S-glutathionylation and S-nitrosylation may be performed by GSNO, the generation of which depends on the GSH content, the GSH/GSSG ratio, and the cellular redox state. An important role in the regulation of these processes is played by Trx family enzymes , the activity of which is determined by the cellular redox status and depends on the GSH/GSSG ratio. In this review, we analyze data concerning the role of GSH/GSSG in the modulation of S-glutathionylation and S-nitrosylation and their relationship for the maintenance of cell viability. The nditions . In addi•), or S-nitrosyl (-SNO) group. Thus, when Pr-SH is oxidized with, for example, H2O2, sulfenic acid (Pr-SOH) is formed and then quickly reacts with GSH to form Pr-SSG:S-glutathionylation can occur when Pr-SH or GSH reacts with an oxidized derivative of the protein cysteine residue; for example, sulfenic acid (-SOH), thiyl radical (-S2H) and eventually to sulfonic acid (Pr-SO3H), the formation of which, as a rule, leads to the irreversible deactivation of the protein. Thus, the S-glutathionylation of sulfenic acid can prevent the oxidation of protein cysteine residues + is transferred to the target protein with the formation of Pr-SSG, while Grx is again capable of catalyzing the reaction. For this process, the possibility of the reversible formation of Grx-S2 from Grx-SSG is also noted phenyl} 1- diazen-1-ium-1,2-diolate] led to a dose-dependent increase in intracellular NO followed by S-nitrosylation, the level of which was extremely low. However, a very high level of S-glutathionylation was detected for some proteins, including β-lactate dehydrogenase, Rho GDP-dissociation inhibitor β, ATP synthase, elongation factor 2, PDI, nucleophosmin-1, actin, protein tyrosine phosphatase 1B, and glucosidase II [In vitro studies have shown that proteins, such as papain, creatine phosphokinase, and glyceraldehyde 3-phosphate dehydrogenase are sensitive to both S-nitrosylation and S-glutathionylation by GSNO, while alcohol dehydrogenase, bovine serum albumin, and actin apparently become S-nitrosylated . In addiidase II ,152. Theidase II .The fact that S-nitrosylation of cysteine residues can serve as a transient intermediate has been evidenced by studies on rat smooth muscle cells treated with CysNO. The total pool of reduced thiols did not change significantly, while the level of reduced non-protein thiols—in particular, GSH—decreased. A decrease in GSH content prior to the use of CysNO by pre-treatment with BSO, which is an inhibitor of gamma-glutamylcysteine synthetase (a key enzyme of GSH de novo synthesis), led to a significant increase in S-nitrosylated proteins, indicating a violation of the ability to reduce Pr-SNO due to S-glutathionylation . In contCellular redox status, as the state of oxidant/antioxidant balance, largely determines the viability of cells, including the processes of proliferation, differentiation, bioenergetics, and apoptosis. To a large extent, this is facilitated by the redox-dependent post-translational modifications of proteins, which alter their functional activity. Among these reactions, S-glutathionylation and S-nitrosylation are the best studied, although their relationship and regulation are still questionable. The function of a connecting link and a "switch" between S-glutathionylation and S-nitrosylation can be performed by GSNO, the level of which depends on the GSH content, the GSH/GSSG ratio, and RONS levels.The algorithms that determine the ratio of S-glutathionylation and S-nitrosylation will be the subject of further study; however, we can already say that they are determined, on one hand, by the RONS ratio and, on the other, by GSH/GSSG and largely depend on the conditions that maintain the intracellular level of GSH, including its de novo synthesis and the rate of GSSG recovery. Trx family enzymes  play an important role in the regulation of these processes, the activity of which is determined by the cellular redox status and the GSH/GSSG level.Taken together, this allows us to emphasize the important role of GSH in protein redox modulation—through S-glutathionylation and S-nitrosylation—in order to determine the contribution of these processes to the maintenance and regulation of cellular redox homeostasis.