Human pluripotent stem cell-derived inner ear organoids recapitulate otic development in vitro.

Our molecular understanding of the early stages of human inner ear development has been limited by the difficulty in accessing fetal samples at early gestational stages. As an alternative, previous studies have shown that inner ear morphogenesis can be partially recapitulated using induced pluripotent stem cells directed to differentiate into inner ear organoids (IEOs). Once validated and benchmarked, these systems could represent unique tools to complement and refine our understanding of human otic differentiation and model developmental defects. Here, we provide the first direct comparisons of the early human embryonic otocyst and fetal sensory organs with human IEOs. We use multiplexed immunostaining and single-cell RNA-sequencing to characterize IEOs at three key developmental steps, providing a new and unique signature of in vitro-derived otic placode, epithelium, neuroblasts and sensory epithelia. In parallel, we evaluate the expression and localization of crucial markers at these equivalent stages in human embryos. Together, our data indicate that the current state-of-the-art protocol enables the specification of bona fide otic tissue, supporting the further application of IEOs to inform inner ear biology and disease.

miR-203 drives breast cancer cell differentiation.

A hallmark of many malignant tumors is dedifferentiated (immature) cells bearing slight or no resemblance to the normal cells from which the cancer originated. Tumor dedifferentiated cells exhibit a higher capacity to survive to chemo and radiotherapies and have the ability to incite tumor relapse. Inducing cancer cell differentiation would abolish their self-renewal and invasive capacity and could be combined with the current standard of care, especially in poorly differentiated and aggressive tumors (with worst prognosis). However, differentiation therapy is still in its early stages and the intrinsic complexity of solid tumor heterogeneity demands innovative approaches in order to be efficiently translated into the clinic. We demonstrate here that microRNA 203, a potent driver of differentiation in pluripotent stem cells (ESCs and iPSCs), promotes the differentiation of mammary gland tumor cells. Combining mouse in vivo approaches and both mouse and human-derived tridimensional organoid cultures, we report that miR-203 influences the self-renewal capacity, plasticity and differentiation potential of breast cancer cells and prevents tumor cell growth in vivo. Our work sheds light on differentiation-based antitumor therapies and offers miR-203 as a promising tool for directly confronting the tumor-maintaining and regeneration capability of cancer cells.

Decadal soil warming decreased vascular plant above and belowground production in a subarctic grassland by inducing nitrogen limitation.

Below and aboveground vegetation dynamics are crucial in understanding how climate warming may affect terrestrial ecosystem carbon cycling. In contrast to aboveground biomass, the response of belowground biomass to long-term warming has been poorly studied. Here, we characterized the impacts of decadal geothermal warming at two levels (on average +3.3°C and +7.9°C) on below and aboveground plant biomass stocks and production in a subarctic grassland. Soil warming did not change standing root biomass and even decreased fine root production and reduced aboveground biomass and production. Decadal soil warming also did not significantly alter the root-shoot ratio. The linear stepwise regression model suggested that following 10 yr of soil warming, temperature was no longer the direct driver of these responses, but losses of soil N were. Soil N losses, due to warming-induced decreases in organic matter and water retention capacity, were identified as key driver of the decreased above and belowground production. The reduction in fine root production was accompanied by thinner roots with increased specific root area. These results indicate that after a decade of soil warming, plant productivity in the studied subarctic grassland was affected by soil warming mainly by the reduction in soil N.

Fine-tuned nitric oxide and hormone interface in plant root development and regeneration.

Plant root growth and developmental capacities reside in a few stem cells of the root apical meristem (RAM). Maintenance of these stem cells requires regenerative divisions of the initial stem cell niche (SCN) cells, self-maintenance, and proliferative divisions of the daughter cells. This ensures sufficient cell diversity to guarantee the development of complex root tissues in the plant. Damage in the root during growth involves the formation of a new post-embryonic root, a process known as regeneration. Post-embryonic root development and organogenesis processes include primary root development and SCN maintenance, plant regeneration, and the development of adventitious and lateral roots. These developmental processes require a fine-tuned balance between cell proliferation and maintenance. An important regulator during root development and regeneration is the gasotransmitter nitric oxide (NO). In this review we have sought to compile how NO regulates cell rate proliferation, cell differentiation, and quiescence of SCNs, usually through interaction with phytohormones, or other molecular mechanisms involved in cellular redox homeostasis. NO exerts a role on molecular components of the auxin and cytokinin signaling pathways in primary roots that affects cell proliferation and maintenance of the RAM. During root regeneration, a peak of auxin and cytokinin triggers specific molecular programs. Moreover, NO participates in adventitious root formation through its interaction with players of the brassinosteroid and cytokinin signaling cascade. Lately, NO has been implicated in root regeneration under hypoxia conditions by regulating stem cell specification through phytoglobins.

Effect of melatonin and nitric oxide on capacitation and apoptotic changes induced by epidermal growth factor in ram sperm.

CONTEXT: Apart from the canonical cAMP-PKA pathway, ram sperm capacitation can be achieved by the MAPK ERK1/2 signalling cascade, activated by epidermal growth factor (EGF). AIMS: This study aims to investigate the effect of melatonin and nitric oxide (NO·) on capacitation and apoptotic-like changes in EGF-capacitated ram spermatozoa. METHODS: In vitro capacitation was induced by EGF in the absence or presence of melatonin (100pM or 1µM). Also, a NO· precursor, L-arginine, or a NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), were added to capacitation media to study the interaction of NO· and melatonin during EGF-capacitation. Sperm functionality parameters (motility, viability, capacitation state), apoptotic markers (caspase activation and DNA damage), NO· levels, and phosphorylated c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (assessed by Western blot), were evaluated in swim-up and capacitated samples with EGF. KEY RESULTS: NO· levels and the apoptotic-related markers were raised after EGF incubation. Melatonin had a bimodal role on sperm EGF-capacitation, preventing it at high concentration and promoting acrosome reaction at low concentration, but neither of the two concentrations prevented the increase in apoptotic-like markers or NO· levels. However, melatonin at 1µM prevented the activation of JNK. CONCLUSIONS: NO· metabolism does not seem to modulate the apoptosis-like events in ram spermatozoa. Melatonin at 1µM prevents ram sperm capacitation induced by EGF independently from nitric oxide metabolism, and it could be exerted by limiting the JNK mitogen-activated protein kinase (MAPK) activation. IMPLICATIONS: This study improvesour understanding of the biochemical mechanisms involved in sperm capacitation, and ultimately, fertility.

PTH1R translocation to primary cilia in mechanically-stimulated ostecytes prevents osteoclast formation via regulation of CXCL5 and IL-6 secretion.

Osteocytes respond to mechanical forces controlling osteoblast and osteoclast function. Mechanical stimulation decreases osteocyte apoptosis and promotes bone formation. Primary cilia have been described as potential mechanosensors in bone cells. Certain osteogenic responses induced by fluid flow (FF) in vitro are decreased by primary cilia inhibition in MLO-Y4 osteocytes. The parathyroid hormone (PTH) receptor type 1 (PTH1R) modulates osteoblast, osteoclast, and osteocyte effects upon activation by PTH or PTH-related protein (PTHrP) in osteoblastic cells. Moreover, some actions of PTH1R seem to be triggered directly by mechanical stimulation. We hypothesize that PTH1R forms a signaling complex in the primary cilium that is essential for mechanotransduction in osteocytes and affects osteocyte-osteoclast communication. MLO-Y4 osteocytes were stimulated by FF or PTHrP (1-37). PTH1R and primary cilia signaling were abrogated using PTH1R or primary cilia specific siRNAs or inhibitors, respectively. Conditioned media obtained from mechanically- or PTHrP-stimulated MLO-Y4 cells inhibited the migration of preosteoclastic cells and osteoclast differentiation. Redistribution of PTH1R along the entire cilium was observed in mechanically stimulated MLO-Y4 osteocytic cells. Preincubation of MLO-Y4 cells with the Gli-1 antagonist, the adenylate cyclase inhibitor (SQ22536), or with the phospholipase C inhibitor (U73122), affected the migration of osteoclast precursors and osteoclastogenesis. Proteomic analysis and neutralizing experiments showed that FF and PTH1R activation control osteoclast function through the modulation of C-X-C Motif Chemokine Ligand 5 (CXCL5) and interleukin-6 (IL-6) secretion in osteocytes. These novel findings indicate that both primary cilium and PTH1R are necessary in osteocytes for proper communication with osteoclasts and show that mechanical stimulation inhibits osteoclast recruitment and differentiation through CXCL5, while PTH1R activation regulate these processes via IL-6.

Analysis of the Pallial Amygdala in Anurans: Derivatives and Cellular Components.

The amygdaloid complex plays a crucial role in socio-emotional conduct, learning, survival, and reproductive behaviors. It is constituted by a set of nuclei presenting a great cellular heterogeneity and embryonic origin diversity (pallial, subpallial, and even extra-telencephalic). In the last two decades, the tetrapartite pallial paradigm defined the pallial portion of the amygdala as a derivative of the lateroventral pallium. However, the pallial conception is currently being reanalyzed and one of these new proposals is to consider the mouse pallial amygdala as a radial histogenetic domain independent from the rest of the pallial subdomains. In anamniotes, and particularly in amphibian anurans, the amygdaloid complex was described as a region with pallial and subpallial components similar to those described in amniotes. In the present study carried out in Xenopus laevis, after a detailed analysis of the orientation of the amygdalar radial glia, we propose an additional amygdala derived from the pallial region. It is independent of the vomeronasal/olfactory amygdaloid nuclei described in anurans, expresses markers such as Lhx9 present in the mammalian pallial amygdala, and lacks Otp-expressing cells, detected in the adjacent medial amygdala. Further studies are needed to clarify the functional involvement of this area, and whether it is a derivative of the adjacent ventral pallium or an independent pallial domain.

Analysis of the Expression Pattern of Cajal-Retzius Cell Markers in the Xenopus laevis Forebrain.

Cajal-Retzius cells are essential for cortical development in mammals, and their involvement in the evolution of this structure has been widely postulated, but very little is known about their progenitor domains in non-mammalian vertebrates. Using in situhybridization and immunofluorescence techniques we analyzed the expression of some of the main Cajal-Retzius cell markers such as Dbx1, Ebf3, ER81, Lhx1, Lhx5, p73, Reelin, Wnt3a, Zic1, and Zic2 in the forebrain of the anuran Xenopus laevis, because amphibians are the only class of anamniote tetrapods and show a tetrapartite evaginated pallium, but no layered or nuclear organization. Our results suggested that the Cajal-Retzius cell progenitor domains were comparable to those previously described in amniotes. Thus, at dorsomedial telencephalic portions a region comparable to the cortical hem was defined in Xenopus based on the expression of Wnt3a, p73, Reelin, Zic1, and Zic2. In the septum, two different domains were observed: a periventricular dorsal septum, at the limit between the pallium and the subpallium, expressing Reelin, Zic1, and Zic2, and a related septal domain, expressing Ebf3, Zic1, and Zic2. In the lateral telencephalon, the ventral pallium next to the pallio-subpallial boundary, the lack of Dbx1 and the unique expression of Reelin during development defined this territory as the most divergent with respect to mammals. Finally, we also analyzed the expression of these markers at the prethalamic eminence region, suggested as Cajal-Retzius progenitor domain in amniotes, observing there Zic1, Zic2, ER81, and Lhx1 expression. Our data show that in anurans there are different subtypes and progenitor domains of Cajal-Retzius cells, which probably contribute to the cortical regional specification and territory-specific properties. This supports the notion that the basic organization of pallial derivatives in vertebrates follows a comparable fundamental arrangement, even in those that do not have a sophisticated stratified cortical structure like the mammalian cerebral cortex.

Challenges and potential strategies utilizing external data for efficacy evaluation in small-sized clinical trials.

In clinical trials for diseases with very small patient populations, trial investigators may encounter recruitment difficulties. It can be challenging to conduct clinical trials with enough power to detect a treatment effect, and randomization may not be feasible due to timeline, budget, and ethical concerns. To bring breakthrough therapies to the market quickly, it is important to come up with efficient approaches to utilizing individual patient data through improved study design and sound statistical methods. Emerging topics in this area include the use of Bayesian approaches to flexibly incorporate prior information into the current clinical trials, the use of historical controls to efficiently conduct trials that will reduce the number of subjects recruited and ease ethical considerations, and the use of innovative study designs, such as a platform design, to improve the efficiency and speed of the medical therapy development progress. In this paper, we describe three scenarios which highlight some of the challenges encountered in small-sized clinical trial development and provide potential statistical approaches to overcome the aforementioned challenges.

Evaluation of Axillary Lymph Node Marking with Magseed® before and after Neoadjuvant Systemic Therapy in Breast Cancer Patients: MAGNET Study.

Background: Due to the high false negative rate (FNR) associated with sentinel lymph node biopsy (SLNB) after neoadjuvant systemic therapy (NAST), the standard surgical treatment for patients with an initially positive axilla and indicated for NAST is axillary lymph node dissection (ALND). To avoid unnecessary ALND, this multicenter, prospective, observational study aimed to determine the effectiveness and ease of using magnetic seeds (Magseed®) for targeted axillary dissection (TAD) when the seeds are placed before or after NAST. Materials and Methods: We recruited 81 patients diagnosed with T1-T3 breast cancer, with clinically/radiologically positive nodal involvement (cN1, 75 patients with 1-3 nodes suspected nodes and 6 patients with up to 4 suspected nodes) prior to NAST. Positive nodes detected by fine-needle aspiration biopsy or core needle biopsy were marked with a stainless steel marker coil and after NAST with Magseed® prior to surgery (Post-NAST group), or directly with Magseed® before NAST (Pre-NAST group). The correlation between lymph nodes marked with Magseed® (MLNs) and sentinel lymph nodes (SLNs) was calculated based on pathologic assessment with the OSNA assay (Sysmex Corporation, Kobe) or conventional sectioning and staining techniques according to the standard protocols of each center. Results: All magnetic seeds were successfully identified and retrieved in just over 10 minutes of surgery, guided by the Sentimag® magnetometer system. The overall concordance rate between MLNs and SLNs was 81.5%, and the concordance between MLNs and SLNs with metastasis was 93.8%. Metastasis was detected in 54.3% of the MLNs compared with 48.1% of SLNs. In cases that presented negative MLN and negative SLN (negative TAD), the FNR was 0%. No significant differences were found between the Post-NAST and Pre-NAST groups. Conclusions: Our results validate the use of Magseed® for long-term marking of axillary lymph nodes and show that when used in combination with SLNB for TAD, a FNR of 0% can be achieved, avoiding unnecessary ALND.

The Impact of Sexism and Gender Stereotypes on the Legitimization of Women's Low Back Pain.

BACKGROUND: Low back pain is the worldwide leading cause of disability and, even though women's pain experience is more severe, frequent, and enduring, female patients are often underdiagnosed and undertreated. Health professionals' gender stereotypes and social norms may underlie the downgrading of pain. AIM: This pilot study aimed to examine the legitimation of low back pain by health professionals in relation to the sex of the patient as well as their gender awareness and the relationship between them. METHOD: This study had a cross-sectional design. Eighty health professionals and students selected by convenience answered a 4-part online questionnaire. The eligibility criteria for participants were: aged >18 years, students in the last course of nursing/medicine or a physician/nurse, and Spanish-speaking. The questionnaire comprises: (1) a between-subjects virtual clinical low back pain case with four random versions (female/male patient and evidence/non-evidence of pathology); (2) the Spanish version of Nijmegen Gender Awareness Scale (S-NGAMS); (3) Ambivalent Sexism Inventory (ASI); and (4) Ambivalence toward Men Inventory (AMI). RESULTS: The total score of legitimation of low back pain correlated negatively with gender role ideology and sexism scales (when the virtual patient was female), as well as the subscales of willingness to offer support and credibility. CONCLUSIONS: Both sexism and gender role ideology could undermine the legitimation of low back pain, the willingness to offer support, and credibility only in female patients. The results showed a possible gender bias in low back pain assessment in health professionals. Low gender sensitivity and high sexism must be treated as modifiable risk factors for health inequities in pain care.

Gender awareness is also nurses' business: Measuring sensitivity and role ideology towards patients.

AIM: This study aims to validate the Nijmegen Gender Awareness in Medicine Scale, which assesses gender sensitivity and gender-role ideology towards patients in the Spanish language for use among physicians and nurses. BACKGROUND: Women are more likely to suffer pain, delays and health consequences related to low therapeutic effort. Health professionals' gender awareness may minimize such bias; however, the only instrument to assess such awareness is limited to physicians and lacks a Spanish version. METHODS: After using the back-translation method, a sample of 167 Spanish nurses and nursing students completed the instrument. In order to obtain additional validity evidence, 98 health professionals filled in gender sensitivity and gender-role ideology towards patients' subscales and the short versions of the Ambivalent Sexism Inventory. RESULTS: Gender-role ideology towards patients correlated strongly with sexist attitudes, demonstrating convergent validity, and Cronbach's alpha coefficients showed an adequate internal consistency. CONCLUSIONS: Nijmegen Gender Awareness in Medicine Scale perfectly applies to nurse population, and this adaptation also broadens its use for Spanish professionals. IMPLICATIONS FOR NURSING MANAGEMENT: Nurse managers and educators can use this applicable tool to treat low gender awareness levels as a modifiable risk factor and promote a gender-sensitive caring culture.

Cerebral Microbleeds in Fragile X-Associated Tremor/Ataxia Syndrome.

BACKGROUND: Fragile X-associated tremor/ataxia syndrome is a neurodegenerative disease of late onset developed by carriers of the premutation in the fragile x mental retardation 1 (FMR1) gene. Pathological features of neurodegeneration in fragile X-associated tremor/ataxia syndrome include toxic levels of FMR1 mRNA, ubiquitin-positive intranuclear inclusions, white matter disease, iron accumulation, and a proinflammatory state. OBJECTIVE: The objective of this study was to analyze the presence of cerebral microbleeds in the brains of patients with fragile X-associated tremor/ataxia syndrome and investigate plausible causes for cerebral microbleeds in fragile X-associated tremor/ataxia syndrome. METHODS: We collected cerebral and cerebellar tissue from 15 fragile X-associated tremor/ataxia syndrome cases and 15 control cases carrying FMR1 normal alleles. We performed hematoxylin and eosin, Perls and Congo red stains, ubiquitin, and amyloid ß protein immunostaining. We quantified the number of cerebral microbleeds, amount of iron, presence of amyloid ß within the capillaries, and number of endothelial cells containing intranuclear inclusions. We evaluated the relationships between pathological findings using correlation analysis. RESULTS: We found intranuclear inclusions in the endothelial cells of capillaries and an increased number of cerebral microbleeds in the brains of those with fragile X-associated tremor/ataxia syndrome, both of which are indicators of cerebrovascular dysfunction. We also found a suggestive association between the amount of capillaries that contain amyloid ß in the cerebral cortex and the rate of disease progression. CONCLUSION: We propose microangiopathy as a pathologic feature of fragile X-associated tremor/ataxia syndrome. © 2021 International Parkinson and Movement Disorder Society.

The acute systemic toxicity of thallium in rats produces oxidative stress: attenuation by metallothionein and Prussian blue.

Thallium (TI) is one of the most toxic heavy metals. Human exposure to Tl occurs through contaminated drinking water and from there to food, a threat to health. Recently, environmental contamination by Tl has been reported in several countries, urging the need for studies to determine the impact of endogenous and exogenous mechanisms preventing thallium toxicity. The cytoprotective effect of metallothionein (MT), a protein with high capacity to chelate metals, at two doses (100 and 600 µg/rat), was tested. Prussian blue (PB) (50 mg/kg) was administered alone or in combination with MT. A dose of Tl (16mg/kg) was injected i.p. to Wistar rats. Antidotes were administered twice daily, starting 24h after Tl injection, for 4 days. Tl concentrations diminished in most organs (p < 0.05) by effect of PB, alone or in combination with MT, whereas MT alone decreased Tl concentrations in testis, spleen, lung and liver. Likewise, brain thallium also diminished (p < 0.05) by effect of PB and MT alone or in combination in most of the regions analyzed (p < 0.05). The greatest diminution of Tl was achieved when the antidotes were combined. Plasma markers of renal damage increased after Tl administration, while PB and MT, either alone or in combination, prevented the raise of those markers. Only MT increased the levels of reduced glutathione (GSH) in the kidney. Finally, increased Nrf2 was observed in liver and kidney, after treatment with MT alone or in combination with PB. Results showed that MT alone or in combination with PB is cytoprotective after thallium exposure.

Microbial carbon limitation: The need for integrating microorganisms into our understanding of ecosystem carbon cycling.

Numerous studies have demonstrated that fertilization with nutrients such as nitrogen, phosphorus, and potassium increases plant productivity in both natural and managed ecosystems, demonstrating that primary productivity is nutrient limited in most terrestrial ecosystems. In contrast, it has been demonstrated that heterotrophic microbial communities in soil are primarily limited by organic carbon or energy. While this concept of contrasting limitations, that is, microbial carbon and plant nutrient limitation, is based on strong evidence that we review in this paper, it is often ignored in discussions of ecosystem response to global environment changes. The plant-centric perspective has equated plant nutrient limitations with those of whole ecosystems, thereby ignoring the important role of the heterotrophs responsible for soil decomposition in driving ecosystem carbon storage. To truly integrate carbon and nutrient cycles in ecosystem science, we must account for the fact that while plant productivity may be nutrient limited, the secondary productivity by heterotrophic communities is inherently carbon limited. Ecosystem carbon cycling integrates the independent physiological responses of its individual components, as well as tightly coupled exchanges between autotrophs and heterotrophs. To the extent that the interacting autotrophic and heterotrophic processes are controlled by organisms that are limited by nutrient versus carbon accessibility, respectively, we propose that ecosystems by definition cannot be 'limited' by nutrients or carbon alone. Here, we outline how models aimed at predicting non-steady state ecosystem responses over time can benefit from dissecting ecosystems into the organismal components and their inherent limitations to better represent plant-microbe interactions in coupled carbon and nutrient models.

OpenCASA: A new open-source and scalable tool for sperm quality analysis.

In the field of assisted reproductive techniques (ART), computer-assisted sperm analysis (CASA) systems have proved their utility and potential for assessing sperm quality, improving the prediction of the fertility potential of a seminal dose. Although most laboratories and scientific centers use commercial systems, in the recent years certain free and open-source alternatives have emerged that can reduce the costs that research groups have to face. However, these open-source alternatives cannot analyze sperm kinetic responses to different stimuli, such as chemotaxis, thermotaxis or rheotaxis. In addition, the programs released to date have not usually been designed to encourage the scalability and the continuity of software development. We have developed an open-source CASA software, called OpenCASA, which allows users to study three classical sperm quality parameters: motility, morphometry and membrane integrity (viability) and offers the possibility of analyzing the guided movement response of spermatozoa to different stimuli (useful for chemotaxis, thermotaxis or rheotaxis studies) or different motile cells such as bacteria, using a single software. This software has been released in a Version Control System at Github. This platform will allow researchers not only to download the software but also to be involved in and contribute to further developments. Additionally, a Google group has been created to allow the research community to interact and discuss OpenCASA. For validation of the OpenCASA software, we analysed different simulated sperm populations (for chemotaxis module) and evaluated 36 ejaculates obtained from 12 fertile rams using other sperm analysis systems (for motility, membrane integrity and morphology modules). The results were compared with those obtained by Open-CASA using the Pearson's correlation and Bland-Altman tests, obtaining a high level of correlation in all parameters and a good agreement between the different used methods and the OpenCASA. With this work, we propose an open-source project oriented to the development of a new software application for sperm quality analysis. This proposed software will use a minimally centralized infrastructure to allow the continued development of its modules by the research community.

Does Melatonin Exert Its Effect on Ram Sperm Capacitation Through Nitric Oxide Synthase Regulation?

Nitric oxide (NO·), synthesized from L-arginine by nitric oxide synthase (NOS), is involved in sperm functionality. NOS isoforms have been detected in spermatozoa from different species, and an increment in NOS activity during capacitation has been reported. This work aims to determine the presence and localization of NOS isoforms in ram spermatozoa and analyse their possible changes during in vitro capacitation. Likewise, we investigated the effect of melatonin on the expression and localization of NOS and NO· levels in capacitated ram spermatozoa. Western blot analysis revealed protein bands associated with neuronal NOS (nNOS) and epithelial NOS (eNOS) but not with inducible NOS (iNOS). However, the three isoforms were detected by indirect immunofluorescence (IFI), and their immunotypes varied over in vitro capacitation with cAMP-elevating agents. NO· levels (evaluated by DAF-2-DA/PI staining) increased after in vitro capacitation, and the presence of L-arginine in the capacitating medium raised NO· production and enhanced the acrosome reaction. Incubation in capacitating conditions with a high-cAMP medium with melatonin modified the NOS distribution evaluated by IFI, but no differences in Western blotting were observed. Melatonin did not alter NO· levels in capacitating conditions, so we could infer that its role in ram sperm capacitation would not be mediated through NO· metabolism.

Investigation of pesticides and their transformation products in the Júcar River Hydrographical Basin (Spain) by wide-scope high-resolution mass spectrometry screening.

The Water Framework Directive 2000/60/EC implemented by the European Union established as the main objectives to achieve a "good ecological and chemical status" of the surface water and a "good quantitative and chemical status" of groundwater bodies. One of the major pressures affecting water bodies comes from the use of pesticides and their potential presence in the water ecosystems. For this purpose, the reliable determination of pesticides and their transformation products (TPs) in natural waters (both surface and groundwater) is required. The high number of compounds potentially reaching the aquatic environment makes extraordinary difficult, if not impossible, to investigate all these compounds even using the most powerful analytical techniques. Among these, liquid chromatography coupled to high-resolution mass spectrometry is emphasized due to its strong potential for detection and identification of many organic contaminants thanks to the accurate-mass full spectrum acquisition data. This work focuses on wide-scope screening of many pesticides and their TPs in surface water and groundwater samples, collected between March and May 2017, in the Júcar River Hydrographical Basin, Spain. For this purpose, a home-made database containing more than 500 pesticides and TPs was employed. Analyses performed by liquid chromatography coupled to quadrupole-time of flight mass spectrometry (LC-QTOF MS) allowed the identification of up to 27 pesticides and 6 TPs. The most detected compounds in groundwater were the herbicides atrazine, simazine, terbuthylazine, and their TPs (atrazine-desethyl, terbumeton-desethyl and terbuthylazine-desethyl). Regarding surface water, the fungicides carbendazim, thiabendazole and imazalil, the herbicide terbutryn and the TP terbumeton-desethyl were also detected. These results illustrate the wide use of these compounds (in the present or in the recent past) in the area under study and the vulnerability of the water bodies, and are in accordance with previous findings in other water bodies of the different Spanish Hydrographic systems.

Utilizing shared internal control arms and historical information in small-sized platform clinical trials.

Recruitment of patients in concurrent control arms can be very challenging for clinical trials for pediatric and rare diseases. Innovative approaches, such as platform trial designs, including shared internal control arm(s), can potentially reduce the needed sample size, improving the efficiency and speed of the drug development program. Furthermore, historical borrowing, which involves leveraging information from control arms in previous relevant clinical trials, may further enhance a clinical trial's efficiency. In this paper, we discuss platform trials highlighting their advantages and limitations. We then compare various strategies that borrow historical data or information, such as pooling data from different studies, analyzing data from studies separately, test-then-pool, dynamic pooling, and Bayesian hierarchical modeling, which focuses on the meta-analytic-predictive (MAP) prior. We further propose a procedure to illustrate the feasibility of utilizing historical controls under a platform setting and describe the statistical performance of our method via simulations.

Functional mechanism of tracheal relaxation, antiasthmatic, and toxicological studies of 6-hydroxyflavone.

Previously, we described tracheal rat rings relaxation by several flavonoids, being 6-hydroxyflavone (6-HOF) the most active derivative of the series. Thus, its mechanism of action was determined in an ex vivo tracheal rat ring bioassay. The anti-asthmatic effect was assayed in in vivo OVAlbumin (OVA)-sensitized guinea pigs. Finally, the toxicological profile of 6-HOF was studied based on Organization of Economic Cooperation and Development guidelines with modifications. 6-HOF-induced relaxation appears to be related with receptor-operated calcium channel and voltage-operated calcium channel blockade as the main mechanism of action, and also through the production of relaxant second messengers NO and cGMP. Molecular docking supports that 6-HOF acts as calcium channel blocker and by activation of nitric oxide synthase. In addition, the in vivo anti-asthmatic experiments demonstrate the dose-dependent significant anti-allergic effect of 6-HOF induced by OVA, with best activity at 50 /kg. Finally, toxicological studies determined a LD50 > 2,000 mg/kg and, after 28 day of treatment with 6-HOF (50 mg/kg) by intragastric route, mice did not exhibit evidence of any significant toxicity. In conclusion, experiments showed that 6-HOF exerts significant relaxant activity through calcium channel blockade, and possibly, by NO/cGMP-system stimulation on rat trachea, which interferes with the contraction mechanism of smooth muscle cells in the airways. In addition, the flavonoid shows potential anti-asthmatic properties in an anti-allergic pathway. Furthermore, because the pharmacological and safety evidence, we propose this flavonoid as lead for the development of a novel therapeutic agent for the treatment of asthma and related respiratory diseases.