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Abstract: The palaeontological site of Langebaanweg (LBW) is internationally renowned for its prolific, diverse and exceptionally well preserved Mio-Pliocene vertebrate faunas. The site is located on the southern West Coast of South Africa which represents a passive intraplate, trailing edge setting. The southern African subcontinent is also removed from glacial influence and has experienced no Cenozoic volcanic activity. Rates of vertical crustal motion are consequently low and Late Cenozoic shoreline datums at LBW chiefly reflect glacio-eustatic sea level history. The primary aim of this study is to clarify the chronology as well as the regional and global context of LBW and to review previous work on these aspects. LBW is ideally situated to document the complex interactions of ocean, atmosphere and land and their respective influence on climate evolution, given its location near the coast and mix of marine, estuarine and terrestrial faunas and depositional settings. This paper also provides a background to the study of the vast existing faunal collections and a guide to undiscovered fossil deposits. Towards these ends, the first detailed geological/topographic maps of the site and surrounds, accompanied by a summary stratigraphic column are provided. Virtual geological modelling using a subsurface database has clarified the spatial and temporal relationships of sedimentary facies, as well as their depositional settings. The geological and palaeontological record at LBW tracks and documents the major regional and global climatic/oceanographic events of the Late Cenozoic. During the Oligocene drawdown in sea levels, the landscape was etched by river incision. Fluctuating sea levels of the Neogene periodically reversed the trend from erosion to deposition, preserving contemporary faunas and floras in the Oligocene palaeovalleys. Earlier Miocene pollen from fluvial facies indicates a humid sub-tropical climate, reflecting a warm southern Atlantic Ocean. The abrupt late Middle Miocene global cooling (Monterey Excursion) coincided with intensified cold upwelling in the Benguela Current and extensive phosphate authigenesis. A globally documented Early Pliocene highstand possibly related to the shoaling of the Isthmus of Panama reached ~90m above sea level (asl), implying extensive melting of the cryosphere. Palaeomagnetic data in tandem with global sea level reconstructions suggested an age of ~5.15±0.1Ma for the faunas and a correlation with the earlier part of this transgression. A subtropical C3 vegetation is indicated by the faunas and floras, but with a significant contribution by sclerophytic fynbos pointing to a cooler and more seasonal climate than in the Miocene. A mid-Pliocene highstand to ~50m asl truncated the Early Pliocene succession at LBW and the globally documented Late Pliocene highstand to ~30m asl saw the Atlantic shoreline approaching LBW for the last time. With the progressive climatic cooling and instability of the terminal Pliocene, culminating in the growth of the Arctic ice cap, strengthening southerly winds driven by a tighter coiled South Atlantic Anticyclone deposited extensive coastal dune fields over the region. [Copyright &y& Elsevier]

Data from stalagmites in the Makapansgat Valley, South Africa, document regional climatic change in southern Africa in the Late Pleistocene and Holocene. A new TIMS U-series dated stalagmite indicates speleothem growth from 24.4 to 12.7 ka and from 10.2 to 0 ka, interrupted by a 2.5 ka hiatus. High-resolution oxygen and carbon stable isotope data suggest that postglacial warming was first initiated ∼17 ka, was interrupted by cooling, probably associated with the Antarctic Cold Reversal, and was followed by strong warming after 13.5 ka. The Early Holocene experienced warm, evaporative conditions with fewer C4 grasses. Cooling is evident from ∼6 to 2.5 ka, followed by warming between 1.5 and 2.5 ka and briefly at ∼AD 1200. Maximum Holocene cooling occurred at AD 1700. The new stalagmite largely confirms results from shorter Holocene stalagmites reported earlier. The strongest variability superimposed on more general trends has a quasi-periodicity between 2.5 and 4.0 ka. Also present are weaker ∼1.0 ka and ∼100-year oscillations, the latter probably solar induced. Given similarities to the Antarctic records, the proximate driving force producing millennial- and centennial-scale changes in the Makapansgat record is postulated to be atmospheric circulation changes associated with change in the Southern Hemisphere circumpolar westerly wind vortex. [Copyright &y& Elsevier]

Far'ah II is an open-air site in the north western Negev desert (Israel). Previous excavations in the 1970's revealed a rich, in situ Middle Paleolithic (MP) assemblage composed of flint and limestone artifacts, animal bones and charcoal. Renewed excavation at the site were undertaken in 2017, to re-date it and provide a more accurate constrain to the sites' age, as well as collect samples for paleoclimatic proxies. Our new Optically Stimulated Luminescence and 14C ages together with the stable oxygen isotope signature of the loess sediments, constrain the age of the upper archaeological horizon to <49 ka. This age agrees with the younger limit of 60–50 ka, obtained by Electron Spin Resonance ages, measured in the 1990's. The heavy δ18O values in carbonates point to cooler climatic conditions than those that prevailed during the preceding short, warm episode between 58 and 49 ka. The fauna, pollen and charcoal collected during the excavation portray a savanna-like environment with a mix of Irano-Turanian and Saharo-Arabian elements and a minor Mediterranean component. The lithic assemblage exhibits large technological variability typical of the Late MP (LMP), amongst which are technological traits that are clearly present in the Initial Upper Paleolithic (IUP) assemblages at the nearby, and roughly contemporaneous, site of Boker Tachtit. If population replacement was the trigger to the MP-UP transition, we would have expected to see a clear break in the cultural material at the onset of the Upper Paleolithic. The semi-arid north western Negev, as an interim region between the Mediterranean and Saharo-Arabian ecozones could have facilitated interaction between populations moving north or south due to fluctuating climatic conditions and changes in population pressures, possibly leading to the emergence of new technological traditions that are observed in the later UP. • Combining 14C and OSL dating with stable isotope analysis the age of Far'ah II is constrained to < 49 ka. • Late MP technological variability at Far'ah II displays traits present in IUP assemblages. • Fauna, pollen and charcoal collected portray an open semi-arid environment. • Heavy δ18O values in carbonates point to cooler climatic conditions. [ABSTRACT FROM AUTHOR]

We conducted the first dynamic downscaling of Last Glacial Maximum (LGM) climate over southern Africa using a regional climate model. Eight coupled global climate model (CGCM) projections of LGM climate were downscaled to 8 km resolution, and compared to a downscaling of present-day climate. It is projected that temperatures were significantly lower during the LGM compared to the present-day, with annual average temperatures 4–6 °C lower along the eastern escarpment, south-north aligned Cape Fold Mountains and western escarpment. Southern Africa is projected to have been generally wetter during the LGM, with a significant extension in the northward reach of frontal rainfall. The largest rainfall increases are projected for the south-north aligned Cape Fold Mountains and the western escarpment of South Africa, but with rainfall decreases projected for the Cape south coast region. Rainfall seasonality is projected to have been significantly different from that of the present-day, with an all-year rainfall region plausibly extending as far north and east as the present-day Free State and Gauteng provinces of South Africa. Evaluations of the downscalings against key published proxy records for the LGM from southern Africa suggest good agreement and few deviations. • This pioneering study is the first to use a dynamic regional climate model to downscale LGM climate over southern Africa. • It provides a new source of information towards the reconstruction of LGM climate over southern Africa. • The simulations are indicative of a northward reach of frontal rainfall, confirming the interpretation of the proxy record. • The simulations provide an explanation for the observed zonal gradient in plant diversity within the GCFR in South Africa. • If suitably bias-corrected, the simulations may be used to reconstruct and interpret the palaeoscape of southern Africa. [ABSTRACT FROM AUTHOR]

Translation fidelity is crucial for prokaryotes and eukaryotic nuclear‐encoded proteins; however, little is known about the role of mistranslation in mitochondria and its potential effects on metabolism. We generated yeast and mouse models with error‐prone and hyper‐accurate mitochondrial translation, and found that translation rate is more important than translational accuracy for cell function in mammals. Specifically, we found that mitochondrial mistranslation causes reduced overall mitochondrial translation and respiratory complex assembly rates. In mammals, this effect is compensated for by increased mitochondrial protein stability and upregulation of the citric acid cycle. Moreover, this induced mitochondrial stress signaling, which enables the recovery of mitochondrial translation via mitochondrial biogenesis, telomerase expression, and cell proliferation, and thereby normalizes metabolism. Conversely, we show that increased fidelity of mitochondrial translation reduces the rate of protein synthesis without eliciting a mitochondrial stress response. Consequently, the rate of translation cannot be recovered and this leads to dilated cardiomyopathy in mice. In summary, our findings reveal mammalian‐specific signaling pathways that respond to changes in the fidelity of mitochondrial protein synthesis and affect metabolism. Synopsis: Experimental manipulation of the fidelity of mitochondrial protein synthesis reveals that translation rate is more critical than translational accuracy. Error‐prone translation elicits stress responses to rescue energy metabolism, while increased fidelity cannot induce such a compensatory response. Mitochondrial mistranslation causes changes in mitochondrial metabolism in mouse liver that recover over time.Stress signalling enables recovery from mistranslation by stimulating cell proliferation and mitochondrial biogenesis.High‐fidelity translation reduces mitochondrial protein synthesis without eliciting a compensatory response, resulting in dilated cardiomyopathy. [ABSTRACT FROM AUTHOR]

Chronic inflammation is a constitutive component of many age-related diseases, including age-related macular degeneration (AMD). Here, we identified interleukin-1 receptor-associated kinase M (IRAK-M) as a key immunoregulator in retinal pigment epithelium (RPE) that declines during the aging process. Rare genetic variants of IRAK3 , which encodes IRAK-M, were associated with an increased likelihood of developing AMD. In human samples and mouse models, IRAK-M abundance in the RPE declined with advancing age or exposure to oxidative stress and was further reduced in AMD. Irak3 -knockout mice exhibited an increased incidence of outer retinal degeneration at earlier ages, which was further exacerbated by oxidative stressors. The absence of IRAK-M led to a disruption in RPE cell homeostasis, characterized by compromised mitochondrial function, cellular senescence, and aberrant cytokine production. IRAK-M overexpression protected RPE cells against oxidative or immune stressors. Subretinal delivery of adeno-associated virus (AAV)-expressing human IRAK3 rescued light-induced outer retinal degeneration in wild-type mice and attenuated age-related spontaneous retinal degeneration in Irak3 -knockout mice. Our data show that replenishment of IRAK-M in the RPE may redress dysregulated pro-inflammatory processes in AMD, suggesting a potential treatment for retinal degeneration.

The heterodimeric transcription factor, hypoxia inducible factor-1 (HIF-1), is an important anticancer target as it supports the adaptation and response of tumors to hypoxia. Here, we optimized the repressed transactivator yeast two-hybrid system to further develop it as part of a versatile yeast-based drug discovery platform and validated it using HIF-1. We demonstrate both fluorescence-based and auxotrophy-based selections that could detect HIF-1α/HIF-1β dimerization inhibition. The engineered genetic selection is tunable and able to differentiate between strong and weak interactions, shows a large dynamic range, and is stable over different growth phases. Furthermore, we engineered mechanisms to control for cellular activity and off-target drug effects. We thoroughly characterized all parts of the biosensor system and argue this tool will be generally applicable to a wide array of protein-protein interaction targets. We anticipate this biosensor will be useful as part of a drug discovery platform, particularly when screening DNA-encoded new modality drugs.

The global impact of synthetic biology has been accelerating, because of the plummeting cost of DNA synthesis, advances in genetic engineering, growing understanding of genome organization, and explosion in data science. However, much of the discipline's application in the pharmaceutical industry remains enigmatic. In this review, we highlight recent examples of the impact of synthetic biology on target validation, assay development, hit finding, lead optimization, and chemical synthesis, through to the development of cellular therapeutics. We also highlight the availability of tools and technologies driving the discipline. Synthetic biology is certainly impacting all stages of drug discovery and development, and the recognition of the discipline's contribution can further enhance the opportunities for the drug discovery and development value chain.

It remains unresolved how retinal pigment epithelial cell metabolism is regulated following immune activation to maintain retinal homeostasis and retinal function. We exposed retinal pigment epithelium (RPE) to several stress signals, particularly Toll-like receptor stimulation, and uncovered an ability of RPE to adapt their metabolic preference on aerobic glycolysis or oxidative glucose metabolism in response to different immune stimuli. We have identified interleukin-33 (IL-33) as a key metabolic checkpoint that antagonizes the Warburg effect to ensure the functional stability of the RPE. The identification of IL-33 as a key regulator of mitochondrial metabolism suggests roles for the cytokine that go beyond its extracellular "alarmin" activities. IL-33 exerts control over mitochondrial respiration in RPE by facilitating oxidative pyruvate catabolism. We have also revealed that in the absence of IL-33, mitochondrial function declined and resultant bioenergetic switching was aligned with altered mitochondrial morphology. Our data not only shed new light on the molecular pathway of activation of mitochondrial respiration in RPE in response to immune stressors but also uncover a potentially novel role of nuclear intrinsic IL-33 as a metabolic checkpoint regulator.

Many conventional, modern genome engineering tools cannot be used to study mitochondrial genetics due to the unusual structure and physiology of the mitochondrial genome. Here, we review a number of newly developed, synthetic biology-based approaches for altering levels of mutant mammalian mitochondrial DNA and mitochondrial RNAs, including transcription activator-like effector nucleases, zinc finger nucleases and engineered RNA-binding proteins. These approaches allow researchers to manipulate and visualize mitochondrial processes and may provide future therapeutics. This article is part of the theme issue 'Linking the mitochondrial genotype to phenotype: a complex endeavour'.

Intrinsic protein properties that may not be apparent by only examining three-dimensional structures can be revealed by careful analysis of mutant protein variants. Deep mutational scanning is a technique that allows the functional analysis of millions of protein variants in a single experiment. To enable this high-throughput technique, the mutant genotype of protein variants must be coupled to a selectable function. This chapter outlines how artificial genetic circuits in the yeast Saccharomyces cerevisiae can maintain the genotype-phenotype link, thus enabling the general application of this approach. To do this, we describe how to engineer genetic selections in yeast, methods to construct mutant libraries, and how to analyze sequencing data. We investigate the structure-function relationships of the antimicrobial resistance protein TetX to illustrate this process. In doing so, we demonstrate that deep mutational scanning is a powerful method to dissect the importance of individual residues for the inactivation of antibiotic analogues, with consequences for the rational design of new drugs to combat antimicrobial resistance., (© 2020 Elsevier Inc. All rights reserved.)

Background/aims: Hypoxia of the retina is a common pathogenic drive leading to vision loss as a result of tissue ischemia, increased vascular permeability and ultimately retinal neovascularisation. Here we tested the hypothesis that Müller cells stabilize the neurovascular unit, microvasculature by suppression of HIF-1α activation as a result of hypoxic preconditioning., Methods: Tube Formation Assay and In vitro Vascular Permeability Image Assay were used to analyze angiogenesis and vascular integrity. Seahorse XF Cell Mito Stress Test was used to measure mitochondrial respiration. Gene and protein expression were examined by qRTPCR, ELISA and western blot., Results: Hypoxic insult induces a significant induction of proangiogenic factors including vascular endothelial growth factor (VEGF) and angiopoietinlike 4 (ANGPTL-4) resulting in angiogenesis and increased vascular permeability of vascular endothelial cells. Hypoxic preconditioning of a human retinal Müller glia cell line significantly attenuates HIF-1α activation through the inhibition of mTOR and concomitant induction of aerobic glycolysis, stabilizing endothelial cells., Conclusion: Hypoxic preconditioning of Müller cells confers a robust protection to endothelial cells, through the suppression of HIF1α activation and its downstream regulation of VEGF and ANGPTL-4., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Understanding the molecular mechanisms underlying antibiotic resistance requires concerted efforts in enzymology and medicinal chemistry. Here we describe a new synthetic biology approach to antibiotic development, where the presence of tetracycline antibiotics is linked to a life-death selection in Saccharomyces cerevisiae. This artificial genetic circuit allowed the deep mutational scanning of the tetracycline inactivating enzyme TetX, revealing key functional residues. We used both positive and negative selections to confirm the importance of different residues for TetX activity, and profiled activity hotspots for different tetracyclines to reveal substrate-specific activity determinants. We found that precise positioning of FAD and hydrophobic shielding of the tetracycline are critical for enzymatic inactivation of doxycycline. However, positioning of FAD is suboptimal in the case of anhydrotetracycline, potentially explaining its comparatively poor degradation and potential as an inhibitor for this family of enzymes. By combining artificial genetic circuits whose function can be modulated by antimicrobial resistance determinants, we establish a framework to select for the next generation of antibiotics.

IL-1 family members regulate innate immune responses, are produced by gestation-associated tissues, and have a role in healthy and adverse pregnancy outcomes. To better understand their role at the materno-fetal interface we used a human tissue explant model to map lipopolysaccharide (LPS)-stimulated production of IL-1α, IL-1β, IL-18, IL-33, IL-1Ra, IL-18BPa, ST2 and IL-1RAcP by placenta, choriodecidua and amnion. Caspase-dependent processing of IL-1α, IL-1β, IL-18, and IL-33 and the ability of IL-1α, IL-1β, IL-18, and IL-33 to regulate the production of IL-1RA, IL-18BPa, ST2 and IL-1RAcP was also determined. LPS acted as a potent inducer of IL-1 family member expression especially in the placenta and choriodecidua with the response by the amnion restricted to IL-1β. Caspases-1, 4 and 8 contributed to LPS-stimulated production of IL-1β and IL-18, whereas calpain was required for IL-1α production. Exogenous administration of IL-1α, IL-1β, IL-18, and IL-33 lead to differential expression of IL-1Ra, IL-18BPa, ST2 and IL-1RAcP across all tissues examined. Most notable were the counter-regulatory effect of LPS on IL-1β and IL-1Ra in the amnion and the broad responsiveness of the amnion to IL-1 family cytokines for increased production of immunomodulatory peptides and soluble receptors. The placenta and membranes vary not only in their output of various IL-1 family members but also in their counter-regulatory mechanisms through endogenous inhibitory peptides, processing enzymes and soluble decoy receptors. This interactive network of inflammatory mediators likely contributes to innate defence mechanisms at the materno-fetal interface to limit, in particular, the detrimental effects of microbial invasion., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Mitochondrial gene expression is predominantly regulated at the post-transcriptional level and mitochondrial ribonucleic acid (RNA)-binding proteins play a key role in RNA metabolism and protein synthesis. The AU-binding homolog of enoyl-coenzyme A (CoA) hydratase (AUH) is a bifunctional protein with RNA-binding activity and a role in leucine catabolism. AUH has a mitochondrial targeting sequence, however, its role in mitochondrial function has not been investigated. Here, we found that AUH localizes to the inner mitochondrial membrane and matrix where it associates with mitochondrial ribosomes and regulates protein synthesis. Decrease or overexpression of the AUH protein in cells causes defects in mitochondrial translation that lead to changes in mitochondrial morphology, decreased mitochondrial RNA stability, biogenesis and respiratory function. Because of its role in leucine metabolism, we investigated the importance of the catalytic activity of AUH and found that it affects the regulation of mitochondrial translation and biogenesis in response to leucine., (© The Author(s) 2014. Published by Oxford University Press.)