Specification and survival of post-metamorphic branchiomeric neurons in a non-vertebrate chordate.

Tunicates are the sister group to the vertebrates, yet most species have a life cycle split between swimming larva and sedentary adult phases. During metamorphosis, larval neurons are replaced by adult-specific ones. The regulatory mechanisms underlying this replacement remain largely unknown. Using tissue-specific CRISPR/Cas9-mediated mutagenesis in the tunicate Ciona, we show that orthologs of conserved hindbrain and branchiomeric neuron regulatory factors Pax2/5/8 and Phox2 are required to specify the 'neck', a cellular compartment set aside in the larva to give rise to cranial motor neuron-like neurons post-metamorphosis. Using bulk and single-cell RNA-sequencing analyses, we characterize the transcriptome of the neck downstream of Pax2/5/8. We present evidence that neck-derived adult ciliomotor neurons begin to differentiate in the larva and persist through metamorphosis, contrary to the assumption that the adult nervous system is formed after settlement and the death of larval neurons during metamorphosis. Finally, we show that FGF signaling during the larval phase alters the patterning of the neck and its derivatives. Suppression of FGF converts neck cells into larval neurons that fail to survive metamorphosis, whereas prolonged FGF signaling promotes an adult neural stem cell-like fate.

Breast adipose tissue-derived extracellular vesicles from obese women alter tumor cell metabolism.

Breast adipose tissue is an important contributor to the obesity-breast cancer link. Extracellular vesicles (EVs) are nanosized particles containing selective cargo, such as miRNAs, that act locally or circulate to distant sites to modulate target cell functions. Here, we find that long-term education of breast cancer cells with EVs obtained from breast adipose tissue of women who are overweight or obese (O-EVs) results in increased proliferation. RNA-seq analysis of O-EV-educated cells demonstrates increased expression of genes involved in oxidative phosphorylation, such as ATP synthase and NADH: ubiquinone oxidoreductase. O-EVs increase respiratory complex protein expression, mitochondrial density, and mitochondrial respiration in tumor cells. The mitochondrial complex I inhibitor metformin reverses O-EV-induced cell proliferation. Several miRNAs-miR-155-5p, miR-10a-3p, and miR-30a-3p-which promote mitochondrial respiration and proliferation, are enriched in O-EVs relative to EVs from lean women. O-EV-induced proliferation and mitochondrial activity are associated with stimulation of the Akt/mTOR/P70S6K pathway, and are reversed upon silencing of P70S6K. This study reveals a new facet of the obesity-breast cancer link with human breast adipose tissue-derived EVs causing metabolic reprogramming of breast cancer cells.

Specification and survival of post-metamorphic branchiomeric neurons in the hindbrain of a non-vertebrate chordate.

Tunicates are the sister group to the vertebrates, yet most species have a life cycle split between swimming larva and sedentary adult phases. During metamorphosis, larval neurons are largely replaced by adult-specific ones. Yet the regulatory mechanisms underlying this neural replacement remain largely unknown. Using tissue-specific CRISPR/Cas9-mediated mutagenesis in the tunicate Ciona, we show that orthologs of conserved hindbrain and branchiomeric neuron regulatory factors Pax2/5/8 and Phox2 are required to specify the "Neck", a compartment of cells set aside in the larva to give rise to cranial motor neuron-like neurons in the adult. Using bulk and single-cell RNAseq analyses, we also characterize the transcriptome of the Neck downstream of Pax2/5/8. Surprisingly, we find that Neck-derived adult ciliomotor neurons begin to differentiate in the larva, contrary to the long-held assumption that the adult nervous system is formed only after settlement and the death of larval neurons during metamorphosis. Finally, we show that manipulating FGF signaling during the larval phase alters the patterning of the Neck and its derivatives. Suppression of FGF converts Neck cells into larval neurons that fail to survive metamorphosis, while prolonged FGF signaling promotes an adult neural stem cell-like fate instead.

Breast adipose tissue-derived extracellular vesicles from women with obesity stimulate mitochondrial-induced dysregulated tumor cell metabolism.

Breast adipose tissue is an important contributor to the obesity-breast cancer link. Dysregulated cell metabolism is now an accepted hallmark of cancer. Extracellular vesicles (EVs) are nanosized particles containing selective cargo, such as miRNAs, that act locally or circulate to distant sites to modulate target cell functions. Here, we found that long-term education of breast cancer cells (MCF7, T47D) with EVs from breast adipose tissue of women who are overweight or obese (O-EVs) leads to sustained increased proliferative potential. RNA-Seq of O-EV-educated cells demonstrates increased expression of genes, such as ATP synthase and NADH: ubiquinone oxidoreductase, involved in oxidative phosphorylation. O-EVs increase respiratory complex protein expression, mitochondrial density, and mitochondrial respiration in tumor cells. Mitochondrial complex I inhibitor, metformin, reverses O-EV-induced cell proliferation. Several miRNAs, miR-155-5p, miR-10a-3p, and miR-30a-3p, which promote mitochondrial respiration and proliferation, are enriched in O-EVs relative to EVs from lean women. O-EV-induced proliferation and mitochondrial activity are associated with stimulation of the Akt/mTOR/P70S6K pathway, and are reversed upon silencing of P70S6K. This study reveals a new facet of the obesity-breast cancer link with human breast adipose tissue-derived EVs causing the metabolic reprogramming of ER+ breast cancer cells.

Using Clinical Measurements to Predict Breast Skin Necrosis: A Quantitative Analysis.

BACKGROUND: Breast skin necrosis can lead to poor healing, reoperation, and unaesthetic reconstructive outcomes after mastectomy. Furthermore, the prolonged recovery can delay adjuvant oncologic regimens. This study aims to explore the role of breast surface area as a risk factor for mastectomy skin flap necrosis and to identify predictive clinical measurements. METHODS: The authors retrospectively identified patients who underwent immediate breast reconstruction (N = 926 breasts) by 2 surgeons at a single institution between 2011 and 2021. Preoperative breast measurements such as nipple-notch (NN) distance, nipple-inframammary fold (NF) distance, chest width (CW), breast circumference (BC), and breast height (BH) were used to estimate breast surface area. Univariate analysis and receiver operating characteristic curves were used to determine predictive measurements and optimal cutoff values. RESULTS: When approximated using either a cone without base or a half ellipsoid, larger surface area was a significant risk factor for mastectomy skin flap necrosis (P = 0.027 and P = 0.022, respectively). Larger NN, NF, CW, BC, and BH measurements were significant predictors of necrosis (P < 0.05). Surface area (cone without base) greater than 212 cm2, surface area (half ellipsoid) greater than 308 cm2, NN distance greater than 27 cm, NF greater than 8.5 cm, CW greater than 15 cm, BC greater than 29 cm, and BH greater than 10.5 cm are all values shown to increase the incidence of necrosis. CONCLUSIONS: Larger breast surface area is an independent risk factor for breast skin necrosis. Preoperative breast measurements can be a useful adjunct for predicting necrosis in postmastectomy patients.

A Comparative Analysis of Risk Factors for Breast Skin Necrosis following Autologous versus Device-Based Breast Reconstruction.

BACKGROUND: Mastectomy skin flap necrosis often necessitates prolonged wound care, surgical re-excision, and it increases the risk for infection. This study aims to compare rates of skin flap necrosis between autologous and device-based reconstructions and identify risk factors. METHODS: The authors retrospectively identified patients who underwent immediate breast reconstruction using either the deep inferior epigastric perforator (DIEP) flap (n = 373 breasts, 41%) or tissue expanders (n = 529 breasts, 59%) by two surgeons at a single institution between 2011 and 2021. The rate of skin flap necrosis between autologous and device-based reconstructions was compared and multivariate regression analysis was performed to identify risk factors. RESULTS: There was no significant difference in rates of skin flap necrosis between the two cohorts (26.8 vs. 15.5%, p = 0.052). Across all patients, hypertension and body mass index >30 were significant predictors of necrosis (p = 0.024 and p <0.001, respectively). Within our DIEP cohort, mastectomy specimen weight was a significant risk factor for necrosis (p = 0.001). The DIEP flap weight itself did not confer a higher risk for necrosis (p = 0.8). CONCLUSION: Immediate autologous reconstruction does not place patients at higher risk of skin necrosis. Hypertension and obesity (body mass index >30) were independent risk factors for necrosis in all patients. Mastectomy specimen weight was a significant predictor of necrosis in DIEP flap patients while the DIEP flap weight itself did not increase the risk for necrosis.

Ep300 sequestration to functionally distinct glucocorticoid receptor binding loci underlie rapid gene activation and repression.

The rapid transcriptional response to the transcription factor, glucocorticoid receptor (GR), including gene activation or repression, is mediated by the spatial association of genes with multiple GR binding sites (GBSs) over large genomic distances. However, only a minority of the GBSs have independent GR-mediated activating capacity, and GBSs with independent repressive activity were rarely reported. To understand the positive and negative effects of GR we mapped the regulatory environment of its gene targets. We show that the chromatin interaction networks of GR-activated and repressed genes are spatially separated and vary in the features and configuration of their GBS and other non-GBS regulatory elements. The convergence of the KLF4 pathway in GR-activated domains and the STAT6 pathway in GR-repressed domains, impose opposite transcriptional effects to GR, independent of hormone application. Moreover, the ROR and Rev-erb transcription factors serve as positive and negative regulators, respectively, of GR-mediated gene activation. We found that the spatial crosstalk between GBSs and non-GBSs provides a physical platform for sequestering the Ep300 co-activator from non-GR regulatory loci in both GR-activated and -repressed gene compartments. While this allows rapid gene repression, Ep300 recruitment to GBSs is productive specifically in the activated compartments, thus providing the basis for gene induction.

A Comparison Between Immediate and Babysitter Deep Inferior Epigastric Perforator Flap Breast Reconstruction in Postoperative Outcomes.

BACKGROUND: Delayed-immediate, or "babysitter," deep inferior epigastric perforator (DIEP) flap reconstruction, defined as immediate tissue expander or implant placement at the time of mastectomy followed by eventual exchange for DIEP flap, is becoming increasingly popular in breast cancer patients anticipated to receive adjuvant radiotherapy. In this study, we aim to compare delayed-immediate to immediate DIEP flap patients in postoperative outcomes including major complications and surgical site morbidity. METHODS: A retrospective cohort study between immediate and delayed-immediate DIEP flap patients was performed. Patient demographics, comorbidities, and preoperative cancer treatment were compared between the 2 cohorts. Clinical outcomes of interest included dehiscence, necrosis, and infection of the breast, abdomen, and umbilicus in the 90-day postoperative period as well as breast hematoma, anastomotic failure, flap loss, and venous thromboembolism. RESULTS: Of the 248 patients (443 breasts) included in this study, 193 women (344 breasts) and 55 women (99 breasts) were in the immediate and delayed-immediate cohorts, respectively. The 2 cohorts were comparable in age, body mass index, and comorbidities (P > 0.05). Despite significantly higher rates of preoperative cancer treatment (P < 0.05), delayed-immediate patients were not at an elevated risk for major complications. The 2 cohorts were also comparable in surgical site outcomes, with the exception of breast skin necrosis, which was significantly higher in incidence in the immediate cohort (16.0% vs 2.0%, P < 0.001). CONCLUSIONS: This study is the first to directly compare delayed-immediate to immediate DIEP flap reconstruction in postoperative outcomes. Our findings show that babysitter DIEP flaps are a safe option for patients, even in those expected to undergo breast irradiation preoperatively.

Explantation in Tissue Expander and Direct-to-Implant Reconstruction with Acellular Dermal Matrix: How to Avoid Early Reconstructive Failures.

BACKGROUND: In the United States, approximately 57,000 tissue expander/implant-based breast reconstructions are performed each year. Complete submuscular tissue expander coverage affords the best protection against implant exposure but can restrict lower pole expansion. The benefits of using acellular dermal matrix are enticing, but questions remain as to whether or not its presence increases reconstructive failures. The purpose of this study was to investigate predictors of explantation in those patients with acellular dermal matrix reconstructions and to discuss salvage techniques. METHODS: An approved retrospective review was conducted of 137 patients undergoing 234 individual breast reconstructions over 4 years performed by a single plastic surgeon (J.D.) at a single institution. Patients who underwent implant-based reconstruction with either immediate placement of a tissue expander that was subsequently exchanged for a permanent implant at a second operation or immediate placement of a permanent implant when indicated were included. RESULTS: One hundred thirty-seven patients who underwent 234 implant-based breast reconstructions using acellular dermal matrix met criteria. There was an overall 23 percent complication rate, including any cellulitis, seroma, skin necrosis, and hematoma formation. Significant preoperative risk factors for any postoperative complication included body mass index greater than 25 kg/m2 and a history of radiation therapy before acellular dermal matrix placement. Radiation therapy was found to be a significant risk factor for postoperative skin necrosis. Of explantations, many fluid cultures grew Gram-negative bacteria. CONCLUSIONS: Skin necrosis is a serious risk factor for explantation in implant-based reconstruction with acellular dermal matrix. The reconstructive surgeon should consider early excision of any skin necrosis as soon as it is identified. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

Genomic retargeting of p53 and CTCF is associated with transcriptional changes during oncogenic HRas-induced transformation.

Gene transcription is regulated by distant regulatory elements via combinatorial binding of transcription factors. It is increasingly recognized that alterations in chromatin state and transcription factor binding in these distant regulatory elements may have key roles in cancer development. Here we focused on the first stages of oncogene-induced carcinogenic transformation, and characterized the regulatory network underlying transcriptional changes associated with this process. Using Hi-C data, we observe spatial coupling between differentially expressed genes and their differentially accessible regulatory elements and reveal two candidate transcription factors, p53 and CTCF, as determinants of transcriptional alterations at the early stages of oncogenic HRas-induced transformation in human mammary epithelial cells. Strikingly, the malignant transcriptional reprograming is promoted by redistribution of chromatin binding of these factors without major variation in their expression level. Our results demonstrate that alterations in the regulatory landscape have a major role in driving oncogene-induced transcriptional reprogramming.

Muscle Flap Closure following Complex Spine Surgery: A Decade of Experience.

BACKGROUND: Advances in surgical technology and adjuvant therapies along with an aging and increasingly morbid U.S. population have led to an increase in complex spine surgery. With this increase comes an elevated risk of complications, including those related to the surgical wound, with some studies demonstrating wound complication incidences approaching 45 percent. The authors hypothesize that immediate muscle flap closure improves outcomes in high-risk patients. METHODS: Three hundred one consecutive index cases of spinal wound closure using local muscle flaps performed by the senior author at a single institution between 2006 and 2018 were reviewed. The primary outcome was major wound complication (reoperation and/or readmission because of surgical-site infection, late infection, dehiscence, seroma, or hematoma). Logistic regression analysis was performed to identify predictors of this endpoint. RESULTS: Major wound complications occurred in 6.6 percent of patients (reoperation, 3.6 percent; readmission, 3.0 percent), with a 6.0 percent infection rate and five cases requiring instrumentation removal because of infection. Risk factors identified included radiotherapy (OR, 5.9; p = 0.004), age 65 years or older (OR, 2.8; p = 0.046), and prior spine surgery (OR, 4.3; p = 0.027). The incidence of major wound complication increased dramatically with each additional risk factor. Mean drain dwell duration was 21.1 ± 10.0 days and not associated with major wound complications, including infection (OR, 1.04; p = 0.112). CONCLUSIONS: Immediate local muscle flap closure following complex spine surgery on high-risk patients is associated with an acceptable rate of wound complications and, as these data demonstrate, is safe and effective. Consideration should be given to immediate muscle flap closure in appropriately selected patients. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

The de-coders: A historical perspective of the genetic counseling profession.

The genetic counseling profession is a relatively new medical specialty that has evolved over time in keeping with advanced in the medical genetics field. Genetic counselors are master's-level trained individuals who apply their skills to assist patients in understanding the genetic etiology of disease, assessing risk for genetic disease, and providing support to those who are encountering a genetic disorder in themselves or family member. Formal training of genetic counselors began nearly 50 years, and since then, the field has been expanding and advancing. Defining the tenets and practice of the genetic counseling profession has also similarly changed over time; however, the core guiding principles of the profession have remained rooted in the concept of patient autonomy and client-centered care. Competency in the field is achieved by successfully graduating from an accredited master's-level training program in which the curriculum is based on standards set forth by the Accreditation Council of Genetic Counseling. Curricula are developed so that trainees are exposed to didactic work, hands-on clinical fieldwork, and research so that at the end of one's training they have become competent in The Practice Based Competencies that have been established for the profession. Upon graduation, genetic counselors find themselves working in a number of professional settings and subspecialties in genetics. As the underlying genetic cause of disease has become clearer, the areas in which genetic counselors practice have expanded and allows for diverse clinical practice. The profession has, and will, continue to adapt along with the advances in molecular genetics.

Practices in synagogues regarding Jewish genetic disease education.

Approximately one in three Ashkenazi Jews are carriers for an autosomal recessive Jewish genetic disease (JGD). However, studies indicate that most Jews are uneducated on this topic and obstetricians do not routinely offer carrier screening to Jewish patients. Both the Reform and Conservative movements of Judaism call for JGD education to take place within the synagogue; however, little is known about the extent of this education occurring today. An online survey was created for Reform and Conservative rabbis to assess the types of JGD education taking place within the synagogue. Additionally, the survey included questions to assess JGD knowledge and possible factors that could predict counseling activity and knowledge level. Of the 94 participants, 91% had provided education about JGDs to congregants, with 98.8% providing this education during premarital counseling sessions. For most respondents, explaining recessive inheritance pattern and carrier screening was the extent of the discussion. Additionally, the majority of rabbis scored below 50% on the knowledge portion of the survey, with an average score of 1.9/4. There were no statistically significant differences between JGD education in Reform vs. Conservative synagogues, and there were no statistically significant predictors of knowledge score or JGD education frequency. In conclusion, while the number of rabbis discussing this topic is encouraging, discussion topics were found to be limited and their knowledge of JGDs was found to be poor.

The medial sural artery perforator flap: A better option in complex head and neck reconstruction?

OBJECTIVES: The medial sural artery perforator (MSAP) free flap is an uncommonly utilized soft tissue flap in head and neck reconstruction. It is a thin, pliable, fasciocutaneous flap that provides significant pedicle length. The donor site can be closed primarily, and its location is more aesthetically pleasing to patients. We aim to describe the MSAP flap and compare it to other commonly used free flaps in the head and neck. STUDY DESIGN: Retrospective case series. METHODS: A retrospective review of all MSAP cases performed at New York University Langone Health was performed from July 2016 to November 2017. We examined the patients' age, diagnosis, history of prior radiation therapy, and comorbidities, as well as flap-specific information and recipient site. RESULTS: Twenty-one patients underwent a variety of different head and neck procedures with coverage using an MSAP flap. Recipient sites included tongue, cheek, soft and hard palate, cervical esophagus, and pharynx. Pedicle length ranged from 8 cm to 12 cm. The smallest surface area harvested was 24 cm2 (6 cm × 4 cm), and the largest was 120 cm2 (15 cm × 8 cm). The flaps ranged from 5 to 12 mm in thickness. Venous coupler size ranged from 2.0 to 3.5 mm. Primary closure of the donor site was achieved in 18 of 21 flaps. Twenty of 21 flaps were transferred successfully. CONCLUSION: The MSAP flap is a highly versatile and reliable option for a thin, pliable soft tissue flap with a donor site that may be preferable over the radial forearm free flap and anterolateral thigh flap in complex head and neck reconstruction. LEVEL OF EVIDENCE: 4 Laryngoscope, 129:1330-1336, 2019.

Implementation Science and Prevention in Action: Application in a Post-Permanency World.

This article describes how implementation science and intervention research guided the process of selecting and implementing an evidence-informed intervention (Tuning in to Teens; TINT). TINT was provided as a selective prevention effort offered to families with youth aged 10 to 13 years old, with characteristics that suggest an elevated risk for post-permanency discontinuity. Usability testing findings: Contact was made with 54% of families, and 12% participated in the intervention. Multivariate results found no statistically significant differences between families who responded to outreach efforts and those who did not; families who participated in TINT and those who did not. Implications: Large public child welfare systems wanting to implement evidence-informed interventions can follow the steps detailed in this paper for selecting, adapting and implementing an intervention. Further, providers that seek to offer post adoption and guardianship services, a growing service need, may gain some insights into activities that promote service usage with this population.

Local muscle flaps minimize post-operative wound morbidity in patients with neoplastic disease of the spine.

OBJECTIVES: Patients with neoplastic disease involving the spine either from primary or metastatic disease present a unique challenge given these patients' frequent poor nutritional status at the time of surgery, the delivery of early post-operative chemotherapy or radiation, and placement of large amounts of hardware and avascular bone graft into a wound bed that is atrophic, previously operated or irradiated. As a result, wound morbidity has traditionally been high in this cohort of patients. Herein we review the outcomes of patients at our institution who underwent local muscle flap closure following spinal tumor extirpation. PATIENTS AND METHODS: Between 2007 and 2017, 55 patients with oncologic disease of the spine underwent 60 spine surgeries and concomitant muscle flap reconstruction. Charts were retrospectively reviewed for diagnosis and indications for surgery, as well as risk factors for poor wound healing including diabetes, steroid use, body mass index (BMI), history of pre-operative chemo and or radiation therapy, preoperative albumin and hemoglobin levels. Outcomes were postoperative wound related complications including surgical site infection, wound dehiscence and/or need for reoperation. RESULTS: 60 reconstructions were included in 55 patients. Median follow up was 253 days. Paraspinous muscle flaps were used in all cases. There were 2 major complications (3.3%) related to wound infections which required reoperation and 10 minor wound complications (16.7%), of which 9 were subcutaneous seromas aspirated in the office, that did not require return to the operating room. Median postoperative stay in the hospital was 10 days. Closed suction drains placed at the end of the reconstruction were removed at a median of 17.5 days. Regression analysis found patient BMI to be a significant risk predictor for wound related post-operative complications. CONCLUSIONS: Post-operative wound specific complications that required return to the operating room were uncommon despite the high-risk profile of this subset of patients. These data indicate that muscle flap closure should be routinely practiced in this high-risk cohort of patients.

An assessment of health, social, communication, and daily living skills of adults with Down syndrome.

Adults with Down syndrome (DS) are surviving longer, yet data delineating life skills are lacking. As providers are encouraged to provide a "balanced" description of DS to family members/caregivers, more quantitative data are required to accurately describe the abilities and potential of adults with DS. This study assessed health, social, communication, and daily living skills of adults with DS to describe the range of abilities and to show how increasing age contributes to functional abilities. Caregivers of an adult with DS 20 years of age or older participated in an online questionnaire. Descriptive statistics and scores from scales assessed relationships between the number of health issues reported and functional abilities, and how the abilities changed as age increased. Of 188 participants, 157 completed the survey with partial results included. Communication, independence, and social activity scores were compared to the number of congenital and non-congenital health issues reported. Linear regression results showed those with more health issues were significantly less likely to be independent and social. However, only current health issues affected communication skills. No significant correlation occurred between the number of congenital abnormalities and scores for independence/life skills as an adult. T-test by age group found decreasing abilities after 40 years of age. In conclusion, quantitative data and information from this study is beneficial for providers in order to describe the potential for an individual with DS and to assist caregivers to plan accordingly for the future of their adult with DS.