Methods for investigating STAT3 regulation of lysosomal function in mammary epithelial cells.

The transcription factor STAT3 is activated by multiple cytokines and other extrinsic factors. It plays a key role in immune and inflammatory responses and, when dysregulated, in tumourigenesis. STAT3 is also an indispensable mediator of the cell death process that occurs during post-lactational regression of the mammary gland, one of the most dramatic examples of physiological cell death in adult mammals. During this involution of the gland, STAT3 powerfully enhances the lysosomal system to efficiently remove superfluous milk-producing mammary epithelial cells via a lysosomal-mediated programmed cell death pathway. The lysosome is a membrane-enclosed  cytoplasmic organelle that digests and recycles cellular waste, with an important role as a signalling centre that monitors cellular metabolism. Here, we describe key strategies for investigating the role of STAT3 in regulating lysosomal function using a mammary epithelial cell culture model system. These include protocols for lysosome enrichment and enzyme activity assays, in addition to microscopic analyses of the vesicular compartment in cell lines. Collectively, these approaches provide the tools to investigate multiple aspects of lysosome biogenesis and function, and to define both direct and indirect roles for STAT3.

Highly efficient multiplex genetic engineering of porcine primary fetal fibroblasts.

BACKGROUND: Genetically engineered porcine donors are a potential solution for the shortage of human organs for transplantation. Incompatibilities between humans and porcine donors are largely due to carbohydrate xenoantigens on the surface of porcine cells, provoking an immune response which leads to xenograft rejection. MATERIALS AND METHODS: Multiplex genetic knockout of GGTA1, ß4GalNT2, and CMAH is predicted to increase the rate of xenograft survival, as described previously for GGTA1. In this study, the clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 system was used to target genes relevant to xenotransplantation, and a method for highly efficient editing of multiple genes in primary porcine fibroblasts was described. RESULTS: Editing efficiencies greater than 85% were achieved for knockout of GGTA1, ß4GalNT2, and CMAH. CONCLUSION: The high-efficiency protocol presented here reduces scale and cost while accelerating the production of genetically engineered primary porcine fibroblast cells for in vitro studies and the production of animal models.

Three decades of progress from surgery to medical therapy for isolated neuroaxis BRAF V600E-positive Langerhans cell histiocytosis management: illustrative case.

BACKGROUND: "Langerhans cell histiocytosis" (LCH) is a term that encompasses single-system or multisystem disorders traditionally characterized by a proliferation of clonal CD1a+/CD207+ myeloid-derived histiocytes. In most cases of LCH, mitogen-activated protein kinase (MAPK) pathway somatic mutations lead to near universal upregulation of phosphorylated extracellular signal-regulated kinase expression. The clinical manifestations of LCH are numerous, but bone involvement is common. Intracranial lesions, especially as isolated manifestations, are rare. OBSERVATIONS: The authors presented the case of a long-term survivor of exclusive intracranial LCH that manifested with isolated craniofacial bone and intraparenchymal central nervous system recurrences, which were managed with 3 decades of multimodal therapy. The patient was initially diagnosed with LCH at age 2 years, and the authors documented the manifestations of disease and treatment for 36 years. Most of the patient's treatment course occurred before the discovery of BRAF V600E. Treatments initially consisted of chemotherapy, radiosurgery, and open resections for granulomatous LCH lesions. Into young adulthood, the patient had a minimal disease burden but still required additional radiosurgical procedures and open resections. LESSONS: Surgical treatments alleviated the patient's immediate symptoms and allowed for tumor burden control. However, surgical interventions did not cure the underlying, aggressive disease. In the current era, access to systemic MAPK inhibitor therapy for histiocytic lesions may offer improved outcomes.

Predictors and Outcomes of Ischemic Stroke After Cardiac Surgery.

BACKGROUND: Stroke is a major complication after cardiac surgery causing increased morbidity and mortality. There are limited data on outcomes of patients with large vessel occlusion after cardiac surgery. METHODS: Patients who underwent index cardiac surgeries as defined by The Society of Thoracic Surgeons (STS) from 2010 to 2017 were reviewed from a prospectively maintained database. All patients with neurologic deficits were identified, and only patients with ischemic strokes were included. RESULTS: A total of 10,250 patients underwent cardiac surgical procedures. Postoperative stroke with neurologic deficits occurred in 221 patients (2.16%). Of these, 53 patients (24%) had large vessel occlusion. Patients who had a postoperative stroke were older and more likely to be female. These patients had higher STS predicted mortality and longer bypass time, cross-clamp time, total intensive care unit stay, and total hospital stay. Operative mortality was significantly higher for patients who had postoperative stroke (14.93% vs 2.15%, P < .001). Kaplan-Meier survival estimates demonstrated worse survival for the large vessel occlusion cohort at 1 year (54.72% vs 75%, P = .002). Predictors of stroke included increasing age, known cerebrovascular disease, diabetes mellitus, and emergent operative status. The most significant predictors of operative mortality included emergent operative status and New York Heart Association stage IV heart failure. There was no difference in 30-day, 1-year, and 5-year mortality between the intervention group and the medically managed patients in the large vessel occlusion cohort. CONCLUSIONS: Stroke is a devastating complication after cardiac surgery that increases operative morbidity and mortality. Stroke with large vessel occlusion was associated with worse survival. However, early intervention did not impart a survival benefit.

Evaluation of a simulation-based mini-elective on medical student interest in cardiac surgery.

BACKGROUND: Integrated cardiothoracic (CT) surgery training programs are an increasingly popular pathway to train CT surgeons. Identifying and engaging medical students early is important to generate interest and ensure highly qualified applicants are aware of opportunities provided by a career in CT surgery. METHODS: An optional CT surgery "mini-elective" was developed for preclinical medical students consisting of five 2-hour sessions covering major procedures in cardiac surgery. Each session had an inital 1 hour lecture immediatly followed by a hands on simulation component. Sessions were taught by CT surgery faculty and residents. A precourse and postcourse survey was administered to identify interest in and awareness of the field of CT surgery. RESULTS: There were 22 students enrolled in the course who provided precourse surveys, while 21 provided postcourse surveys. CT surgery was a career consideration for 95.4% of students who took the mini-elective. nine percent of the students who had either scrubbed or observed a CT case precourse, increased to 33.3% postcourse (P = .11). With regards to mentorship, 23.8% felt they could easily find a mentor in CT surgery precourse, increasing to 66.7% postcourse (P = .01). Eighty-one percent of students reported that the mini-elective significantly increased their CT knowledge over the standard cardiovascular curriculum, and 100% of those completing the course were "extremely satisfied" with the experience. CONCLUSIONS: A CT surgery mini-elective increased awareness and interest in the field among preclinical medical students. Longitudinal exposure and mentorship provided in programs such as this will be key to the continued recruitment of high-quality medical students to the field.

Late Presentation of a 9 mm Bullet in the Ureteropelvic Junction Causing Acute Renal Failure in a Solitary Functioning Left Kidney.

Background: Urinary obstruction as a result of a late complication of a gunshot wound is rarely reported. Bullet shell fragments may migrate from their initial location into an area causing obstruction. In this study, we present a case of a left renal calculus surrounding a 9 mm gun shell in a patient with a solitary functioning left kidney. Case Presentation: A 53-year-old man presented with left hydronephrosis found on an urgent CT scan following complaints of left flank pain and signs of acute renal failure 10 years after suffering a gunshot wound to the abdomen. Urgent cystoscopy and bilateral retrograde pyelograms revealed a left ureteropelvic junction calculus surrounding a 9 mm bullet fragment that was ultimately removed percutaneously. Conclusion: Abdominal gunshot shell fragments may migrate over time causing urinary obstruction. In this case the past medical history of the previous gunshot wounds provided insight into the etiology of the patient's actual clinical presentation. This led to the best endoscopic approach for the effective treatment of this unique case.

Lung Cancer Screening Guidelines: How Readable Are Internet-Based Patient Education Resources?

OBJECTIVE: Following the findings of the National Lung Screening Trial, several national societies from multiple disciplines have endorsed the use of low-dose chest CT to screen for lung cancer. Online patient education materials are an important tool to disseminate information to the general public regarding the proven health benefits of lung cancer screening. This study aims to evaluate the reading level at which these materials related to lung cancer screening are written. MATERIALS AND METHODS: The four terms "pulmonary nodule," "radiation," "low-dose CT," and "lung cancer screening" were searched on Google, and the first 20 online resources for each term were downloaded, converted into plain text, and analyzed using 10 well-established readability scales. If the websites were not written specifically for patients, they were excluded. RESULTS: The 80 articles were written at a 12.6 ± 2.7 (mean ± SD) grade level, with grade levels ranging from 4.0 to 19.0. Of the 80 articles, 62.5% required a high school education to comprehend, and 22.6% required a college degree or higher (≥ 16th grade) to comprehend. Only 2.5% of the analyzed articles adhered to the recommendations of the National Institutes of Health and American Medical Association that patient education materials be written at a 3rd- to 7th-grade reading level. CONCLUSION: Commonly visited online lung cancer screening-related patient education materials are written at a level beyond the general patient population's ability to comprehend and may be contributing to a knowledge gap that is inhibiting patients from improving their health literacy.

An Engineered Human Adipose/Collagen Model for In Vitro Breast Cancer Cell Migration Studies.

Adipocytes are one of the major stromal cell components of the human breast. These cells play a key role in the development of the gland and are implicated in breast tumorigenesis. Frequently, directional stromal collagen I fibers are found surrounding aggressive breast tumors. These fibers enhance breast cancer cell migration and are associated with poor patient prognosis. We sought to recapitulate these stromal components in vitro to provide a three-dimensional (3D) model comprising human adipose tissue and anisotropic collagen fibers. We developed a human mesenchymal stem cell (hMSC) cell line capable of undergoing differentiation into mature adipocytes by immortalizing hMSCs, isolated from breast reduction mammoplasties, through retroviral transduction. These immortalized hMSCs were seeded in engineered collagen I scaffolds with directional internal architecture, and adipogenesis was chemically induced, resulting in human adipose tissue being synthesized in vitro in an architectural structure associated with breast tumorigenesis. Subsequently, fluorescently labeled cells from an established breast cancer cell line were seeded into this model, cocultured for 7 days and imaged using multiphoton microscopy. Enhanced breast cancer cell migration was observed in the adipose-containing model over empty scaffold controls, demonstrating an adipocyte-mediated influence on breast cancer cell migration. Thus, this 3D in vitro model recapitulates the migratory effects of adipocytes observed on breast cancer cells and suggests that it could have utility with fresh breast tumor biopsies as an assay for cancer therapeutic efficacy in personalized medicine strategies.

Stat3-mediated alterations in lysosomal membrane protein composition.

Lysosome function is essential in cellular homeostasis. In addition to its recycling role, the lysosome has recently been recognized as a cellular signaling hub. We have shown in mammary epithelial cells, both in vivo and in vitro, that signal transducer and activator of transcription 3 (Stat3) modulates lysosome biogenesis and can promote the release of lysosomal proteases that culminates in cell death. To further investigate the impact of Stat3 on lysosomal function, we conducted a proteomic screen of changes in lysosomal membrane protein components induced by Stat3 using an iron nanoparticle enrichment strategy. Our results show that Stat3 activation not only elevates the levels of known membrane proteins but results in the appearance of unexpected factors, including cell surface proteins such as annexins and flotillins. These data suggest that Stat3 may coordinately regulate endocytosis, intracellular trafficking, and lysosome biogenesis to drive lysosome-mediated cell death in mammary epithelial cells. The methodologies described in this study also provide significant improvements to current techniques used for the purification and analysis of the lysosomal proteome.

Quantitative analysis of the level of readability of online emergency radiology-based patient education resources.

PURPOSE: The vast amount of information found on the internet, combined with its accessibility, makes it a widely utilized resource for Americans to find information pertaining to medical information. The field of radiology is no exception. In this paper, we assess the readability level of websites pertaining specifically to emergency radiology. METHODS: Using Google, 23 terms were searched, and the top 10 results were recorded. Each link was evaluated for its readability level using a set of ten reputable readability scales. The search terms included the following: abdominal ultrasound, abdominal aortic aneurysm, aortic dissection, appendicitis, cord compression, CT abdomen, cholecystitis, CT chest, diverticulitis, ectopic pregnancy, epidural hematoma, dural venous thrombosis, head CT, MRI brain, MR angiography, MRI spine, ovarian torsion, pancreatitis, pelvic ultrasound, pneumoperitoneum, pulmonary embolism, subarachnoid hemorrhage, and subdural hematoma. Any content that was not written for patients was excluded. RESULTS: The 230 articles that were assessed were written, on average, at a 12.1 grade level. Only 2 of the 230 articles (1%) were written at the third to seventh grade recommended reading level set forth by the National Institutes of Health (NIH) and American Medical Association (AMA). Fifty-two percent of the 230 articles were written so as to require a minimum of a high school education (at least a 12th grade level). Additionally, 17 of the 230 articles (7.3%) were written at a level that exceeded an undergraduate education (at least a 16th grade level). CONCLUSIONS: The majority of websites with emergency radiology-related patient education materials are not adhering to the NIH and AMA's recommended reading levels, and it is likely that the average reader is not benefiting fully from these information outlets. With the link between health literacy and poor health outcomes, it is important to address the online content in this area of radiology, allowing for patient to more fully benefit from their online searches.

NFPA 99 Chapter 13 and healthcare security management.

The new NFPA 99 Security Management Standard for healthcare facilities requires a Security Vulnerability Analysis be conducted annually, the author reports. This will usually uncover little change from year to year, he says, but by using the right model the analysis can be used to win back security additions that had previously been rejected by the C-Suite.

Infectious disease protection for healthcare security officers.

Healthcare Security should be considered an active component in an infectious disease event, the authors maintain, and security officers must be included in an Employee Health screening and N95 fit testing initiative to safely welcome the incoming infected patients. In this article, they spell out the different levels of precautions officers should become familiar with in order to protect themselves.

Analysis of Perforin Assembly by Quartz Crystal Microbalance Reveals a Role for Cholesterol and Calcium-independent Membrane Binding.

Perforin is an essential component in the cytotoxic lymphocyte-mediated cell death pathway. The traditional view holds that perforin monomers assemble into pores in the target cell membrane via a calcium-dependent process and facilitate translocation of cytotoxic proteases into the cytoplasm to induce apoptosis. Although many studies have examined the structure and role of perforin, the mechanics of pore assembly and granzyme delivery remain unclear. Here we have employed quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate binding and assembly of perforin on lipid membranes, and show that perforin monomers bind to the membrane in a cooperative manner. We also found that cholesterol influences perforin binding and activity on intact cells and model membranes. Finally, contrary to current thinking, perforin efficiently binds membranes in the absence of calcium. When calcium is added to perforin already on the membrane, the QCM-D response changes significantly, indicating that perforin becomes membranolytic only after calcium binding.

Assembly of streptolysin O pores assessed by quartz crystal microbalance and atomic force microscopy provides evidence for the formation of anchored but incomplete oligomers.

Streptolysin O (SLO) is a bacterial pore forming protein that is part of the cholesterol dependent cytolysin (CDC) family. We have used quartz crystal microbalance with dissipation monitoring (QCM-D) to examine SLO membrane binding and pore formation. In this system, SLO binds tightly to cholesterol-containing membranes, and assembles into partial and complete pores confirmed by atomic force microscopy. SLO binds to the lipid bilayer at a single rate consistent with the Langmuir isotherm model of adsorption. Changes in dissipation illustrate that SLO alters the viscoelastic properties of the bilayer during pore formation, but there is no loss of material from the bilayer as reported for small membrane-penetrating peptides. SLO mutants were used to further dissect the assembly and insertion processes by QCM-D. This shows the signature of SLO in QCM-D changes when pore formation is inhibited, and that bound and inserted SLO forms can be distinguished. Furthermore a pre-pore locked SLO mutant binds reversibly to lipid, suggesting that the partially complete wtSLO forms observed by AFM are anchored to the membrane.

Development of an audiovisual speech perception app for children with autism spectrum disorders.

Perception of spoken language requires attention to acoustic as well as visible phonetic information. This article reviews the known differences in audiovisual speech perception in children with autism spectrum disorders (ASD) and specifies the need for interventions that address this construct. Elements of an audiovisual training program are described. This researcher-developed program delivered via an iPad app presents natural speech in the context of increasing noise, but supported with a speaking face. Children are cued to attend to visible articulatory information to assist in perception of the spoken words. Data from four children with ASD ages 8-10 are presented showing that the children improved their performance on an untrained auditory speech-in-noise task.