Avoiding ecosystem and social impacts of hydropower, wind, and solar in Southern Africa's low-carbon electricity system.

The scale at which low-carbon electricity will need to be deployed to meet economic growth, electrification, and climate goals in Africa is unprecedented, yet the potential land use and freshwater impacts from this massive build-out of energy infrastructure is poorly understood. In this study, we characterize low-impact onshore wind, solar photovoltaics, and hydropower potential in Southern Africa and identify the cost-optimal mix of electricity generation technologies under different sets of socio-environmental land use and freshwater constraints and carbon targets. We find substantial wind and solar potential after applying land use protections, but about 40% of planned or proposed hydropower projects face socio-environmental conflicts. Applying land and freshwater protections results in more wind, solar, and battery capacity and less hydropower capacity compared to scenarios without protections. While a carbon target favors hydropower, the amount of cost-competitively selected hydropower is at most 45% of planned or proposed hydropower capacity in any scenario-and is only 25% under socio-environmental protections. Achieving both carbon targets and socio-environmental protections results in system cost increases of 3-6%. In the absence of land and freshwater protections, environmental and social impacts from new hydropower development could be significant.

Release of Histone H3K4-reading transcription factors from chromosomes in mitosis is independent of adjacent H3 phosphorylation.

Histone modifications influence the recruitment of reader proteins to chromosomes to regulate events including transcription and cell division. The idea of a histone code, where combinations of modifications specify unique downstream functions, is widely accepted and can be demonstrated in vitro. For example, on synthetic peptides, phosphorylation of Histone H3 at threonine-3 (H3T3ph) prevents the binding of reader proteins that recognize trimethylation of the adjacent lysine-4 (H3K4me3), including the TAF3 component of TFIID. To study these combinatorial effects in cells, we analyzed the genome-wide distribution of H3T3ph and H3K4me2/3 during mitosis. We find that H3T3ph anti-correlates with adjacent H3K4me2/3 in cells, and that the PHD domain of TAF3 can bind H3K4me2/3 in isolated mitotic chromatin despite the presence of H3T3ph. Unlike in vitro, H3K4 readers are still displaced from chromosomes in mitosis in Haspin-depleted cells lacking H3T3ph. H3T3ph is therefore unlikely to be responsible for transcriptional downregulation during cell division.

Acute brain slice elastic modulus decreases over time.

A common benchmark in the brain tissue mechanics literature is that the properties of acute brain slices should be measured within 8 h of the experimental animal being sacrificed. The core assumption is that-since there is no substantial protein degradation during this time-there will be no change to elastic modulus. This assumption overlooks the possibility of other effects (such as osmotic swelling) that may influence the mechanical properties of the tissue. To achieve consistent and accurate analysis of brain mechanics, it is important to account for or mitigate these effects. Using atomic force microscopy (AFM), tissue hydration and volume measurements, we find that acute brain slices in oxygenated artificial cerebrospinal fluid (aCSF) with a standard osmolarity of 300 mOsm/l experience rapid swelling, softening, and increases in hydration within the first 2 hours after slicing. Reductions in elastic modulus can be partly mitigated by addition of chondroitinase ABC enzyme (CHABC). Increasing aCSF osmolarity to 400 mOsm/l does not prevent softening but may hasten equilibration of samples to a point where measurements of relative elastic modulus are consistent across experiments.

Pharmacological approaches to understanding protein kinase signaling networks.

Protein kinases play vital roles in controlling cell behavior, and an array of kinase inhibitors are used successfully for treatment of disease. Typical drug development pipelines involve biological studies to validate a protein kinase target, followed by the identification of small molecules that effectively inhibit this target in cells, animal models, and patients. However, it is clear that protein kinases operate within complex signaling networks. These networks increase the resilience of signaling pathways, which can render cells relatively insensitive to inhibition of a single kinase, and provide the potential for pathway rewiring, which can result in resistance to therapy. It is therefore vital to understand the properties of kinase signaling networks in health and disease so that we can design effective multi-targeted drugs or combinations of drugs. Here, we outline how pharmacological and chemo-genetic approaches can contribute to such knowledge, despite the known low selectivity of many kinase inhibitors. We discuss how detailed profiling of target engagement by kinase inhibitors can underpin these studies; how chemical probes can be used to uncover kinase-substrate relationships, and how these tools can be used to gain insight into the configuration and function of kinase signaling networks.

Robustness of NanoBiT luciferase complementation technology in the presence of widely used kinase inhibitors.

Bioluminescence assays using luciferase enzymes are widely used in research to monitor gene expression and an array of other cell properties, and split luciferase enzymes can be used to measure protein interactions in biochemical assays and in living cells. When these methods are employed in chemical library screening efforts, it is vital that the activity of the luciferase enzyme itself is not strongly influenced by library components. Here, we developed a NanoBiT split luciferase assay to measure phosphorylation of Histone H3 peptides and used it to test the robustness of split luciferase to interference from two libraries of commonly used kinase inhibitors, including the Kinase Chemogenomic Set (KCGS). We found that NanoBiT luciferase is not significantly affected by the great majority of kinase inhibitors tested. However, the weak inhibition observed for a small minority of kinase inhibitors encourages the inclusion of suitable controls in NanoBiT (or NanoLuc) assays.

Dissecting the roles of Haspin and VRK1 in histone H3 phosphorylation during mitosis.

Protein kinases that phosphorylate histones are ideally-placed to influence the behavior of chromosomes during cell division. Indeed, a number of conserved histone phosphorylation events occur prominently during mitosis and meiosis in most eukaryotes, including on histone H3 at threonine-3 (H3T3ph). At least two kinases, Haspin and VRK1 (NHK-1/ballchen in Drosophila), have been proposed to carry out this modification. Phosphorylation of H3 by Haspin has defined roles in mitosis, but the significance of VRK1 activity towards histones in dividing cells has been unclear. Here, using in vitro kinase assays, KiPIK screening, RNA interference, and CRISPR/Cas9 approaches, we were unable to substantiate a direct role for VRK1, or its paralogue VRK2, in the phosphorylation of threonine-3 or serine-10 of Histone H3 in mitosis, although loss of VRK1 did slow cell proliferation. We conclude that the role of VRKs, and their more recently identified association with neuromuscular disease and importance in cancers of the nervous system, are unlikely to involve mitotic histone kinase activity. In contrast, Haspin is required to generate H3T3ph during mitosis.

Reducing adverse impacts of Amazon hydropower expansion.

Proposed hydropower dams at more than 350 sites throughout the Amazon require strategic evaluation of trade-offs between the numerous ecosystem services provided by Earth's largest and most biodiverse river basin. These services are spatially variable, hence collective impacts of newly built dams depend strongly on their configuration. We use multiobjective optimization to identify portfolios of sites that simultaneously minimize impacts on river flow, river connectivity, sediment transport, fish diversity, and greenhouse gas emissions while achieving energy production goals. We find that uncoordinated, dam-by-dam hydropower expansion has resulted in forgone ecosystem service benefits. Minimizing further damage from hydropower development requires considering diverse environmental impacts across the entire basin, as well as cooperation among Amazonian nations. Our findings offer a transferable model for the evaluation of hydropower expansion in transboundary basins.

PCTAIRE1 promotes mitotic progression and resistance against antimitotic and apoptotic signals.

PCTAIRE1 (also known as CDK16) is a serine-threonine kinase implicated in physiological processes like neuronal development, vesicle trafficking, spermatogenesis and cell proliferation. However, its exact role in cell division remains unclear. In this study, using a library screening approach, we identified PCTAIRE1 among several candidates that resisted mitotic arrest and mitotic cell death induced by polyomavirus small T (PolST) expression in mammalian cells. Our study showed that PCTAIRE1 is a mitotic kinase that localizes at centrosomes during G2 and at spindle poles as the cells enter mitosis, and then at the midbody during cytokinesis. We also report that PCTAIRE1 protein levels fluctuate through the cell cycle and reach their peak at mitosis, during which there is an increase in PCTAIRE1 phosphorylation as well. Interestingly, knockdown of PCTAIRE1 resulted in aberrant mitosis by interfering with spindle assembly and chromosome segregation. Further, we found that PCTAIRE1 promotes resistance of cancer cells to antimitotic drugs, and this underscores the significance of PCTAIRE1 as a potential drug target for overcoming chemotherapeutic resistance. Taken together, these studies establish PCTAIRE1 as a critical mediator of mitotic progression and highlight its role in chemotherapeutic resistance. This article has an associated First Person interview with the first author of the paper.

Simultaneous Measurement of Single-Cell Mechanics and Cell-to-Materials Adhesion Using Fluidic Force Microscopy.

The connection between cells and their substrate is essential for biological processes such as cell migration. Atomic force microscopy nanoindentation has often been adopted to measure single-cell mechanics. Very recently, fluidic force microscopy has been developed to enable rapid measurements of cell adhesion. However, simultaneous characterization of the cell-to-material adhesion and viscoelastic properties of the same cell is challenging. In this study, we present a new approach to simultaneously determine these properties for single cells, using fluidic force microscopy. For MCF-7 cells grown on tissue-culture-treated polystyrene surfaces, we found that the adhesive force and adhesion energy were correlated for each cell. Well-spread cells tended to have stronger adhesion, which may be due to the greater area of the contact between cellular adhesion receptors and the surface. By contrast, the viscoelastic properties of MCF-7 cells cultured on the same surface appeared to have little dependence on cell shape. This methodology provides an integrated approach to better understand the biophysics of multiple cell types.

The Aurora B gradient sustains kinetochore stability in anaphase.

Kinetochores assemble on chromosomes in mitosis to allow microtubules to attach and bring about accurate chromosome segregation. The kinases Cyclin B-Cdk1 and Aurora B are crucial for the formation of stable kinetochores. However, the activity of these two kinases appears to decline dramatically at centromeres during anaphase onset, precisely when microtubule attachments are required to move chromosomes toward opposite poles of the dividing cell. We find that, although Aurora B leaves centromeres at anaphase, a gradient of Aurora B activity centered on the central spindle is still able to phosphorylate kinetochore substrates such as Dsn1 to modulate kinetochore stability in anaphase and to regulate kinetochore disassembly as cells enter telophase. We provide a model to explain how Aurora B co-operates with Cyclin B-Cdk1 to maintain kinetochore function in anaphase.

A prometaphase mechanism of securin destruction is essential for meiotic progression in mouse oocytes.

Successful cell division relies on the timely removal of key cell cycle proteins such as securin. Securin inhibits separase, which cleaves the cohesin rings holding chromosomes together. Securin must be depleted before anaphase to ensure chromosome segregation occurs with anaphase. Here we find that in meiosis I, mouse oocytes contain an excess of securin over separase. We reveal a mechanism that promotes excess securin destruction in prometaphase I. Importantly, this mechanism relies on two phenylalanine residues within the separase-interacting segment (SIS) of securin that are only exposed when securin is not bound to separase. We suggest that these residues facilitate the removal of non-separase-bound securin ahead of metaphase, as inhibiting this period of destruction by mutating both residues causes the majority of oocytes to arrest in meiosis I. We further propose that cellular securin levels exceed the amount an oocyte is capable of removing in metaphase alone, such that the prometaphase destruction mechanism identified here is essential for correct meiotic progression in mouse oocytes.

Prioritizing actions: spatial action maps for conservation.

Spatial prioritization is a critical step in conservation planning, a process designed to ensure that limited resources are applied in ways that deliver the highest possible returns for biodiversity and human wellbeing. In practice, many spatial prioritizations fall short of their potential by focusing on places rather than actions, and by using data of snapshots of assets or threats rather than estimated impacts. We introduce spatial action mapping as an approach that overcomes these shortfalls. This approach produces a spatially explicit view of where and how much a given conservation action is likely to contribute to achieving stated conservation goals. Through seven case examples, we demonstrate simple to complex versions of how this method can be applied across local to global scales to inform decisions about a wide range of conservation actions and benefits. Spatial action mapping can support major improvements in efficient use of conservation resources and will reach its full potential as the quality of environmental, social, and economic datasets converge and conservation impact evaluations improve.

Resolution of R-loops by INO80 promotes DNA replication and maintains cancer cell proliferation and viability.

Collisions between the DNA replication machinery and co-transcriptional R-loops can impede DNA synthesis and are a major source of genomic instability in cancer cells. How cancer cells deal with R-loops to proliferate is poorly understood. Here we show that the ATP-dependent chromatin remodelling INO80 complex promotes resolution of R-loops to prevent replication-associated DNA damage in cancer cells. Depletion of INO80 in prostate cancer PC3 cells leads to increased R-loops. Overexpression of the RNA:DNA endonuclease RNAse H1 rescues the DNA synthesis defects and suppresses DNA damage caused by INO80 depletion. R-loops co-localize with and promote recruitment of INO80 to chromatin. Artificial tethering of INO80 to a LacO locus enabled turnover of R-loops in cis. Finally, counteracting R-loops by INO80 promotes proliferation and averts DNA damage-induced death in cancer cells. Our work suggests that INO80-dependent resolution of R-loops promotes DNA replication in the presence of transcription, thus enabling unlimited proliferation in cancers.

Examining the Effects of Whole Crop Wheat Silage on Ewe Performance during Late Gestation Compared to Traditional Grass Silage across Three Prolific Breed Types.

Provision of adequate nutrient intake in late gestation of the ewe is an important determinant of dam and offspring performance. A 2 × 3 factorial design experiment examining two forage types, whole crop wheat silage (WCWS) or grass silage (GS) offered to one of three prolific breed types, (Belclare X, Lleyn X, Mule (Bluefaced Leicester × Blackface Mountain)), was conducted. Forage type had no impact on dry matter (DM) or metabolizable energy (ME) intake, body weight and body condition score change, or colostrum production (p > 0.05). Ewes offered WCWS had lower crude protein (CP) intake (p < 0.0001) and a lower combined litter weight (p < 0.05). Mule ewes consumed less DM, CP, (p < 0.05), and ME (p < 0.01) compared to Belclare X and Lleyn X ewes however, water intake per kg DM consumed did not differ with breed type (p > 0.05). Colostrum yield over the first 18 h postpartum was lower for Mule ewes compared to other breed types (p < 0.05). In conclusion, results from this study suggest nutrient concentration and balance as opposed to forage type is important for late gestation nutrition and breed type can impact feed intake and colostrum yield.

Kinase inhibition profiles as a tool to identify kinases for specific phosphorylation sites.

There are thousands of known cellular phosphorylation sites, but the paucity of ways to identify kinases for particular phosphorylation events remains a major roadblock for understanding kinase signaling. To address this, we here develop a generally applicable method that exploits the large number of kinase inhibitors that have been profiled on near-kinome-wide panels of protein kinases. The inhibition profile for each kinase provides a fingerprint that allows identification of unknown kinases acting on target phosphosites in cell extracts. We validate the method on diverse known kinase-phosphosite pairs, including histone kinases, EGFR autophosphorylation, and Integrin ß1 phosphorylation by Src-family kinases. We also use our approach to identify the previously unknown kinases responsible for phosphorylation of INCENP at a site within a commonly phosphorylated motif in mitosis (a non-canonical target of Cyclin B-Cdk1), and of BCL9L at S915 (PKA). We show that the method has clear advantages over in silico and genetic screening.

Priming chromatin for segregation: functional roles of mitotic histone modifications.

Posttranslational modifications (PTMs) of histone proteins are important for various cellular processes including regulation of gene expression and chromatin structure, DNA damage response and chromosome segregation. Here we comprehensively review mitotic histone PTMs, in particular phosphorylations, and discuss their interplay and functions in the control of dynamic protein-protein interactions as well as their contribution to centromere and chromosome structure and function during cell division. Histone phosphorylations can create binding sites for mitotic regulators such as the chromosomal passenger complex, which is required for correction of erroneous spindle attachments and chromosome bi-orientation. Other histone PTMs can alter the structural properties of nucleosomes and the accessibility of chromatin. Epigenetic marks such as lysine methylations are maintained during mitosis and may also be important for mitotic transcription as well as bookmarking of transcriptional states to ensure the transmission of gene expression programs through cell division. Additionally, histone phosphorylation can dissociate readers of methylated histones without losing epigenetic information. Through all of these processes, mitotic histone PTMs play a functional role in priming the chromatin for faithful chromosome segregation and preventing genetic instability, one of the characteristic hallmarks of cancer cells.

Aneuploidy in Oocytes Is Prevented by Sustained CDK1 Activity through Degron Masking in Cyclin B1.

Successful mitosis requires that cyclin B1:CDK1 kinase activity remains high until chromosomes are correctly aligned on the mitotic spindle. It has therefore been unclear why, in mammalian oocyte meiosis, cyclin B1 destruction begins before chromosome alignment is complete. Here, we resolve this paradox and show that mouse oocytes exploit an imbalance in the ratio of cyclin B1 to CDK1 to control CDK1 activity; early cyclin B1 destruction reflects the loss of an excess of non-CDK1-bound cyclin B1 in late prometaphase, while CDK1-bound cyclin B1 is destroyed only during metaphase. The ordered destruction of the two forms of cyclin B1 is brought about by a previously unidentified motif that is accessible in free cyclin B1 but masked when cyclin B1 is in complex with CDK1. This protects the CDK1-bound fraction from destruction in prometaphase, ensuring a period of prolonged CDK1 activity sufficient to achieve optimal chromosome alignment and prevent aneuploidy.

CDK1-mediated phosphorylation at H2B serine 6 is required for mitotic chromosome segregation.

Faithful mitotic chromosome segregation is required for the maintenance of genomic stability. We discovered the phosphorylation of histone H2B at serine 6 (H2B S6ph) as a new chromatin modification site and found that this modification occurs during the early mitotic phases at inner centromeres and pericentromeric heterochromatin. This modification is directly mediated by cyclin B1-associated CDK1, and indirectly by Aurora B, and is antagonized by PP1-mediated dephosphorylation. H2B S6ph impairs chromatin binding of the histone chaperone SET (I2PP2A), which is important for mitotic fidelity. Injection of phosphorylation-specific H2B S6 antibodies in mitotic cells caused anaphase defects with impaired chromosome segregation and incomplete cytokinesis. As H2B S6ph is important for faithful chromosome separation, this modification may contribute to the prevention chromosomal instability and aneuploidy which frequently occur in cancer cells.

The live cell DNA stain SiR-Hoechst induces DNA damage responses and impairs cell cycle progression.

SiR-Hoechst (SiR-DNA) is a far-red fluorescent DNA probe being used widely for time-lapse imaging of living cells that is reported to be minimally toxic at concentrations as high as 10-25 µM. However, measuring nuclear import of Cyclin B1, inhibition of mitotic entry, and the induction of γH2AX foci in cultured human cells reveals that SiR-Hoechst induces DNA damage responses and G2 arrest at concentrations well below 1 µM. SiR-Hoechst is useful for live cell imaging, but it should be used with caution and at the lowest practicable concentration.

Mitotic Mysteries: The Case of HP1.

The role of Heterochromatin Protein-1 (HP1) during mitosis has been controversial. Two recent studies in Science and Developmental Cell, from Tanno et al. (2015) and Abe et al. (2016), suggest that the means of HP1 localization and its function at inner centromeres are altered in cancer cells with chromosomal instability.