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Expression levels of the lactate–H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4–CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4–CD147 complex is co-delivered to invadopodia with MMP14., Expression levels of the lactate–H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4–CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4–CD147 complex is co-delivered to invadopodia with MMP14.

Despite the physiological and pathophysiological significance of microenvironmental gradients, e.g., for diseases such as cancer, tools for generating such gradients and analyzing their impact are lacking. Here, we present an integrated microfluidic-based workflow that mimics extracellular pH gradients characteristic of solid tumors while enabling high-resolution live imaging of, e.g., cell motility and chemotaxis, and preserving the capacity to capture the spatial transcriptome. Our microfluidic device generates a pH gradient that can be rapidly controlled to mimic spatiotemporal microenvironmental changes over cancer cells embedded in a 3D matrix. The device can be reopened allowing immunofluorescence analysis of selected phenotypes, as well as the transfer of cells and matrix to a Visium slide for spatially resolved analysis of transcriptional changes across the pH gradient. This workflow is easily adaptable to other gradients and multiple cell types and can therefore prove invaluable for integrated analysis of roles of microenvironmental gradients in biology., Despite the physiological and pathophysiological significance of microenvironmental gradients, e.g., for diseases such as cancer, tools for generating such gradients and analyzing their impact are lacking. Here, we present an integrated microfluidic-based workflow that mimics extracellular pH gradients characteristic of solid tumors while enabling high-resolution live imaging of, e.g., cell motility and chemotaxis, and preserving the capacity to capture the spatial transcriptome. Our microfluidic device generates a pH gradient that can be rapidly controlled to mimic spatiotemporal microenvironmental changes over cancer cells embedded in a 3D matrix. The device can be reopened allowing immunofluorescence analysis of selected phenotypes, as well as the transfer of cells and matrix to a Visium slide for spatially resolved analysis of transcriptional changes across the pH gradient. This workflow is easily adaptable to other gradients and multiple cell types and can therefore prove invaluable for integrated analysis of roles of microenvironmental gradients in biology.

G protein-coupled receptors (GPCRs) are seven transmembrane receptors that respond to external stimuli and undergo conformational changes to activate G proteins and modulate cellular processes leading to biological outcomes. To prevent overstimulation and prolonged exposure to stimuli, GPCRs are regulated by internalization. While the canonical GPCR internalization mechanism in mammalian cells is arrestin-dependent, clathrin-mediated endocytosis, more diverse GPCR internalization mechanisms have been described over the years. However, there is a lack of consistent methods used in the literature making it complicated to determine a receptor's internalization pathway. Here, we utilized a highly efficient time-resolved Förster resonance energy transfer (TR-FRET) internalization assay to determine the internalization profile of nine distinct GPCRs representing the GPCR classes A, B and C and with different G protein coupling profiles. This technique, coupled with clustered regularly interspaced palindromic repeats (CRISPR) engineered knockout cells allows us to effectively study the involvement of heterotrimeric G proteins and non-visual arrestins. We found that all the nine receptors internalized upon agonist stimulation in a concentration-dependent manner and six receptors showed basal internalization. Yet, there is no correlation between the receptor class and primary G protein coupling to the arrestin and G protein dependence for GPCR internalization. Overall, this study presents a platform for studying internalization that is applicable to most GPCRs and may even be extended to other membrane proteins. This method can be easily applicable to other endocytic machinery of interest and ultimately will lend itself towards the construction of comprehensive receptor internalization profiles.

Expression levels of the lactate–H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4–CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4–CD147 complex is co-delivered to invadopodia with MMP14., Expression levels of the lactate–H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4–CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4–CD147 complex is co-delivered to invadopodia with MMP14.

Despite the physiological and pathophysiological significance of microenvironmental gradients, e.g., for diseases such as cancer, tools for generating such gradients and analyzing their impact are lacking. Here, we present an integrated microfluidic-based workflow that mimics extracellular pH gradients characteristic of solid tumors while enabling high-resolution live imaging of, e.g., cell motility and chemotaxis, and preserving the capacity to capture the spatial transcriptome. Our microfluidic device generates a pH gradient that can be rapidly controlled to mimic spatiotemporal microenvironmental changes over cancer cells embedded in a 3D matrix. The device can be reopened allowing immunofluorescence analysis of selected phenotypes, as well as the transfer of cells and matrix to a Visium slide for spatially resolved analysis of transcriptional changes across the pH gradient. This workflow is easily adaptable to other gradients and multiple cell types and can therefore prove invaluable for integrated analysis of roles of microenvironmental gradients in biology., Despite the physiological and pathophysiological significance of microenvironmental gradients, e.g., for diseases such as cancer, tools for generating such gradients and analyzing their impact are lacking. Here, we present an integrated microfluidic-based workflow that mimics extracellular pH gradients characteristic of solid tumors while enabling high-resolution live imaging of, e.g., cell motility and chemotaxis, and preserving the capacity to capture the spatial transcriptome. Our microfluidic device generates a pH gradient that can be rapidly controlled to mimic spatiotemporal microenvironmental changes over cancer cells embedded in a 3D matrix. The device can be reopened allowing immunofluorescence analysis of selected phenotypes, as well as the transfer of cells and matrix to a Visium slide for spatially resolved analysis of transcriptional changes across the pH gradient. This workflow is easily adaptable to other gradients and multiple cell types and can therefore prove invaluable for integrated analysis of roles of microenvironmental gradients in biology.

G protein-coupled receptors (GPCRs) are seven transmembrane receptors that respond to external stimuli and undergo conformational changes to activate G proteins and modulate cellular processes leading to biological outcomes. To prevent overstimulation and prolonged exposure to stimuli, GPCRs are regulated by internalization. While the canonical GPCR internalization mechanism in mammalian cells is arrestin-dependent, clathrin-mediated endocytosis, more diverse GPCR internalization mechanisms have been described over the years. However, there is a lack of consistent methods used in the literature making it complicated to determine a receptor's internalization pathway. Here, we utilized a highly efficient time-resolved Förster resonance energy transfer (TR-FRET) internalization assay to determine the internalization profile of nine distinct GPCRs representing the GPCR classes A, B and C and with different G protein coupling profiles. This technique, coupled with clustered regularly interspaced palindromic repeats (CRISPR) engineered knockout cells allows us to effectively study the involvement of heterotrimeric G proteins and non-visual arrestins. We found that all the nine receptors internalized upon agonist stimulation in a concentration-dependent manner and six receptors showed basal internalization. Yet, there is no correlation between the receptor class and primary G protein coupling to the arrestin and G protein dependence for GPCR internalization. Overall, this study presents a platform for studying internalization that is applicable to most GPCRs and may even be extended to other membrane proteins. This method can be easily applicable to other endocytic machinery of interest and ultimately will lend itself towards the construction of comprehensive receptor internalization profiles.

Despite the physiological and pathophysiological significance of microenvironmental gradients, e.g., for diseases such as cancer, tools for generating such gradients and analyzing their impact are lacking. Here, we present an integrated microfluidic-based workflow that mimics extracellular pH gradients characteristic of solid tumors while enabling high-resolution live imaging of, e.g., cell motility and chemotaxis, and preserving the capacity to capture the spatial transcriptome. Our microfluidic device generates a pH gradient that can be rapidly controlled to mimic spatiotemporal microenvironmental changes over cancer cells embedded in a 3D matrix. The device can be reopened allowing immunofluorescence analysis of selected phenotypes, as well as the transfer of cells and matrix to a Visium slide for spatially resolved analysis of transcriptional changes across the pH gradient. This workflow is easily adaptable to other gradients and multiple cell types and can therefore prove invaluable for integrated analysis of roles of microenvironmental gradients in biology.

Background The lactate receptor GPR81 contributes to cancer development through unclear mechanisms. Here, we investigate the roles of GPR81 in three-dimensional (3D) and in vivo growth of breast cancer cells and study the molecular mechanisms involved. Methods GPR81 was stably knocked down (KD) in MCF-7 human breast cancer cells which were subjected to RNA-seq analysis, 3D growth, in situ- and immunofluorescence analyses, and cell viability- and motility assays, combined with KD of key GPR81-regulated genes. Key findings were additionally studied in other breast cancer cell lines and in mammary epithelial cells. Results GPR81 was upregulated in multiple human cancer types and further upregulated by extracellular lactate and 3D growth in breast cancer spheroids. GPR81 KD increased spheroid necrosis, reduced invasion and in vivo tumor growth, and altered expression of genes related to GO/KEGG terms extracellular matrix, cell adhesion, and Notch signaling. Single cell in situ analysis of MCF-7 cells revealed that several GPR81-regulated genes were upregulated in the same cell clusters. Notch signaling, particularly the Notch ligand Delta-like-4 (DLL4), was strikingly downregulated upon GPR81 KD, and DLL4 KD elicited spheroid necrosis and inhibited invasion in a manner similar to GPR81 KD. Conclusions GPR81 supports breast cancer aggressiveness, and in MCF-7 cells, this occurs at least in part via DLL4. Our findings reveal a new GPR81-driven mechanism in breast cancer and substantiate GPR81 as a promising treatment target., Background: The lactate receptor GPR81 contributes to cancer development through unclear mechanisms. Here, we investigate the roles of GPR81 in three-dimensional (3D) and in vivo growth of breast cancer cells and study the molecular mechanisms involved. Methods: GPR81 was stably knocked down (KD) in MCF-7 human breast cancer cells which were subjected to RNA-seq analysis, 3D growth, in situ- and immunofluorescence analyses, and cell viability- and motility assays, combined with KD of key GPR81-regulated genes. Key findings were additionally studied in other breast cancer cell lines and in mammary epithelial cells. Results: GPR81 was upregulated in multiple human cancer types and further upregulated by extracellular lactate and 3D growth in breast cancer spheroids. GPR81 KD increased spheroid necrosis, reduced invasion and in vivo tumor growth, and altered expression of genes related to GO/KEGG terms extracellular matrix, cell adhesion, and Notch signaling. Single cell in situ analysis of MCF-7 cells revealed that several GPR81-regulated genes were upregulated in the same cell clusters. Notch signaling, particularly the Notch ligand Delta-like-4 (DLL4), was strikingly downregulated upon GPR81 KD, and DLL4 KD elicited spheroid necrosis and inhibited invasion in a manner similar to GPR81 KD. Conclusions: GPR81 supports breast cancer aggressiveness, and in MCF-7 cells, this occurs at least in part via DLL4. Our findings reveal a new GPR81-driven mechanism in breast cancer and substantiate GPR81 as a promising treatment target.

Atherosclerosis is a chronic inflammatory disease of the larger arteries that may lead to cardiovascular events. Identification of patients at highest risk of cardiovascular events is challenging, but molecular imaging using positron emission tomography (PET) may prove useful. The aim of this study was to evaluate and compare head-to-head three different PET tracers. Furthermore, tracer uptake is compared to gene expression alterations of the arterial vessel wall. Male New Zealand White rabbits (control group; n = 10, atherosclerotic group; n = 11) were used for the study. Vessel wall uptake was assessed with the three different PET tracers: [18F]FDG (inflammation), Na[18F]F (microcalcification), and [64Cu]Cu-DOTA-TATE (macrophages), using PET/computed tomography (CT). Tracer uptake was measured as standardized uptake value (SUV), and arteries from both groups were analyzed ex vivo by autoradiography, qPCR, histology, and immunohistochemistry. In rabbits, the atherosclerotic group showed significantly higher uptake of all three tracers compared to the control group [18F]FDG: SUVmean 1.50 ± 0.11 versus 1.23 ± 0.09, p = 0.025; Na[18F]F: SUVmean 1.54 ± 0.06 versus 1.18 ± 0.10, p = 0.006; and [64Cu]Cu-DOTA-TATE: SUVmean 2.30 ± 0.27 versus 1.65 ± 0.16; p = 0.047. Of the 102 genes analyzed, 52 were differentially expressed in the atherosclerotic group compared to the control group and several genes correlated with tracer uptake. In conclusion, we demonstrated the diagnostic value of [64Cu]Cu-DOTA-TATE and Na[18F]F for identifying atherosclerosis in rabbits. The two PET tracers provided information distinct from that obtained with [18F]FDG. None of the three tracers correlated significantly to each other, but [64Cu]Cu-DOTA-TATE and Na[18F]F uptake both correlated with markers of inflammation. [64Cu]Cu-DOTA-T

Aquaporin (AQP) water channels facilitate water transport across cellular membranes and are essential in regulation of body water balance. Moreover, several AQPs are overexpressed or ectopically expressed in breast cancer. Interestingly, several in vitro studies have suggested that AQPs can affect the response to conventional anticancer chemotherapies. Therefore, we took a systematic approach to test how AQP1, AQP3 and AQP5, which are often over-/ectopically expressed in breast cancer, affect total viability of 3-dimensional (3D) breast cancer cell spheroids when treated with the conventional anticancer chemotherapies Cisplatin, 5-Fluorouracil (5-FU) and Doxorubicin, a Combination of the three drugs as well as the Combination plus the Ras inhibitor Salirasib. Total viability of spheroids overexpressing AQP1 were decreased by all treatments except for 5-FU, which increased total viability by 20% compared to DMSO treated controls. All treatments reduced viability of spheroids overexpressing AQP3. In contrast, only Doxorubicin, Combination and Combination + Salirasib reduced total viability of spheroids overexpressing AQP5. Thus, this study supports a significant role of AQPs in the response to conventional chemotherapies. Evaluating the role of individual proteins that contribute to resistance to chemotherapies is essential in advancing personalized medicine in breast carcinomas., Aquaporin (AQP) water channels facilitate water transport across cellular membranes and are essential in regulation of body water balance. Moreover, several AQPs are overexpressed or ectopically expressed in breast cancer. Interestingly, several in vitro studies have suggested that AQPs can affect the response to conventional anticancer chemotherapies. Therefore, we took a systematic approach to test how AQP1, AQP3 and AQP5, which are often over-/ectopically expressed in breast cancer, affect total viability of 3-dimensional (3D) breast cancer cell spheroids when treated with the conventional anticancer chemotherapies Cisplatin, 5-Fluorouracil (5-FU) and Doxorubicin, a Combination of the three drugs as well as the Combination plus the Ras inhibitor Salirasib. Total viability of spheroids overexpressing AQP1 were decreased by all treatments except for 5-FU, which increased total viability by 20% compared to DMSO treated controls. All treatments reduced viability of spheroids overexpressing AQP3. In contrast, only Doxorubicin, Combination and Combination + Salirasib reduced total viability of spheroids overexpressing AQP5. Thus, this study supports a significant role of AQPs in the response to conventional chemotherapies. Evaluating the role of individual proteins that contribute to resistance to chemotherapies is essential in advancing personalized medicine in breast carcinomas.

Forests offer a range of amenity values besides the production value of timber. We know from existing studies, that the trees' height and species affect the public's recreational values, but it is less clear how much the joy of owning a property with forest matters, and whether the joy depends on characteristics like tree height and species. In this paper, we estimate landowners' private amenity value of forests. We do this by estimating four hedonic land value models for different sizes of property. In the models, forests are described by the size of forest area with a canopy height above three meters, the size of forest area with a canopy height below three meters, mean canopy height, and the share of broadleaf forest. The value of an additional hectare of forest is predicted using the hedonic model and compared with the forest's expectation value based on accounting statistics for forest income, including hunting rents, discounted using an average real market interest rate of 1.7%. We find a difference of 6500–9900 EUR/ha between the estimated land value models and the production values for properties larger than 10 ha. We interpret this difference as the forest owner's willingness to pay for forest amenities in addition to the timber value. The results offer insight into the value of owning forests for the large majority of private forest owners who own small patches of land. They can be valuable in the design of land-use policies aiming to increase forest area and ecosystem service provision on private land that benefits both the public and the private owner.

One key communication block in 5G and 6G radios is the active phased array (APA). To ensure reliable operation, efficient and timely fault diagnosis of APAs on-site is crucial. To date, fault diagnosis has relied on measurement of frequency domain radiation patterns using costly equipment and multiple strictly controlled measurement probes, which are time-consuming, complex, and therefore infeasible for on-site deployment. This paper proposes a novel method exploiting a Deep Neural Network (DNN) tailored to extract the features hidden in the baseband in-phase and quadrature signals for classifying the different faults. It requires only a single probe in one measurement point for fast and accurate diagnosis of the faulty elements and components in APAs. Validation of the proposed method is done using a commercial 28 GHz APA. Accuracies of 99% and 80% have been demonstrated for single- and multi-element failure detection, respectively. Three different test scenarios are investigated: on-off antenna elements, phase variations, and magnitude attenuation variations. In a low signal to noise ratio of 4 dB, stable fault detection accuracy above 90% is maintained. This is all achieved with a detection time of milliseconds (e.g 6~ms), showing a high potential for on-site deployment., Comment: 10 pages

Aquaporin (AQP) water channels facilitate water transport across cellular membranes and are essential in regulation of body water balance. Moreover, several AQPs are overexpressed or ectopically expressed in breast cancer. Interestingly, several in vitro studies have suggested that AQPs can affect the response to conventional anticancer chemotherapies. Therefore, we took a systematic approach to test how AQP1, AQP3 and AQP5, which are often over-/ectopically expressed in breast cancer, affect total viability of 3-dimensional (3D) breast cancer cell spheroids when treated with the conventional anticancer chemotherapies Cisplatin, 5-Fluorouracil (5-FU) and Doxorubicin, a Combination of the three drugs as well as the Combination plus the Ras inhibitor Salirasib. Total viability of spheroids overexpressing AQP1 were decreased by all treatments except for 5-FU, which increased total viability by 20% compared to DMSO treated controls. All treatments reduced viability of spheroids overexpressing AQP3. In contrast, only Doxorubicin, Combination and Combination + Salirasib reduced total viability of spheroids overexpressing AQP5. Thus, this study supports a significant role of AQPs in the response to conventional chemotherapies. Evaluating the role of individual proteins that contribute to resistance to chemotherapies is essential in advancing personalized medicine in breast carcinomas., Aquaporin (AQP) water channels facilitate water transport across cellular membranes and are essential in regulation of body water balance. Moreover, several AQPs are overexpressed or ectopically expressed in breast cancer. Interestingly, several in vitro studies have suggested that AQPs can affect the response to conventional anticancer chemotherapies. Therefore, we took a systematic approach to test how AQP1, AQP3 and AQP5, which are often over-/ectopically expressed in breast cancer, affect total viability of 3-dimensional (3D) breast cancer cell spheroids when treated with the conventional anticancer chemotherapies Cisplatin, 5-Fluorouracil (5-FU) and Doxorubicin, a Combination of the three drugs as well as the Combination plus the Ras inhibitor Salirasib. Total viability of spheroids overexpressing AQP1 were decreased by all treatments except for 5-FU, which increased total viability by 20% compared to DMSO treated controls. All treatments reduced viability of spheroids overexpressing AQP3. In contrast, only Doxorubicin, Combination and Combination + Salirasib reduced total viability of spheroids overexpressing AQP5. Thus, this study supports a significant role of AQPs in the response to conventional chemotherapies. Evaluating the role of individual proteins that contribute to resistance to chemotherapies is essential in advancing personalized medicine in breast carcinomas.

Forests offer a range of amenity values besides the production value of timber. We know from existing studies, that the trees' height and species affect the public's recreational values, but it is less clear how much the joy of owning a property with forest matters, and whether the joy depends on characteristics like tree height and species. In this paper, we estimate landowners' private amenity value of forests. We do this by estimating four hedonic land value models for different sizes of property. In the models, forests are described by the size of forest area with a canopy height above three meters, the size of forest area with a canopy height below three meters, mean canopy height, and the share of broadleaf forest. The value of an additional hectare of forest is predicted using the hedonic model and compared with the forest's expectation value based on accounting statistics for forest income, including hunting rents, discounted using an average real market interest rate of 1.7%. We find a difference of 6500–9900 EUR/ha between the estimated land value models and the production values for properties larger than 10 ha. We interpret this difference as the forest owner's willingness to pay for forest amenities in addition to the timber value. The results offer insight into the value of owning forests for the large majority of private forest owners who own small patches of land. They can be valuable in the design of land-use policies aiming to increase forest area and ecosystem service provision on private land that benefits both the public and the private owner.

Background The lactate receptor GPR81 contributes to cancer development through unclear mechanisms. Here, we investigate the roles of GPR81 in three-dimensional (3D) and in vivo growth of breast cancer cells and study the molecular mechanisms involved. Methods GPR81 was stably knocked down (KD) in MCF-7 human breast cancer cells which were subjected to RNA-seq analysis, 3D growth, in situ- and immunofluorescence analyses, and cell viability- and motility assays, combined with KD of key GPR81-regulated genes. Key findings were additionally studied in other breast cancer cell lines and in mammary epithelial cells. Results GPR81 was upregulated in multiple human cancer types and further upregulated by extracellular lactate and 3D growth in breast cancer spheroids. GPR81 KD increased spheroid necrosis, reduced invasion and in vivo tumor growth, and altered expression of genes related to GO/KEGG terms extracellular matrix, cell adhesion, and Notch signaling. Single cell in situ analysis of MCF-7 cells revealed that several GPR81-regulated genes were upregulated in the same cell clusters. Notch signaling, particularly the Notch ligand Delta-like-4 (DLL4), was strikingly downregulated upon GPR81 KD, and DLL4 KD elicited spheroid necrosis and inhibited invasion in a manner similar to GPR81 KD. Conclusions GPR81 supports breast cancer aggressiveness, and in MCF-7 cells, this occurs at least in part via DLL4. Our findings reveal a new GPR81-driven mechanism in breast cancer and substantiate GPR81 as a promising treatment target., Background: The lactate receptor GPR81 contributes to cancer development through unclear mechanisms. Here, we investigate the roles of GPR81 in three-dimensional (3D) and in vivo growth of breast cancer cells and study the molecular mechanisms involved. Methods: GPR81 was stably knocked down (KD) in MCF-7 human breast cancer cells which were subjected to RNA-seq analysis, 3D growth, in situ- and immunofluorescence analyses, and cell viability- and motility assays, combined with KD of key GPR81-regulated genes. Key findings were additionally studied in other breast cancer cell lines and in mammary epithelial cells. Results: GPR81 was upregulated in multiple human cancer types and further upregulated by extracellular lactate and 3D growth in breast cancer spheroids. GPR81 KD increased spheroid necrosis, reduced invasion and in vivo tumor growth, and altered expression of genes related to GO/KEGG terms extracellular matrix, cell adhesion, and Notch signaling. Single cell in situ analysis of MCF-7 cells revealed that several GPR81-regulated genes were upregulated in the same cell clusters. Notch signaling, particularly the Notch ligand Delta-like-4 (DLL4), was strikingly downregulated upon GPR81 KD, and DLL4 KD elicited spheroid necrosis and inhibited invasion in a manner similar to GPR81 KD. Conclusions: GPR81 supports breast cancer aggressiveness, and in MCF-7 cells, this occurs at least in part via DLL4. Our findings reveal a new GPR81-driven mechanism in breast cancer and substantiate GPR81 as a promising treatment target.

Atherosclerosis is a chronic inflammatory disease of the larger arteries that may lead to cardiovascular events. Identification of patients at highest risk of cardiovascular events is challenging, but molecular imaging using positron emission tomography (PET) may prove useful. The aim of this study was to evaluate and compare head-to-head three different PET tracers. Furthermore, tracer uptake is compared to gene expression alterations of the arterial vessel wall. Male New Zealand White rabbits (control group; n = 10, atherosclerotic group; n = 11) were used for the study. Vessel wall uptake was assessed with the three different PET tracers: [18F]FDG (inflammation), Na[18F]F (microcalcification), and [64Cu]Cu-DOTA-TATE (macrophages), using PET/computed tomography (CT). Tracer uptake was measured as standardized uptake value (SUV), and arteries from both groups were analyzed ex vivo by autoradiography, qPCR, histology, and immunohistochemistry. In rabbits, the atherosclerotic group showed significantly higher uptake of all three tracers compared to the control group [18F]FDG: SUVmean 1.50 ± 0.11 versus 1.23 ± 0.09, p = 0.025; Na[18F]F: SUVmean 1.54 ± 0.06 versus 1.18 ± 0.10, p = 0.006; and [64Cu]Cu-DOTA-TATE: SUVmean 2.30 ± 0.27 versus 1.65 ± 0.16; p = 0.047. Of the 102 genes analyzed, 52 were differentially expressed in the atherosclerotic group compared to the control group and several genes correlated with tracer uptake. In conclusion, we demonstrated the diagnostic value of [64Cu]Cu-DOTA-TATE and Na[18F]F for identifying atherosclerosis in rabbits. The two PET tracers provided information distinct from that obtained with [18F]FDG. None of the three tracers correlated significantly to each other, but [64Cu]Cu-DOTA-TATE and Na[18F]F uptake both correlated with markers of inflammation. [64Cu]Cu-DOTA-T

The Third Generation Partnership Project (3GPP) is accelerating 5G new radio (NR) global standards aimed at significant enhancement of wireless system performance for higher date rate, better energy efficiency, and higher reliability than the current 4G cellular systems. The operators, manufacturers, and test equipment vendors have worked together to develop standardized over-the-air (OTA) test methodologies for the overall performance evaluation of 5G NR devices. 3GPP is taking the lead in standardizing the OTA testing of 5G NR under fading channel conditions. In 3GPP specifications, test methods have been studied to verify the multiple-input multiple-output (MIMO) performance of 5G NR user equipments (UEs) in OTA mode. This article follows the 3GPP standardization work and discusses the MIMO OTA test methodologies for 5G NR UEs working at frequency range 1 (FR1) and FR2, with a focus on its new challenges and solutions compared to 4G MIMO OTA testing methods. Then the OTA throughput testing results of real 5G NRUEs are demonstrated under the standard channel models. Finally, the challenges and limitations of standard 5G MIMO OTA test solutions are also highlighted.

Background and aims: Urokinase-type plasminogen activator receptor (uPAR) is associated with extracellular matrix (ECM) degradation and cancer aggressiveness. Its role in arterial atherogenesis as a molecular imaging target is not well-established. The aim of this study was to non-invasively visualize uPAR expression in atherosclerosis by a novel uPAR-targeting positron emission tomography (PET) tracer [64Cu]Cu-DOTA-AE105. Methods: We used molecular biology to investigate uPAR expression by analyzing human atherosclerotic plaques and cultured cells. A retrospective analysis was performed on patients, who underwent combined PET/CT (n = 10) to measure [64Cu]Cu-DOTA-AE105 uptake in five large arteries, divided into a high and low-risk group based on coronary artery calcium score (CAC score). Results: The in vitro assay for THP-1 monocytes displayed a significantly upregulated uPAR expression upon stimulation (5.2-fold upregulation, p < 0.0001 by a one-way ANOVA followed by Tukey's test) by single-cell flowcytometric analysis. Freshly excised human atherosclerotic plaques underwent flow cytometric and microarray analyses manifesting 73.9 ± 2.9% of mononuclear phagocyte system (MPS) cells expressing uPAR and had a greater than 7-fold higher gene expression of plasminogen activator urokinase receptor (PLAUR, p = 0.002), integrin subunit alpha X (ITGAX, p = 0.0008), and cluster of differentiation 163 (CD163, p < 0.0001). The tissue-to-background ratios (TBRmax) in five large arteries showed a higher [64Cu]Cu-DOTA-AE105 uptake in the group with high CAC score compared to the group with low CAC score (2.4 ± 0.1 vs 1.7 ± 0.1, p = 0.057), significantly higher in the ascending aorta (2.7 ± 0.1 vs 2.0 ± 0.1, p = 0.038) and the abdominal aorta (3.2 ± 0.2 vs 2.0 ± 0.2, p = 0.038) by a non-parametric Mann-Whitney test. C

Temporal synchrony between facial motion and acoustic modulations is a hallmark feature of audiovisual speech. The moving face and mouth during natural speech is known to be correlated with low-frequency acoustic envelope fluctuations (below 10 Hz), but the precise rates at which envelope information is synchronized with motion in different parts of the face are less clear. Here, we used regularized canonical correlation analysis (rCCA) to learn speech envelope filters whose outputs correlate with motion in different parts of the speakers face. We leveraged recent advances in video-based 3D facial landmark estimation allowing us to examine statistical envelope-face correlations across a large number of speakers (*4000). Specifically, rCCA was used to learn modulation transfer functions (MTFs) for the speech envelope that significantly predict correlation with facial motion across different speakers. The AV analysis revealed bandpass speech envelope filters at distinct temporal scales. A first set of MTFs showed peaks around 3-4 Hz and were correlated with mouth movements. A second set of MTFs captured envelope fluctuations in the 1-2 Hz range correlated with more global face and head motion. These two distinctive timescales emerged only as a property of natural AV speech statistics across many speakers. A similar analysis of fewer speakers performing a controlled speech task highlighted only the well-known temporal modulations around 4 Hz correlated with orofacial motion. The different bandpass ranges of AV correlation align notably with the average rates at which syllables (3-4 Hz) and phrases (1-2 Hz) are produced in natural speech. Whereas periodicities at the syllable rate are evident in the envelope spectrum of the speech signal itself, slower 1-2 Hz regularities thus only become prominent when considering crossmodal signal statistics. This may indicate a motor origin of temporal regularities at the timescales of syllables and phrases in natural speech.

Background The current report aims to describe the nutrient loads from land to sea around the year 1900. The nutrient loads are calculated considering the various factors affecting the nutrient inputs and transport based on available data from that time, literature, comparative analysis methods and modelling tools. The main factors investigated are climate, hydrology, land use, agricultural practices and drainage, urban development and landscape (e.g. nutrient retention in groundwater, wetlands, lakes and streams). The ambition was to use as much data and information from the time as possible, taking into consideration the quality and representativeness, and use modelling and GIS tools to provide a geographically distributed estimate of total nitrogen (TN) and total phosphorus (TP) concentrations and loads from the root zone to the sea. To be able to do this, the datasets on climate and hydrology were analysed and expanded as part of the project, and runoff was modelled using the national water resources model (DK- model). The agricultural practices and land use in different areas of the country were analysed in detail, and the root zone concentration of nitrogen was determined for different land use categories. This was based on old farmland statistics from Denmark and Northern Germany, which at the time included land areas that are now Danish, and root zone nitrogen concentrations extracted from farming experiments resembling year 1900 farm management practices. Phosphorus inputs to the surface waters from the landscape, including agricultural activities, were estimated for the main input categories: soil drainage, land reclamation, grazing animals, soil erosion and manure storage. Furthermore, a detailed analysis of the contribution of nutrients from cities (point sources) was included. This analysis estimated nitrogen as well as phosphorus inputs. To model the concentration and transport of nitrogen in the freshwater load

Background and aims: Urokinase-type plasminogen activator receptor (uPAR) is associated with extracellular matrix (ECM) degradation and cancer aggressiveness. Its role in arterial atherogenesis as a molecular imaging target is not well-established. The aim of this study was to non-invasively visualize uPAR expression in atherosclerosis by a novel uPAR-targeting positron emission tomography (PET) tracer [64Cu]Cu-DOTA-AE105. Methods: We used molecular biology to investigate uPAR expression by analyzing human atherosclerotic plaques and cultured cells. A retrospective analysis was performed on patients, who underwent combined PET/CT (n = 10) to measure [64Cu]Cu-DOTA-AE105 uptake in five large arteries, divided into a high and low-risk group based on coronary artery calcium score (CAC score). Results: The in vitro assay for THP-1 monocytes displayed a significantly upregulated uPAR expression upon stimulation (5.2-fold upregulation, p < 0.0001 by a one-way ANOVA followed by Tukey's test) by single-cell flowcytometric analysis. Freshly excised human atherosclerotic plaques underwent flow cytometric and microarray analyses manifesting 73.9 ± 2.9% of mononuclear phagocyte system (MPS) cells expressing uPAR and had a greater than 7-fold higher gene expression of plasminogen activator urokinase receptor (PLAUR, p = 0.002), integrin subunit alpha X (ITGAX, p = 0.0008), and cluster of differentiation 163 (CD163, p < 0.0001). The tissue-to-background ratios (TBRmax) in five large arteries showed a higher [64Cu]Cu-DOTA-AE105 uptake in the group with high CAC score compared to the group with low CAC score (2.4 ± 0.1 vs 1.7 ± 0.1, p = 0.057), significantly higher in the ascending aorta (2.7 ± 0.1 vs 2.0 ± 0.1, p = 0.038) and the abdominal aorta (3.2 ± 0.2 vs 2.0 ± 0.2, p = 0.038) by a non-parametric Mann-Whitney test. Conclusions: uPAR is abundantly expressed by MPS cells in atherosclerotic plaques and can be visualized by the n

Solid tumors comprise two major components: the cancer cells and the tumor stroma. The stroma is a mixture of cellular and acellular components including fibroblasts, mesenchymal and cancer stem cells, endothelial cells, immune cells, extracellular matrix, and tumor interstitial fluid. The insufficient tumor perfusion and the highly proliferative state and dysregulated metabolism of the cancer cells collectively create a physicochemical microenvironment characterized by altered nutrient concentrations and varying degrees of hypoxia and acidosis. Furthermore, both cancer and stromal cells secrete numerous growth factors, cytokines, and extracellular matrix proteins which further shape the tumor microenvironment (TME), favoring cancer progression. Transport proteins expressed by cancer and stromal cells localize at the interface between the cells and the TME and are in a reciprocal relationship with it, as both sensors and modulators of TME properties. It has been amply demonstrated how acid-base and nutrient transporters of cancer cells enable their growth, presumably by contributing both to the extracellular acidosis and the exchange of metabolic substrates and waste products between cells and TME. However, the TME also impacts other transport proteins important for cancer progression, such as multidrug resistance proteins. In this review, we summarize current knowledge of the cellular and acellular components of solid tumors and their interrelationship with key ion transport proteins. We focus in particular on acid-base transport proteins with known or proposed roles in cancer development, and we discuss their relevance for novel therapeutic strategies.

Background and aims: Urokinase-type plasminogen activator receptor (uPAR) is associated with extracellular matrix (ECM) degradation and cancer aggressiveness. Its role in arterial atherogenesis as a molecular imaging target is not well-established. The aim of this study was to non-invasively visualize uPAR expression in atherosclerosis by a novel uPAR-targeting positron emission tomography (PET) tracer [64Cu]Cu-DOTA-AE105. Methods: We used molecular biology to investigate uPAR expression by analyzing human atherosclerotic plaques and cultured cells. A retrospective analysis was performed on patients, who underwent combined PET/CT (n = 10) to measure [64Cu]Cu-DOTA-AE105 uptake in five large arteries, divided into a high and low-risk group based on coronary artery calcium score (CAC score). Results: The in vitro assay for THP-1 monocytes displayed a significantly upregulated uPAR expression upon stimulation (5.2-fold upregulation, p < 0.0001 by a one-way ANOVA followed by Tukey's test) by single-cell flowcytometric analysis. Freshly excised human atherosclerotic plaques underwent flow cytometric and microarray analyses manifesting 73.9 ± 2.9% of mononuclear phagocyte system (MPS) cells expressing uPAR and had a greater than 7-fold higher gene expression of plasminogen activator urokinase receptor (PLAUR, p = 0.002), integrin subunit alpha X (ITGAX, p = 0.0008), and cluster of differentiation 163 (CD163, p < 0.0001). The tissue-to-background ratios (TBRmax) in five large arteries showed a higher [64Cu]Cu-DOTA-AE105 uptake in the group with high CAC score compared to the group with low CAC score (2.4 ± 0.1 vs 1.7 ± 0.1, p = 0.057), significantly higher in the ascending aorta (2.7 ± 0.1 vs 2.0 ± 0.1, p = 0.038) and the abdominal aorta (3.2 ± 0.2 vs 2.0 ± 0.2, p = 0.038) by a non-parametric Mann-Whitney test. Conclusions: uPAR is abundantly expressed by MPS cells in atherosclerotic plaques and can be visualized by the n

The National Danish Inguinal Hernia Database (1997) and Ventral Hernia Database (2007), together the Danish Hernia Database, was launched to monitor and optimize surgical quality and outcomes after hernia repairs. The purpose of the present qualitative review was to present five "highlight" publications from the 123 published/in press (October 2020) original publications from the Danish Hernia Database. Two international hernia experts independently nominated 10 publications from the Database publication list published between 2010 and 2020. Each of the 10 members of the database steering group ranked the 10 publications according to the following three categories: (I) originality, (II) methodology, and (III) clinical impact. The publications were ranked as 1= good, 2= very good, 3= outstanding. The five publications with the highest score were depicted as highlights. The publications dealt with: (I) long-term mesh-related complications after incisional hernia repair, (II) outcomes after parastomal hernia repairs, (III) mesh or suture repair of ventral hernias in women having subsequent pregnancy, (IV) reoperation for recurrence versus clinical recurrence rates, and (V) recurrence rates after resorbable versus non-resorbable tackers for laparoscopic ventral hernia repair. Data from the Danish Hernia Database is internationally acknowledged. The nationwide Danish data supplements the higher-ranking evidence by randomized controlled trials (RCTs) by adding evidence-based answers to scientifically clinically relevant questions. The Danish Hernia Database is still active after more than 20 years.

The National Danish Inguinal Hernia Database (1997) and Ventral Hernia Database (2007), together the Danish Hernia Database, was launched to monitor and optimize surgical quality and outcomes after hernia repairs. The purpose of the present qualitative review was to present five "highlight" publications from the 123 published/in press (October 2020) original publications from the Danish Hernia Database. Two international hernia experts independently nominated 10 publications from the Database publication list published between 2010 and 2020. Each of the 10 members of the database steering group ranked the 10 publications according to the following three categories: (I) originality, (II) methodology, and (III) clinical impact. The publications were ranked as 1= good, 2= very good, 3= outstanding. The five publications with the highest score were depicted as highlights. The publications dealt with: (I) long-term mesh-related complications after incisional hernia repair, (II) outcomes after parastomal hernia repairs, (III) mesh or suture repair of ventral hernias in women having subsequent pregnancy, (IV) reoperation for recurrence versus clinical recurrence rates, and (V) recurrence rates after resorbable versus non-resorbable tackers for laparoscopic ventral hernia repair. Data from the Danish Hernia Database is internationally acknowledged. The nationwide Danish data supplements the higher-ranking evidence by randomized controlled trials (RCTs) by adding evidence-based answers to scientifically clinically relevant questions. The Danish Hernia Database is still active after more than 20 years.

Objectives: Patient-reported outcome measures (PROMs) are evaluated in randomized controlled trials (RCTs) in patients with systemic lupus erythematosus (SLE), but not widely used in clinical practice. However, interest in incorporating PROMs into the management of SLE is increasing as PROMs provide a unique insight into the patient’s perception of lupus disease activity. The objective was to assess agreement in PROMs answered using a web app versus an outpatient touchscreen among patients with SLE. Methods: In a crossover RCT, SLE patients answered the following PROMs in a random order using the web app and the outpatient touchscreen: Systemic Lupus Erythematosus Activity Questionnaire (SLAQ) Global Health, SLAQ Symptom, SLAQ Total, SLAQ Worsening, Pain Visual Analog Scale (VAS), Fatigue VAS, Patient Global Health VAS, Health Assessment Questionnaire Disability Index (HAQ-DI), Patient Acceptable Symptom State (PASS), and an Anchoring Question. Equivalence between the two device types was demonstrated if the 95% confidence interval (95% CI) of the difference in PROM scores was within the prespecified equivalence margin. Agreement between the two device types was assessed using mixed linear models. Results: Thirty-four patients with SLE were included. Equivalence was demonstrated between the two device types for SLAQ Global Health with a difference of −0.21 (95% CI: −0.65 to 0.23). Moreover, equivalence was also found for HAQ-DI, Pain VAS, and Fatigue VAS whereas only comparability within the limits of the Minimal Clinically Important Difference (MCID) was demonstrated for VAS Patient Global Health. Statistical comparability was demonstrated for SLAQ Total, SLAQ Worsening, PASS, and Anchoring Question (no predefined MCID/equivalence margins available). However, a statistically significant difference between device types was observed for the SLAQ Symptom of −0.56 (95% CI: −1.10 to −0.01). The difference was, however, very small when considering the scale range of 0

Transport of ions and nutrients is a core mitochondrial function, without which there would be no mitochondrial metabolism and ATP production. Both ion homeostasis and mitochon-drial phenotype undergo pervasive changes during cancer development, and both play key roles in driving the malignancy. However, the link between these events has been largely ignored. This review comprehensively summarizes and critically discusses the role of the reciprocal relationship between ion transport and mitochondria in crucial cellular functions, including metabolism, signal-ing, and cell fate decisions. We focus on Ca2+, H+, and K+, which play essential and highly intercon-nected roles in mitochondrial function and are profoundly dysregulated in cancer. We describe the transport and roles of these ions in normal mitochondria, summarize the changes occurring during cancer development, and discuss how they might impact tumorigenesis.

Transport of ions and nutrients is a core mitochondrial function, without which there would be no mitochondrial metabolism and ATP production. Both ion homeostasis and mitochon-drial phenotype undergo pervasive changes during cancer development, and both play key roles in driving the malignancy. However, the link between these events has been largely ignored. This review comprehensively summarizes and critically discusses the role of the reciprocal relationship between ion transport and mitochondria in crucial cellular functions, including metabolism, signal-ing, and cell fate decisions. We focus on Ca2+, H+, and K+, which play essential and highly intercon-nected roles in mitochondrial function and are profoundly dysregulated in cancer. We describe the transport and roles of these ions in normal mitochondria, summarize the changes occurring during cancer development, and discuss how they might impact tumorigenesis.

In the brain, α-synuclein (aSN) partitions between free unbound cytosolic and membrane bound forms modulating both its physiological and pathological role and complicating its study due to structural heterogeneity. Here, we use an interdisciplinary, synergistic approach to characterize the properties of aSN:lipid mixtures, isolated aSN:lipid co-structures, and aSN in mammalian cells. Enabled by the isolation of the membrane-bound state, we show that within the previously described N-terminal membrane anchor, membrane interaction relies both on an N-terminal tail (NTT) head group layer insertion of 14 residues and a folded-upon-binding helix at the membrane surface. Both binding events must be present; if, for example, the NTT insertion is lost, the membrane affinity of aSN is severely compromised and formation of aSN:lipid co-structures hampered. In mammalian cells, compromised cooperativity results in lowered membrane association. Thus, avidity within the N-terminal anchor couples N-terminal insertion and helical surface binding, which is crucial for aSN membrane interaction and cellular localization, and may affect membrane fusion.

Most G protein-coupled receptors (GPCRs) recruit β-arrestins and internalize upon agonist stimulation. For the μ-opioid receptor (μ-OR), this process has been linked to development of opioid tolerance. GPCR kinases (GRKs), particularly GRK2 and GRK3, have been shown to be important for μ-OR recruitment of β-arrestin and internalization. However, the contribution of GRK2 and GRK3 to β-arrestin recruitment and receptor internalization, remain to be determined in their complete absence. Using CRISPR/Cas9-mediated genome editing we established HEK293 cells with knockout of GRK2, GRK3 or both to dissect their individual contributions in β-arrestin2 recruitment and μ-OR internalization upon stimulation with four different agonists. We showed that GRK2/3 removal reduced agonist-induced μ-OR internalization and β-arrestin2 recruitment substantially and we found GRK2 to be more important for these processes than GRK3. Furthermore, we observed a sustained and GRK2/3 independent component of β-arrestin2 recruitment to the plasma membrane upon μ-OR activation. Rescue expression experiments restored GRK2/3 functions. Inhibition of GRK2/3 using the small molecule inhibitor CMPD101 showed a high similarity between the genetic and pharmacological approaches, cross-validating the specificity of both. However, off-target effects were observed at high CMPD101 concentrations. These GRK2/3 KO cell lines should prove useful for a wide range of studies on GPCR function.

Dynamic interactions of proteins with lipid membranes are essential regulatory events in biology, but remain rudimentarily understood and particularly overlooked in membrane proteins. The ubiquitously expressed membrane protein Na+/H+-exchanger 1 (NHE1) regulates intracellular pH (pHi) with dysregulation linked to e.g. cancer and cardiovascular diseases. NHE1 has a long, regulatory cytosolic domain carrying a membrane-proximal region described as a lipid-interacting domain (LID), yet, the LID structure and underlying molecular mechanisms are unknown. Here we decompose these, combining structural and biophysical methods, molecular dynamics simulations, cellular biotinylation- and immunofluorescence analysis and exchanger activity assays. We find that the NHE1-LID is intrinsically disordered and, in presence of membrane mimetics, forms a helical αα-hairpin co-structure with the membrane, anchoring the regulatory domain vis-a-vis the transport domain. This co-structure is fundamental for NHE1 activity, as its disintegration reduced steady-state pHi and the rate of pHi recovery after acid loading. We propose that regulatory lipid-protein co-structures may play equally important roles in other membrane proteins.

Dynamic interactions of proteins with lipid membranes are essential regulatory events in biology, but remain rudimentarily understood and particularly overlooked in membrane proteins. The ubiquitously expressed membrane protein Na+/H+-exchanger 1 (NHE1) regulates intracellular pH (pHi) with dysregulation linked to e.g. cancer and cardiovascular diseases. NHE1 has a long, regulatory cytosolic domain carrying a membrane-proximal region described as a lipid-interacting domain (LID), yet, the LID structure and underlying molecular mechanisms are unknown. Here we decompose these, combining structural and biophysical methods, molecular dynamics simulations, cellular biotinylation- and immunofluorescence analysis and exchanger activity assays. We find that the NHE1-LID is intrinsically disordered and, in presence of membrane mimetics, forms a helical αα-hairpin co-structure with the membrane, anchoring the regulatory domain vis-a-vis the transport domain. This co-structure is fundamental for NHE1 activity, as its disintegration reduced steady-state pHi and the rate of pHi recovery after acid loading. We propose that regulatory lipid-protein co-structures may play equally important roles in other membrane proteins.

The Na+/H+ exchanger-1 (NHE1) supports tumour growth, making NHE1 inhibitors of interest in anticancer therapy, yet their molecular effects are incompletely characterized. Here, we demonstrate that widely used pyrazinoylguanidine-type NHE1 inhibitors potently inhibit growth and survival of cancer cell spheroids, in a manner unrelated to NHE1 inhibition. Cancer and non-cancer cells were grown as 3-dimensional (3D) spheroids and treated with pyrazinoylguanidine-type (amiloride, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), 5-(N,N-dimethyl)-amiloride (DMA), and 5-(N,N-hexamethylene)-amiloride (HMA)) or benzoylguanidine-type (eniporide, cariporide) NHE1 inhibitors for 2-7 days, followed by analyses of viability, compound accumulation, and stress- and death-associated signalling. EIPA, DMA and HMA dose-dependently reduced breast cancer spheroid viability while cariporide and eniporide had no effect. Although both compound types inhibited NHE1, the toxic effects were NHE1-independent, as inhibitor-induced viability loss was unaffected by NHE1 CRISPR/Cas9 knockout. EIPA and HMA accumulated extensively in spheroids, and this was associated with marked vacuolization, apparent autophagic arrest, ER stress, mitochondrial- and DNA damage and poly-ADP-ribose-polymerase (PARP) cleavage, indicative of severe stress and paraptosis-like cell death. Pyrazinoylguanidine-induced cell death was partially additive to that induced by conventional anticancer therapies and strongly additive to extracellular-signal-regulated-kinase (ERK) pathway inhibition. Thus, in addition to inhibiting NHE1, pyrazinoylguanidines exert potent, NHE1-independent cancer cell death, pointing to a novel relevance for these compounds in anticancer therapy.

The Na+/H+ exchanger-1 (NHE1) supports tumour growth, making NHE1 inhibitors of interest in anticancer therapy, yet their molecular effects are incompletely characterized. Here, we demonstrate that widely used pyrazinoylguanidine-type NHE1 inhibitors potently inhibit growth and survival of cancer cell spheroids, in a manner unrelated to NHE1 inhibition. Cancer and non-cancer cells were grown as 3-dimensional (3D) spheroids and treated with pyrazinoylguanidine-type (amiloride, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), 5-(N,N-dimethyl)-amiloride (DMA), and 5-(N,N-hexamethylene)-amiloride (HMA)) or benzoylguanidine-type (eniporide, cariporide) NHE1 inhibitors for 2-7 days, followed by analyses of viability, compound accumulation, and stress- and death-associated signalling. EIPA, DMA and HMA dose-dependently reduced breast cancer spheroid viability while cariporide and eniporide had no effect. Although both compound types inhibited NHE1, the toxic effects were NHE1-independent, as inhibitor-induced viability loss was unaffected by NHE1 CRISPR/Cas9 knockout. EIPA and HMA accumulated extensively in spheroids, and this was associated with marked vacuolization, apparent autophagic arrest, ER stress, mitochondrial- and DNA damage and poly-ADP-ribose-polymerase (PARP) cleavage, indicative of severe stress and paraptosis-like cell death. Pyrazinoylguanidine-induced cell death was partially additive to that induced by conventional anticancer therapies and strongly additive to extracellular-signal-regulated-kinase (ERK) pathway inhibition. Thus, in addition to inhibiting NHE1, pyrazinoylguanidines exert potent, NHE1-independent cancer cell death, pointing to a novel relevance for these compounds in anticancer therapy.

Post-translational modifications (PTMs) greatly expand the function and potential for regulation of protein activity, and O-glycosylation is among the most abundant and diverse PTMs. Initiation of O-GalNAc glycosylation is regulated by 20 distinct GalNAc-transferases (GalNAc-Ts), and deficiencies in individual GalNAc-Ts are associated with human disease, causing subtle but distinct phenotypes in model organisms. Here, we generate a set of isogenic keratinocyte cell lines lacking either of the three dominant and differentially expressed GalNAc-Ts. Through the ability of keratinocytes to form epithelia, we investigate the phenotypic consequences of the loss of individual GalNAc-Ts. Moreover, we probe the cellular responses through global transcriptomic, differential glycoproteomic, and differential phosphoproteomic analyses. We demonstrate that loss of individual GalNAc-T isoforms causes distinct epithelial phenotypes through their effect on specific biological pathways; GalNAc-T1 targets are associated with components of the endomembrane system, GalNAc-T2 targets with cell–ECM adhesion, and GalNAc-T3 targets with epithelial differentiation. Thus, GalNAc-T isoforms serve specific roles during human epithelial tissue formation.

Membrane proteins exist in lipid bilayers and mediate solute transport, signal transduction, cell-cell communication and energy conversion. Their activities are fundamental for life, which make them prominent subjects of study, but access to only a limited number of high-resolution structures complicates their mechanistic understanding. The absence of such structures relates mainly to difficulties in expressing and purifying high quality membrane protein samples in large quantities. An additional layer of complexity stems from the presence of intra- and/or extra-cellular domains constituted by unstructured intrinsically disordered regions (IDR), which can be hundreds of residues long. Although IDRs form key interaction hubs that facilitate biological processes, these are regularly removed to enable structural studies. To advance mechanistic insight into intact intrinsically disordered membrane proteins, we have developed a protocol for their purification. Using engineered yeast cells for optimized expression and purification, we have purified to homogeneity two very different human membrane proteins each with >300 residues long IDRs; the sodium proton exchanger 1 and the growth hormone receptor. Subsequent to their purification we have further explored their incorporation into membrane scaffolding protein nanodiscs, which will enable future structural studies.

Understanding the nature of active sites is central to controlling (electro)catalytic activity. Here we employed surface X-ray scattering coupled with density functional theory and surface-enhanced infrared absorption spectroscopy to examine the oxygen evolution reaction on RuO2 surfaces as a function of voltage. At 1.5 VRHE, our results suggest that there is an –OO group on the coordinatively unsaturated ruthenium (RuCUS) site of the (100) surface (and similarly for (110)), but adsorbed oxygen on the RuCUS site of (101). Density functional theory results indicate that the removal of –OO from the RuCUS site, which is stabilized by a hydrogen bond to a neighbouring –OH (–OO–H), could be the rate-determining step for (100) (similarly for (110)), where its reduced binding on (100) increased activity. A further reduction in binding energy on the RuCUS site of (101) resulted in a different rate-determining step (–O + H2O – (H+ + e−) → –OO–H) and decreased activity. Our study provides molecular details on the active sites, and the influence of their local coordination environment on activity. [Figure not available: see fulltext.]

The flow of non-living carbon (detritus) is considered an important process because it connects ecosystems and fuels benthic communities. In Norwegian kelp forests, 90% of the kelp production is exported to adjacent ecosystems where it can play a significant role in shaping benthic communities. We quantified the major structural and functional traits of an Arctic deep-sea ecosystem associated with kelp exports and assessed the ecological role of kelp export into the deep-sea system. We first developed a food-web model using the Ecopath with Ecosim (EwE) approach to represent the state of the deep (450 m) ecosystem of the Malangen fjord (Northern Norway) in 2017. Subsequently, we used the temporal dynamic model Ecosim to explore the structure and functioning traits of a theoretical deep-sea ecosystem projecting a decrease of kelp detritus biomass reaching the deep-sea ecosystem. Overall, our findings reveal that kelp detritus from shallow coastal areas has a small but noticeable role structuring the deep-sea ecosystem of Malangen. The temporal simulations show important differences depending on the application of mediating effects, which allow considering the detritus as a mediating group in prey-predator interaction, in addition to its direct role in trophic relationships. When mediating effects are applied, biomass increases for benthopelagic shrimps and suprabenthos groups and decreases for rays and skates, velvet belly, rabbitfish and other commercial demersal fishes under the low kelp detritus scenarios. Biomass-based and trophic-based indicators reveal a noticeable impact on the deep-sea ecosystem structure due to depletion of kelp detritus. To further assess future changes of the Arctic deep-sea ecosystems, dependencies with adjacent ecosystems, such as kelp detritus production, should be included

Post-translational modifications (PTMs) greatly expand the function and potential for regulation of protein activity, and O-glycosylation is among the most abundant and diverse PTMs. Initiation of O-GalNAc glycosylation is regulated by 20 distinct GalNAc-transferases (GalNAc-Ts), and deficiencies in individual GalNAc-Ts are associated with human disease, causing subtle but distinct phenotypes in model organisms. Here, we generate a set of isogenic keratinocyte cell lines lacking either of the three dominant and differentially expressed GalNAc-Ts. Through the ability of keratinocytes to form epithelia, we investigate the phenotypic consequences of the loss of individual GalNAc-Ts. Moreover, we probe the cellular responses through global transcriptomic, differential glycoproteomic, and differential phosphoproteomic analyses. We demonstrate that loss of individual GalNAc-T isoforms causes distinct epithelial phenotypes through their effect on specific biological pathways; GalNAc-T1 targets are associated with components of the endomembrane system, GalNAc-T2 targets with cell–ECM adhesion, and GalNAc-T3 targets with epithelial differentiation. Thus, GalNAc-T isoforms serve specific roles during human epithelial tissue formation.

Membrane proteins exist in lipid bilayers and mediate solute transport, signal transduction, cell-cell communication and energy conversion. Their activities are fundamental for life, which make them prominent subjects of study, but access to only a limited number of high-resolution structures complicates their mechanistic understanding. The absence of such structures relates mainly to difficulties in expressing and purifying high quality membrane protein samples in large quantities. An additional layer of complexity stems from the presence of intra- and/or extra-cellular domains constituted by unstructured intrinsically disordered regions (IDR), which can be hundreds of residues long. Although IDRs form key interaction hubs that facilitate biological processes, these are regularly removed to enable structural studies. To advance mechanistic insight into intact intrinsically disordered membrane proteins, we have developed a protocol for their purification. Using engineered yeast cells for optimized expression and purification, we have purified to homogeneity two very different human membrane proteins each with >300 residues long IDRs; the sodium proton exchanger 1 and the growth hormone receptor. Subsequent to their purification we have further explored their incorporation into membrane scaffolding protein nanodiscs, which will enable future structural studies.

The Na+/H+ exchanger-1 (NHE1) supports tumour growth, making NHE1 inhibitors of interest in anticancer therapy, yet their molecular effects are incompletely characterized. Here, we demonstrate that widely used pyrazinoylguanidine-type NHE1 inhibitors potently inhibit growth and survival of cancer cell spheroids, in a manner unrelated to NHE1 inhibition. Cancer and non-cancer cells were grown as 3-dimensional (3D) spheroids and treated with pyrazinoylguanidine-type (amiloride, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), 5-(N,N-dimethyl)-amiloride (DMA), and 5-(N,N-hexamethylene)-amiloride (HMA)) or benzoylguanidine-type (eniporide, cariporide) NHE1 inhibitors for 2-7 days, followed by analyses of viability, compound accumulation, and stress- and death-associated signalling. EIPA, DMA and HMA dose-dependently reduced breast cancer spheroid viability while cariporide and eniporide had no effect. Although both compound types inhibited NHE1, the toxic effects were NHE1-independent, as inhibitor-induced viability loss was unaffected by NHE1 CRISPR/Cas9 knockout. EIPA and HMA accumulated extensively in spheroids, and this was associated with marked vacuolization, apparent autophagic arrest, ER stress, mitochondrial- and DNA damage and poly-ADP-ribose-polymerase (PARP) cleavage, indicative of severe stress and paraptosis-like cell death. Pyrazinoylguanidine-induced cell death was partially additive to that induced by conventional anticancer therapies and strongly additive to extracellular-signal-regulated-kinase (ERK) pathway inhibition. Thus, in addition to inhibiting NHE1, pyrazinoylguanidines exert potent, NHE1-independent cancer cell death, pointing to a novel relevance for these compounds in anticancer therapy.

BackgroundThe International Parkinson and Movement Disorders Society criteria for mild cognitive impairment in PD need validation. The objectives of this present study were to evaluate prognostic validity of level I (abbreviated) International Parkinson and Movement Disorders Society mild cognitive impairment in PD criteria for development of PD dementia and compared them with level II (comprehensive) criteria.MethodsWe analyzed data from 8 international studies (1045 patients) from our consortium that included baseline data on demographics, motor signs, depression, detailed neuropsychological testing, and longitudinal follow-up for conversion to Parkinson's disease dementia. Survival analysis evaluated their contribution to the hazard of Parkinson's disease dementia.ResultsLevel I mild cognitive impairment in PD, increasing age, male sex, and severity of PD motor signs independently increased the hazard of Parkinson's disease dementia. Level I and level II mild cognitive impairment in PD classification had similar discriminative ability with respect to the time to Parkinson's disease dementia.ConclusionsLevel I mild cognitive impairment in PD classification independently contributes to the hazard of Parkinson's disease dementia. This finding supports the prognostic validity of the abbreviated mild cognitive impairment in PD criteria. © 2019 International Parkinson and Movement Disorder Society.

BackgroundThe International Parkinson and Movement Disorders Society criteria for mild cognitive impairment in PD need validation. The objectives of this present study were to evaluate prognostic validity of level I (abbreviated) International Parkinson and Movement Disorders Society mild cognitive impairment in PD criteria for development of PD dementia and compared them with level II (comprehensive) criteria.MethodsWe analyzed data from 8 international studies (1045 patients) from our consortium that included baseline data on demographics, motor signs, depression, detailed neuropsychological testing, and longitudinal follow-up for conversion to Parkinson's disease dementia. Survival analysis evaluated their contribution to the hazard of Parkinson's disease dementia.ResultsLevel I mild cognitive impairment in PD, increasing age, male sex, and severity of PD motor signs independently increased the hazard of Parkinson's disease dementia. Level I and level II mild cognitive impairment in PD classification had similar discriminative ability with respect to the time to Parkinson's disease dementia.ConclusionsLevel I mild cognitive impairment in PD classification independently contributes to the hazard of Parkinson's disease dementia. This finding supports the prognostic validity of the abbreviated mild cognitive impairment in PD criteria. © 2019 International Parkinson and Movement Disorder Society.

Antenna correlation is an important measure for designing multiple-input multiple-output (MIMO) antenna systems. A lower antenna correlation indicates a better MIMO performance that can be achieved with the underlying antenna systems. In the antenna design community, it is very common to evaluate the antenna correlation with isotropic or non-isotropic (e.g. Gaussian-distributed) angular power spectrum (APS) as baselines. On the other hand, antenna correlation can also be evaluated via channel transfer function (CTF) under the a given propagation channel, e.g. drawn from the bi-directional geometrybased stochastic channel model. In this paper, the analytic forms for the antenna correlation based on the APS and the CTF are derived, respectively, with their similarities and differences explained. Moreover, a numerical example is also given with a standard channel model to support our findings.

Key points: Exogenous Na+/H+ exchanger 1 (NHE1) expression stimulated the collective migration of epithelial cell sheets Stimulation with epidermal growth factor, a key morphogen, primarily increased migration of the front row of cells, whereas NHE1 increased that of submarginal cell rows, and the two stimuli were additive Accordingly, NHE1 localized not only to the leading edges of leader cells, but also in cryptic lamellipodia in submarginal cell rows NHE1 expression disrupted the morphology of epithelial cell sheets and three-dimensional cysts. Abstract: Collective cell migration plays essential roles in embryonic development, in normal epithelial repair processes, and in many diseases including cancer. The Na+/H+ exchanger 1 (NHE1, SLC9A1) is an important regulator of motility in many cells and has been widely studied for its roles in cancer, although its possible role in collective migration of normal epithelial cells has remained unresolved. In the present study, we show that NHE1 expression in MDCK-II kidney epithelial cells accelerated collective cell migration. NHE1 localized to the leading edges of leader cells, as well as to cryptic lamellipodia in submarginal cell rows. Epidermal growth factor, a kidney morphogen, increased displacement of the front row of collectively migrating cells and reduced the number of migration fingers. NHE1 expression increased the number of migration fingers and increased displacement of submarginal cell rows, resulting in additive effects of NHE1 and epidermal growth factor. Finally, NHE1 expression resulted in disorganized development of MDCK-II cell cysts. Thus, NHE1 contributes to collective migration and epithelial morphogenesis, suggesting roles for the transporter in embryonic and early postnatal development.