Summary Sex work occurs in many forms and sex workers of all genders have been affected by HIV epidemics worldwide. The determinants of HIV risk associated with sex work occur at several levels, including individual biological and behavioural, dyadic and network, and community and social environmental levels. Evidence indicates that effective HIV prevention packages for sex workers should include combinations of biomedical, behavioural, and structural interventions tailored to local contexts, and be led and implemented by sex worker communities. A model simulation based on the South African heterosexual epidemic suggests that condom promotion and distribution programmes in South Africa have already reduced HIV incidence in sex workers and their clients by more than 70%. Under optimistic model assumptions, oral pre-exposure prophylaxis together with test and treat programmes could further reduce HIV incidence in South African sex workers and their clients by up to 40% over a 10-year period. Combining these biomedical approaches with a prevention package, including behavioural and structural components as part of a community-driven approach, will help to reduce HIV infection in sex workers in different settings worldwide.

Abstract Nuclear pores have an approximately uniform distribution in the nuclear envelope of most living cells. Hence, the morphology of the nuclear envelope is a spherical stomatocyte with a high genus. We have investigated the morphology of high-genus vesicles under pore-size constraint using dynamically triangulated membrane simulations. Bending-energy minimization without volume or other constraints produces a circular-cage stomatocyte, where the pores are aligned in a circular line on an oblate bud. As the pore radius is reduced, the circular-pore alignment is more stabilized than a random pore distribution on a spherical bud. However, we have clarified the conditions for the formation of a spherical stomatocyte: a small perinuclear volume, osmotic pressure within nucleoplasm, and repulsion between the pores. When area-difference elasticity is taken into account, the formation of cylindrical or budded tubules from the stomatocyte and discoidal stomatocyte is found.

Abstract Platinum-containing molecules are widely used as anticancer drugs. These molecules exert cytotoxic effects by binding to DNA through various mechanisms. The binding between DNA and platinum-based drugs hinders the opening of DNA, and therefore, DNA duplication and transcription are severely hampered. Overall, impeding the above-mentioned important DNA mechanisms results in irreversible DNA damage and the induction of apoptosis. Several molecules, including multinuclear platinum compounds, belong to the family of platinum drugs, and there is a body of research devoted to developing more efficient and less toxic versions of these compounds. In this study, we combined different biophysical methods, including single-molecule assays (magnetic tweezers) and bulk experiments (ultraviolet absorption for thermal denaturation) to analyze the differential stability of double-stranded DNA in complex with either cisplatin or multinuclear platinum agents. Specifically, we analyzed how the binding of BBR3005 and BBR3464, two representative multinuclear platinum-based compounds, to DNA affects its stability as compared with cisplatin binding. Our results suggest that single-molecule approaches can provide insights into the drug-DNA interactions that underlie drug potency and provide information that is complementary to that generated from bulk analysis; thus, single-molecule approaches have the potential to facilitate the selection and design of optimized drug compounds. In particular, relevant differences in DNA stability at the single-molecule level are demonstrated by analyzing nanomechanically induced DNA denaturation. On the basis of the comparison between the single-molecule and bulk analyses, we suggest that transplatinated drugs are able to locally destabilize small portions of the DNA chain, whereas other regions are stabilized.

Abstract In this article, we present PolNet, an open-source software tool for the study of blood flow and cell-level biological activity during vessel morphogenesis. We provide an image acquisition, segmentation, and analysis protocol to quantify endothelial cell polarity in entire in vivo vascular networks. In combination, we use computational fluid dynamics to characterize the hemodynamics of the vascular networks under study. The tool enables, to our knowledge for the first time, a network-level analysis of polarity and flow for individual endothelial cells. To date, PolNet has proven invaluable for the study of endothelial cell polarization and migration during vascular patterning, as demonstrated by two recent publications. Additionally, the tool can be easily extended to correlate blood flow with other experimental observations at the cellular/molecular level. We release the source code of our tool under the Lesser General Public License.

Abstract Contractions of lymphangions, i.e., the segment between two one-way lymphatic valves, generate the pressure gradients that propel lymph back to the circulation. Each lymphangion is comprised of an inner sheet of lymphatic endothelial cells circumscribed by one or more layers of lymphatic muscle cells (LMCs). Each contraction is produced by an LMC action potential (AP) that propagates via gap junctions along the lymphangion. Yet, electrical coupling within and between cell layers and the impact on AP waves is poorly understood. Here, we combine studies in rat and mouse lymphatic vessels with mathematical modeling to show that initiation of AP waves depends on high input resistance (low current drain), whereas propagation depends on morphology and sufficient LMC:LMC coupling. Simulations show that 1) myoendothelial coupling is insignificant to facilitate AP generation and sustain an experimentally measured cross-junctional potential difference of 25 mV, i.e., AP waves propagate along the LMC layer only; 2) LMC:LMC resistance is estimated around 2–10 MΩ but depends on vessel structure and cell-cell coupling, e.g., some degree of LMC overlap protects AP waves against LMC decoupling; 3) the propensity of AP wave initiation is highest around the valves, where the density of LMCs is low; and 4) a single pacemaker cell embedded in the LMC layer must be able to generate very large currents to overcome the current drain from the layer. However, the required current generation to initiate an AP wave is reduced upon stimulation of multiple adjacent LMCs. With stimulation of all LMCs, AP waves can also arise from heterogeneity in the electrical activity of LMCs. The findings advance our understanding of the electrical constraints that underlie initiation of APs in the LMC layer and make testable predictions about how morphology, LMC excitability, and LMC:LMC electrical coupling interact to determine the ability to initiate and propagate AP waves in small lymphatic vessels.

Abstract The viscosity is a highly important parameter within the cell membrane, affecting the diffusion of small molecules and, hence, controlling the rates of intracellular reactions. There is significant interest in the direct, quantitative assessment of membrane viscosity. Here we report the use of fluorescence lifetime imaging microscopy of the molecular rotor BODIPY C10 in the membranes of live Escherichia coli bacteria to permit direct quantification of the viscosity. Using this approach, we investigated the viscosity in live E. coli cells, spheroplasts, and liposomes made from E. coli membrane extracts. For live cells and spheroplasts, the viscosity was measured at both room temperature (23°C) and the E. coli growth temperature (37°C), while the membrane extract liposomes were studied over a range of measurement temperatures (5–40°C). At 37°C, we recorded a membrane viscosity in live E. coli cells of 950 cP, which is considerably higher than that previously observed in other live cell membranes (e.g., eukaryotic cells, membranes of Bacillus vegetative cells). Interestingly, this indicates that E. coli cells exhibit a high degree of lipid ordering within their liquid-phase plasma membranes.

Abstract One of the fundamental features that govern the cooperativity of multiple dyneins during cargo trafficking in cells is the spatial distribution of these dyneins on the cargo. Geometric considerations and recent experiments indicate that clustered distributions of dyneins are required for effective cooperation on micron-sized cargos. However, very little is known about the spatial distribution of dyneins and their cooperativity on smaller cargos, such as vesicles or endosomes <200 nm in size, which are not amenable to conventional immunostaining and optical trapping methods. In this work, we present evidence that dyneins can dynamically be clustered on endosomes in response to load. Using a darkfield imaging assay, we measured the repeated stalls and detachments of retrograde axonal endosomes under load with <10 nm localization accuracy at imaging rates up to 1 kHz for over a timescale of minutes. A three-dimensional stochastic model was used to simulate the endosome motility under load to gain insights on the mechanochemical properties and spatial distribution of dyneins on axonal endosomes. Our results indicate that 1) the distribution of dyneins on endosomes is fluid enough to support dynamic clustering under load and 2) the detachment kinetics of dynein on endosomes differs significantly from the in vitro measurements possibly due to an increase in the unitary stall force of dynein on endosomes.

Abstract Superwarfarins are modified analogs of warfarin with additional lipophilic aromatic rings, up to 100-fold greater potency, and longer biological half-lives. We hypothesized that increased hydrophobicity allowed interactions with amphiphilic membranes and modulation of biological responses. We find that superwarfarins brodifacoum and difenacoum increase lactate production and cell death in neuroblastoma cells. In contrast, neither causes changes in glioma cells that have higher cholesterol content. After choleterol depletion, lactate production was increased and cell viability was reduced. Drug-membrane interactions were examined by surface X-ray scattering using Langmuir monolayers of dipalmitoylphosphatidylcholine and/or cholesterol. Specular X-ray reflectivity data revealed that superwarfarins, but not warfarin, intercalate between dipalmitoylphosphatidylcholine molecules, whereas grazing incidence X-ray diffraction demonstrated changes in lateral crystalline order of the film. Neither agent showed significant interactions with monolayers containing >20% cholesterol. These findings demonstrate an affinity of superwarfarins to biomembranes and suggest that cellular responses to these agents are regulated by cholesterol content.

Abstract Fluid-filled interstitial gaps are a common feature of compact tissues held together by cell-cell adhesion. Although such gaps can in principle be the result of weak, incomplete cell attachment, adhesion is usually too strong for this to occur. Using a mechanical model of tissue cohesion, we show that, instead, a combination of local prevention of cell adhesion at three-cell junctions by fluidlike extracellular material and a reduction of cortical tension at the gap surface are sufficient to generate stable gaps. The size and shape of these interstitial gaps depends on the mechanical tensions between cells and at gap surfaces, and on the difference between intracellular and interstitial pressures that is related to the volume of the interstitial fluid. As a consequence of the dependence on tension/tension ratios, the presence of gaps does not depend on the absolute strength of cell adhesion, and similar gaps are predicted to occur in tissues of widely differing cohesion. Tissue mechanical parameters can also vary within and between cells of a given tissue, generating asymmetrical gaps. Within limits, these can be approximated by symmetrical gaps.

Summary The third UN High-Level Meeting on Non-Communicable Diseases (NCDs) on Sept 27, 2018, will review national and global progress towards the prevention and control of NCDs, and provide an opportunity to renew, reinforce, and enhance commitments to reduce their burden. NCD Countdown 2030 is an independent collaboration to inform policies that aim to reduce the worldwide burden of NCDs, and to ensure accountability towards this aim. In 2016, an estimated 40·5 million (71%) of the 56·9 million worldwide deaths were from NCDs. Of these, an estimated 1·7 million (4% of NCD deaths) occurred in people younger than 30 years of age, 15·2 million (38%) in people aged between 30 years and 70 years, and 23·6 million (58%) in people aged 70 years and older. An estimated 32·2 million NCD deaths (80%) were due to cancers, cardiovascular diseases, chronic respiratory diseases, and diabetes, and another 8·3 million (20%) were from other NCDs. Women in 164 (88%) and men in 165 (89%) of 186 countries and territories had a higher probability of dying before 70 years of age from an NCD than from communicable, maternal, perinatal, and nutritional conditions combined. Globally, the lowest risks of NCD mortality in 2016 were seen in high-income countries in Asia-Pacific, western Europe, and Australasia, and in Canada. The highest risks of dying from NCDs were observed in low-income and middle-income countries, especially in sub-Saharan Africa, and, for men, in central Asia and eastern Europe. Sustainable Development Goal (SDG) target 3.4—a one-third reduction, relative to 2015 levels, in the probability of dying between 30 years and 70 years of age from cancers, cardiovascular diseases, chronic respiratory diseases, and diabetes by 2030—will be achieved in 35 countries (19%) for women, and 30 (16%) for men, if these countries maintain or surpass their 2010–2016 rate of decline in NCD mortality. Most of these are high-income countries with already-low NCD mortality, and countries in central and eastern Europe. An additional 50 (27%) countries for women and 35 (19%) for men are projected to achieve such a reduction in the subsequent decade, and thus, with slight acceleration of decline, could meet the 2030 target. 86 (46%) countries for women and 97 (52%) for men need implementation of policies that substantially increase the rates of decline. Mortality from the four NCDs included in SDG target 3.4 has stagnated or increased since 2010 among women in 15 (8%) countries and men in 24 (13%) countries. NCDs and age groups other than those included in the SDG target 3.4 are responsible for a higher risk of death in low-income and middle-income countries than in high-income countries. Substantial reduction of NCD mortality requires policies that considerably reduce tobacco and alcohol use and blood pressure, and equitable access to efficacious and high-quality preventive and curative care for acute and chronic NCDs.

Summary Sex work occurs in many forms and sex workers of all genders have been affected by HIV epidemics worldwide. The determinants of HIV risk associated with sex work occur at several levels, including individual biological and behavioural, dyadic and network, and community and social environmental levels. Evidence indicates that effective HIV prevention packages for sex workers should include combinations of biomedical, behavioural, and structural interventions tailored to local contexts, and be led and implemented by sex worker communities. A model simulation based on the South African heterosexual epidemic suggests that condom promotion and distribution programmes in South Africa have already reduced HIV incidence in sex workers and their clients by more than 70%. Under optimistic model assumptions, oral pre-exposure prophylaxis together with test and treat programmes could further reduce HIV incidence in South African sex workers and their clients by up to 40% over a 10-year period. Combining these biomedical approaches with a prevention package, including behavioural and structural components as part of a community-driven approach, will help to reduce HIV infection in sex workers in different settings worldwide.

Abstract Nuclear pores have an approximately uniform distribution in the nuclear envelope of most living cells. Hence, the morphology of the nuclear envelope is a spherical stomatocyte with a high genus. We have investigated the morphology of high-genus vesicles under pore-size constraint using dynamically triangulated membrane simulations. Bending-energy minimization without volume or other constraints produces a circular-cage stomatocyte, where the pores are aligned in a circular line on an oblate bud. As the pore radius is reduced, the circular-pore alignment is more stabilized than a random pore distribution on a spherical bud. However, we have clarified the conditions for the formation of a spherical stomatocyte: a small perinuclear volume, osmotic pressure within nucleoplasm, and repulsion between the pores. When area-difference elasticity is taken into account, the formation of cylindrical or budded tubules from the stomatocyte and discoidal stomatocyte is found.

Abstract Platinum-containing molecules are widely used as anticancer drugs. These molecules exert cytotoxic effects by binding to DNA through various mechanisms. The binding between DNA and platinum-based drugs hinders the opening of DNA, and therefore, DNA duplication and transcription are severely hampered. Overall, impeding the above-mentioned important DNA mechanisms results in irreversible DNA damage and the induction of apoptosis. Several molecules, including multinuclear platinum compounds, belong to the family of platinum drugs, and there is a body of research devoted to developing more efficient and less toxic versions of these compounds. In this study, we combined different biophysical methods, including single-molecule assays (magnetic tweezers) and bulk experiments (ultraviolet absorption for thermal denaturation) to analyze the differential stability of double-stranded DNA in complex with either cisplatin or multinuclear platinum agents. Specifically, we analyzed how the binding of BBR3005 and BBR3464, two representative multinuclear platinum-based compounds, to DNA affects its stability as compared with cisplatin binding. Our results suggest that single-molecule approaches can provide insights into the drug-DNA interactions that underlie drug potency and provide information that is complementary to that generated from bulk analysis; thus, single-molecule approaches have the potential to facilitate the selection and design of optimized drug compounds. In particular, relevant differences in DNA stability at the single-molecule level are demonstrated by analyzing nanomechanically induced DNA denaturation. On the basis of the comparison between the single-molecule and bulk analyses, we suggest that transplatinated drugs are able to locally destabilize small portions of the DNA chain, whereas other regions are stabilized.

Abstract In this article, we present PolNet, an open-source software tool for the study of blood flow and cell-level biological activity during vessel morphogenesis. We provide an image acquisition, segmentation, and analysis protocol to quantify endothelial cell polarity in entire in vivo vascular networks. In combination, we use computational fluid dynamics to characterize the hemodynamics of the vascular networks under study. The tool enables, to our knowledge for the first time, a network-level analysis of polarity and flow for individual endothelial cells. To date, PolNet has proven invaluable for the study of endothelial cell polarization and migration during vascular patterning, as demonstrated by two recent publications. Additionally, the tool can be easily extended to correlate blood flow with other experimental observations at the cellular/molecular level. We release the source code of our tool under the Lesser General Public License.

Abstract Contractions of lymphangions, i.e., the segment between two one-way lymphatic valves, generate the pressure gradients that propel lymph back to the circulation. Each lymphangion is comprised of an inner sheet of lymphatic endothelial cells circumscribed by one or more layers of lymphatic muscle cells (LMCs). Each contraction is produced by an LMC action potential (AP) that propagates via gap junctions along the lymphangion. Yet, electrical coupling within and between cell layers and the impact on AP waves is poorly understood. Here, we combine studies in rat and mouse lymphatic vessels with mathematical modeling to show that initiation of AP waves depends on high input resistance (low current drain), whereas propagation depends on morphology and sufficient LMC:LMC coupling. Simulations show that 1) myoendothelial coupling is insignificant to facilitate AP generation and sustain an experimentally measured cross-junctional potential difference of 25 mV, i.e., AP waves propagate along the LMC layer only; 2) LMC:LMC resistance is estimated around 2–10 MΩ but depends on vessel structure and cell-cell coupling, e.g., some degree of LMC overlap protects AP waves against LMC decoupling; 3) the propensity of AP wave initiation is highest around the valves, where the density of LMCs is low; and 4) a single pacemaker cell embedded in the LMC layer must be able to generate very large currents to overcome the current drain from the layer. However, the required current generation to initiate an AP wave is reduced upon stimulation of multiple adjacent LMCs. With stimulation of all LMCs, AP waves can also arise from heterogeneity in the electrical activity of LMCs. The findings advance our understanding of the electrical constraints that underlie initiation of APs in the LMC layer and make testable predictions about how morphology, LMC excitability, and LMC:LMC electrical coupling interact to determine the ability to initiate and propagate AP waves in small lymphatic vessels.

Abstract The viscosity is a highly important parameter within the cell membrane, affecting the diffusion of small molecules and, hence, controlling the rates of intracellular reactions. There is significant interest in the direct, quantitative assessment of membrane viscosity. Here we report the use of fluorescence lifetime imaging microscopy of the molecular rotor BODIPY C10 in the membranes of live Escherichia coli bacteria to permit direct quantification of the viscosity. Using this approach, we investigated the viscosity in live E. coli cells, spheroplasts, and liposomes made from E. coli membrane extracts. For live cells and spheroplasts, the viscosity was measured at both room temperature (23°C) and the E. coli growth temperature (37°C), while the membrane extract liposomes were studied over a range of measurement temperatures (5–40°C). At 37°C, we recorded a membrane viscosity in live E. coli cells of 950 cP, which is considerably higher than that previously observed in other live cell membranes (e.g., eukaryotic cells, membranes of Bacillus vegetative cells). Interestingly, this indicates that E. coli cells exhibit a high degree of lipid ordering within their liquid-phase plasma membranes.

Abstract One of the fundamental features that govern the cooperativity of multiple dyneins during cargo trafficking in cells is the spatial distribution of these dyneins on the cargo. Geometric considerations and recent experiments indicate that clustered distributions of dyneins are required for effective cooperation on micron-sized cargos. However, very little is known about the spatial distribution of dyneins and their cooperativity on smaller cargos, such as vesicles or endosomes <200 nm in size, which are not amenable to conventional immunostaining and optical trapping methods. In this work, we present evidence that dyneins can dynamically be clustered on endosomes in response to load. Using a darkfield imaging assay, we measured the repeated stalls and detachments of retrograde axonal endosomes under load with <10 nm localization accuracy at imaging rates up to 1 kHz for over a timescale of minutes. A three-dimensional stochastic model was used to simulate the endosome motility under load to gain insights on the mechanochemical properties and spatial distribution of dyneins on axonal endosomes. Our results indicate that 1) the distribution of dyneins on endosomes is fluid enough to support dynamic clustering under load and 2) the detachment kinetics of dynein on endosomes differs significantly from the in vitro measurements possibly due to an increase in the unitary stall force of dynein on endosomes.

Abstract Superwarfarins are modified analogs of warfarin with additional lipophilic aromatic rings, up to 100-fold greater potency, and longer biological half-lives. We hypothesized that increased hydrophobicity allowed interactions with amphiphilic membranes and modulation of biological responses. We find that superwarfarins brodifacoum and difenacoum increase lactate production and cell death in neuroblastoma cells. In contrast, neither causes changes in glioma cells that have higher cholesterol content. After choleterol depletion, lactate production was increased and cell viability was reduced. Drug-membrane interactions were examined by surface X-ray scattering using Langmuir monolayers of dipalmitoylphosphatidylcholine and/or cholesterol. Specular X-ray reflectivity data revealed that superwarfarins, but not warfarin, intercalate between dipalmitoylphosphatidylcholine molecules, whereas grazing incidence X-ray diffraction demonstrated changes in lateral crystalline order of the film. Neither agent showed significant interactions with monolayers containing >20% cholesterol. These findings demonstrate an affinity of superwarfarins to biomembranes and suggest that cellular responses to these agents are regulated by cholesterol content.

Abstract Fluid-filled interstitial gaps are a common feature of compact tissues held together by cell-cell adhesion. Although such gaps can in principle be the result of weak, incomplete cell attachment, adhesion is usually too strong for this to occur. Using a mechanical model of tissue cohesion, we show that, instead, a combination of local prevention of cell adhesion at three-cell junctions by fluidlike extracellular material and a reduction of cortical tension at the gap surface are sufficient to generate stable gaps. The size and shape of these interstitial gaps depends on the mechanical tensions between cells and at gap surfaces, and on the difference between intracellular and interstitial pressures that is related to the volume of the interstitial fluid. As a consequence of the dependence on tension/tension ratios, the presence of gaps does not depend on the absolute strength of cell adhesion, and similar gaps are predicted to occur in tissues of widely differing cohesion. Tissue mechanical parameters can also vary within and between cells of a given tissue, generating asymmetrical gaps. Within limits, these can be approximated by symmetrical gaps.

Summary The third UN High-Level Meeting on Non-Communicable Diseases (NCDs) on Sept 27, 2018, will review national and global progress towards the prevention and control of NCDs, and provide an opportunity to renew, reinforce, and enhance commitments to reduce their burden. NCD Countdown 2030 is an independent collaboration to inform policies that aim to reduce the worldwide burden of NCDs, and to ensure accountability towards this aim. In 2016, an estimated 40·5 million (71%) of the 56·9 million worldwide deaths were from NCDs. Of these, an estimated 1·7 million (4% of NCD deaths) occurred in people younger than 30 years of age, 15·2 million (38%) in people aged between 30 years and 70 years, and 23·6 million (58%) in people aged 70 years and older. An estimated 32·2 million NCD deaths (80%) were due to cancers, cardiovascular diseases, chronic respiratory diseases, and diabetes, and another 8·3 million (20%) were from other NCDs. Women in 164 (88%) and men in 165 (89%) of 186 countries and territories had a higher probability of dying before 70 years of age from an NCD than from communicable, maternal, perinatal, and nutritional conditions combined. Globally, the lowest risks of NCD mortality in 2016 were seen in high-income countries in Asia-Pacific, western Europe, and Australasia, and in Canada. The highest risks of dying from NCDs were observed in low-income and middle-income countries, especially in sub-Saharan Africa, and, for men, in central Asia and eastern Europe. Sustainable Development Goal (SDG) target 3.4—a one-third reduction, relative to 2015 levels, in the probability of dying between 30 years and 70 years of age from cancers, cardiovascular diseases, chronic respiratory diseases, and diabetes by 2030—will be achieved in 35 countries (19%) for women, and 30 (16%) for men, if these countries maintain or surpass their 2010–2016 rate of decline in NCD mortality. Most of these are high-income countries with already-low NCD mortality, and countries in central and eastern Europe. An additional 50 (27%) countries for women and 35 (19%) for men are projected to achieve such a reduction in the subsequent decade, and thus, with slight acceleration of decline, could meet the 2030 target. 86 (46%) countries for women and 97 (52%) for men need implementation of policies that substantially increase the rates of decline. Mortality from the four NCDs included in SDG target 3.4 has stagnated or increased since 2010 among women in 15 (8%) countries and men in 24 (13%) countries. NCDs and age groups other than those included in the SDG target 3.4 are responsible for a higher risk of death in low-income and middle-income countries than in high-income countries. Substantial reduction of NCD mortality requires policies that considerably reduce tobacco and alcohol use and blood pressure, and equitable access to efficacious and high-quality preventive and curative care for acute and chronic NCDs.