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The intramolecular carbolithiation-cyclization-electrophilic substitution sequence proves to be a promising strategy for synthetic organic chemists. Our current research in this area focuses on the one-pot halogen/lithium exchange of 2-bromophenyl-3-phenylprop-2-enyl ether, followed by intramolecular carbolithiation, and trapping of the new lithiated cyclic intermediate by several electrophiles, affording 3-substituted 2,3-dihydrobenzofurans with some diastereoselectivity. Within this context, a study on the product distribution solvent dependence was carried out using different types of solvents, namely: polar coordinating, polar non-coordinating, and non-polar solvents. The results show that the coordinating features of the solvent affect specially the carbolithiation step, whereas the halogen/lithium exchange seems to be barely affected. Theoretical calculations were carried out to investigate the unexpected diastereoselectivity of the tandem reaction, where two stereocenters are generated. Insights gained from our mechanistic investigations enabled us to propose an inversion of configuration at the lithiated intermediate prior to the reaction, being the electrophile the likely cause for the observed distereoselectivity. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

We evaluated the population growth responses of three species of Brachionus (B. angularis, B. havanaensis and B. rubens on Chlorella vulgaris at three densities (0.0625×106, 0.125×106 and 0.25×106 cells mL-1) offered in three forms (live, frozen and heat-killed). When raised on live Chlorella, increase in algal concentration resulted in increased population abundances of all the three brachionid rotifers. For B. angularis and B. rubens at any given food density live Chlorella resulted in higher population densities than when fed in frozen or heat-killed form. However, for B. havanaensis, heat-killed alga supported higher population densities than on live or frozen Chlorella. B. havanaensis failed to grow on frozen alga regardless of the food density, while the other two rotifers showed positive growth, especially under higher diet levels. The rate of population increase (r) varied from 0.45 ±0.03 to -0.58±0.01 per day, depending on the brachionid species, algal density and the diet form. Statistically, the r of B. angularis and B. havanaensis was significantly (p<0.05) affected by algal form and the density as well as their interaction. However, the r of B. rubens was not significantly affected by the algal form (p>0.05), although algal concentration and its interaction with diet type were significantly p<0.001). Thus, our study showed that the response of tested species of Brachionus varied depending not only on the form in which Chlorella was offered but also on its concentration. [ABSTRACT FROM AUTHOR]

The aim of this study was to have a better understanding of the influence of the coronavirus disease 2019 (COVID-19) pandemic in people with epilepsy (PWE) and to assess whether there have been changes in seizure control during the current COVID-19 outbreak, exploring the possible causes thereof. This is an observational, retrospective study based on prospective data collection of 100 successive patients who attended an epilepsy outpatient clinic either face-to-face or telephonically during the months of the COVID-19 outbreak and national state of emergency. One hundred patients were included, 52% women, mean age 42.4 years. During the COVID-19 period, 27% of the patients presented an increase of > 50% of seizure frequency. An increase of stress/anxiety (odds ratios (OR): 5.78; p = 0.008) and a prior higher seizure frequency (OR: 12.4; p = 0.001) were associated with worsening of seizures. Other risk factors were exacerbation of depression, sleep deprivation, less physical activity, and history of epilepsy surgery. Three patients had status epilepticus (SE) and one a cluster of seizures. Likewise, 9% of patients improved their seizure control. Reduction in stress/anxiety (OR: 0.05; p = 0.03) and recent adjustment of antiepileptics (OR: 0.07; p = 0.01) acted as protecting factors. A high proportion of PWE suffered a significant worsening of their seizure control during the months of the COVID-19 pandemic. Emotional distress due to home confinement was the main factor for the change in seizure control. Promoting physical activity and adequate sleep may minimize the potential impact of the pandemic in PWE. Ensuring correct follow-up can prevent decompensation in those PWE at high risk. • During COVID-19 pandemic, seizure control worsened in 27% of people with epilepsy. • Emotional distress was the most important risk factor for seizure control worsening. • Promoting physical activity and adequate sleep can minimize seizures exacerbation. • Ensuring correct follow-up can prevent decompensations in people with epilepsy. [ABSTRACT FROM AUTHOR]

We have identified an unexpected role for netrin1, a canonical axonal guidance cue, as a suppressor of bone morphogenetic protein (Bmp) signaling in the developing dorsal spinal cord. Using a combination of gain- and loss-of-function approaches in chicken and mouse embryonic models, as well as mouse embryonic stem cells (mESCs), we have observed that manipulating the level of netrin1 specifically alters the patterning of the Bmp-dependent dorsal interneurons (dIs), dI1-dI3. Altered netrin1 levels also change Bmp signaling activity, as assessed using bioinformatic approaches, as well as monitoring phosophoSmad1/5/8 activation, the canonical intermediate of Bmp signaling, and Id levels, a known Bmp target. Together, these studies support the hypothesis that netrin1 acts from the intermediate spinal cord to regionally confine Bmp signaling to the dorsal spinal cord. Thus, netrin1 has reiterative activities shaping dorsal spinal circuits, first by regulating cell fate decisions and then acting as a guidance cue to direct axon extension., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Infective endocarditis (IE) poses a diagnostic challenge due to its diverse clinical presentations, especially among high-risk groups. Diagnosis relies on integrating clinical presentation, blood cultures and imaging findings. Advanced imaging techniques enhance diagnostic accuracy, particularly in complex cases. Treatment involves antimicrobial therapy and surgery in complicated cases, with early intervention crucial for optimal outcomes. Coordinated care by an Endocarditis Team ensures tailored treatment plans, prompt complication management and long-term monitoring after discharge. The authors present a case of subacute IE presenting initially with back pain in a patient with a complex medical history, highlighting diagnostic and management approaches., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Improving models of species' distributions is essential for conservation, especially in light of global change. Species distribution models (SDMs) often rely on mean environmental conditions, yet species distributions are also a function of environmental heterogeneity and filtering acting at multiple spatial scales. Geodiversity, which we define as the variation of abiotic features and processes of Earth's entire geosphere (inclusive of climate), has potential to improve SDMs and conservation assessments, as they capture multiple abiotic dimensions of species niches, however they have not been sufficiently tested in SDMs. We tested a range of geodiversity variables computed at varying scales using climate and elevation data. We compared predictive performance of MaxEnt SDMs generated using CHELSA bioclimatic variables to those also including geodiversity variables for 31 mammalian species in Colombia. Results show the spatial grain of geodiversity variables affects SDM performance. Some variables consistently exhibited an increasing or decreasing trend in variable importance with spatial grain, showing slight scale-dependence and indicating that some geodiversity variables are more relevant at particular scales for some species. Incorporating geodiversity variables into SDMs, and doing so at the appropriate spatial scales, enhances the ability to model species-environment relationships, thereby contributing to the conservation and management of biodiversity. This article is part of the Theo Murphy meeting issue 'Geodiversity for science and society'.

Cardiopulmonary exercise testing (CPET) provides important information for the assessment and management of patients with heart failure. This testing measures the respiratory and cardiac responses to exercise and allows measurement of the oxygen uptake (V˙O 2 ) max and the relationship between minute ventilation (V˙E) and carbon dioxide excretion (V˙CO 2 ). These two parameters help classify patients into categories that help predict prognosis, and patients with a V˙O 2 < 14 mL/kg/min and V˙E/V˙CO 2 slope >35 have a poor prognosis. This testing has been used in drug trials to determine complex physiologic responses to medications, such as angiotensin-converting enzyme inhibitors. For example, a study with enalapril demonstrated that the peak V˙O 2 was 14.6 ± 1.6 mL/kg/min on placebo and 15.8 ± 2.0 mL/kg/min on enalapril after 15 days of treatment. The V˙E/V˙CO 2 slopes were 43 ± 8 on placebo and 39 ± 7 on enalapril. Chronic heart failure and reduced physical activity measured by cardiopulmonary exercise testing are associated with increases in BNP, and several studies have demonstrated that cardiac rehabilitation is associated with reductions in BNP and increases in V˙O 2 . Therefore, BNP measurements can help determine the benefits of cardiac rehabilitation and provide indirect estimates of changes in V˙O 2 . In addition, measurement of microRNAs can determine the status of skeletal muscle used during physical activity and the changes associated with rehabilitation. However, CPET requires complicated technology, and simpler methods to measure physical activity could help clinicians to manage their patients. Recent advances in technology have led to the development of portable cardiopulmonary exercise testing equipment, which can be used in various routine physical activities, such as walking upstairs, sweeping the floor, and making the bed, to provide patients and clinicians a better understanding of the patient's current symptoms. Finally, current smart watches can provide important information about the cardiorespiratory system, identify unexpected clinical problems, and help monitor the response to treatment. The organized use of these devices could contribute to the management of certain aspects of these patients' care, such as monitoring the treatment of atrial fibrillation. This review article provides a comprehensive overview of the current use of CPET in heart failure patients and discusses exercise principles, methods, clinical applications, and prognostic implications.

In this work, we aimed at investigating cell and tissue responses of the apple snail Pomacea canaliculata , following the inoculation of the zoonotic pathogen Mycobacterium marinum . Different doses were tested (10, 20, 65, and 100 M CFU) and the mortality rate was negligible. The histopathogenesis was followed at 4, 9, and 28 days after inoculation. Overt histopathological lesions were consistently observed after the two largest doses only. In the lung, marked hemocyte aggregations, including intravascular nodule formation, were observed within the large blood veins that run along the floor and roof of this organ. Hemocyte aggregations were found occluding many of the radial sinuses supplying the respiratory lamina. Acid-fast bacilli were contained in the different hemocyte aggregations. In addition, hemocytes were observed infiltrating the storage tissue, which makes up most of the lung wall, and the connective tissue of the mantle edge. Additionally, signs of degradation in the storage tissue were observed in the lung wall on day 28. In the kidney, nodules were formed associated with the constitutive hemocyte islets and with the subpallial hemocoelic space, in whose hemocytes the acid-fast structures were found. Electron microscopy analysis revealed the presence of bacteria-containing phagosomes within hemocytes located in the surface zone of the islets. Additionally, electron-dense spheroidal structures, which are likely remnants of digested mycobacteria, were observed in close proximity to the hemocytes' nuclei. The size attained by the hemocyte nodules varied during the observation period, but there was no clear dependence on dose or time after inoculation. Nodules were also formed subpallially. Some of these nodules showed 2-3 layers with different cellular composition, suggesting they may also form through successive waves of circulating cells reaching them. Nodular cores, including those formed intravascularly in the lung, would exhibit signs of hemocyte dedifferentiation, possibly proliferation, and death. Hemocyte congestion was observed in the hemocoelic spaces surrounding the pallial ends of the renal crypts, and the renal crypts themselves showed de-epithelization, particularly on day 28. The diverse cellular responses of P. canaliculata to M. marinum inoculation and the high resilience of this snail to the pathogen make it a suitable species for studying mycobacterial infections and their effects on cellular and physiological processes., Competing Interests: 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., (Copyright © 2023 Cruz-Flores, Rodriguez, Giai, Vega and Castro-Vazquez.)

Objective: The purpose of this study was to evaluate the impact of COVID-19 pandemic public health restrictions on our drip and ship mechanical thrombectomy program in Santiago Chile., Materials and Methods: This was a retrospective analysis of a prospectively collected database comparing two cohorts, one during a two-year period before COVID-19 and the second during the two years of the pandemic at our metropolitan stroke program., Results: A total of 100 patients were included in the pre COVID-19 cohort (cohort 1) and 121 in the COVID-19 cohort (cohort 2). There was a significant difference between cohorts, with older patients, different occlusion sites and higher door to arterial puncture time during the COVID-19 period. A non-significant trend for worse 90-day outcomes and higher mortality was present in cohort 2. There were no statistical differences in safety treatment parameters., Conclusions: COVID-19 pandemic has had a measurable impact on our mechanical thrombectomy program. Results showed similarities to other reported Latin American series, where less robust health systems could adapt less efficiently compared to developed countries. After two years of public health restrictions, there were changes in the treatment population characteristics, delay in some internal management metrics and a non-significant trend to worse 90-day outcomes and higher mortality., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Infectious keratitis is a vision-threatening microbial infection. The increasing antimicrobial resistance and the fact that severe cases often evolve into corneal perforation necessitate the development of alternative therapeutics for effective medical management. Genipin, a natural crosslinker, was recently shown to exert antimicrobial effects in an ex vivo model of microbial keratitis, highlighting its potential to serve as a novel treatment for infectious keratitis. This study aimed to evaluate the antimicrobial and anti-inflammatory effects of genipin in an in vivo model of Staphylococcus aureus ( S. aureus ) and Pseudomonas aeruginosa ( P. aeruginosa ) keratitis. Clinical scores, confocal microscopy, plate count, and histology were carried out to evaluate the severity of keratitis. To assess the effect of genipin on inflammation, the gene expression of pro- and anti-inflammatory factors, including matrix metalloproteinases (MMPs), were evaluated. Genipin treatment alleviated the severity of bacterial keratitis by reducing bacterial load and repressing neutrophil infiltration. The expression of interleukin 1B (IL1B), interleukin 6 (IL6), interleukin 8 (IL8), interleukin 15 (IL15), tumor necrosis factor-α (TNF-α), and interferon γ (IFNγ), as well as MMP2 and MMP9, were significantly reduced in genipin-treated corneas. Genipin promoted corneal proteolysis and host resistance to S. aureus and P. aeruginosa infection by suppressing inflammatory cell infiltration, regulating inflammatory mediators, and downregulating the expression of MMP2 and MMP9.

States of natural dormancy include estivation and hibernation. Ampullariids are exemplary because they undergo estivation when deprived of water or hibernation when exposed to very low temperatures. Regardless of the condition, ampullariids show increased endogenous antioxidant defenses, anticipating the expected respiratory burst during reoxygenation after reactivation, known as "Preparation for Oxidative Stress (POS)". In this work, we tested the POS hypothesis for changes in the blood and hemocytes of the bimodal breather Pomacea canaliculata (Ampullariidae) induced at experimental estivation and hibernation. We described respiratory (hemocyanin, proteins, lactate), antioxidant (GSH, uric acid, SOD, CAT, GST), and immunological (hemocyte levels, ROS production) parameters. We showed that, although the protein level remains unchanged in all experimental groups, hemocyanin increases in response to estivation. Furthermore, lactate remains unchanged in challenged snails, suggesting an aerobic metabolism during short-term challenges. Blood uric acid increases during estivation and arousal from estivation or hibernation, supporting the previously proposed antioxidant role. Regarding hemocytes, we showed that the total population increases with all challenges, and granulocytes increase during hibernation. We further showed that hibernation affects ROS production by hemocytes, possibly through mitochondrial inhibition. This study contributed to the knowledge of the adaptive strategies of ampullariids to tolerate adverse environmental conditions.

We stand as dissenters against the acceptance of scientific knowledge that has not been built on empirical data. With this in mind, this review synthesizes selected aspects of the immunobiology of gastropods and of apple snails (Ampullariidae) in particular, from morphological to molecular and "omics" studies. Our trip went through more than two centuries of history and was guided by an evo-devo hypothesis: that the gastropod immune system originally developed in the mesenchymal connective tissue of the reno-pericardial complex, and that in that tissue some cells differentiated into hematopoietically committed progenitor cells that integrate constitutive hemocyte aggregations in the reno-pericardial territory, whether concentrated in the pericardium or the kidney in a species-specific manner. However, some of them may be freed from those aggregations, circulate in the blood, and form distant contingent aggregations anywhere in the body, but always in response to intruders (i.e., pathogens or any other immune challenge). After that, we reviewed the incipient immunology of the Ampullariidae by critically revising the findings in Pomacea canaliculata and Marisa cornuarietis , the only ampullariid species that have been studied in this respect, and we attempted to identify the effectors and the processes in which they are involved. Particularly for P. canaliculata , which is by far the most studied species, we ask which hemocytes are involved, in which tissues or organs are integrated, and what cellular reactions to intruders this species has in common with other animals. Furthermore, we wondered what humoral factors could also integrate its internal defense system. Among the cellular defenses, we give an outstanding position to the generation of hemocyte nodules, which seems to be an important process for these snails, serving the isolation and elimination of intruders. Finally, we discuss hematopoiesis in apple snails. There have been contrasting views about some of these aspects, but we envision a hematopoietic system centered in the constitutive hemocyte islets in the ampullariid kidney., Competing Interests: 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., (Copyright © 2022 Rodriguez, Vega and Castro-Vazquez.)

The COVID-19 pandemic has affected the lives of all Americans, but the severity of the pandemic has been experienced unevenly across space and time. Some states saw sharp rises in COVID-19 cases in early March, whereas case counts rose much later in the rest of the country. In this article, we examine the relationship between exposure to COVID-19 and citizens' views on what type of measures are required to deal with the crises and how experience with and exposure to COVID-19 is associated with greater partisan polarization. We find consistent evidence of partisan divergence in pandemic-response policy preferences across the first six months of the COVID-19 pandemic: Republicans support national control measures whereas Democrats support welfare policies, and interparty differences grow over time. We find only limited evidence that exposure or experience moderates these partisan differences. Our findings are consistent with the view that Americans interpret the COVID-19 pandemic in fundamentally partisan manner, and that objective pandemic conditions play at most a minor role in shaping mass preferences., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. Political Psychology published by Wiley Periodicals LLC on behalf of International Society of Political Psychology.)

Long-term estivation (45 days) in the apple snail Pomacea canaliculata induces an increase of non-enzymatic antioxidants, such as uric acid and reduced glutathione (GSH), which constitutes an alternative to the adaptive physiological strategy of preparation for oxidative stress (POS). Here, we studied markers of oxidative stress damage, uric acid levels, and non-enzymatic antioxidant capacity, enzymatic antioxidant defenses, such as superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST), and transcription factors expression [forkhead box protein O (FOXO), hypoxia-inducible factor-1 alpha (HIF1α), and nuclear factor erythroid 2-related factor 2 (Nrf2)] in control active animals, 7-day estivating and aroused snails, in digestive gland, gill, and lung tissue samples. In the digestive gland, SOD and CAT activities significantly increased after estivation and decreased during arousal. Meanwhile, GST activity decreased significantly during the activity-estivation-arousal cycle. Gill CAT activity increased significantly at 7 days of estivation, and it decreased during arousal. In the lung, the CAT activity level increased significantly during the cycle. FOXO upregulation was observed in the studied tissues, decreasing its expression only in the gill of aroused animals during the cycle. HIF1α and Nrf2 transcription factors decreased their expression during estivation in the gill, while in the lung and the digestive gland, both transcription factors did not show significant changes. Our results showed that the short-term estivation induced oxidative stress in different tissues of P. canaliculata thereby increasing overall antioxidant enzymes activity and highlighting the role of FOXO regulation as a possible underlying mechanism of the POS strategy., Competing Interests: 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., (Copyright © 2022 Giraud-Billoud, Campoy-Diaz, Dellagnola, Rodriguez and Vega.)

Purpose: To determine whether genipin (a natural crosslinker) could reduce the colonization and proliferation of bacteria and fungi in an ex vivo model of corneal infection., Methods: This study, using an ex vivo model of bacterial and fungal keratitis, investigated the antimicrobial efficacy of genipin crosslinking. Excised corneoscleral buttons were wounded by scalpel incision and subsequently infected with Staphylococcus aureus, Pseudomonas aeruginosa, or Candida albicans. After inoculation, corneas were treated with genipin for 24 hours at 37°C. Histologic examinations were carried out, and the number of viable colony-forming units (CFU)/cornea was determined., Results: Genipin exerts bactericidal action against S. aureus and P. aeruginosa, as well as fungicidal action against C. albicans and significantly reduced the CFU compared to contralateral eyes that received saline treatment (P < 0.05)., Conclusions: These data identify genipin as a novel ocular antimicrobial agent that has the potential to be incorporated into the therapeutic armamentarium against microbial keratitis., Translational Relevance: This study provided evidence for the antimicrobial and antifungal properties of genipin as an alternative crosslinker that could be used in the management of infectious keratitis.

The freshwater caenogastropod family Ampullariidae is emerging as a model for a variety of studies, among them, the evolution of terrestriality. A common character of the family is that all its members bear a lung while retaining the ancestral gill. This ensures that many ampullariids are able to inhabit poorly oxygenated waters, to bury in the mud during estivation, and to temporarily leave the water, in some species for oviposition. To these characters Pomacea canaliculata (Caenogastropoda, Ampullariidae) adds that is an obligate air-breather. In a recent paper, we showed the gill epithelium of P. canaliculata has a set of characteristics that suggest its role for oxygen uptake may be less significant than its role in ionic/osmotic regulation and immunity. We complement here our morphological investigation on the respiratory organs of P. canaliculata by studying the lung of this species at the anatomical (3D reconstructions of the blood system and nerve supply), histological and ultrastructural levels. The circulation of the gill and the lung are interconnected so that the effluence of blood from the gill goes to the lung where it completes oxygenation. Besides that, we found the lung cavity is lined by a pavement epithelium that encloses an anastomosing network of small blood spaces resting over a fibromuscular layer, which altogether form the respiratory lamina. The pavement cells form a blood-gas barrier that is 80-150 nm thick and thus fulfils the requirements for an efficient gas exchanger. Tufts of ciliary cells, together with some microvillar and secretory cells, are interspersed in the respiratory lamina. Rhogocytes, which have been proposed to partake in metal depuration and in the synthesis of hemocyanin in other gastropods, were found below the respiratory lamina, in close association with the storage cell tissue. In light of these findings, we discuss the functional role of the lung in P. canaliculata and compare it with that of other gastropods. Finally, we point to some similarities in the pattern of the evolution of air dependence in this family., Competing Interests: The authors declare that they have no competing interests., (© 2021 Rodriguez et al.)

Background & Aims: The short-term efficacy of RPC4046, a monoclonal antibody against interleukin-13, has been shown in patients with eosinophilic esophagitis (EoE). We investigated the long-term efficacy and safety of RPC4046 in an open-label, long-term extension (LTE) study in adults with EoE., Methods: We analyzed data from 66 patients who completed the 16-week, double-blind, induction portion of a phase 2 study of RPC4046 (180 mg or 360 mg/wk) vs placebo and then completed a 52-week LTE, receiving open-label RPC4046 360 mg/wk. The study was conducted at 28 centers in 3 countries; patients were enrolled between September 2014 and January 2017. Outcomes were stratified by double-blind dose group and included esophageal eosinophil counts, EoE endoscopic reference score, EoE histologic scoring system score, symptom-based EoE activity index score, and safety., Results: By week 12 of the LTE, esophageal eosinophil mean and peak counts, total EoE endoscopic reference scores, and EoE histologic scoring system grade and stage scores did not differ considerably between patients who originally received placebo vs RPC4046. Most patients maintained responses through week 52. Symptom remission (symptom-based EoE activity index score, ≤20) increased from 14% at LTE entry to 67% at LTE week 52 in placebo‒RPC4046 patients and from 30% to 54% in RPC4046‒RPC4046 (either dose) patients. Of the 28 patients who did not have a histologic response to RPC4046 during the double-blind induction phase, 10 patients (36%) achieved response during the LTE. The most common adverse events were upper respiratory tract infection (21%) and nasopharyngitis (14%)., Conclusions: One year of treatment with RPC4046 is generally well tolerated and results in continued improvement and/or maintenance of endoscopic, histologic, and clinical measures of EoE disease activity relative to baseline., Trial Registration: NCT02098473., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Gastropod hematopoiesis occurs at specialized tissues in some species, but the evidence also suggests that hemocyte generation is maybe widespread in the connective tissues or the blood system in others. In Ampullariidae (Caenogastropoda), both the kidney and the lung contain putative hematopoietic cells, which react to immune challenges. In the current study, we wanted to explore if hematopoiesis occurs in the blood of Pomacea canaliculata. Thus, we obtained circulating hemocytes from donor animals and tested their ability to proliferate in the blood of conspecific recipients. We tracked cell proliferation by labeling the donors' hemocytes with the fluorescent cell proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE). Transferred CFSE-labeled hemocytes survived and proliferated into the recipients' circulation for at least 17 days. We also determined the cell cycle status of circulating hemocytes by using the propidium iodide (PI) and acridine orange (AO) staining methods. Flow cytometry analyses showed that most PI-stained hemocytes were in the G1 phase (~96%), while a lower proportion of cells were through the G2/S-M transition (~4%). When we instead used AO-staining, we further distinguished a subpopulation of cells (~5%) of low size, complexity-granularity, and RNA content. We regarded this subpopulation as quiescent cells. In separate experimental sets, we complemented these findings by assessing in circulating hemocytes two evolutionary conserved features of quiescent, undifferentiated cells. First, we used JC-1 staining to determine the mitochondrial membrane potential (Ψ m ) of circulating hemocytes, which is expected to be low in quiescent cells. Most hemocytes (~87%) showed high aggregation of JC-1, which indicates a high Ψ m . Besides that, a small hemocyte subpopulation (~11%) showed low aggregation of the dye, thus indicating a low Ψ m . It is known that the transition from a quiescent to a proliferating state associates with an increase of the Ψ m . The specificity of these changes was here controlled by membrane depolarization with the Ψ m disruptor CCCP. Second, we stained hemocytes with Hoechst33342 dye to determine the efflux activity of ABC transporters, which participate in the multixenobiotic resistance system characteristic of undifferentiated cells. Most hemocytes (>99%) showed a low dye-efflux activity, but a small proportion of cells (0.06-0.12%) showed a high dye-efflux activity, which was significantly inhibited by 100 and 500 μM verapamil, and thus is indicative of an undifferentiated subpopulation of circulating hemocytes. Taken together, our results suggest that, among circulating hemocytes, there are cells with the ability to proliferate or to stay in a quiescent state and behave as progenitor cells later, either in the circulation or the hematopoietic tissues/organs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Fibrostenosis, the most serious eosinophilic esophagitis (EoE) complication, is mediated by epithelial-mesenchymal transition (EMT). Transitioned cells contribute to pathogenesis by overproducing extracellular matrix., Objective: Our aim was to determine whether RPC4046 (anti‒IL-13 mAb) modulates EMT biomarkers in biopsy samples from adults with active EoE in a substudy of a double-blind, placebo-controlled phase 2 trial., Methods: Baseline and week 16 esophageal biopsy samples were taken from 69 patients who were randomized to weekly treatment with subcutaneous RPC4046, 180 mg (n = 19), 360 mg (n = 26), or placebo (n = 24). Duplex immunofluorescence slides stained for E-cadherin and vimentin were digitally analyzed by mapping each epithelial cell and recording fluorescence intensities. End points included change from baseline to week 16 in percentage of vimentin-positive epithelial cells (primary), total E-cadherin expression, and vimentin-to-E-cadherin ratio per cell (an average of 47,000 cells per biopsy sample analyzed)., Results: The mean percentage of vimentin-positive cells decreased by 0.94%, 2.75%, and 4.24% in the placebo, low-dose, and high-dose groups, respectively (P =.032 for the high-dose vs placebo group). Mean E-cadherin expression per cell increased 5.6-fold in both dose groups versus in the placebo group (high-dose group P = .047). The increases in E-cadherin expression per cell from baseline to week 16 were correlated with improvements in histology, eosinophil counts, endoscopic findings, and symptoms., Conclusion: RPC4046 significantly reduced EMT markers in adults with active EoE, with greater effects at 360 mg. Together with results for eosinophil density and clinical end points from the main trial, these data support the hypothesis that pharmacologic IL-13 inhibition ameliorates both inflammatory and remodeling pathways and could potentially reduce the risk of fibrostenotic complications., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The DAF-12 receptor is a ligand-activated transcription factor that in its ligand-bound form allows the expression of target genes needed to support the reproductive life cycle of the free-living nematode Caenorhabditis elegans, whereas unbound DAF-12 receptor leads to the developmentally arrested "dauer larvae", specialized for survival and dispersal. The endogenous ligands of the DAF-12 receptor are 3-keto-cholestenoic acids dubbed dafachronic acids. In a previous publication we reported that oxysterols with a shorter side chain (C 24 ) modulate the DAF-12 receptor activity either as partial agonists or, in the case of the C 24 alcohol 24-hydroxy-4-cholen-3-one, as an antagonist both in vitro and in vivo. Preliminary structure-activity relationships suggested that this activity profile could be improved with more lipophilic and less acidic functional groups at the end of the side chain. Thus, we have now synthesized two fluorine containing analogues in which the C-24 hydroxyl was replaced by a difluoromethyl group (regarded as a "lipophilic hydroxyl") or a difluoromethylidene group with similar lipophilicity but lacking the hydrogen bond donor capacity. Activity was evaluated in vitro using transactivation cell-based assays and in vivo by the effect on the development of wild-type C. elegans. The 24-difluoromethyl analogue retained the antagonist activity in vitro, being completely devoid of agonist activity and exhibited improved activity in vivo. The difluoromethylidene showed a slight antagonist tendency in vitro (statistically not significant), in the concentration range tested and was weakly active in vivo. None of the compounds were toxic, as treated worms recovered to normal development, when transferred to fresh media without added steroids., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Liver X receptors (LXRs) are nuclear receptors that play central roles in the transcriptional control of lipid metabolism. The ability of LXRs to integrate metabolic and inflammation signalling makes them attractive targets for intervention in human metabolic diseases. Several oxidized metabolites of cholesterol (oxysterols) are endogenous LXR ligands, that modulate their transcriptional responses. While 25R-cholestenoic acid is an agonist of the LXRs, the synthetic analogue 27-norcholestenoic acid that lacks the 25-methyl is an inverse agonist. This change in the activity profile is triggered by a disruption of a key interaction between residues His435 and Trp457 that destabilizes the H11-H12 region of the receptor and favors the binding of corepressors. The introduction of fluorine atoms on the oxysterol side chain can favor both hydrophobic interactions as well as hydrogen bonds with the fluorine atoms and may thus induce changes in the receptor that may lead to changes in the activity profile. To evaluate these effects we have synthesized two fluorinated 27-nor-steroids, analogues of 27-norcholestenoic acid, the 25,25-difluoroacid and the corresponding 26-alcohol. The key step was a Reformatsky reaction on the C-24 cholenaldehyde, with ethyl bromodifluoroacetate under high intensity ultrasound (HIU) irradiation, followed by a Barton-McCombie type deoxygenation. Activity was evaluated in a luciferase reporter assay in the human HEK293T cells co-transfected with full length human LXRβ expression vector. The 25,25-difluoro-27-norcholestenoic acid was an inverse agonist and antagonist similar to its non-fluorinated analogue while its reduced derivative 25,25-difluoro-27-norcholest-5-ene-3β,26-diol was an agonist. Molecular dynamics simulation of the ligand-receptor complexes showed that the difluoroacid disrupted the His435-Trp457 interaction although the resulting conformational changes were different from those induced by the non-fluorinated analogue. In the case of the difluoroalcohol, the fluorine atoms actively participated in the interaction with several residues in the ligand binding pocket leading to a stabilization of the active receptor conformation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Bcl-2 is overexpressed in about a half of human cancers and 50-70% of breast cancer patients, thereby conferring resistance to conventional therapies and making it an excellent therapeutic target. Small interfering RNA (siRNA) offers novel and powerful tools for specific gene silencing and molecularly targeted therapy. Here, we show that therapeutic silencing of Bcl-2 by systemically administered nanoliposomal (NL)-Bcl-2 siRNA (0.15 mg siRNA/kg, intravenous) twice a week leads to significant antitumor activity and suppression of growth in both estrogen receptor-negative (ER(-)) MDA-MB-231 and ER-positive (+) MCF7 breast tumors in orthotopic xenograft models (P < 0.05). A single intravenous injection of NL-Bcl-2-siRNA provided robust and persistent silencing of the target gene expression in xenograft tumors. NL-Bcl-2-siRNA treatment significantly increased the efficacy of chemotherapy when combined with doxorubicin in both MDA-MB-231 and MCF-7 animal models (P < 0.05). NL-Bcl-2-siRNA treatment-induced apoptosis and autophagic cell death, and inhibited cyclin D1, HIF1α and Src/Fak signaling in tumors. In conclusion, our data provide the first evidence that in vivo therapeutic targeting Bcl-2 by systemically administered nanoliposomal-siRNA significantly inhibits growth of both ER(-) and ER(+) breast tumors and enhances the efficacy of chemotherapy, suggesting that therapeutic silencing of Bcl-2 by siRNA is a viable approach in breast cancers.Molecular Therapy-Nucleic Acids (2013) 2, e121; doi:10.1038/mtna.2013.45; published online 10 September 2013.