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This article explains how participatory approaches can promote civic engagement among young people, resulting from their active involvement in the research process, namely identifying their communities' priorities and problems. Five project‐building sessions were held with young people from five contexts located in the border regions of mainland Portugal. The data supporting this article were collected during sessions dedicated to identifying and exploring community‐based problems and priorities and designing projects to address those local challenges. The results show the importance of contextualising young people's experiences and priorities, here related to their own community and its well‐being and development. It is here that using participatory methodologies can create opportunities for young people to participate in processes of community change. [ABSTRACT FROM AUTHOR]

Variation in apolipoprotein E (APOE) has been shown to have the strongest genetic effect on human longevity. The aim of this study was to unravel the evolutionary history of the three major APOE alleles in Europe by analysing ancient samples up to 12,000 years old. We detected significant allele frequency shifts between populations and over time. Our analyses indicated that selection led to large frequency differences between the earliest European populations (i.e., hunter‐gatherers vs. first farmers), possibly due to changes in diet/lifestyle. In contrast, the allele distributions in populations from ~4000 BCE onward can mainly be explained by admixture, suggesting that it also played an important role in shaping current APOE variation. In any case, the resulting allele frequencies strongly influence the predisposition for longevity today, likely as a consequence of past adaptations and demographic processes. [ABSTRACT FROM AUTHOR]

This work explores the effect of convective self‐aggregation on extreme rainfall intensities through an analysis at several stages of the cloud lifecycle. In addition to increases in 3‐hourly extremes consistent with previous studies, we find that instantaneous rainrates increase significantly (+30%). We mainly focus on instantaneous extremes and, using a recent framework, relate their increase to increased precipitation efficiency: the local increase in relative humidity drives larger accretion efficiency and lower re‐evaporation. An in‐depth analysis based on an adapted scaling for precipitation extremes reveals that the dynamic contribution decreases (−25%) while the thermodynamic is slightly enhanced (+5%) with convective self‐aggregation, leading to lower condensation rates. When the atmosphere is more organized into a moist convecting region and a dry convection‐free region, deep convective updrafts are surrounded by a warmer environment which reduces convective instability and thus the dynamic contribution. The moister boundary‐layer explains the positive thermodynamic contribution. The microphysic contribution is increased by +50% with aggregation. The latter is partly due to reduced evaporation of rain falling through a moister near‐cloud environment, but also to the associated larger accretion efficiency. Thus, a potential change in convective organization regimes in a warming climate could lead to an evolution of tropical precipitation extremes significantly different than that expected from thermodynamical considerations. The relevance of self‐aggregation to the real tropics is still debated. Improved fundamental understanding of self‐aggregation, its sensitivity to warming and connection to precipitation extremes, is hence crucial to achieve accurate rainfall projections in a warming climate. Plain Language Summary: Heavy precipitation and floods are frequent in the tropics. The spatial organization of weather systems is often associated with these events. Our study investigates the case of convective self‐aggregation which is a particular type of cloud systems' organization observed in idealized numerical simulations. We find that convective self‐aggregation tends to increase rainfall intensities by 30%–70%. There are several processes involved in the formation of heavy rainfall: vertical motion air, condensation into cloud droplets, growth of these droplets into precipitating drops, collection of other cloud‐droplets through their descent and partial re‐evaporation between cloud base and the ground. We examine the contribution of each of these processes and find that the increase in rain rates with convective self‐aggregation is related to both lower rain re‐evaporation and more efficient cloud droplet collection by rain drops through their descent. It is still unclear how heavy rainfall will evolve in a warming climate. While the relationship between temperature and water vapor suggests that heavy rainfall will increase by 7% per 1K, our result shows that a hypothetic change in the organization of weather systems could potentially lead to more dramatic changes in heavy rainfall in a future warming climate. Key Points: Convective self‐aggregation may increase both accumulated and instantaneous rainfall extremesThis increased precipitation is related to reduced rain re‐evaporation and enhanced accretion efficiency with convective self‐aggregationExtreme convective updrafts within self‐aggregated convection are weaker due to warmer environment [ABSTRACT FROM AUTHOR]

The western Maritime Continent (MC) and Southeast Asia lie at the heart of the largest area of high precipitation on Earth. Extreme precipitation is one of the major high‐impact weather events to affect the population of over 500 million in this region. The deep convection associated with this extreme precipitation is difficult to forecast, even with modern high‐resolution numerical weather prediction with explicit convection. However, larger‐scale organised convective systems, such as the Madden–Julian Oscillation (MJO), can be skilfully predicted to 3–5 weeks lead time. The MJO has a well‐known precipitation signal, and it is likely that it also modulates extreme precipitation. Here, the extreme precipitation signal of the MJO is analysed in detail for the western MC and Southeast Asia using 19 years of high‐resolution Integrated Multi‐satellitE Retrievals for Global Precipitation Measurement (GPM IMERG) data. The probability of experiencing extreme precipitation increases robustly by a factor of two, and decreases by a factor of half, dependent on location and the phase of the MJO. The spatial pattern of these changes in extreme precipitation does not describe a smooth eastward propagation, but shows rapid variation over short distances, tied to the complex distribution of land and sea within the archipelago. There is also a seasonal dependence of this MJO modulation in some locations. A more detailed analysis of the effect of the MJO on extreme precipitation is presented for the major cities in the region. Extreme precipitation days over the MC are generally linked with an amplification of the diurnal cycle. However, although an active MJO increases the frequency of extreme precipitation days and therefore an amplified diurnal cycle, there was no further amplification of the diurnal cycle in the active MJO, compared with extreme precipitation days during non‐active MJO periods. [ABSTRACT FROM AUTHOR]

Aerosols affect atmospheric dynamics through their direct and semi-direct effects as well as through their effects on cloud microphysics (indirect effects). The present study investigates the indirect effects of aerosols on summer precipitation in the Euro-Mediterranean region, which is located at the crossroads of air masses carrying both natural and anthropogenic aerosols. While it is difficult to disentangle the indirect effects of aerosols from the direct and semidirect effects in reality, a numerical sensitivity experiment is carried out using the Weather Research and Forecasting (WRF) model, which allows us to isolate indirect effects, all other effects being equal. The Mediterranean hydrological cycle has often been studied using regional climate model (RCM) simulations with parameterized convection, which is the approach we adopt in the present study. For this purpose, the Thompson aerosol-aware microphysics scheme is used in a pair of simulations run at 50 km resolution with extremely high and low aerosol concentrations. An additional pair of simulations has been performed at a convection-permitting resolution (3.3 km) to examine these effects without the use of parameterized convection. While the reduced radiative flux due to the direct effects of the aerosols is already known to reduce precipitation amounts, there is still no general agreement on the sign and magnitude of the aerosol indirect forcing effect on precipitation, with various processes competing with each other. Although some processes tend to enhance precipitation amounts, some others tend to reduce them. In these simulations, increased aerosol loads lead to weaker precipitation in the parameterized (low-resolution) configuration. The fact that a similar result is obtained for a selected area in the convection-permitting (high-resolution) configuration allows for physical interpretations. By examining the key variables in the model outputs, we propose a causal chain that links the aerosol effects on microphysics to their simulated effect on precipitation, essentially through reduction of the radiative heating of the surface and corresponding reductions of surface temperature, resulting in increased atmospheric stability in the presence of high aerosol loads. [ABSTRACT FROM AUTHOR]

Due to their potential role in organizing tropical mesoscale convective systems, a better understanding of cold pool (CP) dynamics in such regions is critical, particularly over land where the diurnal cycle further concentrates convective activity. Numerical models help disentangle the processes involved but often lack observational benchmarks. To close this gap, we analyze nearly 43 years of five‐minute resolution near‐surface timeseries, recorded from 12 automatic weather stations across equatorial Africa during 2019–2023. We identify 4,218 CPs based on criteria for temperature and wind. The identified CP gust fronts, which exhibit respective median temperature and specific humidity decreases of 5.3 K and 2.8 g kg−1, closely correlate with satellite‐observed brightness temperature discontinuities. Despite weak diurnal variation in precipitation, observed CP occurrence shows a pronounced diurnal cycle with an afternoon peak — a feature we attribute to low‐level moisture conditions. Our findings can serve as observational benchmark to improve simulations of CP organization. Plain Language Summary: Convective cold pools form when rain evaporates underneath thunderstorm clouds. The evaporation causes the air to cool and sink toward the ground, where it is deflected horizontally. Cold pools are thus associated with strong gusty winds, and over tropical land, they can be especially vigorous. Cold pools are also suggested to contribute to the organization of thunderstorm clouds into large clusters of rain‐producing areas. The widespread, heavy rainfall can then cause flooding. To better predict such flooding in numerical weather models, having a precise observational basis for cold pool properties is essential — yet currently missing in equatorial Africa. We here provide such an observational benchmark by analyzing thousands of cold pools using timeseries of near‐surface temperature, wind, humidity and precipitation. We additionally show that the cold pools can even be detected from satellite data when analyzing abrupt changes in cloud top temperature. Such satellite‐based detection could open for cold pool studies across all tropical land areas — of great practical relevance to the prediction of thunderstorm clusters. Key Points: 4,218 cold pools are identified across equatorial Africa based on temperature and wind criteriaThe occurrence and intensity of the observed cold pools are related to low‐level moisture conditions and the depth of convectionThe identified cold pool gust fronts closely correlate with satellite‐observed brightness temperature discontinuities [ABSTRACT FROM AUTHOR]

This short paper is a Reply to ‘Medical altruism in mainstream health economics: theoretical and political paradoxes. COMMENTS’. [ABSTRACT FROM PUBLISHER]

Macrophages (MΦ) and dendritic cells (DCs) are heterogeneous families of functionally and developmentally related immune cells that play crucial roles in tissue homeostasis and the regulation of immune responses. During the past 5 years, immunologists have generated a considerable amount of data that challenge dogmas about the ontogeny and functions of these highly versatile cells. The male excurrent duct system plays a critical role in the establishment of fertility by allowing sperm maturation, transport and storage. In addition, it is challenged by pathogens and must establish a protective and tolerogenic environment for a continuous flow of autoantigenic spermatozoa. The post-testicular environment and, in particular, the epididymis contain an intricate network of DCs and MΦ; however, the immunophysiology of this intriguing and highly specialized mucosal system is poorly understood. This review summarizes the current trends in mouse MΦ and DC biology and speculates about their roles in the steady-state epididymis. Unraveling immune cell functions in the male reproductive tract is an essential prerequisite for the design of innovative strategies aimed at controlling male fertility and treating infertility. [ABSTRACT FROM AUTHOR]

In the field of healthcare, ethical considerations are omnipresent. The problem is that it is not clear how to introduce professional ethics within the frontiers demarcated by economic rationality. In mainstream economics, medical altruism is defined as the inclusion of the patient's welfare in the doctor's utility function. This definition presents two serious problems that we develop in this paper. The first problem is that mainstream theory does not propose a model of authentic altruism because it reduces otherness to a source of utility like any other. The second problem is that ethical and altruistic (instrumental or otherwise) behaviour should not be conflated. By reducing ethics to altruism, mainstream theory prevents any genuine discussion of medical ethics. Then, the thesis of the paper is that the attempt to introduce altruism into the standard framework creates theoretical paradoxes that create policy dilemmas. [ABSTRACT FROM AUTHOR]

Luminal acidification in the epididymis is critical for sperm maturation and storage. Clear cells express the vacuolar H+-ATPase (V-ATPase) in their apical membrane and are major contributors to proton secretion. We showed that this process is regulated via recycling of V-ATPase-containing vesicles. We now report that RhoA and its effector ROCKII are enriched in rat epididymal clear cells. In addition, cortical F-actin was detected beneath the apical membrane and along the lateral membrane of "resting" clear cells using a pan-actin antibody or phalloidin-TRITC. In vivo luminal perfusion of the cauda epididymal tubule with the ROCK inhibitors Y27632 (10-30 μM) and HA1077 (30 μM) or with the cell-permeable Rho inhibitor Clostridium botulinum C3 transferase (3.75 μg/ml) induced the apical membrane accumulation of V-ATPase and extension of V-ATPase-labeled microvilli in clear cells. However, these newly formed microvilli were devoid of ROCKII. In addition, Y27632 (30 μM) or HA1077 (30 μM) decreased the ratio of F-actin to G-actin detected by Western blot analysis in epididymal epithelial cells, and Y27632 also decreased the ratio of F-actin to G-actin in clear cells isolated by fluorescence activated cell sorting from B1-enhanced green fluorescence protein (EGFP) transgenic mice. These results provide evidence that depolymerization of the cortical actin cytoskeleton via inhibition of RhoA or its effector ROCKII favors the recruitment of V-ATPase from the cytosolic compartment into the apical membrane in clear cells. In addition, our data suggest that the RhoA-ROCKII pathway is not locally involved in the elongation of apical microvilli. We propose that inhibition of RhoA-ROCKII might be part of the intracellular signaling cascade that is triggered upon agonist-induced apical membrane V-ATPase accumulation. [ABSTRACT FROM AUTHOR]

Proton-transporting cells are located in several tissues where they acidify the extracellular environment. These cells express the vacuolar H+-ATPase (V-ATPase) B1 subunit (ATP6V1B1) in their plasma membrane. We provide here a comprehensive catalog of the proteins that are expressed in these cells, after their isolation by enzymatic digestion and fluorescence-activated cell sorting (FACS) from transgenic B1-enhanced green fluorescent protein (EGFP) mice. In these mice, type A and B intercalated cells and connecting segment cells of the kidney, and narrow and clear cells of the epididymis, which all express ATP6V1B1, also express EGFP, while all other cell types are negative. The proteome of renal and epididymal EGFP-positive (EGFP+) cells was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and compared with their respective EGFP-negative (EGFP-) cell populations. A total of 2.297 and 1,564 proteins were detected in EGFP+ cells from the kidney and epididymis, respectively. Out of these proteins, 202 and 178 were enriched by a factor greater than 1.5 in EGFP+ cells compared with EGFP- cells, in the kidney and epididymis respectively, and included subunits of the V-ATPase (B1, a4, and A). In addition, several proteins involved in intracellular trafficking, signaling, and cytoskeletal dynamics were identified. A novel common protein that was enriched in renal and epididymal EGFP+ cells is the progesterone receptor, which might be a potential candidate for the regulation of V-ATPase-dependent proton transport. These proteomic databases provide a framework for comprehensive future analysis of the common and distinct functions of V-ATPase-B1-expressing cells in the kidney and epididymis. [ABSTRACT FROM AUTHOR]

Extracellular purinergic agonists regulate a broad range of physiological functions via P1 and P2 receptors. Using the epididymis as a model system in which luminal acidification is essential for sperm maturation and storage, we show here that extracellular ATP and its hydrolysis product adenosine trigger the apical accumulation of vacuolar H+-ATPase (V-ATPase) in acidifying clear cells. We demonstrate that the epididymis can hydrolyze luminal ATP into other purinergic agonists such as ADP via the activity of nucleotidases located in the epididymal fluid and in the apical membrane of epithelial cells. Alkaline phosphatase activity and abundant ecto-5'-nucleotidase protein were detected in the apical pole of principal cells. In addition, we show that nine nucleotidase genes (Nt5e, Alp!, Alpp, Enppl, 2, and 3, and Entpd 2, 4, and 5), seven ATP P2 receptor genes (P2X1, P2X2, P2X3, P2X4, P2X6, P2Y2, P2Y5), and three adenosine P1 receptor genes (Al, A2B, and A3) are expressed in epithelial cells isolated by laser cut microdissection (LCM). The calcium chelator BAPTA-AM abolished the apical VATPase accumulation induced by ATP, supporting the contribution of P2X or P2Y in this response. The PKA inhibitor myristoylated protein kinase inhibitor (mPKI) inhibited adenosine-dependent V-ATPase apical accumulation, indicating the participation of the P1 A2B receptor. Altogether, these results suggest that the activation of P1 and P2 purinergic receptors by ATP and adenosine might play a significant role in luminal acidification in the epididymis, a process that is crucial for the establishment of male fertility. [ABSTRACT FROM AUTHOR]

Summary: Basal cells are by definition located on the basolateral side of several epithelia, and they have never been observed reaching the lumen. Using high-resolution 3D confocal imaging, we report that basal cells extend long and slender cytoplasmic projections that not only reach toward the lumen but can cross the tight junction barrier in some epithelia of the male reproductive and respiratory tracts. In this way, the basal cell plasma membrane is exposed to the luminal environment. In the epididymis, in which luminal acidification is crucial for sperm maturation and storage, these projections contain the angiotensin II type 2 receptor (AGTR2). Activation of AGTR2 by luminal angiotensin II, increases proton secretion by adjacent clear cells, which are devoid of AGTR2. We propose a paradigm in which basal cells scan and sense the luminal environment of pseudostratified epithelia and modulate epithelial function by a mechanism involving crosstalk with other epithelial cells. [Copyright &y& Elsevier]

Water and solute transport across the plasma membrane of cells is a crucial biological function that is mediated mainly by aquaporins and aquaglyceroporins. The regulation of these membrane proteins is stifi incompletely understood. Using the male reproductive tract as a model system in which water and glycerol transport are critical for the establishment of fertility, we now report a novel pathway for the regulation of aquaporin 9 (AQP9) permeability. AQP9 is the major aquaglyceroporin of the epididymis, liver, and peripheral leukocytes, and its COOH-terminal portion contains a putative PDZ binding motif (SVIM). Here we show that NHERF1, cystic fibrosis transmembrane conductance regulator (CFTR), and AQP9 co-localize in the apical membrane of principal cells of the epididymis and the vas deferens, and that both NHERF1 and CFTR co-immunoprecipitate with AQP9. Overlay assays revealed that AQP9 binds to both the PDZ1 and PDZ2 domains of NHERF1, with an apparently higher affinity for PDZ1 versus PDZ2. Pull-down assays showed that the AQP9 COOH-terminal SVIM motif is essential for interaction with NHERF1. Functional assays on isolated tubules perfused in vitro showed a high permeabffity of the apical membrane to glycerol, which is inhibited by the AQP9 inhibitor, phioretin, and is markedly activated by cAMP. The CFTR inhibitors DPC, G1yH-101 and CFTR1nh-172 all significantly reduced the cAMP-activated glycerol-induced cell sweffing. We propose that CFTR is an important regulator of AQP9 and that the interaction between AQP9, NHERF1, and CFTR may facilitate the activation of AQP9 by cAMP. [ABSTRACT FROM AUTHOR]

Activation of soluble adenylyl cyclase (sAC) by bicarbonate causes local cAMP generation, indicating that sAC might act as a pH andlor bicarbonate sensor in kidney cells involved in acid-base homeostasis. Therefore, we examined the expression of sAC in renal acid-base transporting intercalated cells (IC) and compared its distribution to that of the vacuolar proton pumping ATPase (V-ATPase) under different conditions. In all IC, sAC and V-ATPase showed considerable overlap under basal conditions, but sAC staining was also found in other cellular locations in the absence of V-ATPase. In type A-IC, both sAC and V-ATPase were apically and subapically located, whereas in type B-IC, significant basolateral colocalization of sAC and the V-ATPase was seen. When apical membrane insertion of the V-ATPase was stimulated by treatment of rats with acetazolamide, sAC was also concentrated in the apical membrane of A-IC. In mice that lack a functional B! subunit of the V-ATPase, sAC was colocalized apically in A-IC along with V-ATPase containing the alternative B2 subunit isoform. The close association between these two enzymes was confirmed by coimmuno-precipitation of sAC from kidney homogenates using anti-V-ATPase antibodies. Our data show that sAC and the V-ATPase colocalize in IC, that they are concentrated in the IC plasma membrane under conditions that "activate" these proton secretory cells, and that they are both present in an immunoprecipitated complex. This suggests that these enzymes have a close association and could be part of a protein complex that is involved in regulating renal distal proton secretion. [ABSTRACT FROM AUTHOR]

An acidic luminal pH in the epididymis contributes to maintaining sperm quiescent during their maturation and storage. The vacuolar H+ATPase (V-ATPase), located in narrow and clear cells, is a major contributor to luminal acidification. Mutations in one of the V-ATPase subunits, ATP6v1B1 (B1), cause distal renal tubular acidosis in humans but surprisingly, B1-/- mice do not develop metabolic acidosis and are fertile. While B1 is located in the apical membrane of narrow and clear cells, the B2 subunit localizes to subapical vesicles in wild-type mouse, rat and human epididymis. However, a marked increase (84%) in the mean pixel intensity of B2 staining was observed in the apical pole of clear cells by conventional immunofluorescence, and relocalization into their apical membrane was detected by confocal microscopy in B1-/- mice compared with B1+/+. Immunogold electron microscopy showed abundant B2 in the apical microvilli of clear cells in B1-/- mice. B2 mRNA expression, determined by real time RT-PCR using laser-microdissected epithelial cells, was identical in both groups. Semiquantitative Western blots from whole epididymis and cauda epididymidis showed no variation of B2 expression. Finally, the luminal pH of the cauda epididymidis was the same in B1-/- mice as in B1+/+ (pH 6.7). These data indicate that whereas overall expression of B2 is not affected in B1-/- mice, significant redistribution of B2-containing complexes occurs from intracellular compartments into the apical membrane of clear cells in B1-/- mice. This relocation compensates for the absence of functional B1 and maintains the luminal pH in an acidic range that is compatible with fertility. [ABSTRACT FROM AUTHOR]

Abstract: The male reproductive tract and accessory glands comprise a complex but interrelated system of tissues that are composed of many distinct cell types, all of which contribute to the ability of spermatozoa to carry out their ultimate function of fertilizing an oocyte. Spermatozoa undergo their final steps of maturation as they pass through the male excurrent duct, which includes efferent ducts, the epididymis and the vas deferens. The composition of the luminal environment in these organs is tightly regulated. Major fluid reabsorption occurs in efferent ducts and in the epididymis, and leads to a significant increase in sperm concentration. In the distal epididymis and vas deferens, fluid secretion controls the final fluidity of the luminal content. Therefore, the process of water movement in the excurrent duct is a crucial step for the establishment of male fertility. Aquaporins contribute to transepithelial water transport in many tissues, including the kidney, the brain, the eye and the respiratory tract. The present article reviews our current knowledge regarding the distribution and function of aquaporins in the male excurrent duct. [Copyright &y& Elsevier]

B1 and B2 are two highly homologous isoforms of the vacuolar H+-ATPase (V-ATPase) 56-kDa B subunit. We investigated whether the B2 subunit is expressed alongside B1 in proton-secreting cells of the rodent kidney collecting duct (intercalated cells, IC) and epididymis (clear cells) by using antibodies against distinct COOH-terminal peptides from the two B isoforms. B2 was detected not only in the kidney proximal tubule, thick ascending limb, distal convoluted tubule, and connecting segment but also in A- and B-type IC of collecting ducts (CD) in both rat and mouse. B2 had a predominant cytoplasmic localization in most IC but was clearly located in a tighter apical band together with the V-ATPase 31-kDa E subunit in some A-IC, especially in the medulla. Apical membrane staining was confirmed by immunogold electron microscopy. B2 was very weakly expressed on the basolateral membranes of B-IC in control kidney CD, but some connecting segment B-IC had more distinct basolateral staining. In response to chronic carbonic anhydrase inhibition by acetazolamide, many A-IC showed a strong apical membrane localization of B2, where it colocalized with E and BI. In rat and mouse epididymis, B2 isoform expression was detected in clear cells, where it was concentrated in subapical vesicles. Unlike B1, B2 did not colocalize with the E subunit in the apical microvilli. These findings indicate that in addition to its role in the acidification of intracellular organelles, the B2 isoform could also contribute to transepithelial proton secretion and the maintenance of acid-base homeostasis. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β (TGF-β) is known to induce α-smooth muscle actin (α-SMA) in fibroblasts and is supposed to play a role in myofibroblast differentiation and tumor desmoplasia. Our objective was to elucidate the impact of TGF-β1 on α-SMA expression in fibroblasts in a threedimensional (3-D) vs. two-dimensional (2-D) environment. In monolayer culture, all fibroblast cultures responded in a similar fashion to TGF-β1 with regard to α-SMA expression. In fibroblast spheroids, α-SMA expression was reduced and induction by TGF-β1 was highly variable. This difference correlated with a differential regulation in the TGF-β receptor (TGFβR) expression, in particular with a reduction in TGF-βRII in part of the fibroblast types. Our data indicate that 1) sensitivity to TGF-β1-induced α-SMA expression in a 3-D environment is fibroblast-type specific, 2) fibroblast typeindependent regulatory mechanisms, such as a general reduction/loss in TGF-βRIII, contribute to an altered TGFβR expression profile in spheroid compared with monolayer culture, and 3) fibroblast type-specific alterations in TGFβR types I and II determine the sensitivity to TGF-β1-induced α-SMA expression in the 3-D setting. We suggest that fibroblasts that can be induced by TGF-β1 to produce α-SMA in spheroid culture reflect a "premyofibroblastic" phenotype. [ABSTRACT FROM AUTHOR]

Human CD43 is an abundant, heavily glycosylated molecule expressed exclusively on the surface of leucocytes. When leucocytes are at rest, CD43 acts to prevent intercellular interaction but during leucocyte differentiation such cell–cell interaction is facilitated by CD43. This change in the function of CD43 is mediated in part by a reduction in its level of expression. Previous studies have implicated proteolytic cleavage events at the cell surface in causing such reduction. Here, we report that, in an in vitro model of leucocyte differentiation, CD43 mRNA levels were also subject to reduction. Specifically, we demonstrated that within 48 h of the cell line U937 being induced to differentiate along the monocytic pathway, CD43 mRNA levels were reduced by 69%. This decline coincided with a decrease in the activity of the CD43 gene promoter mediated by the single-stranded DNA binding protein Purα. Previously, we have demonstrated that Purα mediates induction of the CD11c β2 integrin promoter during U937 differentiation. Consequently, Purα represents a potential means by which the induction of pro-adhesive molecules and the repression of anti-adhesive molecules is co-ordinated during leucocyte differentiation. [ABSTRACT FROM AUTHOR]

INTRODUCTION:: Efficacy of differentiating agents requires that their specific cellular targets are still expressed and functional in the leukemic cells. One hypothesis to target sensitive cells is to select leukemic clones which harbor disrupted transcription factors. CBFα and CBFβ are core-binding proteins which have been identified as transcription regulators of hematopoietic genes and shown to be altered in numerous leukemias. In M2 AML, the t(8;21) translocation, CBFα (AML1) is altered and produced as the AML1-ETO fusion protein. The fusion protein blocks transcription and differentiation mediated by G-CSF. Interestingly, AML1-ETO leukemic cell lines are sensitive to numerous cytokines in vitro and can be induced to differentiate in the presence of G-CSF and PMA. MATERIALS AND METHODS:: As in the APL differentiation model, primary culture provides a useful tool for therapeutic screening of differentiation inducers, we analysed the in vitro sensitivity of 10 fresh M2 AML t(8;21) leukemic samples to G-CSF and the functionality of G-CSF intracellular pathways. In vitro data were compared with in vivo data from four patients treated with rhG-CSF at the dosage of 5 μg/kg/day i.v. for two to three weeks before the initiation of AML induction chemotherapy and immunophenotypic analysis performed weekly to monitor in vivo differentiation. RESULTS:: In vitro, an increase in CD34+ cells expressing differentiation antigens (CD11b, CD13 or CD15) was noted along with a decrease of immature CD34+/differentiation antigen negative cells. After two weeks of a daily rhG-CSF administration in vivo, a significant, albeit transient, decrease of blast count was achieved, concomitant with an increase in differentiated leukemic cells suggesting that in vivo differentiation occurs. Fresh t(8;21) leukemic cells possess functional G-CSF signaling pathways as normal activity and kinetics of STAT1 and STAT3 binding was observed. Furthermore, differentiation... [ABSTRACT FROM AUTHOR]

We have created entirely biological tissue-engineered vascular grafts (TEVGs) using sheets of cell-assembled extracellular matrix (CAM) produced by human fibroblasts in vitro. A large animal TEVG would allow long-term pre-clinical studies in a clinically relevant setting (graft size and allogeneic setting). Therefore, canine, porcine, ovine, and human skin fibroblasts were compared for their ability to form CAM sheets. Serum sourcing greatly influenced CAM production in a species-dependent manner. Ovine cells produced the most homogenous and strongest animal CAM sheets but remained ≈3-fold weaker than human sheets despite variations of serum, ascorbate, insulin, or growth factor supplementations. Key differences in cell growth dynamics, tissue development, and tissue architecture and composition were observed between human and ovine. This study demonstrates critical species-to-species differences in fibroblast behavior and how they pose a challenge when attempting to substitute animal cells for human cells during the development of tissue-engineered constructs that require long-term cultures. [ABSTRACT FROM AUTHOR]

We have created entirely biological tissue-engineered vascular grafts (TEVGs) using sheets of cell-assembled extracellular matrix (CAM) produced by human fibroblasts in vitro. A large animal TEVG would allow long-term pre-clinical studies in a clinically relevant setting (graft size and allogeneic setting). Therefore, canine, porcine, ovine, and human skin fibroblasts were compared for their ability to form CAM sheets. Serum sourcing greatly influenced CAM production in a species-dependent manner. Ovine cells produced the most homogenous and strongest animal CAM sheets but remained ≈3-fold weaker than human sheets despite variations of serum, ascorbate, insulin, or growth factor supplementations. Key differences in cell growth dynamics, tissue development, and tissue architecture and composition were observed between human and ovine. This study demonstrates critical species-to-species differences in fibroblast behavior and how they pose a challenge when attempting to substitute animal cells for human cells during the development of tissue-engineered constructs that require long-term cultures. [ABSTRACT FROM AUTHOR]

We present a robust radiocarbon (14C) chronology for burials at Sakhtysh, in European Russia, where nearly 180 inhumations of Lyalovo and Volosovo pottery-using hunter-gatherer- fishers represent the largest known populations of both groups. Past dating attempts were restricted by poor understanding of dietary 14C reservoir effects (DREs). We developed a DRE correction approach that uses multiple linear regression of differences in 14C, d13C, and d15N between bones and teeth of the same individuals to predict DREs of up to approximately 900 years. Our chronological model dates Lyalovo burials to the early fifth millennium BCE, and Volosovo burials to the mid-fourth to early third millennium. It reveals a change in the subsistence economy at approximately 3300 BCE, coinciding with a reorientation of trade networks, and dates the final burial to the early Fatyanovo period, the regional expression of the Yamnaya/Corded Ware expansion. Our approach is applicable when freshwater 14C reservoir effects are poorly constrained and grave goods cannot be dated directly. [ABSTRACT FROM AUTHOR]

[Display omitted] • X-ray fluorescence can be used to classify soybean according to protein content; • XRF measurements are rapid, user-friendly, and compatible with in-field analysis; • S and Mn Kα emission lines can be used as proxies to infer soybean protein content. The purpose of this research was to evaluate performance of an energy-dispersive X-ray fluorescence (XRF) sensor to classify soybean based on protein content. The hypothesis was that sulfur signals and other XRF spectral features can be used as proxies to infer soybean protein content. Sample preparation and equipment settings to optimize detection of S and other specific emission lines were tested for this application. A logistic regression model for classifying soybean as high- or low-protein was developed based on XRF spectra and protein contents. Additionally, the model was validated with an independent set of samples. Global accuracies of the method were 0.83 (training set) and 0.81 (test set) and the corresponding kappa indices were 0.66 and 0.61, respectively. These numbers indicated satisfactory performance of the sensor, suggesting that XRF spectral features can be applied for screening protein content in soybean. [ABSTRACT FROM AUTHOR]

Mutations in CFTR lead to dysfunction of tubular organs, which is currently attributed to impairment of its conductive properties. We now show that CFTR regulates tight junction assembly and epithelial cell differentiation through modulation of the ZO-1- ZONAB pathway. CFTR colocalizes with ZO-1 at the tight junctions of trachea and epididymis, and is expressed before ZO- 1 in Wolffian ducts. CFTR interacts with ZO-1 through the CTFR PDZ-binding domain. In a three-dimensional (3D) epithelial cell culture model, CFTR regulates tight junction assembly and is required for tubulogenesis. CFTR inhibition or knockdown reduces ZO-1 expression and induces the translocation of the transcription factor ZONAB (also known as YBX3) from tight junctions to the nucleus, followed by upregulation of the transcription of CCND1 and downregulation of ErbB2 transcription. The epididymal tubules of cftr22 and cftrDF508 mice have reduced ZO-1 levels, increased ZONAB nuclear expression, and decreased epithelial cell differentiation, illustrated by the reduced expression of apical AQP9 and V-ATPase. This study provides a new paradigm for the etiology of diseases associated with CFTR mutations, including cystic fibrosis. [ABSTRACT FROM AUTHOR]

Mice deficient in the ATP6V1B1 ("B 1") subunit of the vacuolar proton-pumping ATPase (V-ATPase) maintain body acid-base homeostasis under normal conditions, but not when exposed to an acid load. Here, compensatory mechanisms involving the alternate ATP6V1B2 ("B2") isoform were examined to explain the persistence of baseline pH regulation in these animals. By immunocytochemistry, the mean pixel intensity of apical B2 immunostaining in medullary A intercalated cells (A-ICs) was twofold greater in B 1-/- mice than in B 1+1+ animals, and B2 was colocalized with other V-ATPase subunits. No significant upregulation of B2 mRNA or protein expression was detected in B 1-1- mice compared with wild-type controls. We conclude that increased apical B2 staining is due to relocalization of B2-containing V-ATPase complexes from the cytosol to the plasma membrane. Recycling of B2-containing holoenzymes between these domains was confirmed by the intracellular accumulation of B 1 - deficient V-ATPases in response to the microtubule-disrupting drug colchicine. V-ATPase membrane expression is further supported by the presence of "rod-shaped" intramembranous particles seen by freeze fracture microscopy in apical membranes of normal and B 1 - deficient A-ICs. Intracellular pH recovery assays show that significant (28-40% of normal) V-ATPase function is preserved in medullary ICs from B 1 -/- mice. We conclude that the activity of apical B2-containing V-ATPase holoenzymes in A-ICs is sufficient to maintain baseline acid-base homeostasis in B 1-deficient mice. However, our results show no increase in cell surface V-ATPase activity in response to metabolic acidosis in ICs from these animals, consistent with their inability to appropriately acidify their urine under these conditions. [ABSTRACT FROM AUTHOR]

Key points CFTR is expressed in principal cells but not clear cells in mouse cauda epididymis., Inhibition or knockdown of CFTR inhibits ATP release from mouse epididymal principal cells., Inhibition of CFTR reduces ATP release into the lumen of cauda epididymis in mice in vivo., These results show the involvement of CFTR in the regulation of ATP release from epithelial principal cells in the cauda epididymidis., Defective ATP signalling in the epididymis might contribute to dysfunction of the male reproductive tract associated with CFTR mutations., Abstract Extracellular ATP is essential for the function of the epididymis and spermatozoa, but ATP release in the epididymis remains uncharacterized. We investigated here whether epithelial cells release ATP into the lumen of the epididymis, and we examined the role of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl− and HCO3− conducting ion channel known to be associated with male fertility, in this process. Immunofluorescence labelling of mouse cauda epididymidis showed expression of CFTR in principal cells but not in other epithelial cells. CFTR mRNA was not detectable in clear cells isolated by fluorescence-activated cell sorting (FACS) from B1-EGFP mice, which express enhanced green fluorescent protein (EGFP) exclusively in these cells in the epididymis. ATP release was detected from the mouse epididymal principal cell line (DC2) and increased by adrenaline and forskolin. Inhibition of CFTR with CFTRinh172 and transfection with CFTR-specific siRNAs in DC2 cells reduced basal and forskolin-activated ATP release. CFTR-dependent ATP release was also observed in primary cultures of mouse epididymal epithelial cells. In addition, steady-state ATP release was detected in vivo in mice, by measuring ATP concentration in a solution perfused through the lumen of the cauda epididymidis tubule and collected by cannulation of the vas deferens. Luminal CFTRinh172 reduced the ATP concentration detected in the perfusate. This study shows that CFTR is involved in the regulation of ATP release from principal cells in the cauda epididymidis. Given that mutations in CFTR are a leading cause of male infertility, we propose that defective ATP signalling in the epididymis might contribute to dysfunction of the male reproductive tract associated with these mutations. [ABSTRACT FROM AUTHOR]

Urinary exosomes or microvesicles are being studied intensively to identify potential new biomarkers for renal disease. We sought to identify whether these microvesicles contain nucleic acids. We isolated microvesicles from human urine in the same density range as that previously described for urinary exosomes and found them to have an RNA integrity profile similar to that of kidney tissue, including 18S and 28S rRNA. This profile was better preserved in urinary microvesicles compared with whole cells isolated from urine, suggesting that microvesicles may protect RNA during urine passage. We were able to detect mRNA in the human urinary microvesicles encoding proteins from all regions of the nephron and the collecting duct. Further, to provide a proof of principle, we found that microvesicles isolated from the urine of the V-ATPase B1 subunit knockout mice lacked mRNA of this subunit while containing a normal amount of the B2 subunit and aquaporin 2. The microvesicles were found to be contaminated with extraneous DNA potentially on their surface; therefore, we developed a rapid and reliable means to isolate nucleic acids from within urine microvesicles devoid of this extraneous contamination. Our study provides an experimental strategy for the routine isolation and use of urinary microvesicles as a novel and non-invasive source of nucleic acids to further renal disease biomarker discovery. [ABSTRACT FROM AUTHOR]

Modulation of environmental pH is critical for the function of many biological systems. However, the molecular identity of the pH sensor and its interaction with downstream effector proteins remain poorly understood. Using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage, we now report a novel pathway for pH regulation linking the bicarbonate activated soluble adenylyl cyclase (sAC) to the vacuolar H[sup +]ATPase (V-ATPase). Clear cells of the epididymis and vas deferens contain abundant V-ATPase in their apical pole and are responsible for acidifying the lumen. Proton secretion is regulated via active recycling of V-ATPase. Here we demonstrate that this recycling is regulated by luminal pH and bicarbonate, sAC is highly expressed in clear cells, and apical membrane accumulation of V-ATPase is triggered by a sAC-dependent rise in cAMP in response to alkaline luminal pH. As sAC is expressed in other acid/base transporting epithelia, including kidney and choroid plexus, this cAMP-dependent signal transduction pathway may be a widespread mechanism that allows cells to sense and modulate extracellular pH. [ABSTRACT FROM AUTHOR]

Summary Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11b DTR mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction. [ABSTRACT FROM AUTHOR]