Multimodal Surveillance Model for Enterovirus D68 Respiratory Disease and Acute Flaccid Myelitis among Children in Colorado, USA, 2022.

Surveillance for emerging pathogens is critical for developing early warning systems to guide preparedness efforts for future outbreaks of associated disease. To better define the epidemiology and burden of associated respiratory disease and acute flaccid myelitis (AFM), as well as to provide actionable data for public health interventions, we developed a multimodal surveillance program in Colorado, USA, for enterovirus D68 (EV-D68). Timely local, state, and national public health outreach was possible because prospective syndromic surveillance for AFM and asthma-like respiratory illness, prospective clinical laboratory surveillance for EV-D68 among children hospitalized with respiratory illness, and retrospective wastewater surveillance led to early detection of the 2022 outbreak of EV-D68 among Colorado children. The lessons learned from developing the individual layers of this multimodal surveillance program and how they complemented and informed the other layers of surveillance for EV-D68 and AFM could be applied to other emerging pathogens and their associated diseases.

Analysis of gender representation, authorship inflation, and institutional affiliation in abstract acceptance: a 5-year study.

Purpose: Our goals were to characterize associations of author number, author gender, and institutional affiliation on ratings and acceptances of abstracts submitted to one surgical education conference over 5 years. Methods: We retrospectively reviewed all abstracts submitted between 2017 and 2021 to the annual meeting of the Association for Surgical Education (ASE). Abstract data included average rater scores, acceptance status, author lists, and institutional affiliations. We cross-referenced last author affiliation with top-40 National Institutes of Health (NIH)-funded institutions and used a gender determination software to code first and last author genders. Results: We analyzed 1,162 abstracts. Higher reviewer scores demonstrated positive, weak associations with more authors [r(1160) = 0.191, p < 0.001] and institutions [r(1160) = 0.182, p < 0.001]. Significantly higher scores were noted for abstracts with last authors affiliated with top-40 NIH-funded institutions [4.18 (SD 0.96) vs. 3.72 (SD 1.12), p < 0.001]. Women were first authors (51.8%) (n = 602) and last authors (35.4%) (n = 411) of the time. Abstracts were rated significantly higher with women rather than men as first authors [3.98 (SD 0.99) vs. 3.82 (SD 1.12), p = 0.011] or last [4.01 (SD 1.04) vs. 3.82 (SD 1.10), p = 0.005]. Across all years, abstracts were accepted more often as podium or plenary presentations when submitted by women first [n = 279, 59.7% (p = 0.002)] or last [n = 183, 38.4% (p = 0.095)] authors. Conclusion: Abstracts whose last authors were affiliated with top-40 NIH-funded institutions received significantly higher scores, possibly indicating increased tangible or intangible resources contributing to research efforts. Abstracts with women first and last authors scored higher and were more frequently invited for plenary and podium presentations.

Development and validation of a multiplex real-time PCR assay for detection and quantification of Streptococcus pneumoniae in pediatric respiratory samples.

IMPORTANCE: Streptococcus pneumoniae (Spn) is the world's leading cause of lower respiratory tract infection morbidity and mortality in children. However, current clinical microbiological methods have disadvantages. Spn can be difficult to grow in laboratory conditions if a patient is pre-treated, and Spn antigen testing has unclear clinical utility in children. Syndromic panel testing is less cost-effective than targeted PCR if clinical suspicion is high for a single pathogen. Also, such testing entails a full, expensive validation for each panel target if used for multiple respiratory sources. Therefore, better diagnostic modalities are needed. Our study validates a multiplex PCR assay with three genomic targets for semi-quantitative and quantitative Spn molecular detection from lower respiratory sources for clinical testing and from upper respiratory sources for research investigation.

Qualitative detection of enterovirus D68 from PrimeStore® molecular transport medium: implications for home- and self-collection.

To ensure proper specimen handling for detecting pathogens, like Enterovirus D68 (EV-D68), from home- and self-collection, alternative techniques are needed to ensure safe transport and reliable testing. PrimeStore® Molecular Transport Medium (MTM) may be an option since it does not require cold storage and inactivates virus while preserving RNA for detection. The purpose of this validation study was to demonstrate the ability to detect EV-D68 via rRT-PCR in MTM. Using a quantified EV-D68 positive control standard, MTM limit of detection for EV-D68 RNA is 104 cp/mL and RNA remains stable up to 30 days unfrozen. Positive and negative residual respiratory specimens from the 2018 EV-D68 outbreak were used for clinical testing. There was an 80% positive and 100% negative agreement with samples in MTM compared to reference. This study demonstrates the feasibility of EV-D68 detection from respiratory specimens collected and stored in PrimeStore® MTM, with implications for home- and self-collection.

Identifying Intraoperative Behaviors Associated With Surgical Resident Teachability.

OBJECTIVE: The resident-attending dyad influences the intraoperative training of surgery residents. To better understand the role of trainees within the dyad, we hypothesized there is a measurable concept of "teachability," a combination of the trainee's observed skills and behaviors with their performance. This study aims to define teachability and identify discrete intraoperative behaviors that contribute to this concept. We posit that residents who are active learners as demonstrated by asking questions, proposing next steps, and initiating purposeful actions have higher teachability. DESIGN, SETTING, PARTICIPANTS: Previously recorded videos from 26 laparoscopic inguinal hernia repairs performed by two PGY-5 general surgery residents at a Midwest tertiary care center were qualitatively reviewed for intraoperative behaviors. A summative content analysis identified behaviors associated with increased teachability and improved operative performance assessment scores. RESULTS: Average frequencies of intraoperative behaviors for resident 1 and 2 (R1 and R2) were not significantly different, although R2 asked more medical knowledge and technical questions. While the rate of attending feedback was similar for both residents (x=3.82 vs 3.40, p=0.646), R1 consistently incorporated feedback (x=2.27 vs 0.40, p=0.001) whereas R2 needed frequent prompting (x=2.45 vs 1.55, p=0.239). R1 scored higher in all but one operative performance assessment category, including overall performance (x=4.17 vs 2.93, p=0.007), but R2 had a larger magnitude of overall improvement (+1 vs +2). CONCLUSIONS: Teachability is a dynamic component of the resident-attending dyad. While intraoperative active learning behaviors do not appear to be associated with teachability, asking questions may increase the magnitude of improvement in performance. Most importantly, the ability to incorporate intraoperative feedback in real time seems to be a critical aspect of teachability and warrants further research.

Enhancing Surgical Teaching Effectiveness: A Needs Assessment Study Using a Modified Delphi Process.

INTRODUCTION: As methods of measuring surgical resident competency become more defined, how can faculty know that they are effectively guiding residents toward increasing entrustment? The goal of this study was to use a systematic process to identify effective teaching behaviors, understand discrepancies between learner and teacher perception of behaviors, and provide an insight into areas for improvement in surgical teaching. MATERIALS AND METHODS: A modified Delphi process was used to create a list of critical teaching behaviors for surgical resident education in four domains: Operating Room, Clinic, Inpatient Rounds, and Didactics. Round One surveyed residents and faculty to identify critical teaching behaviors. In Rounds Two and Three, stakeholders narrowed the list to five behaviors in each domain. A needs assessment survey was created and used to identify (1) areas for improvement in residency education and (2) differences in perception of teaching behavior use between faculty and residents. RESULTS: Eighty one faculty and 56 residents in the Department of Surgery completed the survey. All teaching behaviors in the Operating Room, Clinic, and Rounds domains had a significant difference in response distribution between residents and faculty. Except in Didactics, residents perceived that teaching behaviors were performed less often by attending surgeons than was reported by the faculty members. CONCLUSIONS: A modified Delphi process is an effective way to create a needs assessment survey relating to how surgical education is delivered. Future steps will involve directed interventions aimed at improving the use of certain surgical teaching behaviors in our department.

TrustEd: A Tool for Developing Intraoperative Entrustment Skills.

OBJECTIVE: Toolkits to assess progressive resident autonomy are integral to the movement toward competency-based surgical education. OpTrust is one such tool validated for intraoperative assessment of both faculty and resident entrustment behaviors. We developed a supplementary tool to OpTrust that would aid faculty and residents in making meaningful improvements in entrustment behavior by providing talking points and reflection items tailored to different motivational styles as defined by Regulatory Focus Theory (RFT). DESIGN: Existing literature about surgical entrustment was used to build a list of sample dialogue and self-reflection items to use in the operating room. This list was distributed as a survey to individuals familiar with OpTrust and RFT, asking them to categorize each item as Promotion-oriented, Prevention-oriented, or Either. The respondents then met to discuss survey items that did not reach a consensus until the group agreed on their categorization. SETTING: University of Wisconsin, School of Medicine and Public Health, Madison, Wisconsin Michigan Medicine, Ann Arbor, Michigan PARTICIPANTS: Clinician and education researchers familiar with intraoperative entrustment and RFT RESULTS: Eight respondents completed the survey categorizing the talking points and reflection items by RFT (100% response rate). Six of these respondents attended the additional meeting to discuss discordant items. The input from this panel was used to develop "TrustEd," the supplementary tool that faculty and residents can quickly reference before beginning a case. CONCLUSION: Although tools such as OpTrust allow intraoperative entrustment behaviors to be quantified, TrustEd offers concrete strategies for faculty and residents who are interested in improving those behaviors over time. Further study is needed to assess whether the use of TrustEd does in fact lead to durable behavior change and improvement in OpTrust scores.

Simulation-Based Medical Education: Development of an Assessment Tool for Novice Use.

BACKGROUND: Simulation-based medical education, an educational model in which students engage in simulated patient scenarios, improves performance. However, assessment tools including the Oxford Non-Technical Skills (NOTECHS) scale require expert assessors. We modified this tool for novice use. METHODS: Medical students participated in 5 nontechnical simulations. The NOTECHS scale was modified to allow for novice evaluation. Three novices and 2 experts assessed performance, with intraclass correlation used to assess validity. RESULTS: Twenty-two learners participated in the simulations. Novice reviewers had moderate to excellent correlation among evaluations (0.66 < intraclass correlation coefficients [ICC] < 0.95). Novice and expert reviewers had moderate to good correlation among evaluations (0.51 < ICC < 0.88). DISCUSSION: The modified NOTECHS scales can be utilized by novices to evaluate simulation performance. Novice assessment correlates with expert review. These tools may encourage the use of simulation-based medical education.

Gender Differences in Entrustable Professional Activity Evaluations of General Surgery Residents.

OBJECTIVE: To determine differences in entrustable professional activity (EPA) assessments between male and female general surgery residents. SUMMARY BACKGROUND DATA: Evaluations play a critical role in career advancement for physicians. However, female physicians in training receive lower evaluations and underrate their own performance. Competency-based assessment frameworks, such as EPAs, may help address gender bias in surgery by linking evaluations to specific, observable behaviors. METHODS: In this cohort study, EPA assessments were collected from July 2018 to May 2020. The effect of resident sex on EPA entrustment levels was analyzed using multiple linear and ordered logistic regressions. Narrative comments were analyzed using latent dirichlet allocation to identify topics correlated with resident sex. RESULTS: Of the 2480 EPAs, 1230 EPAs were submitted by faculty and 1250 were submitted by residents. After controlling for confounding factors, faculty evaluations of residents were not impacted by resident sex (estimate = 0.09, P = 0.08). However, female residents rated themselves lower by 0.29 (on a 0-4 scale) compared to their male counterparts (P < 0.001). Within narrative assessments, topics associated with resident sex demonstrated that female residents focus on the "guidance" and "supervision" they received while performing an EPA, while male residents were more likely to report "independent" action. CONCLUSIONS: Faculty assessments showed no difference in EPA levels between male and female residents. Female residents rate themselves lower by nearly an entire post graduate year (PGY) level compared to male residents. Latent dirichlet allocation -identified topics suggest this difference in self-assessment is related to differences in perception of autonomy.

Instructional methods of attendings as exhibited during intraoperative takeovers: A pilot study.

BACKGROUND: This pilot study examined intraoperative instructional techniques during "takeovers," defined as the act of an attending taking control of a case from a resident. This work describes what happens during takeovers and identifies possible reasons for takeovers. METHODS: Intraoperative audio-video recordings during 25 laparoscopic inguinal hernia repair procedures were collected. Participants included 2 postgraduate year-5 residents and 5 attendings. Postoperative evaluation forms were completed by attendings. Coding schemes for takeovers during hernia reduction and mesh placement steps were developed using conventional and directed content analysis in an iterative process by study team members, including individuals with expertise in education, surgery, and surgical education. RESULTS: Takeovers occurred in 72% of cases. Frequency of takeovers was not related to case difficulty or differences in resident technical skill levels, nor did they decrease over the duration of the 2-month rotation. Takeovers most commonly occurred when a resident struggled to progress the case. They also occurred when anatomy was unclear or when the attending wanted to teach a specific skill. Differences were identified among attendings regarding frequency of takeovers. The majority of takeover behaviors were directed at instructing residents; however, attendings' teaching techniques did not vary by resident. CONCLUSION: Attending teaching habits appear to be independent of resident skills and depend on the attending's teaching style rather than residents' learning needs. Findings highlight the need for faculty development to help surgical educators learn how to tailor instruction to individual trainees. Additionally, future research is needed to establish the effectiveness of instruction through takeovers in the operating room.

Natural language processing and entrustable professional activity text feedback in surgery: A machine learning model of resident autonomy.

BACKGROUND: Entrustable Professional Activities (EPAs) contain narrative 'entrustment roadmaps' designed to describe specific behaviors associated with different entrustment levels. However, these roadmaps were created using expert committee consensus, with little data available for guidance. Analysis of actual EPA assessment narrative comments using natural language processing may enhance our understanding of resident entrustment in actual practice. METHODS: All text comments associated with EPA microassessments at a single institution were combined. EPA-entrustment level pairs (e.g. Gallbladder Disease-Level 1) were identified as documents. Latent Dirichlet Allocation (LDA), a common machine learning algorithm, was used to identify latent topics in the documents associated with a single EPA. These topics were then reviewed for interpretability by human raters. RESULTS: Over 18 months, 1015 faculty EPA microassessments were collected from 64 faculty for 80 residents. LDA analysis identified topics that mapped 1:1 to EPA entrustment levels (Gammas >0.99). These LDA topics appeared to trend coherently with entrustment levels (words demonstrating high entrustment were consistently found in high entrustment topics, word demonstrating low entrustment were found in low entrustment topics). CONCLUSIONS: LDA is capable of identifying topics relevant to progressive surgical entrustment and autonomy in EPA comments. These topics provide insight into key behaviors that drive different level of resident autonomy and may allow for data-driven revision of EPA entrustment maps.

A Mastery Learning Module on Sterile Technique to Prepare Graduating Medical Students for Internship.

Introduction: Sterile technique is a basic technical skill used for a number of bedside procedures. Proper use of sterile technique improves patient safety by reducing infection risk. Methods: We applied the principles of mastery learning to develop a simulation-based mastery learning module for sterile technique that was used as part of a 2-week internship preparatory course for fourth-year medical students. Forty-one medical students entering surgical or emergency medicine internships completed the module. Learners demonstrated baseline skills with a pretest, watched a didactic online video, participated in supervised deliberate practice sessions, and then completed a posttest. Physicians evaluated performance using a nine-item mastery checklist validated by a multispecialty panel of board-certified physicians. Learners who did not demonstrate mastery by correctly performing all nine checklist items received formative feedback and repeated the posttest as needed until mastery was achieved. Results: No learners demonstrated mastery of sterile technique during pretesting. A total of 100% of learners demonstrated mastery of sterile technique during either their first or second attempt of the posttest. The learners reported statistically significantly higher levels of confidence at the end of the module. Discussion: Our module highlights the skills gap that exists in the transition from undergraduate to graduate medical education and offers a cheap, effective, and easily reproducible curriculum for sterile technique that could be widely adopted for many learner populations.

Entrustable Professional Activities in General Surgery: Trends in Resident Self-Assessment.

OBJECTIVE: Effective self-assessment is a cornerstone of lifelong professional development; however, evidence suggests physicians have a limited ability to self-assess. Novel strategies to improve the accuracy of learner self-assessment are needed. Our institution's surgical entrustable professional activity (EPA) implementation strategy incorporates resident self-assessment to address this issue. This study evaluates the accuracy of resident self-assessment versus faculty assessment across 5 EPAs in general surgery. DESIGN, SETTING, PARTICIPANTS: Within a single academic general surgery residency program, assessment data for 5 surgery EPAs was prospectively collected using a mobile application. Matched assessments (resident and faculty assessments for the same clinical encounter) were identified and the remainder excluded. Assessment scores were compared using Welch's t test. Agreement was analyzed using Cohen's kappa with squared weights. RESULTS: One thousand eight hundred and fifty-seven EPA assessments were collected in 17 months following implementation. One thousand one hundred and fifty-five (62.2%) were matched pairs. Residents under-rated their own performance relative to faculty assessments (2.36 vs 2.65, p < 0.01). This pattern held true for all subsets except for Postgraduate Year (PGY)2 residents and Inguinal Hernia EPAs. There was at least moderate agreement between matched resident and faculty EPA assessment scores (κ = 0.57). This was consistent for all EPAs except Trauma evaluations, which were completed by faculty from 2 different departments. Surgery resident self-assessments more strongly agreed with Surgery faculty assessments than Emergency Medicine faculty assessments (κ = 0.58 vs 0.36). CONCLUSIONS: Resident EPA self-assessments are equivalent or slightly lower than faculty assessments across a wide breadth of clinical scenarios. Resident and faculty matched assessments demonstrate moderate agreement.

Implementation of Entrustable Professional Activities into a General Surgery Residency.

OBJECTIVE: Concerns over resident ability to practice effectively after graduation have led to the competency-based medical education movement. Entrustable professional activities (EPAs) may facilitate competency-based medical education in surgery, but implementation is challenging. This manuscript describes 1 strategy used to implement EPAs into an academic general surgery residency. DESIGN, SETTING, PARTICIPANTS: A mobile application was developed incorporating 5 EPAs developed by the American Board of Surgery; residents and faculty from the Departments of Surgery, Emergency Medicine, and Hospital Medicine at a single tertiary care center were trained in its use. Entrustment levels and free text feedback were collected. Self-assessment was paired with supervisor assessment, and faculty assessments were used to inform clinical competency committee entrustment decisions. Feedback was regularly solicited from app users and results distributed on a monthly basis. RESULTS: One thousand seven hundred and twenty microassessments were collected over the first 16 months of implementation; 898 (47.8%) were performed by faculty with 569 (66.0%) matched pairs. Engagement was skewed with small numbers of high performers in both resident and faculty groups. Continued development of resident and faculty was required to sustain engagement with the program. Nonsurgical specialties contributed significantly to resident assessments (496, 28.8%). CONCLUSIONS: EPAs are being successfully integrated into the assessment framework at our institution. EPA implementation in surgery residency is a long-term process that requires investment, but may address limitations in the current assessment framework.

Parallel quorum-sensing system in Vibrio cholerae prevents signal interference inside the host.

Many bacteria use quorum sensing (QS) to regulate virulence factor production in response to changes in population density. QS is mediated through the production, secretion, and detection of signaling molecules called autoinducers (AIs) to modulate population-wide behavioral changes. Four histidine kinases, LuxPQ, CqsS, CqsR and VpsS, have been identified in Vibrio cholerae as QS receptors to activate virulence gene expression at low cell density. Detection of AIs by these receptors leads to virulence gene repression at high cell density. The redundancy among these receptors is puzzling since any one of the four receptors is sufficient to support colonization of V. cholerae in the host small intestine. It is believed that one of the functions of such circuit architecture is to prevent interference on any single QS receptor. However, it is unclear what natural molecules can interfere V. cholerae QS and in what environment interference is detrimental. We show here mutants expressing only CqsR without the other three QS receptors are defective in colonizing the host large intestine. We identified ethanolamine, a common intestinal metabolite that can function as a chemical source of QS interference. Ethanolamine specifically interacts with the ligand-binding CACHE domain of CqsR and induces a premature QS response in V. cholerae mutants expressing only CqsR without the other three QS receptors. The effect of ethanolamine on QS gene expression and host colonization is abolished by mutations that disrupt CqsR signal sensing. V. cholerae defective in producing ethanolamine is still proficient in QS, therefore, ethanolamine functions only as an external cue for CqsR. Our findings suggest the inhibitory effect of ethanolamine on CqsR could be a possible source of QS interference but is masked by the presence of the other parallel QS pathways, allowing V. cholerae to robustly colonize the host.

Teaching practicing surgeons what not to do: An analysis of instruction fluidity during a simulation-based continuing medical education course.

BACKGROUND: Interest is growing in simulation-based continuing medical education courses for practicing surgeons. However, little research has explored the instruction employed during these courses. This study examines instruction practices used during an annual simulation-based continuing medical education course. METHODS: Audio-video data were collected from surgeon instructors (n = 12) who taught a simulated laparoscopic hernia repair continuing medical education course across 2 years. Surgeon learners (n = 58) were grouped by their self-reported laparoscopic and hernia repair experience. Instructors' transcribed dialogue was automatically coded for 5 types of responses to the following questions: anecdotes, confirming, correcting, guidance, and what not to do. Differences in these responses were measured against the progress of the simulations and across learners with different experience levels. Postcourse interviews with instructors were conducted for additional qualitative validation. RESULTS: Performing t tests of instructor responses revealed that they were significantly more likely to answer in forms coded as anecdotes when responding to relative experts and in forms coded as what not to do when responding to novices. Linear regressions of each code against normalized progressions of each simulation revealed a significant relationship between progression through a simulation and frequency of the what not to do code for less-experienced learners. Postcourse interviews revealed that instructors continuously assess participants throughout a session and modify their teaching strategies. CONCLUSION: Instructors significantly modified the focus of their teaching as a function both of their learners' self-reported experience levels, their assessment of learner needs, and learner progression through the training sessions.

Specificity and complexity in bacterial quorum-sensing systems.

Quorum sensing (QS) is a microbial cell-to-cell communication process that relies on the production and detection of chemical signals called autoinducers (AIs) to monitor cell density and species complexity in the population. QS allows bacteria to behave as a cohesive group and coordinate collective behaviors. While most QS receptors display high specificity to their AI ligands, others are quite promiscuous in signal detection. How do specific QS receptors respond to their cognate signals with high fidelity? Why do some receptors maintain low signal recognition specificity? In addition, many QS systems are composed of multiple intersecting signaling pathways: what are the benefits of preserving such a complex signaling network when a simple linear 'one-to-one' regulatory pathway seems sufficient to monitor cell density? Here, we will discuss different molecular mechanisms employed by various QS systems that ensure productive and specific QS responses. Moreover, the network architectures of some well-characterized QS circuits will be reviewed to understand how the wiring of different regulatory components achieves different biological goals.

Parallel quorum sensing signaling pathways in Vibrio cholerae.

Quorum sensing (QS) is a microbial signaling process for monitoring population density and complexity. Communication among bacterial cells via QS relies on the production, secretion, and detection of small molecules called autoinducers. Many bacteria have evolved their QS systems with different network architectures to incorporate information from multiple signals. In the human pathogen Vibrio cholerae, at least four parallel signaling pathways converge to control the activity of a single regulator to modulate its QS response. By integrating multiple signal inputs, it is believed that Vibrio species can survey intra-species, intra-genus, and inter-species populations and program their gene expression accordingly. Our recent studies suggest that this "many-to-one" circuitry is also important for maintaining the integrity of the input-output relationship of the system and minimizes premature commitment to QS due to signal perturbation. Here we discuss the implications of this specific parallel network setup for V. cholerae intercellular communication and how this system arrangement affects our approach to manipulate the QS response of this clinically important pathogen.

Quadruple quorum-sensing inputs control Vibrio cholerae virulence and maintain system robustness.

Bacteria use quorum sensing (QS) for cell-cell communication to carry out group behaviors. This intercellular signaling process relies on cell density-dependent production and detection of chemical signals called autoinducers (AIs). Vibrio cholerae, the causative agent of cholera, detects two AIs, CAI-1 and AI-2, with two histidine kinases, CqsS and LuxQ, respectively, to control biofilm formation and virulence factor production. At low cell density, these two signal receptors function in parallel to activate the key regulator LuxO, which is essential for virulence of this pathogen. At high cell density, binding of AIs to their respective receptors leads to deactivation of LuxO and repression of virulence factor production. However, mutants lacking CqsS and LuxQ maintain a normal LuxO activation level and remain virulent, suggesting that LuxO is activated by additional, unidentified signaling pathways. Here we show that two other histidine kinases, CqsR (formerly known as VC1831) and VpsS, act upstream in the central QS circuit of V. cholerae to activate LuxO. V. cholerae strains expressing any one of these four receptors are QS proficient and capable of colonizing animal hosts. In contrast, mutants lacking all four receptors are phenotypically identical to LuxO-defective mutants. Importantly, these four functionally redundant receptors act together to prevent premature induction of a QS response caused by signal perturbations. We suggest that the V. cholerae QS circuit is composed of quadruple sensory inputs and has evolved to be refractory to sporadic AI level perturbations.

Tpl2 knockout keratinocytes have increased biomarkers for invasion and metastasis.

Skin cancer is the most common form of cancer in the USA, with an estimated two million cases diagnosed annually. Tumor progression locus 2 (Tpl2), also known as MAP3K8, is a serine/threonine protein kinase in the mitogen-activated protein kinase signal transduction cascade. Tpl2 was identified by our laboratory as having a tumor suppressor function in skin carcinogenesis, with the absence of this gene contributing to heightened inflammation and increased skin carcinogenesis. In this study, we used gene expression profiling to compare expression levels between Tpl2 (+/+) and Tpl2 (-) (/-) keratinocytes. We identified over 2000 genes as being differentially expressed between genotypes. Functional annotation analysis identified cancer, cell growth/proliferation, cell death, cell development, cell movement and cell signaling as the top biological processes to be differentially regulated between genotypes. Further microarray analysis identified several candidate genes, including Mmp1b, Mmp2, Mmp9 and Mmp13, involved in migration and invasion to be upregulated in Tpl2 (-) (/-) keratinocytes. Moreover, Tpl2 (-/-) keratinocytes had a significant downregulation in the matrix metalloproteinase (MMP) inhibitor Timp3. Real-time PCR validated the upregulation of the MMPs in Tpl2 (-/-) keratinocytes and zymography confirmed that MMP2 and MMP9 activity was higher in conditioned media from Tpl2 (-/-) keratinocytes. Immunohistochemistry confirmed higher MMP9 staining in 12-O-tetradecanoylphorbol-13-acetate-treated skin from Tpl2 (-/-) mice and grafted tumors formed from v-ras(Ha) retrovirus-infected Tpl2 (-/-) keratinocytes. Additionally, Tpl2 (-/-) keratinocytes had significantly higher invasion, malignant conversion rates and increased endothelial cell tube formation when compared with Tpl2 (+/+) keratinocytes. In summary, our studies reveal that keratinocytes from Tpl2 (-/-) mice demonstrate a higher potential to be invasive and metastatic.