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.

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.

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.

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.

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.

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.

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.

Multiple developmental mechanisms regulate species-specific jaw size.

Variation in jaw size during evolution has been crucial for the adaptive radiation of vertebrates, yet variation in jaw size during development is often associated with disease. To test the hypothesis that early developmental events regulating neural crest (NC) progenitors contribute to species-specific differences in size, we investigated mechanisms through which two avian species, duck and quail, achieve their remarkably different jaw size. At early stages, duck exhibit an anterior shift in brain regionalization yielding a shorter, broader, midbrain. We find no significant difference in the total number of pre-migratory NC; however, duck concentrate their pre-migratory NC in the midbrain, which contributes to an increase in size of the post-migratory NC population allocated to the mandibular arch. Subsequent differences in proliferation lead to a progressive increase in size of the duck mandibular arch relative to that of quail. To test the role of pre-migratory NC progenitor number in regulating jaw size, we reduced and augmented NC progenitors. In contrast to previous reports of regeneration by NC precursors, we find that neural fold extirpation results in a loss of NC precursors. Despite this reduction in their numbers, post-migratory NC progenitors compensate, producing a symmetric and normal-sized jaw. Our results suggest that evolutionary modification of multiple aspects of NC cell biology, including NC allocation within the jaw primordia and NC-mediated proliferation, have been important to the evolution of jaw size. Furthermore, our finding of NC post-migratory compensatory mechanisms potentially extends the developmental time frame for treatments of disease or injury associated with NC progenitor loss.

Embryonic bauplans and the developmental origins of facial diversity and constraint.

A central issue in biology concerns the presence, timing and nature of phylotypic periods of development, but whether, when and why species exhibit conserved morphologies remains unresolved. Here, we construct a developmental morphospace to show that amniote faces share a period of reduced shape variance and convergent growth trajectories from prominence formation through fusion, after which phenotypic diversity sharply increases. We predict in silico the phenotypic outcomes of unoccupied morphospaces and experimentally validate in vivo that observed convergence is not due to developmental limits on variation but instead from selection against novel trajectories that result in maladaptive facial clefts. These results illustrate how epigenetic factors such as organismal geometry and shape impact facial morphogenesis and alter the locus of adaptive selection to variation in later developmental events.

A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease.

There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small-molecule inhibitor of the WNT pathway discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex-associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a type 1 binding mode involving insertion of the CDK8 C terminus into the ligand binding site. In contrast to type II inhibitors of CDK8 and CDK19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogs alter WNT pathway-regulated gene expression and other on-target effects of modulating CDK8 and CDK19, including expression of genes regulated by STAT1. Consistent with this, we find that phosphorylation of STAT1(SER727) is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors.

Neural crest-mediated bone resorption is a determinant of species-specific jaw length.

Precise control of jaw length during development is crucial for proper form and function. Previously we have shown that in birds, neural crest mesenchyme (NCM) confers species-specific size and shape to the beak by regulating molecular and histological programs for the induction and deposition of cartilage and bone. Here we reveal that a hitherto unrecognized but similarly essential mechanism for establishing jaw length is the ability of NCM to mediate bone resorption. Osteoclasts are considered the predominant cells that resorb bone, although osteocytes have also been shown to participate in this process. In adults, bone resorption is tightly coupled to bone deposition as a means to maintain skeletal homeostasis. Yet, the role and regulation of bone resorption during growth of the embryonic skeleton have remained relatively unexplored. We compare jaw development in short-beaked quail versus long-billed duck and find that quail have substantially higher levels of enzymes expressed by bone-resorbing cells including tartrate-resistant acid phosphatase (TRAP), Matrix metalloproteinase 13 (Mmp13), and Mmp9. Then, we transplant NCM destined to form the jaw skeleton from quail to duck and generate chimeras in which osteocytes arise from quail donor NCM and osteoclasts come exclusively from the duck host. Chimeras develop quail-like jaw skeletons coincident with dramatically elevated expression of TRAP, Mmp13, and Mmp9. To test for a link between bone resorption and jaw length, we block resorption using a bisphosphonate, osteoprotegerin protein, or an MMP13 inhibitor, and this significantly lengthens the jaw. Conversely, activating resorption with RANKL protein shortens the jaw. Finally, we find that higher resorption in quail presages their relatively lower adult jaw bone mineral density (BMD) and that BMD is also NCM-mediated. Thus, our experiments suggest that NCM not only controls bone resorption by its own derivatives but also modulates the activity of mesoderm-derived osteoclasts, and in so doing enlists bone resorption as a key patterning mechanism underlying the functional morphology and evolution of the jaw.

A simple PCR-based strategy for estimating species-specific contributions in chimeras and xenografts.

Many tissue-engineering approaches for repair and regeneration involve transplants between species. Yet a challenge is distinguishing donor versus host effects on gene expression. This study provides a simple molecular strategy to quantify species-specific contributions in chimeras and xenografts. Species-specific primers for reverse transcription quantitative real-time PCR (RT-qPCR) were designed by identifying silent mutations in quail, duck, chicken, mouse and human ribosomal protein L19 (RPL19). cDNA from different pairs of species was mixed in a dilution series and species-specific RPL19 primers were used to generate standard curves. Then quail cells were transplanted into transgenic-GFP chick and resulting chimeras were analyzed with species-specific primers. Fluorescence-activated cell sorting (FACS) confirmed that donor- and host-specific levels of RPL19 expression represent actual proportions of cells. To apply the RPL19 strategy, we measured Runx2 expression in quail-duck chimeras. Elevated Runx2 levels correlated with higher percentages of donor cells. Finally, RPL19 primers also discriminated mouse from human and chick. Thus, this strategy enables chimeras and/or xenografts to be screened rapidly at the molecular level.

Squamous Cell Carcinoma Xenografts: Use of VEGFR2-targeted Microbubbles for Combined Functional and Molecular US to Monitor Antiangiogenic Therapy Effects.

PURPOSE: To assess the ability of vascular endothelial growth factor receptor type 2 (VEGFR2)-targeted and nontargeted ultrasonography (US) to depict antiangiogenic therapy effects and to investigate whether first-pass kinetics obtained with VEGFR2-targeted microbubbles provide independent data about tumor vascularization. MATERIALS AND METHODS: Governmental approval was obtained for animal experiments. Vascularization in response to anti-vascular endothelial growth factor receptor or vehicle-control treatment (10 per group) in HaCaT-ras A-5RT3 xenografts was longitudinally assessed in mice by means of first-pass kinetics of nontargeted microbubbles (BR1, BR38; Bracco, Geneva, Switzerland) and VEGFR2-targeted microbubbles (BR55, Bracco) before and 4, 7, and 14 days after therapy. VEGFR2 expression was determined 8 minutes after BR55 injection with destruction-replenishment analysis. US data were validated with immunohistochemistry. Significant differences were evaluated with the Mann-Whitney test. RESULTS: First-pass analysis with BR1, BR38, and BR55 showed similar tendencies toward decreasing vascularization, with a stronger decrease in tumors treated with anti-VEGF antibody. The median signal intensity (in arbitrary units [au]) of anti-VEGF antibody-treated versus control tumors at day 14 was as follows: BR1, 5.2 au (interquartile range [IQR], 3.2 au) vs 11.3 au (IQR, 10.0 au), respectively; BR38, 6.2 au (IQR, 3.5) vs 10.0 au (IQR, 7.8); and BR55, 9.5 au (IQR, 6.0 au) vs 13.8 au (IQR, 9.8) (P = .0230). VEGFR2 assessment with BR55 demonstrated significant differences between both groups throughout the therapy period (median signal intensity of anti-VEGF antibody-treated vs control tumors: 0.04 au [IQR, 0.1 au] vs 0.14 au [IQR, 0.08 au], respectively, at day 4, P = .0058; 0.04 au [IQR, 0.06 au] vs 0.13 au [IQR, 0.09 au] at day 7, P = .0058; and 0.06 au [IQR, 0.11 au] vs 0.16 au [IQR, 0.15 au] at day 14, P = .0247). Immunohistochemistry confirmed the lower microvessel density and VEGFR2-positive area fraction in tumors treated with anti-VEGF antibody. CONCLUSION: Antiangiogenic therapy effects were detected earlier and more distinctly with VEGFR2-targeted US than with functional US. First-pass analyses with BR55, BR38, and BR1 revealed similar results, with a decrease in vascularization during therapy. Functional data showed that BR55 is not strongly affected by early binding of the microbubbles to VEGFR2. Thus, functional and molecular imaging of angiogenesis can be performed with BR55 within one examination.

Discovery of potent and selective CDK8 inhibitors from an HSP90 pharmacophore.

Here we describe the discovery and optimization of 3-benzylindazoles as potent and selective inhibitors of CDK8, also modulating CDK19, discovered from a high-throughput screening (HTS) campaign sampling the Merck compound collection. The primary hits with strong HSP90 affinity were subsequently optimized to potent and selective CDK8 inhibitors which demonstrate inhibition of WNT pathway activity in cell-based assays. X-ray crystallographic data demonstrated that 3-benzylindazoles occupy the ATP binding site of CDK8 and adopt a Type I binding mode. Medicinal chemistry optimization successfully led to improved potency, physicochemical properties and oral pharmacokinetics. Modulation of phospho-STAT1, a pharmacodynamic biomarker of CDK8, was demonstrated in an APC-mutant SW620 human colorectal carcinoma xenograft model following oral administration.

Divergence of craniofacial developmental trajectories among avian embryos.

BACKGROUND: Morphological divergence among related species involves changes to developmental processes. When such variation arises in development has garnered considerable theoretical interest relating to the broader issue of how development may constrain evolutionary change. The hourglass model holds that while early developmental events may be highly evolvable, there is a phylotypic stage when key developmental events are conserved. Thus, evolutionary divergence among related species should tend to arise after such a stage of reduced evolvability and, consequently, reduced variation among species. We test this prediction by comparing developmental trajectories among three avian species of varying relatedness (chick, quail, and duck) to locate their putative point of divergence. Three-dimensional geometric morphometrics and trajectory analyses were used to measure the significance of the facial shape variation observed among these species. RESULTS: Duck embryos, being more distantly related, differed from the more closely-related chick and quail embryos in the enlargement of their frontonasal prominences. Phenotypic trajectory analyses demonstrated divergence of the three species, most notably, duck. CONCLUSIONS: The results demonstrate that the two more closely related species share similar facial morphologies for a longer time during development, while ducks diverge. This suggests a surprising lability of craniofacial development during early face formation. Developmental Dynamics 244:1158-1167, 2015. © 2015 Wiley Periodicals, Inc.

Evolution of a developmental mechanism: Species-specific regulation of the cell cycle and the timing of events during craniofacial osteogenesis.

Neural crest mesenchyme (NCM) controls species-specific pattern in the craniofacial skeleton but how this cell population accomplishes such a complex task remains unclear. To elucidate mechanisms through which NCM directs skeletal development and evolution, we made chimeras from quail and duck embryos, which differ markedly in their craniofacial morphology and maturation rates. We show that quail NCM, when transplanted into duck, maintains its faster timetable for development and autonomously executes molecular and cellular programs for the induction, differentiation, and mineralization of bone, including premature expression of osteogenic genes such as Runx2 and Col1a1. In contrast, the duck host systemic environment appears to be relatively permissive and supports osteogenesis independently by providing circulating minerals and a vascular network. Further experiments reveal that NCM establishes the timing of osteogenesis by regulating cell cycle progression in a stage- and species-specific manner. Altering the time-course of D-type cyclin expression mimics chimeras by accelerating expression of Runx2 and Col1a1. We also discover higher endogenous expression of Runx2 in quail coincident with their smaller craniofacial skeletons, and by prematurely over-expressing Runx2 in chick embryos we reduce the overall size of the craniofacial skeleton. Thus, our work indicates that NCM establishes species-specific size in the craniofacial skeleton by controlling cell cycle, Runx2 expression, and the timing of key events during osteogenesis.

A tag-free collisionally induced fragmentation approach to detect drug-adducted proteins by mass spectrometry.

RATIONALE: The covalent modification of proteins by toxicants, new chemical entities or drug molecules, either by metabolic activation or the presence of inherently reactive functional groups, is commonly implicated in organ toxicity and idiosyncratic reactions. In efforts to better prosecute protein modifications, we investigated a tag-free technique capable of detecting protein-small molecule adducts based solely on the collision-induced dissociation (CID) of the protein-small molecule complex. Detection of proteins using unique CID small molecule (SM) product ions would mitigate common issues associated with tagging technologies (e.g., altered reactivity/affinity of the protein-SM complex). METHODS: A Waters SYNAPT G2 mass spectrometer (MS) was operated in MS(e) mode with appropriate collision energy conditions during the MS(2) acquisition for fragmentation of protein-small molecule adducts to generate characteristic small molecule product ions. RESULTS: Ibrutinib, an acrylamide-containing small molecule drug, was shown to form adducts with rat serum albumin in ex vivo experiments and these adducts were detected by relying solely on the CID product ions generated from ibrutinib. Additionally, ibrutinib produced three CID product ions, one of which was a selective protein-ibrutinib fragment ion not produced by the compound alone. CONCLUSIONS: Herein we describe a tag-free mass spectral detection technique for protein-small molecule conjugates that relies on the unique product ion fragmentation profile of the small molecule. This technique allows the detection of macromolecular ions containing the adducted small molecule from complex protein matrices through mass range selection for the unique product ions in the CID spectra.

Molecular and cellular changes associated with the evolution of novel jaw muscles in parrots.

Vertebrates have achieved great evolutionary success due in large part to the anatomical diversification of their jaw complex, which allows them to inhabit almost every ecological niche. While many studies have focused on mechanisms that pattern the jaw skeleton, much remains to be understood about the origins of novelty and diversity in the closely associated musculature. To address this issue, we focused on parrots, which have acquired two anatomically unique jaw muscles: the ethmomandibular and the pseudomasseter. In parrot embryos, we observe distinct and highly derived expression patterns for Scx, Bmp4, Tgfß2 and Six2 in neural crest-derived mesenchyme destined to form jaw muscle connective tissues. Furthermore, immunohistochemical analysis reveals that cell proliferation is more active in the cells within the jaw muscle than in surrounding connective tissue cells. This biased and differentially regulated mode of cell proliferation in cranial musculoskeletal tissues may allow these unusual jaw muscles to extend towards their new attachment sites. We conclude that the alteration of neural crest-derived connective tissue distribution during development may underlie the spatial changes in jaw musculoskeletal architecture found only in parrots. Thus, parrots provide valuable insights into molecular and cellular mechanisms that may generate evolutionary novelties with functionally adaptive significance.

Economic and ecological outcomes of flexible biodiversity offset systems.

The commonly expressed goal of biodiversity offsets is to achieve no net loss of specific biological features affected by development. However, strict equivalency requirements may complicate trading of offset credits, increase costs due to restricted offset placement options, and force offset activities to focus on features that may not represent regional conservation priorities. Using the oil sands industry of Alberta, Canada, as a case study, we evaluated the economic and ecological performance of alternative offset systems targeting either ecologically equivalent areas (vegetation types) or regional conservation priorities (caribou and the Dry Mixedwood natural subregion). Exchanging dissimilar biodiversity elements requires assessment via a generalized metric; we used an empirically derived index of biodiversity intactness to link offsets with losses incurred by development. We considered 2 offset activities: land protection, with costs estimated as the net present value of profits of petroleum and timber resources to be paid as compensation to resource tenure holders, and restoration of anthropogenic footprint, with costs estimated from existing restoration projects. We used the spatial optimization tool MARXAN to develop hypothetical offset networks that met either the equivalent-vegetation or conservation-priority targets. Networks that required offsetting equivalent vegetation cost 2-17 times more than priority-focused networks. This finding calls into question the prudence of equivalency-based systems, particularly in relatively undeveloped jurisdictions, where conservation focuses on limiting and directing future losses. Priority-focused offsets may offer benefits to industry and environmental stakeholders by allowing for lower-cost conservation of valued ecological features and may invite discussion on what land-use trade-offs are acceptable when trading biodiversity via offsets. Resultados Económicos y Ecológicos de Sistemas de Compensación de Biodiversidad Flexible Habib et al.

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.