Crossing fibers may underlie the dynamic pulling forces of muscles that attach to cartilage at the tip of the nose.

The present study used microdissection, histology, and microcomputed tomography (micro-CT) with the aims of determining the prevalence and patterns of the depressor septi nasi (DSN) and orbicularis oris (OOr) muscles attached to the footplate of the medial crus (fMC) of the major alar cartilage, focusing on their crossing fibers. The DSN and OOr attached to the fMC of the major alar cartilage were investigated in 76 samples from 38 embalmed Korean adult cadavers (20 males, 18 females; mean age 70 years). The DSN, OOr, or both were attached to the fMC. When the DSN ran unilaterally or was absent, some OOr fibers ascended to attach to the fMC instead of the DSN in 20.6% of the samples. Crossing fibers of the DSN or OOr attached to the fMC were found in 82.4% of the samples. Bilateral and unilateral crossing fibers were found in 32.4% and 50.0%, respectively, and no crossing fibers were found in 17.6%. The DSN and OOr that attached to the fMC could be categorized into six types according to presence of the DSN and the crossing patterns of the DSN and OOr. Anatomical findings of the DSN and OOr that attached to the fMC were confirmed in histology and micro-CT images. These findings offer insights on anatomical mechanisms that may underlie the dynamic pulling forces generated by muscles that attach to the fMCs and on evolutionary variation observed in human facial expressions. They can also provide useful information for guiding rhinoplasty of the nasal tip.

An International Comparison and Review of Self-Induced Intoxication Causing Automatism.

The topic of self-induced intoxication causing automatism is a complex legal question that straddles the border of psychiatry, the law, and social policy. It has been argued that women and children are predominantly positioned as victims of sexual and domestic violence, in which substances often play a part. This consideration sensitizes society to any legal measures that may potentially excuse, mitigate, or absolve perpetrators. The legal systems in Canada, the United States, and the United Kingdom have dealt with these situations as best as they can, sometimes inconsistently and sometimes coming into conflict with the public discourse and subsequent legislation. This article presents a comparison of case law and legislation among these three countries. We review the concept of automatism and self-induced intoxication leading to automatism, and we show how the courts have dealt with this subject.

First report of microsporidians in the non-native shrimp Neocaridina davidi from a temperate European stream.

The release of ornamental pets outside their native range can directly or indirectly impact the recipient community, e.g. via the co-introduction of associated pathogens. However, studies on parasites associated with non-native species, in particular freshwater decapods, have focused mainly on a limited set of pathogens. Here we provide data for the first time on microsporidian parasites of the non-native ornamental shrimp Neocaridina davidi, collected in a stream in Germany. Furthermore, we confirm an ongoing range expansion of the warm-adapted N. davidi from thermally polluted colder water. In the investigated shrimps, the microsporidian parasite Enterocytozoon hepatopenaei and an unknown microsporidian isolate were detected, raising concerns about their transmission potential and pathogenicity on native crustacean species.

Species-specific sensitivity to TGFß signaling and changes to the Mmp13 promoter underlie avian jaw development and evolution.

Precise developmental control of jaw length is critical for survival, but underlying molecular mechanisms remain poorly understood. The jaw skeleton arises from neural crest mesenchyme (NCM), and we previously demonstrated that these progenitor cells express more bone-resorbing enzymes including Matrix metalloproteinase 13 (Mmp13) when they generate shorter jaws in quail embryos versus longer jaws in duck. Moreover, if we inhibit bone resorption or Mmp13, we can increase jaw length. In the current study, we uncover mechanisms establishing species-specific levels of Mmp13 and bone resorption. Quail show greater activation of and sensitivity to transforming growth factor beta (TGFß) signaling than duck; where intracellular mediators like SMADs and targets like Runt-related transcription factor 2 (Runx2), which bind Mmp13, become elevated. Inhibiting TGFß signaling decreases bone resorption, and overexpressing Mmp13 in NCM shortens the duck lower jaw. To elucidate the basis for this differential regulation, we examine the Mmp13 promoter. We discover a SMAD-binding element and single nucleotide polymorphisms (SNPs) near a RUNX2-binding element that distinguish quail from duck. Altering the SMAD site and switching the SNPs abolish TGFß sensitivity in the quail Mmp13 promoter but make the duck promoter responsive. Thus, differential regulation of TGFß signaling and Mmp13 promoter structure underlie avian jaw development and evolution.

Optimization of TEAD P-Site Binding Fragment Hit into In Vivo Active Lead MSC-4106.

The dysregulated Hippo pathway and, consequently, hyperactivity of the transcriptional YAP/TAZ-TEAD complexes is associated with diseases such as cancer. Prevention of YAP/TAZ-TEAD triggered gene transcription is an attractive strategy for therapeutic intervention. The deeply buried and conserved lipidation pocket (P-site) of the TEAD transcription factors is druggable. The discovery and optimization of a P-site binding fragment (1) are described. Utilizing structure-based design, enhancement in target potency was engineered into the hit, capitalizing on the established X-ray structure of TEAD1. The efforts culminated in the optimized in vivo tool MSC-4106, which exhibited desirable potency, mouse pharmacokinetic properties, and in vivo efficacy. In close correlation to compound exposure, the time- and dose-dependent downregulation of a proximal biomarker could be shown.

Anatomical connections among the depressor supercilii, levator labii superioris alaeque nasi, and inferior fibers of orbicularis oculi: Implications for variation in human facial expressions.

The aim of this study was to determine how the depressor supercilii (DS) connects to the levator labii superioris alaeque nasi (LLSAN) and inferior fibers of the orbicularis oculi (OOc INF) in the human midface. While grimacing, contraction of the DS with fibers connecting to the LLSAN and OOc INF can assist in pulling the medial eyebrow downward more than when these connecting fibers are not present. Contraction of these distinct connecting fibers between the DS and the LLSAN can also slightly elevate the nasal ala and upper lip. The DS was examined in 44 specimens of embalmed adult Korean cadavers. We found that the DS connected to the LLSAN or the OOc INF by muscle fibers or thin aponeuroses in 33 (75.0%) of the 44 specimens. The DS was connected to both the LLSAN and OOc INF by muscle fibers or aponeuroses and had no connection to either in 5 (11.4%) and 11 (25.0%) specimens, respectively. The DS was connected to the LLSAN by the muscle fibers and thin aponeuroses in 6 (13.6%) and 4 (9.1%) specimens, respectively. The DS was connected to the OOc INF by the muscle fibers and thin aponeuroses in 5 (11.4%) and 23 (52.3%) specimens, respectively. Our findings regarding the anatomical connections of the glabellar region DS to the midface LLSAN and OOc INF provide insights on the dynamic balance between the brow depressors such as the DS and brow-elevating muscle and contribute to understanding the anatomical origins of individual variation in facial expressions. These results can also improve the safety, predictability, and aesthetics of treatments for the glabellar region with botulinum toxin type A and can be helpful when performing electromyography.

Violence Risk Assessment with the HCR-20V3 in Legal Contexts: A Critical Reflection.

The HCR-20V3 is a violence risk assessment tool that is widely used in forensic clinical practice for risk management planning. The predictive value of the tool, when used in court for legal decision-making, is not yet intensively been studied and questions about legal admissibility may arise. This article aims to provide legal and mental health practitioners with an overview of the strengths and weaknesses of the HCR-20V3 when applied in legal settings. The HCR-20V3 is described and discussed with respect to its psychometric properties for different groups and settings. Issues involving legal admissibility and potential biases when conducting violence risk assessments with the HCR-20V3 are outlined. To explore legal admissibility challenges with respect to the HCR-20V3, we searched case law databases since 2013 from Australia, Canada, Ireland, the Netherlands, New Zealand, the UK, and the USA. In total, we found 546 cases referring to the HCR-20/HCR-20V3. In these cases, the tool was rarely challenged (4.03%), and when challenged, it never resulted in a court decision that the risk assessment was inadmissible. Finally, we provide recommendations for legal practitioners for the cross-examination of risk assessments and recommendations for mental health professionals who conduct risk assessments and report to the court. We conclude with suggestions for future research with the HCR-20V3 to strengthen the evidence base for use of the instrument in legal contexts.

Optimization of a Screening Hit toward M2912, an Oral Tankyrase Inhibitor with Antitumor Activity in Colorectal Cancer Models.

Constitutive activation of the canonical Wnt signaling pathway, in most cases driven by inactivation of the tumor suppressor APC, is a hallmark of colorectal cancer. Tankyrases are druggable key regulators in these malignancies and are considered as attractive targets for therapeutic interventions, although no inhibitor has been progressed to clinical development yet. We continued our efforts to develop tankyrase inhibitors targeting the nicotinamide pocket with suitable drug-like properties for investigating effects of Wnt pathway inhibition on tumor growth. Herein, the identification of a screening hit series and its optimization through scaffold hopping and SAR exploration is described. The systematic assessment delivered M2912, a compound with an optimal balance between excellent TNKS potency, exquisite PARP selectivity, and a predicted human PK compatible with once daily oral dosing. Modulation of cellular Wnt pathway activity and significant tumor growth inhibition was demonstrated with this compound in colorectal xenograft models in vivo.

Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development.

Precisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.

A Scaffold-Free 3-D Co-Culture Mimics the Major Features of the Reverse Warburg Effect In Vitro.

Most tumors consume large amounts of glucose. Concepts to explain the mechanisms that mediate the achievement of this metabolic need have proposed a switch of the tumor mass to aerobic glycolysis. Depending on whether primarily tumor or stroma cells undergo such a commutation, the terms 'Warburg effect' or 'reverse Warburg effect' were coined to describe the underlying biological phenomena. However, current in vitro systems relying on 2-D culture, single cell-type spheroids, or basal-membrane extract (BME/Matrigel)-containing 3-D structures do not thoroughly reflect these processes. Here, we aimed to establish a BME/Matrigel-free 3-D microarray cancer model to recapitulate the metabolic interplay between cancer and stromal cells that allows mechanistic analyses and drug testing. Human HT-29 colon cancer and CCD-1137Sk fibroblast cells were used in mono- and co-cultures as 2-D monolayers, spheroids, and in a cell-chip format. Metabolic patterns were studied with immunofluorescence and confocal microscopy. In chip-based co-cultures, HT-29 cells showed facilitated 3-D growth and increased levels of hexokinase-2, TP53-induced glycolysis and apoptosis regulator (TIGAR), lactate dehydrogenase, and: translocase of outer mitochondrial membrane 20 (TOMM20), when compared with HT-29 mono-cultures. Fibroblasts co-cultured with HT-29 cells expressed higher levels of mono-carboxylate transporter 4, hexokinase-2, microtubule-associated proteins 1A/1B light chain 3, and ubiquitin-binding protein p62 than in fibroblast mono-cultures, in both 2-D cultures and chips. Tetramethylrhodamin-methylester (TMRM) live-cell imaging of chip co-cultures revealed a higher mitochondrial potential in cancer cells than in fibroblasts. The findings demonstrate a crosstalk between cancer cells and fibroblasts that affects cellular growth and metabolism. Chip-based 3-D co-cultures of cancer cells and fibroblasts mimicked features of the reverse Warburg effect.

Routine Optical Clearing of 3D-Cell Cultures: Simplicity Forward.

Three-dimensional cell cultures, such as spheroids and organoids, serve as increasingly important models in fundamental and applied research and start to be used for drug screening purposes. Optical tissue clearing procedures are employed to enhance visualization of fluorescence-stained organs, tissues, and three-dimensional cell cultures. To get a more systematic overview about the effects and applicability of optical tissue clearing on three-dimensional cell cultures, we compared six different clearing/embedding protocols on seven types of spheroid- and chip-based three-dimensional cell cultures of approximately 300 µm in size that were stained with nuclear dyes, immunofluorescence, cell trackers, and cyan fluorescent protein. Subsequent whole mount confocal microscopy and semi-automated image analysis were performed to quantify the effects. Quantitative analysis included fluorescence signal intensity and signal-to-noise ratio as a function of z-depth as well as segmentation and counting of nuclei and immunopositive cells. In general, these analyses revealed five key points, which largely confirmed current knowledge and were quantified in this study. First, there was a massive variability of effects of different clearing protocols on sample transparency and shrinkage as well as on dye quenching. Second, all tested clearing protocols worked more efficiently on samples prepared with one cell type than on co-cultures. Third, z-compensation was imperative to minimize variations in signal-to-noise ratio. Fourth, a combination of sample-inherent cell density, sample shrinkage, uniformity of signal-to-noise ratio, and image resolution had a strong impact on data segmentation, cell counts, and relative numbers of immunofluorescence-positive cells. Finally, considering all mentioned aspects and including a wish for simplicity and speed of protocols - in particular, for screening purposes - clearing with 88% Glycerol appeared to be the most promising option amongst the ones tested.

Demographic and Burn Injury-Specific Variables Associated with Limited Joint Mobility at Discharge in a Multicenter Study.

Burn scar contractures. Existing research on contractures is limited by incomplete analysis of potential contributing variables and differing protocols. This study expands the exploration of contributing variables to include surgery and rehabilitation treatment-related factors. Additionally, this study quantifies direct patient therapy time and patient exposure to rehabilitation prevention therapies. Data from subjects enrolled in the prospective Burn Patient Acuity Demographics, Scar Contractures and Rehabilitation Treatment Related to Patient Outcome Study (ACT) were analyzed to determine variables related to a limited range of motion (limROM) in seven joints and 18 motions (forearm supination) at discharge. Chi-squared and Student's t-test were used accordingly. Multivariate analysis was performed at the patient and joint motion level to control for confounders. Of the 300-member study group, 259 (86.3%) patients had limROM at discharge. Variables independently related to the development of moderate-to-severe limROM on the patient level were larger TBSA, having skin grafted and prolonged bed rest. Variables independently related to moderate-severe limROM on the joint motion level were the percentage of cutaneous functional unit (CFU) burned (P = .044), increase in the length of stay, weight gain, poor compliance with rehabilitation therapy and lower extremity joint burns. Rates of limROM are increased in patients who had larger burns, required surgery, had a greater percentage of the associated CFU burned, and had lower extremity burns. Attention to adequate pain control to ensure rehabilitation tolerance and early ambulation may also decrease limROM at discharge and quicker return to pre-burn activities and employment.

Molecular and cellular mechanisms underlying the evolution of form and function in the amniote jaw.

The amniote jaw complex is a remarkable amalgamation of derivatives from distinct embryonic cell lineages. During development, the cells in these lineages experience concerted movements, migrations, and signaling interactions that take them from their initial origins to their final destinations and imbue their derivatives with aspects of form including their axial orientation, anatomical identity, size, and shape. Perturbations along the way can produce defects and disease, but also generate the variation necessary for jaw evolution and adaptation. We focus on molecular and cellular mechanisms that regulate form in the amniote jaw complex, and that enable structural and functional integration. Special emphasis is placed on the role of cranial neural crest mesenchyme (NCM) during the species-specific patterning of bone, cartilage, tendon, muscle, and other jaw tissues. We also address the effects of biomechanical forces during jaw development and discuss ways in which certain molecular and cellular responses add adaptive and evolutionary plasticity to jaw morphology. Overall, we highlight how variation in molecular and cellular programs can promote the phenomenal diversity and functional morphology achieved during amniote jaw evolution or lead to the range of jaw defects and disease that affect the human condition.

Morphological diversity of integumentary traits in fowl domestication: Insights from disparity analysis and embryonic development.

The domestication of the fowl resulted in a large diversity of integumental structures in chicken breeds. Several integumental traits have been investigated from a developmental genetics perspective. However, their distribution among breeds and their developmental morphology remain unexplored. We constructed a discrete trait-breed matrix and conducted a disparity analysis to investigate the variation of these structures in chicken breeds; 20 integumental traits of 72 chicken breeds and the red junglefowl were assessed. The analyses resulted in slight groupings of breed types comparable to standard breed classification based on artificial selection and chicken type use. The red junglefowl groups together with bantams and European breeds. We provide new data on the red junglefowl and four chicken breeds, demonstrating where and when variation arises during embryonic development. We document variation in developmental timing of the egg tooth and feather formation, as well as other kinds of developmental patterning as in the anlagen of different type of combs. Changes in epithelial-mesenchymal signaling interactions may drive the highly diverse integument in chickens. Experimental and comparative work has revealed that the cranial neural crest mesenchyme mediates its interactions with the overlying epithelium and is the likely source of patterning that generates diversity in integumental structures.

Discovery and Optimization of 2-Arylquinazolin-4-ones into a Potent and Selective Tankyrase Inhibitor Modulating Wnt Pathway Activity.

Tankyrases 1 and 2 (TNKS1/2) are promising pharmacological targets that recently gained interest for anticancer therapy in Wnt pathway dependent tumors. 2-Aryl-quinazolinones were identified and optimized into potent tankyrase inhibitors through SAR exploration around the quinazolinone core and the 4'-position of the phenyl residue. These efforts were supported by analysis of TNKS X-ray and WaterMap structures and resulted in compound 5k, a potent, selective tankyrase inhibitor with favorable pharmacokinetic properties. The X-ray structure of 5k in complex with TNKS1 was solved and confirmed the design hypothesis. Modulation of Wnt pathway activity was demonstrated with this compound in a colorectal xenograft model in vivo.

Liver safety evaluation of endothelin receptor antagonists using HepatoPac® : A single model impact assessment on hepatocellular health, function and bile acid disposition.

Marketed (bosentan, ambrisentan) and discontinued (sitaxsentan, CI-1034) endothelin receptor antagonists were examined in the human micropatterned hepatocyte co-culture (MPCC) model HepatoPac® . Differences across hepatocellular health (cellular adenosine triphosphate/glutathione content), function (urea production/albumin secretion) and taurocholic acid transport (biliary clearance/excretion index) were compared using amiodarone and ciclosporin A as positive controls. Ambrisentan had the weakest potency in all six endpoints, while sitaxsentan, bosentan and CI-1034 had more potent effects on hepatobiliary transport than health/function endpoints. Normalization to clinical Cmax gave the following relative rank order of safety based on margins for each endpoint: ambrisentan ≥ CI-1034 ~ bosentan > sitaxsentan. These data suggested impaired hepatobiliary disposition might contribute to a more prominent role in liver injury associated within sensitive human populations exposed to these compounds than direct hepatocellular toxicity. Rat, dog and monkey MPCCs also showed greater sensitivity potential to disrupted hepatobiliary disposition compared with hepatocellular health/functional endpoints. Drug metabolism competency was exhibited across all species. In vivo, rats and dogs appear more resistant to transaminase elevations and/or histological evidence of liver injury caused by these mechanisms even at exceedingly high systemic exposures relative to sensitive humans. Rats and dogs are resistant to hepatobiliary toxicants due to physiological differences in bile composition/handling. Although traditional animal testing provides adequate safety coverage for advancement of novel pharmaceuticals into clinical trials, supplemental assays employing human MPCCs may strengthen weight-of-evidence predictions for sensitive human populations. Proving the predictive value of this single impact assessment model in advance of clinical trial information for human liver injury risk is needed across more pharmaceuticals.

The Society for Craniofacial Genetics and Developmental Biology 41st Annual Meeting.

The mission of the Society for Craniofacial Genetics and Developmental Biology (SCGDB) is to promote education, research, and communication about normal and abnormal development of the tissues and organs of the head. The SCGDB welcomes as members undergraduate students, graduate students, postdoctoral researchers, medical and dental practitioners, scientists, and academicians who possess an interest in craniofacial biology. Each year our members come together to share their novel findings, build upon, and challenge current knowledge of craniofacial biology.

A novel tankyrase inhibitor, MSC2504877, enhances the effects of clinical CDK4/6 inhibitors.

Inhibition of the PARP superfamily tankyrase enzymes suppresses Wnt/ß-catenin signalling in tumour cells. Here, we describe here a novel, drug-like small molecule inhibitor of tankyrase MSC2504877 that inhibits the growth of APC mutant colorectal tumour cells. Parallel siRNA and drug sensitivity screens showed that the clinical CDK4/6 inhibitor palbociclib, causes enhanced sensitivity to MSC2504877. This tankyrase inhibitor-CDK4/6 inhibitor combinatorial effect is not limited to palbociclib and MSC2504877 and is elicited with other CDK4/6 inhibitors and toolbox tankyrase inhibitors. The addition of MSC2504877 to palbociclib enhances G1 cell cycle arrest and cellular senescence in tumour cells. MSC2504877 exposure suppresses the upregulation of Cyclin D2 and Cyclin E2 caused by palbociclib and enhances the suppression of phospho-Rb, providing a mechanistic explanation for these effects. The combination of MSC2504877 and palbociclib was also effective in suppressing the cellular hyperproliferative phenotype seen in Apc defective intestinal stem cells in vivo. However, the presence of an oncogenic Kras p.G12D mutation in mice reversed the effects of the MSC2504877/palbociclib combination, suggesting one molecular route that could lead to drug resistance.

Neural crest and the origin of species-specific pattern.

For well over half of the 150 years since the discovery of the neural crest, the special ability of these cells to function as a source of species-specific pattern has been clearly recognized. Initially, this observation arose in association with chimeric transplant experiments among differentially pigmented amphibians, where the neural crest origin for melanocytes had been duly noted. Shortly thereafter, the role of cranial neural crest cells in transmitting species-specific information on size and shape to the pharyngeal arch skeleton as well as in regulating the timing of its differentiation became readily apparent. Since then, what has emerged is a deeper understanding of how the neural crest accomplishes such a presumably difficult mission, and this includes a more complete picture of the molecular and cellular programs whereby neural crest shapes the face of each species. This review covers studies on a broad range of vertebrates and describes neural-crest-mediated mechanisms that endow the craniofacial complex with species-specific pattern. A major focus is on experiments in quail and duck embryos that reveal a hierarchy of cell-autonomous and non-autonomous signaling interactions through which neural crest generates species-specific pattern in the craniofacial integument, skeleton, and musculature. By controlling size and shape throughout the development of these systems, the neural crest underlies the structural and functional integration of the craniofacial complex during evolution.

FGF and TGFß signaling link form and function during jaw development and evolution.

How does form arise during development and change during evolution? How does form relate to function, and what enables embryonic structures to presage their later use in adults? To address these questions, we leverage the distinct functional morphology of the jaw in duck, chick, and quail. In connection with their specialized mode of feeding, duck develop a secondary cartilage at the tendon insertion of their jaw adductor muscle on the mandible. An equivalent cartilage is absent in chick and quail. We hypothesize that species-specific jaw architecture and mechanical forces promote secondary cartilage in duck through the differential regulation of FGF and TGFß signaling. First, we perform transplants between chick and duck embryos and demonstrate that the ability of neural crest mesenchyme (NCM) to direct the species-specific insertion of muscle and the formation of secondary cartilage depends upon the amount and spatial distribution of NCM-derived connective tissues. Second, we quantify motility and build finite element models of the jaw complex in duck and quail, which reveals a link between species-specific jaw architecture and the predicted mechanical force environment. Third, we investigate the extent to which mechanical load mediates FGF and TGFß signaling in the duck jaw adductor insertion, and discover that both pathways are mechano-responsive and required for secondary cartilage formation. Additionally, we find that FGF and TGFß signaling can also induce secondary cartilage in the absence of mechanical force or in the adductor insertion of quail embryos. Thus, our results provide novel insights on molecular, cellular, and biomechanical mechanisms that couple musculoskeletal form and function during development and evolution.