Ubiquitin-specific proximity labeling for the identification of E3 ligase substrates.

Protein ubiquitylation controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in disease. Moreover, small-molecule degraders that redirect ubiquitylation activities toward disease targets are an emerging and promising therapeutic class. Over 600 E3 ubiquitin ligases are expressed in humans, but their substrates remain largely elusive, necessitating the development of new methods for their discovery. Here we report the development of E3-substrate tagging by ubiquitin biotinylation (E-STUB), a ubiquitin-specific proximity labeling method that biotinylates ubiquitylated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitylated targets of protein degraders, including collateral targets and ubiquitylation events that do not lead to substrate degradation. It also detects known substrates of E3 ligase CRBN and VHL with high specificity. With the ability to elucidate proximal ubiquitylation events, E-STUB may facilitate the development of proximity-inducing therapeutics and act as a generalizable method for E3-substrate mapping.

Shell shape does not accurately predict self-righting ability in hatchling freshwater turtles.

Flat hydrodynamic shells likely represent an evolutionary trade-off between adaptation to an aquatic lifestyle and the instability of more rounded shells, thought beneficial for self-righting. Trade-offs often result in compromises, this is particularly true when freshwater turtles, with flatter shells, must self-right to avoid the negative effects of inverting. These turtles, theoretically, invest more biomechanical effort to achieve successful and timely self-righting when compared to turtles with rounded carapaces. This increase in effort places these hatchlings in a precarious position; prone to inversion and predation and with shells seemingly maladapted to the act of self-righting. Here, we examine hatchling self-righting performance in three morphologically distinct freshwater turtle species (Apalone spinifera, Chelydra serpentina and Trachemys scripta scripta) that inhabit similar environmental niches. We demonstrate that these hatchlings were capable of rapid self-righting and used considerably less biomechanical effort relative to adult turtles. Despite differences in shell morphology the energetic efficiency of self-righting remained remarkably low and uniform between the three species. Our results confound theoretical predictions of self-righting ability based on shell shape metrics and indicate that other morphological characteristics like neck or tail morphology and shell material properties must be considered to better understand the biomechanical nuances of Testudine self-righting.

Systematic profiling of conditional pathway activation identifies context-dependent synthetic lethalities.

The paradigm of cancer-targeted therapies has focused largely on inhibition of critical pathways in cancer. Conversely, conditional activation of signaling pathways as a new source of selective cancer vulnerabilities has not been deeply characterized. In this study, we sought to systematically identify context-specific gene-activation-induced lethalities in cancer. To this end, we developed a method for gain-of-function genetic perturbations simultaneously across ~500 barcoded cancer cell lines. Using this approach, we queried the pan-cancer vulnerability landscape upon activating ten key pathway nodes, revealing selective activation dependencies of MAPK and PI3K pathways associated with specific biomarkers. Notably, we discovered new pathway hyperactivation dependencies in subsets of APC-mutant colorectal cancers where further activation of the WNT pathway by APC knockdown or direct ß-catenin overexpression led to robust antitumor effects in xenograft and patient-derived organoid models. Together, this study reveals a new class of conditional gene-activation dependencies in cancer.

Biomechanics and the origins of human bipedal walking: The last 50 years.

Motion analysis, as applied to evolutionary biomechanics, has experienced its own evolution over the last 50 years. Here we review how an ever-increasing fossil record, together with continuing advancements in biomechanics techniques, have shaped our understanding of the origin of upright bipedal walking. The original, and long-established hypothesis held by Lamarck (1809), Darwin (1859) and Keith (1934), amongst others, maintained that bipedality originated in an arboreal context. However, the first field studies of gorilla and chimpanzees from the 1960's, highlighted their so-called 'knucklewalking' quadrupedalism, leading scientists to assume, semi-automatically, that knucklewalking must have been the precursor to bipedality. It would not be until the discovery of skeletons of early human relatives Australopithecus afarensis and Australopithecus prometheus, and the inclusion of methods of analysis from computer science, biomechanics, sports science and medicine, that the knucklewalking hypothesis would be most robustly challenged. Their short, but human-like lower limbs and human-like hand indicated that knucklewalking was not part of our ancestral locomotor repertoire. Rather, most current research in evolutionary biomechanics agrees it was a combination of climbing and bipedalism, both in an arboreal context, which facilitated upright, terrestrial, bipedal walking over short distances.

The next big questions in cancer research.

Our understanding of tumorigenesis and cancer progression as well as clinical therapies for different cancer types have evolved dramatically in recent years. However, even with this progress, there are big challenges for scientists and oncologists to tackle, ranging from unpacking the molecular and cellular mechanisms involved to therapeutics and biomarker development to quality of life in the aftermath of therapy. In this article, we asked researchers to comment on the questions that they think are important to address in the coming years.

Investigating the quadrupedal abilities of Scutellosaurus lawleri and its implications for locomotor behavior evolution among dinosaurs.

A reversion to secondary quadrupedality is exceptionally rare in nature, yet the convergent re-evolution of this locomotor style occurred at least four separate times within Dinosauria. Facultative quadrupedality, an intermediate state between obligate bipedality and obligate quadrupedality, may have been an important transitional step in this locomotor shift, and is proposed for a range of basal ornithischians and sauropodomorphs. Advances in virtual biomechanical modeling and simulation have allowed for the investigation of limb anatomy and function in a range of extinct dinosaurian species, yet this technique has not been widely applied to explore facultatively quadrupedal gait generation. This study places its focus on Scutellosaurus, a basal thyreophoran that has previously been described as both an obligate biped and a facultative quadruped. The functional anatomy of the musculoskeletal system (myology, mass properties, and joint ranges of motion) has been reconstructed using extant phylogenetic bracketing and comparative anatomical datasets. This information was used to create a multi-body dynamic locomotor simulation that demonstrates that whil quadrupedal gaits were physically possible, they did not outperform bipedal gaits is any tested metric. Scutellosaurus cannot therefore be described as an obligate biped, but we would predict its use of quadrupedality would be very rare, and perhaps restricted to specific activities such as foraging. This finding suggests that basal thyreophorans are still overwhelmingly bipedal but is perhaps indicative of an adaptive pathway for later evolution of quadrupedality.

Integrated CRISPR screening and drug profiling identifies combination opportunities for EGFR, ALK, and BRAF/MEK inhibitors.

Anti-tumor efficacy of targeted therapies is variable across patients and cancer types. Even in patients with initial deep response, tumors are typically not eradicated and eventually relapse. To address these challenges, we present a systematic screen for targets that limit the anti-tumor efficacy of EGFR and ALK inhibitors in non-small cell lung cancer and BRAF/MEK inhibitors in colorectal cancer. Our approach includes genome-wide CRISPR screens with or without drugs targeting the oncogenic driver ("anchor therapy"), and large-scale pairwise combination screens of anchor therapies with 351 other drugs. Interestingly, targeting of a small number of genes, including MCL1, BCL2L1, and YAP1, sensitizes multiple cell lines to the respective anchor therapy. Data from drug combination screens with EGF816 and ceritinib indicate that dasatinib and agents disrupting microtubules act synergistically across many cell lines. Finally, we show that a higher-order-combination screen with 26 selected drugs in two resistant EGFR-mutant lung cancer cell lines identified active triplet combinations.

Endostructural and periosteal growth of the human humerus.

The growth and development of long bones are of considerable interests in the fields of comparative anatomy and palaeoanthropology, as evolutionary changes and adaptations to specific physical activity patterns are expected to be revealed during bone ontogeny. Traditionally, the cross-sectional geometry of long bones has been examined at discrete locations usually placed at set intervals or fixed percentage distances along the midline axis of the bone shaft. More recently, the technique of morphometric mapping has enabled the continuous analysis of shape variation along the shaft. Here we extend this technique to the full sequence of late fetal and postnatal development of the humeral shaft in a modern human population sample, with the aim of establishing the shape changes during growth and their relationship with the development of the arm musculature and activity patterns. A sample of modern human humeri from individuals of age ranging from 24 weeks in utero to 18 years was imaged using microtomography at multiple resolutions and custom Matlab scripts. Standard biomechanical properties, cortical thickness, surface curvature, and pseudo-landmarks were extracted along radial vectors spaced at intervals of 1° at each 0.5% longitudinal increment measured along the shaft axis. Heat maps were also generated for cortical thickness and surface curvature. The results demonstrate that a whole bone approach to analysis of cross-sectional geometry is more desirable where possible, as there is a continuous pattern of variation along the shaft. It is also possible to discriminate very young individuals and adolescents from other groups by relative cortical thickness, and also by periosteal surface curvature.

Magnesium sulphate intravenously reduces tachycardia side-effects of ß2-agonists.

Intravenous magnesium sulphate allows safer intravenous ß2-agonist delivery in acute-severe and life-threatening asthma attacks https://bit.ly/3veUpfC.

Mediterranean Spur-Thighed Tortoises (Testudo graeca) Have Optimal Speeds at Which They Can Minimise the Metabolic Cost of Transport, on a Treadmill.

Tortoises are famed for their slow locomotion, which is in part related to their herbivorous diet and the constraints imposed by their protective shells. For most animals, the metabolic cost of transport (CoT) is close to the value predicted for their body mass. Testudines appear to be an exception to this rule, as previous studies indicate that, for their body mass, they are economical walkers. The metabolic efficiency of their terrestrial locomotion is explainable by their walking gait biomechanics and the specialisation of their limb muscle physiology, which embodies a predominance of energy-efficient slow-twitch type I muscle fibres. However, there are only two published experimental reports of the energetics of locomotion in tortoises, and these data show high variability. Here, Mediterranean spur-thighed tortoises (Testudo graeca) were trained to walk on a treadmill. Open-flow respirometry and high-speed filming were simultaneously used to measure the metabolic cost of transport and to quantify limb kinematics, respectively. Our data support the low cost of transport previously reported and demonstrate a novel curvilinear relationship to speed in Testudines, suggesting tortoises have an energetically optimal speed range over which they can move in order to minimise the metabolic cost of transport.

Systematic profiling of conditional degron tag technologies for target validation studies.

Conditional degron tags (CDTs) are a powerful tool for target validation that combines the kinetics and reversible action of pharmacological agents with the generalizability of genetic manipulation. However, successful design of a CDT fusion protein often requires a prolonged, ad hoc cycle of construct design, failure, and re-design. To address this limitation, we report here a system to rapidly compare the activity of five unique CDTs: AID/AID2, IKZF3d, dTAG, HaloTag, and SMASh. We demonstrate the utility of this system against 16 unique protein targets. We find that expression and degradation are highly dependent on the specific CDT, the construct design, and the target. None of the CDTs leads to efficient expression and/or degradation across all targets; however, our systematic approach enables the identification of at least one optimal CDT fusion for each target. To enable the adoption of CDT strategies more broadly, we have made these reagents, and a detailed protocol, available as a community resource.

The cost of chewing: The energetics and evolutionary significance of mastication in humans.

Any change in the energetic cost of mammalian mastication will affect the net energy gain from foods. Although the energetic efficiency of masticatory effort is fundamental in understanding the evolution of the human masticatory system, nothing is known currently about the associated metabolic costs of chewing different items. Here, using respirometry and electromyography of the masseter muscle, we demonstrate that chewing by human subjects represents a measurable energy sink. Chewing a tasteless odorless gum elevates metabolic rate by 10 to 15% above basal levels. Energy expenditure increases with gum stiffness and is paid for by greater muscle recruitment. For modern humans, it is likely that mastication represents a small part of the daily energy budget. However, for our ancestors, before the onset of cooking and sophisticated food processing methods, the costs must have been relatively high, adding a previously unexplored energetic dimension to the interpretation of hominin dentofacial fossils.

Comparative optimization of combinatorial CRISPR screens.

Combinatorial CRISPR technologies have emerged as a transformative approach to systematically probe genetic interactions and dependencies of redundant gene pairs. However, the performance of different functional genomic tools for multiplexing sgRNAs vary widely. Here, we generate and benchmark ten distinct pooled combinatorial CRISPR libraries targeting paralog pairs to optimize digenic knockout screens. Libraries composed of dual Streptococcus pyogenes Cas9 (spCas9), orthogonal spCas9 and Staphylococcus aureus (saCas9), and enhanced Cas12a from Acidaminococcus were evaluated. We demonstrate a combination of alternative tracrRNA sequences from spCas9 consistently show superior effect size and positional balance between the sgRNAs as a robust combinatorial approach to profile genetic interactions of multiple genes.

The metabolic cost of turning right side up in the Mediterranean spur-thighed tortoise (Testudo graeca).

Armoured, rigid bodied animals, such as Testudines, must self-right should they find themselves in an inverted position. The ability to self-right is an essential biomechanical and physiological process that influences survival and ultimately fitness. Traits that enhance righting ability may consequently offer an evolutionary advantage. However, the energetic requirements of self-righting are unknown. Using respirometry and kinematic video analysis, we examined the metabolic cost of self-righting in the terrestrial Mediterranean spur-thighed tortoise and compared this to the metabolic cost of locomotion at a moderate, easily sustainable speed. We found that self-righting is, relatively, metabolically expensive and costs around two times the mass-specific power required to walk. Rapid movements of the limbs and head facilitate successful righting however, combined with the constraints of breathing whilst upside down, contribute a significant metabolic cost. Consequently, in the wild, these animals should favour environments or behaviours where the risk of becoming inverted is reduced.

Paralog knockout profiling identifies DUSP4 and DUSP6 as a digenic dependence in MAPK pathway-driven cancers.

Although single-gene perturbation screens have revealed a number of new targets, vulnerabilities specific to frequently altered drivers have not been uncovered. An important question is whether the compensatory relationship between functionally redundant genes masks potential therapeutic targets in single-gene perturbation studies. To identify digenic dependencies, we developed a CRISPR paralog targeting library to investigate the viability effects of disrupting 3,284 genes, 5,065 paralog pairs and 815 paralog families. We identified that dual inactivation of DUSP4 and DUSP6 selectively impairs growth in NRAS and BRAF mutant cells through the hyperactivation of MAPK signaling. Furthermore, cells resistant to MAPK pathway therapeutics become cross-sensitized to DUSP4 and DUSP6 perturbations such that the mechanisms of resistance to the inhibitors reinforce this mechanism of vulnerability. Together, multigene perturbation technologies unveil previously unrecognized digenic vulnerabilities that may be leveraged as new therapeutic targets in cancer.

Are CRISPR Screens Providing the Next Generation of Therapeutic Targets?

CRISPR screens combined with molecular and genetic profiling of large panels of cell lines are helping to systematically identify cancer vulnerabilities. These large-scale screens, together with focused in vivo and isogenic cell line screens, have identified a growing number of promising targets and led directly to numerous target-specific drug discovery programs, several of which have reached clinical testing. However, systematic loss-of-function studies are still in their early stages. Genetic redundancy, the limitation of cell line models for many cancer types, and the difficulty of conducting complex in vitro and in vivo screens remain opportunities for discovery. We expect that over the next few years, efforts like the Cancer Dependency Map along with more focused screens will play a significant role in the creation of a roadmap of oncology therapeutic targets.

Distinct CDK6 complexes determine tumor cell response to CDK4/6 inhibitors and degraders.

CDK4/6 inhibitors (CDK4/6i) are effective in metastatic breast cancer, but they have been only modestly effective in most other tumor types. Here we show that tumors expressing low CDK6 rely on CDK4 function, and are exquisitely sensitive to CDK4/6i. In contrast, tumor cells expressing both CDK4 and CDK6 have increased reliance on CDK6 to ensure cell cycle progression. We discovered that CDK4/6i and CDK4/6 degraders potently bind and inhibit CDK6 selectively in tumors in which CDK6 is highly thermo-unstable and strongly associated with the HSP90/CDC37 complex. In contrast, CDK4/6i and CDK4/6 degraders are ineffective in antagonizing tumor cells expressing thermostable CDK6, due to their weaker binding to CDK6 in these cells. Thus, we uncover a general mechanism of intrinsic resistance to CDK4/6i and CDK4/6i-derived degraders and the need for novel inhibitors targeting the CDK4/6i-resistant, thermostable form of CDK6 for application as cancer therapeutics.

A highly multiplexed quantitative phosphosite assay for biology and preclinical studies.

Reliable methods to quantify dynamic signaling changes across diverse pathways are needed to better understand the effects of disease and drug treatment in cells and tissues but are presently lacking. Here, we present SigPath, a targeted mass spectrometry (MS) assay that measures 284 phosphosites in 200 phosphoproteins of biological interest. SigPath probes a broad swath of signaling biology with high throughput and quantitative precision. We applied the assay to investigate changes in phospho-signaling in drug-treated cancer cell lines, breast cancer preclinical models, and human medulloblastoma tumors. In addition to validating previous findings, SigPath detected and quantified a large number of differentially regulated phosphosites newly associated with disease models and human tumors at baseline or with drug perturbation. Our results highlight the potential of SigPath to monitor phosphoproteomic signaling events and to nominate mechanistic hypotheses regarding oncogenesis, response, and resistance to therapy.

StW 573 Australopithecus prometheus: Its Significance for an Australopith Bauplan.

The StW 573 skeleton of Australopithecus prometheus from Sterkfontein Member 2 is some 93% complete and thus by far the most complete member of that genus yet found. Firmly dated at 3.67 Ma, it is one of the earliest specimens of its genus. A crucial aspect of interpretation of locomotor behaviour from fossil remains is an understanding of the palaeoenvironment in which the individual lived and the manner in which it would have used it. While the value of this ecomorphological approach is largely accepted, it has not been widely used as a stable framework on which to build evolutionary biomechanical interpretations. Here, we collate the available evidence on StW 573's anatomy in order, as far as currently possible, to reconstruct what might have been this individual's realized and potential niche. We explore the concept of a common Australopithecus "bauplan" by comparing the morphology and ecological context of StW 573 to that of paenocontemporaneous australopiths including Australopithecus anamensis and KSD-VP-1/1 Australopithecus afarensis. Each was probably substantially arboreal and woodland-dwelling, relying substantially on arboreal resources. We use a hypothesis-driven approach, tested by: virtual experiments, in the case of extinct species; biomechanical analyses of the locomotor behaviour of living great ape species; and analogical experiments with human subjects. From these, we conclude that the habitual locomotor mode of all australopiths was upright bipedalism, whether on the ground or on branches. Some later australopiths such as Australopithecus sediba undoubtedly became more terrestrial, allowing sacrifice of arboreal stability in favour of manual dexterity. Indeed, modern humans retain arboreal climbing skills but have further sacrificed arboreal effectiveness for enhanced ability to sustain striding terrestrial bipedalism over much greater distances. We compare StW 573's locomotor adaptations to those of living great apes and protohominins, and agree with those earlier observers who suggest that the common panin-hominin last common ancestor was postcranially more like Gorilla than Pan.