Human SRMAtlas: A Resource of Targeted Assays to Quantify the Complete Human Proteome.

The ability to reliably and reproducibly measure any protein of the human proteome in any tissue or cell type would be transformative for understanding systems-level properties as well as specific pathways in physiology and disease. Here, we describe the generation and verification of a compendium of highly specific assays that enable quantification of 99.7% of the 20,277 annotated human proteins by the widely accessible, sensitive, and robust targeted mass spectrometric method selected reaction monitoring, SRM. This human SRMAtlas provides definitive coordinates that conclusively identify the respective peptide in biological samples. We report data on 166,174 proteotypic peptides providing multiple, independent assays to quantify any human protein and numerous spliced variants, non-synonymous mutations, and post-translational modifications. The data are freely accessible as a resource at http://www.srmatlas.org/, and we demonstrate its utility by examining the network response to inhibition of cholesterol synthesis in liver cells and to docetaxel in prostate cancer lines.

Twist1 controls a cell-specification switch governing cell fate decisions within the cardiac neural crest.

Neural crest cells are multipotent progenitor cells that can generate both ectodermal cell types, such as neurons, and mesodermal cell types, such as smooth muscle. The mechanisms controlling this cell fate choice are not known. The basic Helix-loop-Helix (bHLH) transcription factor Twist1 is expressed throughout the migratory and post-migratory cardiac neural crest. Twist1 ablation or mutation of the Twist-box causes differentiation of ectopic neuronal cells, which molecularly resemble sympathetic ganglia, in the cardiac outflow tract. Twist1 interacts with the pro-neural factor Sox10 via its Twist-box domain and binds to the Phox2b promoter to repress transcriptional activity. Mesodermal cardiac neural crest trans-differentiation into ectodermal sympathetic ganglia-like neurons is dependent upon Phox2b function. Ectopic Twist1 expression in neural crest precursors disrupts sympathetic neurogenesis. These data demonstrate that Twist1 functions in post-migratory neural crest cells to repress pro-neural factors and thereby regulate cell fate determination between ectodermal and mesodermal lineages.

An integrated quantification method to increase the precision, robustness, and resolution of protein measurement in human plasma samples.

BACKGROUND: Current quantification methods for mass spectrometry (MS)-based proteomics either do not provide sufficient control of variability or are difficult to implement for routine clinical testing. RESULTS: We present here an integrated quantification (InteQuan) method that better controls pre-analytical and analytical variability than the popular quantification method using stable isotope-labeled standard peptides (SISQuan). We quantified 16 lung cancer biomarker candidates in human plasma samples in three assessment studies, using immunoaffinity depletion coupled with multiple reaction monitoring (MRM) MS. InteQuan outperformed SISQuan in precision in all three studies and tolerated a two-fold difference in sample loading. The three studies lasted over six months and encountered major changes in experimental settings. Nevertheless, plasma proteins in low ng/ml to low µg/ml concentrations were measured with a median technical coefficient of variation (CV) of 11.9% using InteQuan. The corresponding median CV using SISQuan was 15.3% after linear fitting. Furthermore, InteQuan surpassed SISQuan in measuring biological difference among clinical samples and in distinguishing benign versus cancer plasma samples. CONCLUSIONS: We demonstrated that InteQuan is a simple yet robust quantification method for MS-based quantitative proteomics, especially for applications in biomarker research and in routine clinical testing.

EMT inducers catalyze malignant transformation of mammary epithelial cells and drive tumorigenesis towards claudin-low tumors in transgenic mice.

The epithelial-mesenchymal transition (EMT) is an embryonic transdifferentiation process consisting of conversion of polarized epithelial cells to motile mesenchymal ones. EMT-inducing transcription factors are aberrantly expressed in multiple tumor types and are known to favor the metastatic dissemination process. Supporting oncogenic activity within primary lesions, the TWIST and ZEB proteins can prevent cells from undergoing oncogene-induced senescence and apoptosis by abolishing both p53- and RB-dependent pathways. Here we show that they also downregulate PP2A phosphatase activity and efficiently cooperate with an oncogenic version of H-RAS in malignant transformation of human mammary epithelial cells. Thus, by down-regulating crucial tumor suppressor functions, EMT inducers make cells particularly prone to malignant conversion. Importantly, by analyzing transformed cells generated in vitro and by characterizing novel transgenic mouse models, we further demonstrate that cooperation between an EMT inducer and an active form of RAS is sufficient to trigger transformation of mammary epithelial cells into malignant cells exhibiting all the characteristic features of claudin-low tumors, including low expression of tight and adherens junction genes, EMT traits, and stem cell-like characteristics. Claudin-low tumors are believed to be the most primitive breast malignancies, having arisen through transformation of an early epithelial precursor with inherent stemness properties and metaplastic features. Challenging this prevailing view, we propose that these aggressive tumors arise from cells committed to luminal differentiation, through a process driven by EMT inducers and combining malignant transformation and transdifferentiation.

Raising Capacity to Address Mental Health Concerns in Costa Rica.

Background A family medicine team based out of Mayo Clinic, Rochester assembled in 2019 to provide home visits and direct care to underserved populations of patients in La Cruz, Costa Rica. In addition to the provision of direct patient care, our team was interested in conducting a community needs-based assessment to identify an area for provider education efforts and the local providers on a chronic health issue using local feedback and physician data. Suicide awareness and prevention were identified as a priority based on rising suicide rates as well as limited psychiatry services in the area, with some major providences having ~0.60 psychiatrists available per 100,000 people. Our group provided a half-day educational course on mental health topics related to suicide awareness for local health workers. The primary objective of this study was to evaluate any lasting changes in practice, confidence, and knowledge among local health workers attributable to our training and add to the limited research on this topic. Methods Two groups of participants (81) from local hospitals were recruited via local providers and divided into two morning and afternoon groups on a single day. Each group comprised primary care providers, nurses, social workers, and finance officers. Both were given the same educational presentation that could be broadly applied to each various role. Our team provided lectures on mental health, as well as how to improve personal resilience. Locally medically trained translators were used. Pre and post-lecture surveys gathered demographic data, experience with these mental health issues, and confidence in addressing mental health concerns. Pre and post-lecture surveys, including open-ended as well as Likert scale and multiple-choice questions, were handed out at the beginning and end of each lecture to all participants. A four to six months follow-up survey was delivered by email using SurveyMonkey to evaluate retention and impact of educational materials. Results The initial two groups of participants (n = 81) were aged 23-60 years (mean: 43), and 67% (39) were female. Work experience ranged from 0 to 37 years (mean: 14) with 64% (37) doing direct patient care. Preliminary lecture content data from participants (n = 44) demonstrated an overall increase in correct responses by +15.4% from the pre-test (percent correct, 38.1%) to post-test (53.5%, p < 0.01). Individuals (n = 55) with past exposure to suicide were much more likely to report asking patients about suicide than those with no prior exposure (56.3% vs. 8.3%; p < 0.01). At the six-month follow-up with participants (n = 11), when asked about their confidence in learning objectives from the lecture given prior, the rates of low confidence decreased as well as the level of high confidence improved but was not statistically significant. The rate of low confidence of respondents' confidence in asking about mental health concerns decreased from 35.2% to 0% (p < 0.01). Conclusions Our group was able to successfully deliver lectures to a mixed audience of health workers in a region self-identified as struggling with mental health issues in Costa Rica. The surveys suggested learning occurred. A trend suggestive that the educational content improved the participants' confidence and knowledge components over time was noted. Future service trips may be able to build on this initial experience to improve on ways to raise capacity while delivering direct care to regions in need.

Twist1 promotes heart valve cell proliferation and extracellular matrix gene expression during development in vivo and is expressed in human diseased aortic valves.

During embryogenesis the heart valves develop from undifferentiated mesenchymal endocardial cushions (EC), and activated interstitial cells of adult diseased valves share characteristics of embryonic valve progenitors. Twist1, a class II basic-helix-loop-helix (bHLH) transcription factor, is expressed during early EC development and is down-regulated later during valve remodeling. The requirements for Twist1 down-regulation in the remodeling valves and the consequences of prolonged Twist1 activity were examined in transgenic mice with persistent expression of Twist1 in developing and mature valves. Persistent Twist1 expression in the remodeling valves leads to increased valve cell proliferation, increased expression of Tbx20, and increased extracellular matrix (ECM) gene expression, characteristic of early valve progenitors. Among the ECM genes predominant in the EC, Col2a1 was identified as a direct transcriptional target of Twist1. Increased Twist1 expression also leads to dysregulation of fibrillar collagen and periostin expression, as well as enlarged hypercellular valve leaflets prior to birth. In human diseased aortic valves, increased Twist1 expression and cell proliferation are observed adjacent to nodules of calcification. Overall, these data implicate Twist1 as a critical regulator of valve development and suggest that Twist1 influences ECM production and cell proliferation during disease.

Notch and transforming growth factor-beta (TGFbeta) signaling pathways cooperatively regulate vascular smooth muscle cell differentiation.

Notch and transforming growth factor-beta (TGFbeta) play pivotal roles during vascular development and the pathogenesis of vascular disease. The interaction of these two pathways is not fully understood. The present study utilized primary human smooth muscle cells (SMC) to examine molecular cross-talk between TGFbeta1 and Notch signaling on contractile gene expression. Activation of Notch signaling using Notch intracellular domain or Jagged1 ligand induced smooth muscle alpha-actin (SM actin), smooth muscle myosin heavy chain, and calponin1, and the expression of Notch downstream effectors hairy-related transcription factors. Similarly, TGFbeta1 treatment of human aortic smooth muscle cells induced SM actin, calponin1, and smooth muscle protein 22-alpha (SM22alpha) in a dose- and time-dependent manner. Hairy-related transcription factor proteins, which antagonize Notch activity, also suppressed the TGFbeta1-induced increase in SMC markers, suggesting a general mechanism of inhibition. We found that Notch and TGFbeta1 cooperatively activate SMC marker transcripts and protein through parallel signaling axes. Although the intracellular domain of Notch4 interacted with phosphoSmad2/3 in SMC, this interaction was not observed with Notch1 or Notch2. However, we found that CBF1 co-immunoprecipitated with phosphoSmad2/3, suggesting a mechanism to link canonical Notch signaling to phosphoSmad activity. Indeed, the combination of Notch activation and TGFbeta1 treatment led to synergistic activation of a TGFbeta-responsive promoter. This increase corresponded to increased levels of phosphoSmad2/3 interaction at Smad consensus binding sites within the SM actin, calponin1, and SM22alpha promoters. Thus, Notch and TGFbeta coordinately induce a molecular and functional contractile phenotype by co-regulation of Smad activity at SMC promoters.

Identification of responsive cells in the developing somite supports a role for beta-catenin-dependent Wnt signaling in maintaining the DML myogenic progenitor pool.

Somitic beta-catenin is involved in both maintaining a stem cell population and controlling myogenic differentiation. It is unclear how beta-catenin-dependent Wnt signaling accomplishes these disparate roles. The present study shows that only dorsal cells in the early somite respond to beta-catenin-dependent Wnt signaling and as the somites compartmentalize to form the dermomyotome and myotome, responding cells are detected primarily in the dorsomedial lip (DML). Forced activation of Wnt target genes in DML cells prevents their progeny from entering the myotome, while blocking activation allows myotomal entry. This suggests a role for beta-catenin-dependent/Wnt signaling in maintaining progenitor cells in the DML and that if beta-catenin-dependent/Wnt signaling is required to induce myogenesis, the response is transitory and rapidly down-regulated.

Conditional expression of Spry1 in neural crest causes craniofacial and cardiac defects.

BACKGROUND: Growth factors and their receptors are mediators of organogenesis and must be tightly regulated in a temporal and spatial manner for proper tissue morphogenesis. Intracellular regulators of growth factor signaling pathways provide an additional level of control. Members of the Sprouty family negatively regulate receptor tyrosine kinase pathways in several developmental contexts. To gain insight into the role of Spry1 in neural crest development, we analyzed the developmental effects of conditional expression of Spry1 in neural crest-derived tissues. RESULTS: Here we report that conditional expression of Spry1 in neural crest cells causes defects in craniofacial and cardiac development in mice. Spry1;Wnt1-Cre embryos die perinatally and exhibit facial clefting, cleft palate, cardiac and cranial nerve defects. These defects appear to be the result of decreased proliferation and increased apoptosis of neural crest and neural crest-derived cell populations. In addition, the domains of expression of several key transcription factors important to normal craniofacial and cardiac development including AP2, Msx2, Dlx5, and Dlx6 were reduced in Spry1;Wnt1-Cre transgenic embryos. CONCLUSION: Collectively, these data suggest that Spry1 is an important regulator of craniofacial and cardiac morphogenesis and perturbations in Spry1 levels may contribute to congenital disorders involving tissues of neural crest origin.

Making the Connection: Using Concept Mapping to Bring the Basic Sciences to the Diagnosis.

Although medical education has historically emphasized the role and importance of basic science in clinical reasoning, educators have struggled to teach basic science to optimize its use for students. Concept mapping helps students develop relationships between basic and clinical science, which can enhance understanding of the material. Educators at the University of New England College of Osteopathic Medicine developed a weekly concept-mapping activity connecting biomedical principles with clinical signs, symptoms, and laboratory values from a comprehensive clinical case. This activity elicits cross-disciplinary discussion, illustrates content integration by the students, and enhances faculty collaboration across disciplines.

Twist1 homodimers enhance FGF responsiveness of the cranial sutures and promote suture closure.

Haploinsufficiency of the transcription factor TWIST1 is associated with Saethre-Chotzen Syndrome and is manifested by craniosynostosis, which is the premature closure of the calvaria sutures. Previously, we found that Twist1 forms functional homodimers and heterodimers that have opposing activities. Our data supported a model that within the calvaria sutures Twist1 homodimers (T/T) reside in the osteogenic fronts while Twist1/E protein heterodimers (T/E) are in the mid-sutures. Twist1 haploinsufficiency alters the balance between these dimers, favoring an increase in homodimer formation throughout the sutures. The data we present here further supports this model and extends it to integrate the Twist1 dimers with the pathways that are known to regulate cranial suture patency. This data provides the first evidence of a functional link between Twist1 and the FGF pathway, and indicates that differential regulation of FGF signaling by T/T and T/E dimers plays a central role in governing cranial suture patency. Furthermore, we show that inhibition of FGF signaling prevents craniosynostosis in Twist1(+/-) mice, demonstrating that inhibition of a signaling pathway that is not part of the initiating mutation can prevent suture fusion in a relevant genetic model of craniosynostosis.

Medical Biochemistry Without Rote Memorization: Multi-Institution Implementation and Student Perceptions of a Nationally Standardized Metabolic Map for Learning and Assessment.

Despite the growing number of patients worldwide with metabolism-related chronic diseases, medical biochemistry education is commonly perceived as focusing on recall of facts irrelevant for patient care. The authors suggest that this focus on rote memorization of pathways creates excessive cognitive load that may interfere with learners' development of an integrated understanding of metabolic regulation and dysregulation. This cognitive load can be minimized by providing appropriate references during learning and assessment. Biochemistry educators collaborated to develop a medically relevant pathways of human metabolism map (MetMap) that is now being used at many medical schools as a nationally standardized resource during learning and assessments. To assess impact, students from three medical schools were surveyed about its benefits and disadvantages. Responses were obtained from 481 students (84%) and were examined using thematic analysis. Five main themes emerged as perceived benefits of using the MetMap: (1) aids visual and mental organization, (2) promotes deep learning and applied understanding, (3) decreases emphasis on memorization, (4) reduces anxiety on exams, and (5) aids recall. Perceived disadvantages were (1) fear of underpreparation for licensing exams, (2) overwhelming nature of the map, and (3) reduced motivation for and time spent studying. Results affirm that students' perceive use of the MetMap promotes focus on broader metabolic concepts and deep versus surface learning, supporting a shift in cognitive load toward desired goals. Although the long-term impact on learning needs to be further studied, the use of the MetMap represents a step toward open-reference exams that reflect "real-world" practice.

Aberrant expression of Twist1 in diseased articular cartilage and a potential role in the modulation of osteoarthritis severity.

The bHLH transcription factor Twist1 has emerged as a negative regulator of chondrogenesis in skeletal progenitor cells and as an inhibitor of maturation in growth plate chondrocytes. However, its role in articular cartilage remains obscure. Here we examine Twist1 expression during re-differentiation of expanded human articular chondrocytes, the distribution of Twist1 proteins in normal versus OA human articular cartilage, and its role in modulating OA development in mice. High levels of Twist1 transcripts were detected by qPCR analyses of expanded de-differentiated human articular chondrocytes that had acquired mesenchymal-like features. The induction of hallmark cartilage genes by Bmp-2 mediated chondrogenic differentiation was paralleled by the dramatic suppression of Twist1 in vitro. In normal human articular cartilage, Twist1-expressing chondrocytes were most abundant in the superficial zone with little to no expression in the middle and deep zones. However, our analyses revealed a higher proportion of deep zone articular chondrocytes expressing Twist1 in human OA cartilage as compared to normal articular cartilage. Moreover, Twist1 expression was prominent within proliferative cell clusters near fissure sites in more severely affected OA samples. To assess the role of Twist1 in OA pathophysiology, we subjected wild type mice and transgenic mice with gain of Twist1 function in cartilage to surgical destabilization of the medial meniscus. At 12 weeks post-surgery, micro-CT and histological analyses revealed attenuation of the OA phenotype in Twist1 transgenic mice compared to wild type mice. Collectively, the data reveal a role for Twist in articular cartilage maintenance and the attenuation of cartilage degeneration.

The Heterodimeric TWIST1-E12 Complex Drives the Oncogenic Potential of TWIST1 in Human Mammary Epithelial Cells.

The TWIST1 embryonic transcription factor displays biphasic functions during the course of carcinogenesis. It facilitates the escape of cells from oncogene-induced fail-safe programs (senescence, apoptosis) and their consequent neoplastic transformation. Additionally, it promotes the epithelial-to-mesenchymal transition and the initiation of the metastatic spread of cancer cells. Interestingly, cancer cells recurrently remain dependent on TWIST1 for their survival and/or proliferation, making TWIST1 their Achilles' heel. TWIST1 has been reported to form either homodimeric or heterodimeric complexes mainly in association with the E bHLH class I proteins. These complexes display distinct, sometimes even antagonistic, functions during development and unequal prometastatic functions in prostate cancer cells. Using a tethered dimer strategy, we successively assessed the ability of TWIST1 dimers to cooperate with an activated version of RAS in human mammary epithelial cell transformation, to provide mice with the ability to spontaneously develop breast tumors, and lastly to maintain a senescence program at a latent state in several breast cancer cell lines. We demonstrate that the TWIST1-E12 complex, unlike the homodimer, is an oncogenic form of TWIST1 in mammary epithelial cells and that efficient binding of both partners is a prerequisite for its activity. The detection of the heterodimer in human premalignant lesions by a proximity ligation assay, at a stage preceding the initiation of the metastatic cascade, is coherent with such an oncogenic function. TWIST1-E protein heterodimeric complexes may thus constitute the main active forms of TWIST1 with regard to senescence inhibition over the time course of breast tumorigenesis.

A rapid method for manual or automated purification of fluorescently labeled nucleic acids for sequencing, genotyping, and microarrays.

Fluorescent dyes provide specific, sensitive, and multiplexed detection of nucleic acids. To maximize sensitivity, fluorescently labeled reaction products (e.g., cycle sequencing or primer extension products) must be purified away from residual dye-labeled precursors. Successful high-throughput analyses require that this purification be reliable, rapid, and amenable to automation. Common methods for purifying reaction products involve several steps and require processes that are not easily automated. Prolinx, Inc. has devel oped RapXtract superparamagnetic separation technology affording rapid and easy-to-perform methods that yield high-quality product and are easily automated. The technology uses superparamagnetic particles that specifically remove unincorporated dye-labeled precursors. These particles are efficiently pelleted in the presence of a magnetic field, making them ideal for purification because of the rapid separations that they allow. RapXtract-purified sequencing reactions yield data with good signal and high Phred quality scores, and they work with various sequencing dye chemistries, including BigDye and near-infrared fluorescence IRDyes. RapXtract technology can also be used to purify dye primer sequencing reactions, primer extension reactions for genotyping analysis, and nucleic acid labeling reactions for microarray hybridization. The ease of use and versatility of RapXtract technology makes it a good choice for manual or automated purification of fluorescently labeled nucleic acids.

Persistent expression of Twist1 in chondrocytes causes growth plate abnormalities and dwarfism in mice.

Evidence from various in vitro gain and loss of function studies indicate that the bHLH transcription factor Twist1 negatively regulates chondrocyte differentiation; however limited information regarding Twist1 function in postnatal cartilage development and maintenance is available. Twist1 expression within the postnatal growth plate is restricted to immature, proliferating chondrocytes, and is significantly decreased or absent in hypertrophic chondrocytes. In order to examine the effect of maintaining the expression of Twist1 at later stages of chondocyte differentiation, we used type II collagen Cre (Col2-Cre) mice to activate a Cre-inducible Twist1 transgene specifically in chondrocytes (Col2-Twist1). At two weeks, postnatal growth was inhibited in Col2-Twist1 mice, as evidenced by limb shortening. Histological examination revealed abnormal growth plate structure, characterized by poor columnar organization of proliferating cartilaginous cells, decreased cellularity, and expansion of the hypertrophic zone. Moreover, structural defects within the growth plates of Col2-Twist1 transgenic mice included abnormal vascular invasion and focal regions of bony formation. Quantitative analysis of endochondral bone formation via micro-computed topography revealed impaired trabecular bone formation in the hindlimbs of Col2-Twist1 transgenic mice at various timepoints of postnatal development. Taken together, these findings indicate that regulated Twist1 expression contributes to growth plate organization and endochondral ossification to modulate postnatal longitudinal bone growth.

Two novel human NUMB isoforms provide a potential link between development and cancer.

We previously identified four functionally distinct human NUMB isoforms. Here, we report the identification of two additional isoforms and propose a link between the expression of these isoforms and cancer. These novel isoforms, NUMB5 and NUMB6, lack exon 10 and are expressed in cells known for polarity and migratory behavior, such as human amniotic fluid cells, glioblastoma and metastatic tumor cells. RT-PCR and luciferase assays demonstrate that NUMB5 and NUMB6 are less antagonistic to NOTCH signaling than other NUMB isoforms. Immunocytochemistry analyses show that NUMB5 and NUMB6 interact and complex with CDC42, vimentin and the CDC42 regulator IQGAP1 (IQ (motif) GTPase activating protein 1). Furthermore, the ectopic expression of NUMB5 and NUMB6 induces the formation of lamellipodia (NUMB5) and filopodia (NUMB6) in a CDC42- and RAC1-dependent manner. These results are complemented by in vitro and in vivo studies, demonstrating that NUMB5 and NUMB6 alter the migratory behavior of cells. Together, these novel isoforms may play a role in further understanding the NUMB function in development and cancer.

A loss-of-function polymorphism in the propeptide domain of the LOX gene and breast cancer.

The lysyl oxidase (LOX) gene reverted Ras transformation of NIH 3T3 fibroblasts and tumor formation by gastric cancer cells, which frequently carry mutant RAS genes. The secreted lysyl oxidase proenzyme is processed to a propeptide (LOX-PP) and a functional enzyme (LOX). Unexpectedly, the tumor suppressor activity mapped to the LOX-PP domain, which inhibited tumor formation and the invasive phenotype of NF639 breast cancer cells driven by human epidermal growth factor receptor-2/neu, which signals via Ras. A single-nucleotide polymorphism, G473A (rs1800449), resulting in an Arg158Gln substitution in a highly conserved region within LOX-PP, occurs with an average 473A allele carrier frequency of 24.6% in the HapMap database, but was present in many breast cancer cell lines examined. Here, we show that the Arg-to-Gln substitution profoundly impairs the ability of LOX-PP to inhibit the invasive phenotype and tumor formation of NF639 cells in a xenograft model. LOX-PP Gln displayed attenuated ability to oppose the effects of LOX, which promoted a more invasive phenotype. In a case-control study of African American women, a potential association of the Gln-encoding A allele was seen with increased risk of estrogen receptor (ER)-alpha-negative invasive breast cancer in African American women. Consistently, LOX gene expression was higher in ER-negative versus ER-positive primary breast cancers, and LOX-PP Gln was unable to inhibit invasion by ER-negative cell lines. Thus, these findings identify for the first time genetic polymorphism as a mechanism of impaired tumor suppressor function of LOX-PP and suggest that it may play an etiologic role in ER-negative breast cancer.