Disruption of Ca2+/calmodulin:KSR1 interaction lowers ERK activation.

KSR1, a key scaffold protein for the MAPK pathway, facilitates ERK activation upon growth factor stimulation. We recently demonstrated that KSR1 binds the Ca2+-binding protein calmodulin (CaM), thereby providing an intersection between KSR1-mediated and Ca2+ signaling. In this study, we set out to generate a KSR1 point mutant with reduced Ca2+/CaM binding in order to unravel the functional implications of their interaction. To do so, we solved the structural determinants of complex formation. Using purified fragments of KSR1, we showed that Ca2+/CaM binds to the CA3 domain of KSR1. We then used in silico molecular modeling to predict contact residues for binding. This approach identified two possible modes of interaction: (1) binding of extended Ca2+/CaM to a globular conformation of KSR1-CA3 via electrostatic interactions or (2) binding of collapsed Ca2+/CaM to α-helical KSR1-CA3 via hydrophobic interactions. Experimentally, site-directed mutagenesis of the predicted contact residues for the two binding models favored that where collapsed Ca2+/CaM binds to the α-helical conformation of KSR1-CA3. Importantly, replacing KSR1-Phe355 with Asp reduces Ca2+/CaM binding by 76%. The KSR1-F355D mutation also significantly impairs the ability of EGF to activate ERK, which reveals that Ca2+/CaM binding promotes KSR1-mediated MAPK signaling. This work, by uncovering structural insight into the binding of KSR1 to Ca2+/CaM, identifies a KSR1 single-point mutant as a bioreagent to selectively study the crosstalk between Ca2+ and KSR1-mediated signaling.

Efficacy and specificity of melanopsin reporters for retinal ganglion cells.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are specialized retinal output neurons that mediate behavioral, neuroendocrine, and developmental responses to environmental light. There are diverse molecular strategies for marking ipRGCs, especially in mice, making them among the best characterized retinal ganglion cells (RGCs). With the development of more sensitive reporters, new subtypes of ipRGCs have emerged. We therefore tested high-sensitivity reporter systems to see whether we could reveal yet more. Substantial confusion remains about which of the available methods, if any, label all and only ipRGCs. Here, we compared many different methods for labeling of ipRGCs, including anti-melanopsin immunofluorescence, Opn4-GFP BAC transgenic mice, and Opn4cre mice crossed with three different Cre-specific reporters (Z/EG, Ai9, and Ai14) or injected with Cre-dependent (DIO) AAV2. We show that Opn4cre mice, when crossed with sensitive Cre-reporter mice, label numerous ganglion cell types that lack intrinsic photosensitivity. Though other methods label ipRGCs specifically, they do not label the entire population of ipRGCs. We conclude that no existing method labels all and only ipRGCs. We assess the appropriateness of each reporter for particular applications and integrate findings across reporters to estimate that the overall abundance of ipRGCs among mouse RGCs may approach 11%.

A high-throughput, 28-day, microfluidic model of gingival tissue inflammation and recovery.

Nearly half of American adults suffer from gum disease, including mild inflammation of gingival tissue, known as gingivitis. Currently, advances in therapeutic treatments are hampered by a lack of mechanistic understanding of disease progression in physiologically relevant vascularized tissues. To address this, we present a high-throughput microfluidic organ-on-chip model of human gingival tissue containing keratinocytes, fibroblast and endothelial cells. We show the triculture model exhibits physiological tissue structure, mucosal barrier formation, and protein biomarker expression and secretion over several weeks. Through inflammatory cytokine administration, we demonstrate the induction of inflammation measured by changes in barrier function and cytokine secretion. These states of inflammation are induced at various time points within a stable culture window, providing a robust platform for evaluation of therapeutic agents. These data reveal that the administration of specific small molecule inhibitors mitigates the inflammatory response and enables tissue recovery, providing an opportunity for identification of new therapeutic targets for gum disease with the potential to facilitate relevant preclinical drug efficacy and toxicity testing.

Precise Quantification of Behavioral Individuality From 80 Million Decisions Across 183,000 Flies.

Individual animals behave differently from each other. This variability is a component of personality and arises even when genetics and environment are held constant. Discovering the biological mechanisms underlying behavioral variability depends on efficiently measuring individual behavioral bias, a requirement that is facilitated by automated, high-throughput experiments. We compiled a large data set of individual locomotor behavior measures, acquired from over 183,000 fruit flies walking in Y-shaped mazes. With this data set we first conducted a "computational ethology natural history" study to quantify the distribution of individual behavioral biases with unprecedented precision and examine correlations between behavioral measures with high power. We discovered a slight, but highly significant, left-bias in spontaneous locomotor decision-making. We then used the data to evaluate standing hypotheses about biological mechanisms affecting behavioral variability, specifically: the neuromodulator serotonin and its precursor transporter, heterogametic sex, and temperature. We found a variety of significant effects associated with each of these mechanisms that were behavior-dependent. This indicates that the relationship between biological mechanisms and behavioral variability may be highly context dependent. Going forward, automation of behavioral experiments will likely be essential in teasing out the complex causality of individuality.

The structural basis of BCR-ABL recruitment of GRB2 in chronic myelogenous leukemia.

BCR-ABL drives chronic myeloid leukemia (CML). BCR binding to GRB2 transduces signaling via the Ras/MAPK pathway. Despite considerable data confirming the binding, molecular-level understanding of exactly how the two proteins interact, and, especially, what are the determinants of the specificity of the SH2GRB2 domain-phosphorylated BCR (pBCR) recognition are still open questions. Yet, this is vastly important for understanding binding selectivity, and for predicting the phosphorylated receptors, or peptides, that are likely to bind. Here, we uncover these determinants and ascertain to what extent they relate to the affinity of the interaction. Toward this end, we modeled the complexes of the pBCR and SH2GRB2 and other pY/Y-peptide-SH2 complexes and compared their specificity and affinity. We observed that pBCR's 176FpYVNV180 motif is favorable and specific to SH2GRB2, similar to pEGFR, but not other complexes. SH2GRB2 contains two binding pockets: pY-binding recognition pocket triggers binding, and the specificity pocket whose interaction is governed by N179 in pBCR and W121 in SH2GRB2. Our proposed motif with optimal affinity to SH2GRB2 is E/D-pY-E/V-N-I/L. Collectively, we provide the structural basis of BCR-ABL recruitment of GRB2, outline its specificity hallmarks, and delineate a blueprint for prediction of BCR-binding scaffolds and for therapeutic peptide design.

The mechanism of activation of MEK1 by B-Raf and KSR1.

MEK1 interactions with B-Raf and KSR1 are key steps in Ras/Raf/MEK/ERK signaling. Despite this, vital mechanistic details of how these execute signal transduction are still enigmatic. Among these is why, despite B-Raf and KSR1 kinase domains similarity, the B-Raf/MEK1 and KSR1/MEK1 complexes have distinct contributions to MEK1 activation, and broadly, what is KSR1's role. Our molecular dynamics simulations clarify these still unresolved ambiguities. Our results reveal that the proline-rich (P-rich) loop of MEK1 plays a decisive role in MEK1 activation loop (A-loop) phosphorylation. In the inactive B-Raf/MEK1 heterodimer, the collapsed A-loop of B-Raf interacts with the P-rich loop and A-loop of MEK1, minimizing MEK1 A-loop fluctuation and preventing it from phosphorylation. In the active B-Raf/MEK1 heterodimer, the P-rich loop moves in concert with the A-loop of B-Raf as it extends. This reduces the number of residues interacting with MEK1 A-loop, allowing increased A-loop fluctuation, and bringing Ser222 closer to ATP for phosphorylation. B-Raf αG-helix Arg662 promotes MEK1 activation by orienting Ser218 towards ATP. In KSR1/MEK1, the KSR1 αG-helix has Ala826 in place of B-Raf Arg662. This difference results in much fewer interactions between KSR1 αG-helix and MEK1 A-loop, thus a more flexible A-loop. We postulate that if KSR1 were to adopt an active configuration with an extended A-loop as seen in other protein kinases, then the MEK1 P-rich loop would extend in a similar manner, as seen in the active B-Raf/MEK1 heterodimer. This would result in highly flexible MEK1 A-loop, and KSR1 functioning as an active, B-Raf-like, kinase.

Allostery: Allosteric Cancer Drivers and Innovative Allosteric Drugs.

Here, we discuss the principles of allosteric activating mutations, propagation downstream of the signals that they prompt, and allosteric drugs, with examples from the Ras signaling network. We focus on Abl kinase where mutations shift the landscape toward the active, imatinib binding-incompetent conformation, likely resulting in the high affinity ATP outcompeting drug binding. Recent pharmacological innovation extends to allosteric inhibitor (GNF-5)-linked PROTAC, targeting Bcr-Abl1 myristoylation site, and broadly, allosteric heterobifunctional degraders that destroy targets, rather than inhibiting them. Designed chemical linkers in bifunctional degraders can connect the allosteric ligand that binds the target protein and the E3 ubiquitin ligase warhead anchor. The physical properties and favored conformational state of the engineered linker can precisely coordinate the distance and orientation between the target and the recruited E3. Allosteric PROTACs, noncompetitive molecular glues, and bitopic ligands, with covalent links of allosteric ligands and orthosteric warheads, increase the effective local concentration of productively oriented and placed ligands. Through covalent chemical or peptide linkers, allosteric drugs can collaborate with competitive drugs, degrader anchors, or other molecules of choice, driving innovative drug discovery.

Gastrectomy mortality in Australia.

BACKGROUND: Despite advances in medical management and endoscopic therapy, gastrectomy remains an important yet high-risk procedure for a range of benign and malignant upper gastrointestinal pathologies. No study has previously analysed Australian gastrectomy perioperative mortality rate (POMR). This retrospective, population-based cohort study was conducted to determine the Australian national gastrectomy POMR, allowing state-based and regional trends and outcomes to be assessed. METHODS: Logistic regression models were compared using de-identified procedural data between 1 July 2005 and 30 June 2017 from the Australian Institute of Health and Welfare. Codes relating to total and subtotal gastrectomy contained in the Australian Classification of Health Interventions were used to extract patient data. Mortality rates were risk adjusted for age and gender. Temporal trends and differences between states/territories and regions were investigated. RESULTS: The national average POMR throughout the study period was 2.1%. For subtotal gastrectomy, the national mean POMR was 1.1%, decreasing from 2.7% (2005) to 1.3% (2017). For total gastrectomy, the national mean POMR was 2.8%, decreasing from 3.3% (2005) to 1.7% (2017). POMR significantly reduced over time without variation between states or regions. Procedure volume steadily reduced in rural centres with a concomitant increase in metropolitan centres over time. CONCLUSION: Pleasingly, the Australian gastrectomy POMR is favourable when compared to international cohorts. Improved outcomes were consistent between states and territories, and metropolitan and regional centres. Progressive metropolitan centralization of gastrectomy was demonstrated without evidence of improved outcomes.

Mechanism of activation and the rewired network: New drug design concepts.

Precision oncology benefits from effective early phase drug discovery decisions. Recently, drugging inactive protein conformations has shown impressive successes, raising the cardinal questions of which targets can profit and what are the principles of the active/inactive protein pharmacology. Cancer driver mutations have been established to mimic the protein activation mechanism. We suggest that the decision whether to target an inactive (or active) conformation should largely rest on the protein mechanism of activation. We next discuss the recent identification of double (multiple) same-allele driver mutations and their impact on cell proliferation and suggest that like single driver mutations, double drivers also mimic the mechanism of activation. We further suggest that the structural perturbations of double (multiple) in cis mutations may reveal new surfaces/pockets for drug design. Finally, we underscore the preeminent role of the cellular network which is deregulated in cancer. Our structure-based review and outlook updates the traditional Mechanism of Action, informs decisions, and calls attention to the intrinsic activation mechanism of the target protein and the rewired tumor-specific network, ushering innovative considerations in precision medicine.

Neuromodulation and Individuality.

Within populations, individuals show a variety of behavioral preferences, even in the absence of genetic or environmental variability. Neuromodulators affect these idiosyncratic preferences in a wide range of systems, however, the mechanism(s) by which they do so is unclear. I review the evidence supporting three broad mechanisms by which neuromodulators might affect variability in idiosyncratic behavioral preference: by being a source of variability directly upstream of behavior, by affecting the behavioral output of a circuit in a way that masks or accentuates underlying variability in that circuit, and by driving plasticity in circuits leading to either homeostatic convergence toward a given behavior or divergence from a developmental setpoint. I find evidence for each of these mechanisms and propose future directions to further understand the complex interplay between individual variability and neuromodulators.

Ras isoform-specific expression, chromatin accessibility, and signaling.

The anchorage of Ras isoforms in the membrane and their nanocluster formations have been studied extensively, including their detailed interactions, sizes, preferred membrane environments, chemistry, and geometry. However, the staggering challenge of their epigenetics and chromatin accessibility in distinct cell states and types, which we propose is a major factor determining their specific expression, still awaits unraveling. Ras isoforms are distinguished by their C-terminal hypervariable region (HVR) which acts in intracellular transport, regulation, and membrane anchorage. Here, we review some isoform-specific activities at the plasma membrane from a structural dynamic standpoint. Inspired by physics and chemistry, we recognize that understanding functional specificity requires insight into how biomolecules can organize themselves in different cellular environments. Within this framework, we suggest that isoform-specific expression may largely be controlled by the chromatin density and physical compaction, which allow (or curb) access to "chromatinized DNA." Genes are preferentially expressed in tissues: proteins expressed in pancreatic cells may not be equally expressed in lung cells. It is the rule-not an exception, and it can be at least partly understood in terms of chromatin organization and accessibility state. Genes are expressed when they can be sufficiently exposed to the transcription machinery, and they are less so when they are persistently buried in dense chromatin. Notably, chromatin accessibility can similarly determine expression of drug resistance genes.

The associations of factors with previous alcohol use in the Northern Territory compared to other states - an observational study.

BACKGROUND: Data on previous alcohol use in surgical patients who died in the Northern Territory (NT) are lacking and have important public health implications. METHODS: The prevalence of previous alcohol (ab)use among surgical patients who died (n=560) was assessed in patients within the Northern Territory and the remainder of Australia (n=28,245) over nine years. RESULTS: The likelihood of previous alcohol use (21.4%; 120 of 560), was the outcome measured and was higher in the Northern Territory than outside it (5.9%; 1,660 of 28,245). Factors associated with the outcome of previous alcohol use were: male gender (aOR 1.6); Aboriginal and Torres Strait Islander status (aOR 2.0); liver disease (aOR 7.8); comorbidities (aOR 2.5); and trauma (aOR 1.1), in both the Northern Territory (aOR 11.5) and all Australia (aOR 7.8). In the Northern Territory, alcohol use was high in both Aboriginal and Torres Strait Islander people (31%) and non-Aboriginal and Torres Strait Islander (16%) people (p=0.316). CONCLUSION: Of surgical patients who died, the likelihood of being a previous alcohol user was double in the Northern Territory as opposed to other states. Alcohol misuse is widespread across all groups in the Northern Territory. Implications for public health: Previous alcohol (ab)use is a negative factor for survival in any racial group.

The mechanism of activation of monomeric B-Raf V600E.

Oncogenic mutations in the serine/threonine kinase B-Raf, particularly the V600E mutation, are frequent in cancer, making it a major drug target. Although much is known about B-Raf's active and inactive states, questions remain about the mechanism by which the protein changes between these two states. Here, we utilize molecular dynamics to investigate both wild-type and V600E B-Raf to gain mechanistic insights into the impact of the Val to Glu mutation. The results show that the wild-type and mutant follow similar activation pathways involving an extension of the activation loop and an inward motion of the αC-helix. The V600E mutation, however, destabilizes the inactive state by disrupting hydrophobic interactions present in the wild-type structure while the active state is stabilized through the formation of a salt bridge between Glu600 and Lys507. Additionally, when the activation loop is extended, the αC-helix is able to move between an inward and outward orientation as long as the DFG motif adopts a specific orientation. In that orientation Phe595 rotates away from the αC-helix, allowing the formation of a salt bridge between Lys483 and Glu501. These mechanistic insights have implications for the development of new Raf inhibitors.

Elective abdominal wall hernia repair surgical mortality-A systematic review of the literature and peer review of mortality in Australia.

BACKGROUND: This study systematically reviewed the literature regarding perioperative mortality in human adults undergoing elective surgical abdominal wall hernia repair, including an audit of the Royal Australasian College of Surgeons (RACS) Australian and New Zealand Audit of Surgical Mortality (ANZASM) database. METHODS: A systematic review was conducted in accordance with PRISMA guidelines for the reporting of systematic reviews and meta-analysis of observational studies. Cochrane Library, PubMed, MEDLINE and Embase database searches and data extraction were conducted from June 1979 to October 2019. Statistical analysis was undertaken utilising denominator values for elective hernia procedures derived from the Australian Institute of Health and Welfare (AIHW) data. Risk-adjusted perioperative mortality rates for the relevant procedures were also produced, using a binary logistic regression for the risk adjustment. RESULTS: Through systematic review of the literature, it was established that the overall reported perioperative mortality in human adults undergoing elective surgical abdominal wall hernia repair was low (0.1%-0.5%). Using ANZASM and AIHW data, the calculated risk-adjusted mortality rate for Australian patients was found to be significantly lower (0.04%-0.06%, p < 0.001). CONCLUSION: The risk-adjusted mortality rate for elective abdominal wall hernia surgery in Australia is very low and compares favourably to international cohorts. Despite low absolute numbers, the factors which were most significantly associated with increased perioperative mortality in patients undergoing elective surgical abdominal wall hernia repair were increased age, cardiorespiratory co-morbidity and incisional hernia repair.

The mechanism of Raf activation through dimerization.

Raf, a threonine/serine kinase in the Raf/MEK/ERK pathway, regulates cell proliferation. Raf's full activation requires dimerization. Aberrant activation through dimerization is an important therapeutic target. Despite its clinical importance, fundamental questions, such as how the side-to-side dimerization promotes the OFF-to-ON transition of Raf's kinase domain and how the fully activated ON-state kinase domain is stabilized in the dimer for Raf signaling, remain unanswered. Herein, we decipher an atomic-level mechanism of Raf activation through dimerization, clarifying this enigma. The mechanism reveals that the replacement of intramolecular π-π stacking by intermolecular π-π stacking at the dimer interface releases the structural constraint of the αC-helix, promoting the OFF-to-ON transition. During the transition, the inhibitory hydrophobic interactions were disrupted, making the phosphorylation sites in A-loop approach the HRD motif for cis-autophosphorylation. Once fully activated, the ON-state kinase domain can be stabilized by a newly identified functional N-terminal basic (NtB) motif in the dimer for Raf signaling. This work provides atomic level insight into critical steps in Raf activation and outlines a new venue for drug discovery against Raf dimerization.

Decoding Neural Responses to Motion-in-Depth Using EEG.

Two stereoscopic cues that underlie the perception of motion-in-depth (MID) are changes in retinal disparity over time (CD) and interocular velocity differences (IOVD). These cues have independent spatiotemporal sensitivity profiles, depend upon different low-level stimulus properties, and are potentially processed along separate cortical pathways. Here, we ask whether these MID cues code for different motion directions: do they give rise to discriminable patterns of neural signals, and is there evidence for their convergence onto a single "motion-in-depth" pathway? To answer this, we use a decoding algorithm to test whether, and when, patterns of electroencephalogram (EEG) signals measured from across the full scalp, generated in response to CD- and IOVD-isolating stimuli moving toward or away in depth can be distinguished. We find that both MID cue type and 3D-motion direction can be decoded at different points in the EEG timecourse and that direction decoding cannot be accounted for by static disparity information. Remarkably, we find evidence for late processing convergence: IOVD motion direction can be decoded relatively late in the timecourse based on a decoder trained on CD stimuli, and vice versa. We conclude that early CD and IOVD direction decoding performance is dependent upon fundamentally different low-level stimulus features, but that later stages of decoding performance may be driven by a central, shared pathway that is agnostic to these features. Overall, these data are the first to show that neural responses to CD and IOVD cues that move toward and away in depth can be decoded from EEG signals, and that different aspects of MID-cues contribute to decoding performance at different points along the EEG timecourse.

Clustering and Phase Separation in Mixtures of Dipolar and Active Particles in an External Field.

Directing the assembly of colloidal particles through the use of external electric or magnetic fields shows promise for the creation of reconfigurable materials. Self-propelled particles can also be used to dynamically drive colloidal systems to nonequilibrium steady states. We investigate colloidal systems that combine both of these methods of directed assembly, simulating mixtures of passive dipolar colloids and active soft spheres in an external magnetic field using Brownian dynamics in two dimensions. In these systems, the dipolar particles align in the direction of the external field, but the active particles are unaffected by the field. The phase behaviors exhibited included a percolated dipolar network, dipolar string-fluid, isotropic fluid, and phase-separated state. We find that the external field allows the dipolar particles to form a percolated network more easily compared to when no external field is present. Additionally, the mixture phase separates at lower active particle velocity in an external field than when no field is present. Our results suggest that combining multiple methods of directing colloidal assembly could lead to new pathways to fabricate reconfigurable materials.