Several small molecule corrector and potentiator drugs have recently been licensed for Cystic Fibrosis (CF) therapy. However, other aspects of the disease, especially inflammation, are less effectively treated by these drugs. We hypothesized that small molecule drugs could function either alone or as an adjuvant to licensed therapies to treat these aspects of the disease, perhaps emulating the effects of gene therapy in CF cells. The cardiac glycoside digitoxin, which has been shown to inhibit TNFα/NFκB signaling in CF lung epithelial cells, may serve as such a therapy.wildtype]CFTR-treated IB3–1 (S9) cells. We also compared in vitro gene expression signatures with in vivo data from biopsied nasal epithelial cells from digitoxin-treated CF patients.IB3–1 CF lung epithelial cells were treated with different Vertex (VX) drugs, digitoxin, and various drug mixtures, and ELISA assays were used to assess suppression of baseline and TNFα-activated secretion of cytokines and chemokines. Transcriptional responses to these drugs were assessed by RNA-seq and compared with gene expression in AAV-[CF cells exposed to digitoxin exhibited significant suppression of both TNFα/NFκB signaling and downstream secretion of IL-8, IL-6 and GM-CSF, with or without co-treatment with VX drugs. No evidence of drug-drug interference was observed. RNA-seq analysis showed that gene therapy-treated CF lung cells induced changes in 3134 genes. Among these, 32.6% were altered by digitoxin treatment in the same direction. Shared functional gene ontology themes for genes suppressed by both digitoxin and gene therapy included inflammation (84 gene signature), and cell-cell interactions and fibrosis (49 gene signature), while genes elevated by both were enriched for epithelial differentiation (82 gene signature). A new analysis of mRNA data from digitoxin-treated CF patients showed consistent trends in expression for genes in these signatures.Adjuvant gene therapy-emulating activities of digitoxin may contribute to enhancing the efficacy of currently licensed correctors and potentiators in CF patients. CFTR gene [F508del] [G551D] mutation [G551D] gating mutant [F508del] mutation in combination with VX- 770 [F508del] CFTR mutation. However, despite substantial and successful responses in CF patients, there remain other aspects of the disease for which the corrector and potentiator drugs are less effective [Cystic fibrosis (CF) is a life-limiting autosomal recessive genetic disease due to mutations in the FTR gene –3. In thF508del] , 5, and mutation –9, are bmutation –14. For g mutant . A modes VX- 770 . More re VX- 770  and hete VX- 770  for the ffective –22. The ffective ., where it was found to inhibit TNFα/NFκB signaling and downstream IL-8 secretion CFTR gene therapy in CF lung epithelial cells. Finally, re-examination of nasal epithelial cell mRNA expression data from CF patients treated with digitoxin suggests that there are significant clinical parallels with the in vitro data collected in this study. Taken together, these data suggest adjuvant gene therapy-emulating activities of digitoxin may contribute to enhancing the efficacy of currently licensed correctors and potentiators in CF patients.Here, we show that digitoxin is able to General information is given below. Greater detail is included in Additional file F508del/W1282X]), and daughter IB3–1/S9 cells stably transfected with AAV-[wildtype] CFTR CFTR with RNA-seq CFTR gene therapy. We conclude that studies with digitoxin on the CF IB3–1 lung epithelial cell system appear to be clinically associative for mRNA expression in upper airway epithelial cells from CF patients.To test whether our in vitro transcriptional data of cultured digitoxin-treated CF cells had any clinical relevance for CF patients, we interrogated Affymetrix exon array mRNA expression data collected from nasal biopsies of digitoxin-treated CF patients . We calcreduced inflammation, a 49 gene signature for reduced fibrosis, and an 82 gene signature for elevated epithelial differentiation. Consistently, digitoxin also suppressed expression of TGFBR2 protein, the receptor for TGFβ, the master regulator of fibrosis  and [G551D]CFTR proteins fail to bind TRADD, and thus fail to inhibit downstream activation of NFκB. These two different experiments thus indicate that TRADD is a common inflammation-controlling target for both digitoxin and [wildtype]CFTR. It is thus possible that coincident interactions with TRADD may be the basis for the large scale and highly functional transcriptional overlap between digitoxin and gene therapy. Digitoxin thus differs from other candidate NFκB inhibitors with different mechanisms of action in this way, whose renal and CNS side effects are said to have rendered them generally useless for clinical applications  (Orkambi®), and [VX-770/VX-661] (Symdeko®). Based on the ELISA and RNA-seq data, reduction of TNFα/NFκB-driven inflammation is not among the significant functional gene ontology themes for the VX drugs , CFTR mutation.Additional file 6. Functional Gene Ontology themes for [wildtype]CFTR Gene Therapy, and Drug-specific changes in CF lung Epithelial cells.Additional file 7. Genes up-regulated by both digitoxin treatment and gene therapy.Additional file 8. Genes down-regulated by both digitoxin treatment and gene therapy.Additional file 9. Genes upregulated by digitoxin treatment and gene therapy against IB3-1 controls annotated to epithelial differentiation ontologies, and compared in parallel to effects of VX-661, VX-770 and VX-809.Additional file 10. Digitoxin-dependent changes in protein expression for IL-8, IL-6, TGFBR2 and KRT8.Additional file 11. Genes downregulated by digitoxin treatment and gene therapy against IB3-1 controls annotated to inflammatory ontologies, compared in parallel to effects of VX-661, VX-770 and VX-809.Additional file 12. Genes down-regulated by digitoxin treatment and gene therapy against IB3-1 controls annotated to cell-cell interaction/fibrosis ontologies, and compared in parallel with VX-661, VX-770 and VX-809.Additional file 13. Effects of digitoxin on licensed VX-drug and drug combinations on reduced expression of mRNAs common to digitoxin and AAV-[wildtype]CFTR for the functional GO theme of inflammation.Additional file 14. Effects of digitoxin on licensed VX-drug and drug combinations on reduced expression of mRNAs common to digitoxin and AAV-[wildtype]CFTR for the functional GO theme of cell-cell interactions/fibrosis.Additional file 15. Effects of digitoxin on licensed VX-drug and drug combinations on increased expression of genes common to digitoxin and AAV-[wildtype]CFTR for the functional theme of epithelial differentiation.Additional file 16. Comparison of genes down-regulated by digitoxin treatment and gene therapy in IB3-1 cells compared to controls by RNA-seq and annotated to inflammatory processes versus Affymetrix log2 expression fold-changes from CF patoents treated with 0.1 mg daily digitoxin for 28 days . Additional file 17. Comparison of genes down-regulated by digitoxin and gene therapy in IB3-1 cells compared to controls by RNA-seq and annotated to fibrotic processes versus Affymetrix log2 expression fold-changes from CF patients treated with 0.1 mg daily digitoxin for 28 days . Additional file 18. Comparison of genes up-regulated by digitoxin treatment and gene therapy in IB3-1 cells compared to controls by RNA-seq and annotated to epithelial differentiation processes versus Affymetrix log2 expression fold-changes from CF patients treated with 0.1 mg daily digitoxin for 28 days .


Endoscopic biopsy is thought to be the best method to obtain biopsy samples of the gastrointestinal tract. In our case, however, an endoscopic forceps biopsy failed to confirm malignancy of an intramural gastric tumour. Since the tumour, about 4 cm in diameter, was well delineated on the CT scan, the patient was referred for a percutaneous CT-guided needle biopsy, which confirmed a gastrointestinal stromal tumour. Besides abdominal discomfort and arterial hypertension, there were no further relevant signs or symptoms, and her family history was unremarkable. As part of the evaluation, a contrast-enhanced CT scan of the abdomen was performed  was performed, but no hypermetabolic structures were found in either the stomach or the pelvic area. However, an 18F-fludeoxyglucose-negative signet-ring cell carcinoma or a gastrointestinal stromal tumour (GIST) was still considered as a differential diagnosis. The origin of the tumour was still unclear, but as the tumour was well delineated on the contrast-enhanced CT scan, a percutaneous CT-guided needle biopsy was considered to obtain valid histological information. We were encouraged by the successful results published by others,4 and a biopsy was scheduled 4 weeks after the initial diagnosis.Subsequently, gastroscopy demonstrated a bulging submucosal tumour in the fundal area . ForcepsThe patient gave oral and written informed consent before the biopsy. All oral feeds and fluids were stopped overnight. The patient was placed in the supine position, with both arms elevated above the head. First, an unenhanced CT scan (slice thickness 3 mm) of the upper abdomen was obtained during shallow breathing to localize the gastric tumour and plan the best entry site. However, the gastric lumen was partially collapsed and the tumour was barely distinguishable from secretions inside the gastric lumen . In ordeThe epigastrium was then prepared in a sterile manner and local anaesthesia (1% lidocaine) was applied down to the peritoneum and throughout the anterior gastric wall. The white line shows the distance between the skin entry point and the surface of the gastric tumour along the proposed biopsy path. To enable further needle insertion, we then used a CT scan protocol dedicated for biopsy, in which each scan acquired in between the biopsy steps consisted of a series of three slices (thickness 6 mm). The triplet of scan slices was repeated before and after each interventional step while we moved in and out of the scanner room. After a small skin incision was made, a 17 G puncture needle of length 17 cm  was gradually inserted during breath-hold through the anterior gastric wall until the tip of the puncture needle was just at the tumour surface. The inner stylet of the puncture needle was then removed and a semiautomatic 18 G cutting biopsy needle  was inserted. The position of the exposed 20 mm biopsy chamber of the sharp metal stylet within the tumour was confirmed  just bef5CT scans performed immediately after the biopsy excluded any bleeding and pneumoperitoneum. The patient was moved back to the ward with bed rest and no oral feeding or liquids for another 3 h. Vital signs were closely monitored for signs of peritonitis, which might indicate leakage of gastric contents. The patient did well and was discharged the following day. Histological examination revealed a C-kit-positive  GIST with a low Ki67 proliferation rate (< 10%); however, the small sample size showed < 1 mitotic figure per 10 high power field (HPF) and thus did not allow accurate grading of the tumour, which requires evaluation of 50 HPF. Based on a consensus decision of the internal tumour board, the patient underwent a successful laparoscopic resection of the gastric tumour 7 weeks after the initial biopsy and is doing well since then. Post-surgical histopathological examination confirmed the primary histological diagnosis and demonstrated a low-risk intramural GIST (< 5 mitosis per 50 HPF). Based on guidelines, no further therapy was required.2 An endoscopic ultrasound-guided fine needle aspiration may offer a diagnostic accuracy of 60–90% depending upon the site, but is associated with limited diagnostic accuracy for GISTs.4 In these cases, larger calibre cutting needles for endoscopic biopsy are recommended6 but these were not available in our case. We considered percutaneous CT-guided needle biopsy to be suitable, which is a well-recognized method for biopsy of many solid organs, but is less often used for the biopsy of GI lesions. To our knowledge only the following authors described the role and safety of percutaneous gastric biopsy small bowel or colon,4 although a transgastric access is well established for percutaneous gastrostomy.7 Before biopsy, non-invasive assessment of the gastric wall is mandatory. While some authors have recommended up to 1500 ml of water or flavoured methylcellulose as an oral contrast and 20 mg of intravenous scopolamine to expand the gastric wall,8 we used effervescent granules administered orally.Endoscopy is considered the gold standard for evaluating tumorous lesions of the gastrointestinal (GI) tract. In some cases, however, forceps biopsy of an endophytic tumour can be challenging and may fail to provide reliable histology.7 In any case, the puncture should be made with a brief, deliberate thrust so as not to push the anterior gastric wall away from the anterior abdominal wall. While Perez-Johnston et al4 recommended obtaining a biopsy sample from the thickest area of a GI lesion, thus avoiding traversing the wall into the lumen, a gastric lesion located in the fundus or the lesser curvature may require a transgastric approach, as in our case.In order to minimize the risk of haemorrhage, the inferior epigastric artery, which is well seen even on an unenhanced CT scan, should be avoided, as it courses through the junction of the medial two-thirds and lateral one-third of the rectus muscle. To avoid the risk of displacing the gastric wall when pushing the puncture needle into the stomach, a gastropexy device [a 17 G needle preloaded with a Cope suture anchor ] may be used.4 whereas others9 reported a bleeding rate of 3%, and a risk of pneumocolon and peritonitis of 0.3%, following transgression of the bowel with an 18 or 19 G needle. However, bowel transgression can lead to possible abscess formation and should be avoided in any case.In general, percutaneous needle biopsy has a reported overall complication rate of approximately 8%, although 2% require further intervention,10 Similar to percutaneousgastrostomy, limited percutaneous access to the stomach  and uncorrectable coagulopathy are considered contraindications for percutaneous transgastric biopsy.Interestingly, a significantly higher bacterial load in the stomach was observed in patients using a proton pump inhibitor; this emphasizes the need to cease the use of proton pump inhibitors prior to transgastric interventions to avoid infection.2Endoscopic biopsy is thought to be the best method for obtaining valid histological information from the GI tract. However, in specific cases where traditional endoscopic biopsies are not feasible or are non-diagnostic, an image-guided percutaneous biopsy of an intramural gastric tumour may be considered an effective and safe technique with which to obtain a valid histological diagnosis; the morbidity and cost of a surgical biopsy under anaesthesia can thus be avoided.Percutaneous biopsy of a gastric tumour is feasible, safe and effective.Percutaneous gastric biopsy should be offered if endoscopic biopsy fails.Expanding the gastric lumen by having the patient swallow effervescent granules improves the delineation of a bulging gastric tumour.Written informed consent for this case to be published  was obtained from the patient.


The use of social media platforms as an educational tool to promote awareness has become increasingly popular as technology advances. Twitter is a microblogging, social media platform in which users share short, text-based posts (“tweets”) that can contain hyperlinked articles, web-pages, pictures, and more. 79% of the 336 million current monthly twitter users are international, suggesting Twitter serves as a tool allowing international connection via the rapid spread of information worldwide. Simplur Signals (Simplur LLC) was used to perform a retrospective analysis of the use of #Geriatrics on Twitter. Data was collected from Oct. 13th, 2010 through Jun. 5th, 2018. Spam and unknown accounts were excluded from the data set before analysis. Manual analysis was performed to qualitatively assess tweet content of the top 200 Retweets by Impressions. A total of 65,002 tweets were shared during the selected time frame. Tweet activity rose to a high in Year 5  but has declined since. The majority of the top 100 influencers were doctors (57.4%). Regarding tweet content, most discussions focus on increasing awareness and promoting advocacy (30%) as well as sharing research related to the practice of geriatrics (23.5%). With its widespread use and lack of international boundaries, Twitter serves as an effective platform in informing and increasing awareness about geriatrics and other medical specialties.


Obtaining grant funding is a fundamental component to achieving a successful research career. A successful grant application needs to meet specific mechanistic expectations of reviewers and funders. This paper provides an overview of the importance of grant funding within medical education, followed by a stepwise discussion of strategies for creating a successful grant application for medical education-based proposals. The last section includes a list of available medical education research grants. Promotion of faculty in academic institutions is critical and includes service, education, and research agendas. The development of faculty researchers has been a topic of interest and debate. Studies in this arena have primarily focused on leadership practices,5Interestingly, the ability to initially predict successful grant applicants from non-successful applicants based upon their qualifications alone can be difficult, as they often have similar backgrounds and credentials.Grant funding is particularly important in the field of medical education research, where funding is even more scarceCompleting a grant application can be a large and arduous task. Grant applicants will often need several experts to formulate their team. The grant development team should include a principal investigator (PI), a primary mentor who is ideally from the same institution, and at least one content expert in the topic of interest. A content expert could be a clinical, translational, educational, or statistical expert either within or outside your institution. It is also important to include a methodological expert early in the process, particularly if the PI does not have significant expertise with the technique. For example, if an investigator is performing a qualitative study, it would be important to include someone who has significant experience with this approach to ensure that the qualitative approach  and specific methodology is appropriate and rigorous. It is also important to establish strong collaboration with the team, rather than merely listing prominent names without previous collaboration. Therefore, we strongly recommend that the PI start the preliminary work early by assigning specific roles among team members and involving them in the grant process.While the specific structure of individual grants can vary by sponsoring institution, many grants use similar requirements and formats. The specific format typically includes but is not limited to a project description with references, biosketches of the investigative team, a complete budget with justification of the financial expenditures, a description of the facilities and resources, and letters of support. The following sections of this paper provide a brief summary of each of these commonly included elements within grant applications; however, the reader is advised to spend additional time reviewing the individual funders’ websites and application resources to ensure that all required forms are completed appropriately and included within the final grant application. It is also important to check with your institution’s grant office, which often requires approval to submit a grant. This process can take up to a month, so it is important to start the work early on grant applications.https://www.niaid.nih.gov/grants-contracts/draft-specific-aims for those who are interested in learning more. A sample aims section from a successful medical education grant application has also been included as an The project description section serves as the body of the grant and clearly defines the work to be performed. The allotted length varies by the funding mechanism, but often ranges from 6–12 pages. The first page of the application is typically the specific aims page where the project is summarized in two or three paragraphs and the hypotheses and objectives are explicitly defined. Well-designed aims ensure that success is not dependent upon any single outcome and that more than one possible outcome is acceptable. Success of a subsequent aim should not be dependent upon the prior aim. Depending upon the funding mechanism, the specific aims page may or may not be included in the page count for the project description. This section is often considered the most important page in the entire grant application. Significant time should be spent on this page by all members of the grant team to ensure that it clearly and accurately articulates all aspects of the proposal. This page often requires several revisions before reaching the final version that is submitted. A nice review of the approach to writing the aims section is available at The remainder of the project description section is frequently divided into three components: significance, innovation, and approach. The significance section describes the background literature and highlights the knowledge gap that will be addressed by completing the proposed project. Within medical education it is essential to include a discussion of the underlying educational theory or conceptual framework upon which the study is based.Finally, the approach section details how the work will be completed. This should include details on study design, recruitment and sample size, outcome measures, analytic techniques, and consideration of the potential limitations. The clarity and technical accuracy of this section can be an important signal to a reviewer of the probability of an applicant successfully completing their proposed research program. Funders generally place a significant weight on a high likelihood of a successful return on their investment; so concerns that the analytical approach is underdeveloped or flawed can be fatal. Of special consideration for educational researchers is the description of methods less common in clinical or bench research; for example, qualitative research methodology may encounter a negative bias from quantitatively focused scholars.In general, reviewers expect to be able to understand your project without having to read substantially about your methods elsewhere. Including appropriate references is mandatory but making reviewers work harder than they expected to is counterproductive to your goal of achieving funding. A complete bibliography may be included either at the end of the project description section or as a separate document within the application. Once again, it is important to note that the grant reviewer may not be familiar with the specific topic or may be from a different specialty or field. Therefore, it can be valuable to have a non-physician review the application to ensure that it is easily understood.https://grants.nih.gov/grants/forms/biosketch.htm).Biographical sketches, or biosketches, are often required for all key personnel within the investigative team. These differ in format from traditional curriculum vitaes, especially in that there generally is an expectation that they will be formatted per the National Institutes of Health (NIH) requirements. Biosketches consist of the investigator’s professional positions and educational history along with their respective publications that are most relevant to the field of medical education. These publications often include a short description of how they have impacted the relevant field of research. These may also include publications demonstrating the investigator’s experience with the specific methodology proposed for the study . Examples of well-written biosketches can be found in several locations, including on the NIH website . Specific to medical education research, one might also want to include the educational environment, including access to the learners and logistics of training. This can also include the number of learners available and prior medical education research conducted in this location. In addition to the above components, this section must also include support services available within the institution. These resources can include medical librarians to help with literature searches, a clinical translational science institute to assist with study design, or a statistician to assist with data analysis.Finally, this section should discuss the research environment within the department and the greater institution. This may include a list of collaborators, recent publications, and other funded projects as a way to highlight the successful completion of other work. Here again, this section can be an important signal to reviewers that the proposed research project will be successfully completed. Institutions with well-documented histories of successfully completing funded research demonstrate an environment that is conducive to a successful return on the investment made by the funder. This can be especially important for more junior researchers and for educational researchers with whom grant reviewers may be less familiar with their work.Letters of support further illustrate the level of commitment from the investigative team, the leadership of their respective departments, and the universities involved in the project. Depending upon the funding agency, letters of support may be required from key personnel involved in the project . These letters should clearly document the necessary resources, the commitment from the investigative team, and the institutional support to ensure success of the proposed work.Depending upon the type of funding mechanism sought, there are several other important documents that may be required. These may include a project timeline, description of the ethical treatment of subjects, and proof of institutional review board submission or approval. Similarly, for training awards a specific plan for career development is a necessary and vital component of the grant application process. These awards are often fundamentally different from grants that fund a specific medical education research question. Rather, training awards can fund specific professional development programs, such as a fellowship. Applicants seeking a training award must clearly delineate a specific and achievable plan for professional development, as well as how the funds will be used to achieve this plan. This is also required for certain career development (K) awards that are available through the NIH.https://grants.nih.gov/grants/peer/guidelines_general/Review_Criteria_at_a_glance.pdf.Grant applications are evaluated using multiple different methods; however, the most common scoring method is that used by the NIH. The NIH scoring tool evaluates several areas of the grant , with each given a score from 1  to 9 (poor). The reviewer scores are averaged and multiplied by 10, resulting in an overall score.While there are a number of grants available for research, significantly fewer are available in medical education. One of the more significant challenges to obtaining grant funding is awareness of which grants are available. The accompanying The Grant Application Writer’s Workbook by Russell and Morrison if they are interested in learning more. Additionally, many of the recommendations are based upon the authors’ combined experience, as there is limited empirical data on effective grant-writing. However, the authors are experienced grant writers, having received over $4 million in grant funding. Finally, the grant list in the It is important to consider several limitations with respect to the current paper. First, this publication serves as a primer for medical education researchers interested in obtaining grant funding. While this is intended to provide an overview of the major components of grant funding, readers are advised to read Obtaining grant funding is a fundamental component of a successful research career, but it can also be challenging, especially in the field of medical education. Successful applications must meet specific structural requirements. This paper provides an overview of grant opportunities within medical education and strategies for successful grant applications. After reading this paper, researchers should feel more knowledgeable and confident with applying for medical education research grants.


Herpes zoster (HZ) caused by varicella zoster virus (VZV) reactivation is characterized as a vesicular rash of unilateral distribution that can also cause multiple complications; such as post-herpetic neuralgia; ophthalmic zoster; and other neurological issues. VZV can also increase incident hemorrhagic or ischemic complications by causing inflammatory vasculopathy. Thus; emerging epidemiological and clinical data recognizes an association between HZ and subsequent acute strokes or myocardial infarction (MI). This study reviewed published articles to elucidate the association between HZ and cerebrovascular and cardiac events. Individuals exposed to HZ or herpes zoster ophthalmicus had 1.3 to 4-fold increased risks of cerebrovascular events. Higher risks were noted among younger patients (age < 40 years) within one year after an HZ episode. The elevated risk of CV events diminished gradually according to age and length of time after an HZ episode. The putative mechanisms of VZV vasculopathy were also discussed. Several studies showed that the development of herpes zoster and herpes zoster ophthalmicus increased the risks of stroke; transient ischemic attack; and acute cardiac events. The association between VZV infection and cardiovascular events requires further studies to establish the optimal antiviral treatment and zoster vaccination to reduce zoster-associated vascular risk Herpes zoster (HZ) is an infectious disease induced by the reactivation of the varicella zoster virus (VZV) from its latent state in the sensory ganglia. VZV is a double-stranded DNA alpha herpesvirus that can be transmitted by direct airborne transmission. Primary infection with VZV in childhood is known as chickenpox, and then the latent VZV may develop HZ (shingles) in adults. HZ usually starts with a prodromal phase with severe, sharp radicular pain and paresthesia spreading in the affected dermatomes. It then proceeds via vesicular eruption on an erythematous base in the dermatomes within 48–72 h. During the acute phase, the erythematous maculopapular rash evolves into vesicles, pustules, and finally scabs. The lesions exfoliate for approximately 10 days, and the skin usually returns to its intact state after 2–4 weeks. The lesion is mostly in the thoracic region (>50%), but is also possible in the ophthalmic, cervical, and lumbosacral regions. Herpes zoster ophthalmicus (HZO) involves the ophthalmic branch of the trigeminal nerve and can occur in approximately 10% to 20% of HZ cases [Comprehensive searches of electronic databases  were performed  to identify relevant studies written in English. Text headings and medical subject heading (MeSH) terms used in the searches included HZ, shingles, HZO, stroke, TIA, cerebrovascular disease, and MI. Fifteen studies examined the associations between unspecified HZ types and the following: Non-specified stroke ,6,7,8,9,n = 15) from Asia , ,12, n =   ,7,10 conSeveral studies declared that the vasculopathy caused by VZV was associated with the increased risk of future CV outcomes. A clinical case series found that patients with symptoms of HZ and stroke or TIA presented positive for VZV DNA and anti-VZV antibodies from cerebrospinal fluid samples ,26. In aThe potential mechanism of VZV-associated vascular events is shown in Virus-induced inflammation accounts for remodeling of the vessel wall that causes an overly thickened intima and inflammation in the vasa vasorum vessels ,32. VZV The virus sometimes travels from the same trigeminal ganglion along afferent fibers around the carotid artery and its branches . VZV adjVascular events occurring within days of an HZ episode could be due to the associated inflammatory response . For exaThe limitations of the observational studies included a lack of randomization and a risk of bias. The review studies were controlled for important confounders, such as demographics and CVD risk factors. Some studies were flawed by a small number of enrolled subjectof HZ  , few HZOTo summarize the association of HZ and CVD, 15 published epidemiological studies and six meta-analysis reports were reviewed. Individuals exposed to HZ or HZO had 1.3 to 4-fold increased risks of cerebrovascular events. Higher risks were noted among younger patients (age < 40 years) within one year after an HZ episode. The elevated risk of CV events diminished gradually according to age and length of time after an HZ episode. VZV infection of the cerebral arteries with subsequent inflammation leads to vascular remodeling and thickened intima that contributes to vascular occlusion in patients with VZV vasculopathy. Epidemiological studies and the pathology of VZV vasculopathy both indicate that HZ is an important risk factor for stroke within one year after an HZ episode. However, limited reports indicated that antiviral treatment reduces stroke risk. It was also difficult to confirm the relationship between the zoster vaccine and cerebrovascular events because of the small sample size. Emerging studies are needed to determine the effect of optimal antiviral treatment and zoster vaccination on reducing the burden of cerebrovascular and CVD, especially for patients with pre-existing high CVD risk.


Tm) compared with the control. SMG in 4.4% HPMC solution resulted in ΔTm of 6.3 ± 1.21, while other concentration showed no differences in Tm shift on porcine cornea. In ex vivo rabbit cornea, there was a trend toward an increasing cross-linking effect with higher viscosity albeit mild differences. While a significant Tm shift was observed in porcine and rabbit sclera, there was no difference in effect of cross-linking between four HPMC concentrations. Increasing the HPMC concentration does not negatively affect the cross-linking efficacy attributed by SMG and could still be a positive cross-linking enhancer by virtue of increasing tissue contact time in a dynamic biological system. This information will be useful for planning further animal and human studies.Hyperviscosity agents are commonly used in ophthalmic formulations for improving corneal drug penetration by increasing tissue contact time. One such viscosity agent is hydroxypropyl methylcellulose (HPMC). HPMC has been used in riboflavin solutions for photochemical UVA cross-linking (CXL). Sodium hydroxymethylglycinate (SMG) is a small molecule formaldehyde releaser that can function as a therapeutic tissue cross-linker for corneal and scleral applications. The present study was undertaken in order to study formulation factors using HPMC and SMG that could positively influence the cross-linking effect in these ocular tissues. Formulations containing 10 mM SMG and 100 mM sodium bicarbonate were prepared with varying HPMC concentrations from 0 to 4.4%. Their cross-linking effects on porcine and rabbit eyes were measured using differential scanning calorimetry (DSC), expressed as the change/difference in melting temperature (Δ Keratoconus is being managed successfully with ultraviolet-A – riboflavin photochemical corneal cross-linking (known as ‘CXL’) throughout the world, which has revolutionized the field of corneal ectasia and related disorders. The treatment method utilizes riboflavin and UVA radiation . The DrePreviously, our (Babar 2015) screening studies for potential cross-linking agents demonstrated that SMG induces significant cross-linking effects at a pH of 8.5 with an intact epithelium . The smaFor our studies, SMG was incorporated into a formulation containing hydroxy propyl methyl cellulose (HPMC), which is a polymer that has been used to form micro-emulsions to deliver drugs in a more stable manner . HPMC isOphthalmic preparations that are topically applied need to be formulated to have both maximal ocular contact time as well as corneal permeability . A formu3, CAS# 144-55-8) were purchased from Sigma-Aldrich Corp. . Dulbecco’s phosphate-buffered Saline (DPBS) was purchased from VWR . All chemical solutions and buffers were prepared fresh using Millipore water  on the day of the experiment.Sodium hydroxymethylglycinate  solution was obtained from Ashland Inc. . Hydroxypropyl methyl cellulose , protease inhibitor tablets (part# S8820) and sodium bicarbonate  and tested for differences in SMG cross-linking effectiveness on enucleated porcine globes purchased from Clements Food Group . Eyes were kept frozen until the day of the experiment and thawed to room temperature prior to use. As a comparison, cross-linking experiments were performed using on-toxic . Each foFollowing the incubation, dissected corneal and scleral pieces were soaked in protease inhibitor solution. Prior to placement into pre-weighed 50 µl aluminum pan (Perkin-Elmer part# B0169321), dissected pieces were carefully blotted in a standardized, repetitive manner on a double-folded paper towel to remove excess solution. It should be noted that amounts under 2 mg have smaller signal to noise ratios and as such, can complicate thermogram peak analysis. Residual water in the pan can shift the thermogram downward since water has a high heat capacity. Also of importance is a flattening of the sample onto the bottom of the pan. This maximizes uniform heat transfer from the pan to the tissue and can affect the margin of error in readings. Then, pans were immediately hermetically sealed using a DSC pan sealing press (Perkin-Elmer part# B0139005), preventing tissue dehydration due to evaporative losses, and loaded into the DSC Autosampler.Tm) of all samples was measured using a Perkin-Elmer DSC 6000 Autosampler  with Pyris software (version 11.0). The samples were heated from 50 to 80°C at a scan speed of 1°C/min, which was determined to be an optimal scan speed based on preliminary runs. Denaturation curves representing differential heat flow over time were generated. DSC heat flow endotherm data was then analyzed by interpolation of a segment of the curve containing the major endotherm. The maximal transition temperature point was then identified using the Pyris data analysis peak search function. The instrument has an expected margin of error of ±0.3°C based on preliminary runs and is consistent with the manufacturer’s specifications. Less stable fibrils will result in a lower Tm and likewise, more stable fibrils induced by the cross-linking treatment will raise the Tm. Tm has been used as a surrogate assay for tissue cross-linking efficacy, in lieu of mechanical testing, for many decades in the biomaterials world and we have applied to this simple and efficient technique to our therapeutic tissue cross-linking studies previously [Thermal denaturation temperature  eye of a rabbit, then subtracted the average Tm of that rabbit’s control eye from the Tm values of the treated (right) eye. To compare the mean ΔTm between HPMC concentration groups, we fit linear mixed models, where the outcome was the ΔTm and the predictors were indicator variables for the HPMC concentration group. The model included random effects for rabbit. Then to test whether there were any mean differences between HPMC concentration groups, we performed an overall test, using a Wald test. Significance of all statistical tests were based on an alpha value of 0.05 (P ≤ .05). All ΔTm values are reported in the form of mean value followed by standard error.For all porcine eye globe experiments, 3 (buffer) in order to determine the effect/interference of HPMC on tissue cross-linking. For preliminary experimentation, we have used the previously frozen porcine eyes. Cross-linking effect was measured using DSC. As shown in Tm was observed in cornea that was treated with 0% (0.19 ± 0.20), 1.1% (−0.36 ± 0.29 versus 0% P = 0.07) and 2.2% (1.1 ± 0.75 versus 0% P = 0.245) HPMC solutions. ΔTm of 6.3 ± 1.21 was observed using 4.4% HPMC solution (versus 0% P < 0.001), a rather dramatic difference by comparison to the other concentrations of HPMC tested. Sclera tissues were cross-linked more effectively by all of the preparations, resulting ΔTm of 3.93 ± 0.97 with 0%, 3.51 ± 0.87 with 1.1% (versus 0% P = 0.742), 4.16 ± 0.78 with 2.2% (versus 0% P = 0.855) and 5.90 ± 1.05 with 4.4% HPMC solution (versus 0% P = 0.169). There were no statistically significant differences among the four HPMC preparations , 1.00 ± 0.42 with 2.2% (versus 0% P = 0.161) and 1.12 ± 0.27 with 4.4% HPMC solutions (versus 0% P = 0.115). Scleral cross-linking resulted in ΔTm of 9.88 ± 0.66 with 0%, 9.07 ± 0.55 with 1.1% (versus 0% P = 0.459), 8.13 ± 0.34 with 2.2% (versus 0% P = 0.172) and 9.37 ± 0.40 with 4.4% HPMC solutions (versus 0% P = 0.634). Although there was no statistical significance in ex vivo samples, we have observed that higher viscosity solution stays longer in sub-Tenon’s space using ultrasound imaging; therefore, it might be more beneficial in the living animal study (data not shown).These results were further tested using o cornea . There wOur study focuses on the development of a chemically and physiologically sound formulation using SMG, a cross-linking agent with a potential to become an alternative therapy for strengthening corneal and scleral tissues. SMG is an effective compound to induce cross-linking and its effects are directly proportional to its concentration within the formulation. However, maintaining a balance between toxicity and efficacy is key, in order to maximize the positive effects and minimize the adverse effects. This can be done by increasing the contact time between SMG and the tissues that require cross-linking. We have previously shown that 10 mM  SMG is a safe and effective compound that can induce cross-linking and remain non-toxic to endothelial cells on de-epithelialized corneal tissue during a 30-min exposure . This isViscosity is a critical component of ophthalmic preparations. There have been many viscosity enhancing agents used for drug delivery, but HPMC stands out quite clearly as being extremely efficacious and pliable . There aThe high cross-linking effect observed with 4.4% in the porcine cornea might be attributed to various reasons. First, the condition of the epithelial layer may account for the differences in effectiveness in porcine and rabbit samples. The porcine eyes used in preliminary experiments were enucleated and frozen for days before being utilized for experiments. Therefore, the epithelium layer was not well preserved. Alternatively, the rabbit samples were used within an hour post-mortem, preserving the epithelium and clarity of the cornea. The rabbit cornea with an intact epithelium layer should have had a more regulated SMG diffusion to its corneal collagen layer; therefore, porcine cornea with disrupted epithelium was more susceptible to be cross-linked due to increased SMG diffusion. Second, the interspecies differences in the corneal collagen arrangements may be another reason for higher cross-linking effect. Meek and others have shown the variation in structural parameters, including interfibrillar spacing, in various species . FormaldA notable observation during our experiment was the differences in the responsiveness of cross-linking between the corneal and the scleral tissues. The cornea and sclera have similar collagen composition in mammals ; however2 and 370 nm illumination for 30 min [By comparison to the photochemical cross-linking technique, known as UV-riboflavin (CXL), it has been reported that human sclera was not as effectively cross-linked as human cornea with the energy density of 3 mW/cmr 30 min ,36. Usinr 30 min ,36, simir 30 min . The rear 30 min .HPMC has been effective in the delivery of nanocrystals to the eye . Even thOur HPMC experiments using porcine and rabbit eyes indicate that increasing HPMC concentration would not hinder the effectiveness of SMG’s ability to cross-link tissue. This could be useful information in optimizing the formulation. It would be possible to use any viscosity levels (between 0% and 4.4%) to formulate the SMG solution. Considerations should be made to maintain the formulation’s cross-linking effect and also adjust the viscosity depending on the comfort level of the solution to the eye. The least viscous solution (0% HPMC) would have the shortest contact time, whereas a more viscous solution has longer contact time but may cause discomfort on patients’ eyes, causing frequent blinking after application on the cornea . We are


Patients often look to social media as an important tool to gather information about institutions and professionals. Since 1990, United States News and World Report (USNWR) has published annual rankings of hospitals and subspecialty divisions. It remains unknown if social media presence is associated with the USNWR gastroenterology and gastrointestinal (GI) surgery divisional rankings, or how changes in online presence over time affects division ranking.The objective of this study was to determine if social media presence is associated with USNWR gastroenterology and GI surgery divisional rankings and to ascertain how changes in online presence over time affect division rankings.Social media presence among the top 30 institutions listed in the 2014 USNWR gastroenterology and GI surgery divisional rankings were assessed using Pearson’s correlation coefficients and multivariate analysis, controlling for covariates. Linear and logistic regression using data from 2014 and 2016 USNWR rankings were then used to assess the association between institutional ranking or reputation score with any potential changes in numbers of followers over time. Sensitivity analysis was performed by assessing the area under the receiver operating characteristic curve to determine the follower threshold associated with improved or maintained ranking, which was done by dichotomizing changes in followers at values between the 7000 and 12,000 follower mark.P<.001) and reputation score  in 2014. Academic affiliation also independently predicted USNWR division ranking  and reputation score . Between 2014 and 2016, Twitter followers remained significantly associated with improved or maintained rankings . On sensitivity analysis, an 8000 person increase in Twitter followers significantly predicted improved or maintained rankings compared to other cutoffs.Twitter follower count was an independent predictor of divisional ranking (β=.00004; Institutional social media presence is independently associated with USNWR divisional ranking and reputation score. Improvement in social media following was also independently associated with improved or maintained divisional ranking and reputation score, with a threshold of 8000 additional followers as the best predictor of improved or stable ranking. Social media refers to a variety of web-based platforms where individuals create and share information, experiences, and ideas. In recent years, social media has transformed into an important form of communication that has led to significant changes across the globe, including political uprisings. The Office of Health Information Technology within the United States Department of Health and Human Services has recognized the significance of social media in healthcare and the opportunities that lie therein for both patients and providers . MedicalTwitter is one social media platform that allows individuals or organizations to post messages limited to 280 characters or less. According to Twitter’s official website, there are more than 300 million monthly active users around the world and over 500 million tweets per day . IncreasUnited States News and World Report (USNWR) boasts a reach of over 300 million readers . Since 1The aim of this study was to determine the degree to which institutional social media involvement, based on number of followers on Twitter, may be associated with USNWR gastroenterology and gastrointestinal (GI) surgery divisional rankings . A secondary aim was to determine whether social media presence is a predictor of improvement in USNWR rankings, and to attempt to define the growth necessary to achieve improved division rankings.This was a cohort study of the top 30 institutions listed in the 2014 and 2016 USNWR gastroenterology and GI surgery subspecialty rankings. Data from the 2014 rankings were used to address the first aim, and to address the secondary aims a follow-up study compared data based on the 2014 and the 2016 USNWR rankings. Both the numeric ranking and the institutional reputation score were publicly available on the USNWR webpage. The number of Twitter followers corresponding to the 2014 and 2016 USNWR rankings were accessed in June 2015 and November 2016 at time of analysis, respectively, as displayed on each institution’s Twitter account. The official institutional Twitter accounts were identified using standard internet search tools, and hospital-specific Twitter accounts were included. Twitter accounts that appeared to combine hospital with medical school affairs were included. Medical school–specific, or university-specific accounts distinct from a hospital-based Twitter account, were excluded.Spearman’s rank correlation coefficient was calculated to evaluate the association between institutional ranking and social media following. Pearson’s correlation coefficients were calculated to determine the association between social media presence and divisional reputation score. Multivariate analysis using linear regression was performed, controlling for number of hospital beds, academic affiliation, city population, and the number of full-time gastroenterologists on staff. SAS software  was used for statistical analyses. Among the covariates, the number of hospital beds was available online as it was published in the USNWR rankings. Other covariates such as academic affiliation  and number of full-time gastroenterologists on staff were obtained from the institutions’ official websites. Local population size of the city in which the institution is located was obtained from the latest United States Census Bureau figures.t tests were performed to assess the association between 2014 and 2016 institutional rankings, or reputation scores, with changes in numbers of followers between 2014 and 2016. Multivariate analysis was performed using logistic and linear regression to adjust for the aforementioned covariates. Sensitivity analysis was performed by assessing the area under the receiver operating characteristic curve (AUROC) to determine the follower threshold associated with improved or maintained ranking, which was done by dichotomizing changes in followers at the 7000, 8000, 9000, 10,000, and 12,000 follower mark.For our secondary aim, two-tailed, two-sample P<.001). Similarly, there was a positive correlation between Twitter followers and the divisional reputation score  (P<.001) (P=0.03) (P=0.04) and reputation score  on USNWR . Among these Twitter accounts, the number of followers ranged from 0 to 1.7 million as of June 2015. Spearman’s rank correlation coefficient demonstrated a negative correlation between the number of Twitter followers and the numeric divisional ranking  . Twitter(P<.001) . Quantit(P=0.03) . Academion USNWR .P=.03). On logistic regression, change in Twitter followers remained significantly associated with improved or maintained rankings . On sensitivity analysis, an increase of 8000 Twitter followers was associated with significantly higher area under the curve (AUC) (0.765) for prediction of improved or maintained ranking compared to other cutoffs (P=.03) and reputation score . Academic status was also a significant predictor  for reputation score.For the second aim, 39 hospitals were included in the analysis . Of the  cutoffs . On multBased on the 2014 USNWR figures, both institutional Twitter following and academic affiliation are independent predictors of gastroenterology, and GI surgery subspecialty, rankings and reputation scores. Furthermore, gaining Twitter followers between 2014 and 2016 was significantly associated with improved or maintained divisional ranking, with 8000 more followers as the greatest predictor of improved or stable divisional ranking as noted on sensitivity analysis.According to the Pew Research Center, social media usage has seen a nearly 10-fold increase over the past more than ten years since 2005, with 65% of all adults using a social networking site . AccordiMore importantly, many consumers use social media to access health-related consumer reviews, including hospital reviews ,4. BoostThe academic community may question the validity and transparency of the USNWR rankings as an objective representation of hospital quality. One study found USNWR ranked institutions had reportedly demonstrated better clinical outcomes in cardiac care and mortality for acute myocardial infarction when compared to their unranked counterparts . HoweverThe limitations of our study included using a single social media platform and using a single source for institutional rankings (USNWR), both of which may limit the generalizability of our results. Moreover, only institutional Twitter accounts were included in the study. At the time of data collection, there were too few gastroenterology division–specific accounts  and subspecialty- or disease-specific Twitter accounts . Furthermore, any causality cannot be directly established from our results. The differences in hospital rankings could also potentially affect social media following. Further research would therefore be required to elucidate the mechanism of how greater social media presence impacts hospital ranking, or if a higher divisional ranking may lead to a greater social media following. While reputation score determines 27.5% of the overall USNWR specialty score, there is also no existing data evaluating the number of Doximity voters who are active social media users or have significant exposure to dialogue occurring on Twitter . MoreoveInstitutional social media following is independently associated with USNWR divisional ranking and reputation score. Moreover, an increase in social media following was also independently associated with improved or stable divisional ranking and reputation score with a threshold of 8000 additional followers as the best predictor of improved or stable ranking. Institutions hoping to boost their overall and divisional rankings may benefit from strengthening their social media presence by both engaging the public and increasing online visibility through platforms such as Twitter.


The electro-kinetic instability in a pressure driven shear flow near an ion exchange membrane is considered. The electrochemical system, through which an electrical potential drop is applied, consists in a polarization layer in contact with the membrane and a bulk. The numerical investigation contained two aspects: analysis of the instability modes and description of the Lagrangian transport of fluid and ions. Regarding the first aspect, the modes were analyzed as a function of the potential drop. The analysis revealed how the spatial distribution of forces controls the dynamics of vortex association and dissociation. In particular, the birth of a counter-clockwise vortex between two clockwise vortices, and the initiation of clusters constituting one or two envelopes wrapping a vortex group, were examined. In regards to the second aspect, the trajectories were computed with the fourth order Runge Kutta scheme for the time integration and with the biquadratric upstream scheme for the spatial and time interpolation of the fluid velocity and the ion flux. The results for the periodic mode showed two kinds of trajectories: the trochoidal motion and the longitudinal one coupled with a periodic transverse motion. For the aperiodic modes, other mechanisms appeared, such as ejection from the mixing layer, trapping by a growing vortex or merging vortices. The analysis of the local velocity field, the vortices’ shape, the spatial distribution of the forces and the ion flux components explained these trajectories. Ion exchange membranes play an important role for a wide range of engineering applications . These aElectro-convective instability involving an ion exchange membrane was predicted for the first time by Rubinstein and Zaltzman ,14,15,16Three regimes are commonly observed in a current–voltage response when a potential drop (∆Φ) is applied normal to the membrane system see . For a sDirect numerical simulations are necessary to reach a complete understanding of the electro-kinetic instability mechanism. With this aim, the Navier–Stokes equation, coupled with the Poisson–Nernst–Planck systems, has been solved in previous works for the case of one or two membranes immersed in a stationary reservoir. In this context, instability modes, transition to chaotic motion, 3D electro-convective pattern, bifurcation, non-ideal selectivity and chaotic mixing were investigated ,35,36,37Few studies have been published in the case of electro-kinetic instabilities in the presence of an imposed inflow. Kwak et al.  found a Despite the quality of the published results, the mechanisms controlling the instability modes remain only partially understood. The goal of the current work is to intend to explain some processes like vortex dissociation/association, counter-clockwise vortex birth and also time evolution of the cluster structure by considering the spatial distribution of the electric and pressure force. As in previous publications ,33,40,41zL. The bulk is located at xL from the membrane. At the inlet (z = 0), the ionic concentration is Uslip. +, Cl−).The elementary electrodialytic cell consists of a channel between two parallel ion exchange membranes. The electrolyte solution enters one end of the channel and exits from the other. The transverse electric potential drop, imposed at the two outer sides of the membranes, induces two polarization layers at their inner sides . In the The incompressible fluid motion is described by the Stokes equation (Equation (1)) coupled with the continuity equation (Equation (2)). The ionic transport is governed by the Nernst–Planck equation (Equation (3)) and the electric potential is related to the local ionic concentration by the Poisson equation (Equation (4)):P is the pressure, µ is the dynamic viscosity, t is the time, Q is the electric charge density, T are the Boltzmann constant and the temperature, respectively.Here co. Here e is the elementary charge, These equations are scaled as in Druzgalski et al. . The chaHere 2/s. Hence, this value will be used to define For simple ions, the diffusion coefficient is roughly The boundary conditions used at the fluid/membrane interface are the no-slip condition, and the imposed value of the cationic concentration In this expression, boundary . As the At the membrane surface, the no-slip boundary condition is usually applied. However, the slip condition can be justified by the hydrophobic property of membranes. The velocity slip is modeled by the Navier equation, which is characterized by the slip length. The value of this parameter depends on the degree of hydrophobicity and heterogeneity. It is admitted that it is lower than 100 nm for a homogeneous surface and may reach a few micrometers for a grooved surface (superhydrophobicity) ,43. ExpeIn this study, In this equation, A is the area of the computational domain The same discretization method and structured grid refinement as the previous article  were useIn References ,32,40,41Two kinds of dynamic streamlines are computed in this work: the hydrodynamic and the ionic one. As regards to the ions, their local velocity is computed by means of the ratio between the flux density and the concentration: Consider an elementary volume of fluid located at the point Co, Fi and mE mean convective, Fickian and electro-migration contribution, respectively. The interpolated flux is divided by the interpolated concentration to determine the four The Eulerian fluid velocity To validate the numerical method, the results were compared to those published in References ,28,41. TI) and through the membrane surface (mI) as a function of the potential drop. Three series of computations were carried out. Each point of the curves corresponds to the value of the current density averaged in time at a prescribed potential drop. In the Ohmic and limiting regimes, the potential drop increases by a step of 2.5 until it reaches a value of 21. In the over-limiting regime, a first series is carried out with I is the relevant quantity in the present model.These series show that the over-limiting threshold values lie between nditions ,37. In Nnditions , an exprn et al. ):(14)Ilio et al.  because mL [Let us compare the cation profiles obtained in the present work and those published in References ,41. The n layers ,41. Assuyers [Lm ,41. At tΦ=22 see A. Their o et al. ) come fr1/3×10−3 B. The co.6=Lm/7 1 µm correv over Kwak et al.  found a city see . With th23 to 30 , at the v et al.   in the ESC region. Two spots of kinetic energy are located in the second layer and close to the ESC hump. A third spot is located between the two vortices in the upward region due to the convergence of the fluid streamlines. The pressure and potential disequilibrium on both sides of the vortices is explained first by the transverse profile of charge density, which controls the variation of the potential gradient, and also by the equilibrium between the electric force and the pressure one in the transverse direction leading to the quadratic dependence of the pressure on the transverse potential gradient.Before going further, the main results described in the previous article  about thof force A are equz ≈ 5.3 for example, the spot z ≈ 5 and z ≈ 4.7), the pair spot/layer A layer and a spot of the same force sign, located at two different transverse positions, may be linked as shown in Q. The transverse pressure force is opposed to the electric one in the front region, whereas it is negative in the rear region. Three spots are located around the vortex front. At the vortex front, the pressure force dominates . For the sake of simplicity, this time is used as the initial one and the time is expressed in terms of δt in the comments (with t’ = t/δt − 1600).In t’ = 0), a counter-clockwise roll (V1/−) lies between the two clockwise vortices V1/+ and V2/+ located at z = 3.4 and 3.8, respectively. The signs + and − refer to the rotation direction. In First, let us consider t’ = 20), the volume of V1/− increases and its bottom moves to the membrane, so that it is clearly located inside the positive layer In t’ = 40), the negative layer In  process A–E, V2/+t’ = 60), V1/− and the front of V2/+ disappear owing to the decrease in the charge hump in the non-equilibrium EDL. The spot t’ = 80), V2/+, i.e., the charge hump moves downstream this time. The tail is composed of two vortices rotating clockwise separated by a secondary envelope. In t’ = 100), the tail, constituting one roll, is separated completely from the head of V2/+ and the cluster disappears.In The concept of thin film can be extended to the cluster envelope. This film separates the clockwise vortices from the membrane and the surrounding electrolyte. Along the membrane surface, it spreads over a width smaller than the non-equilibrium EDL one. The presence of this film means that a kind of upstream electro-osmotic velocity is imposed between the vortex group and the membrane. However, the immobility of the cluster is not related to this velocity but to the stability of the non-equilibrium EDL.z = 2.1) and V2/+ (z = 2.35). Contrary to In the second series , V1/− ist closer B and thet’ = 40 (z = 1.6) upstream of the cluster moves closer with a decreasing volume. From t’ = 40 to 80  display the real trajectories of the fluid and ions, and point out that the vortices visualized by the SSL are fictitious. In this way, for instance, the DSLs show that, in a periodic regime, the vortices and the region far from the membrane, i.e., the diffusion layer containing high ion concentration, are linked by a mechanism of fluid flux through the vortices. However, in an aperiodic regime, the unsteady size of the inter-vortex regions and the vortices promotes fluid exchange with the diffusion layer and enhances the efficiency of the mixing. The same parameter values as in t’ = 0.t’ = 10, the fluid begins to move upward as it comes closer to the vortex front. Then t’ = 20 (x = 0.033), t’ = 95  with z cancels if Q is positive, Q is very small. On the other side of the vortex, t’ = 190 far from the center of rotation and close to the membrane x>0 see B. Duringt’ = 70 at the position . The loop is located at z = 4.8 . The loop at z = 5 belongs to the trajectory beginning at Now if  t’ = 35 B, the ve= 75 see C. Theref z = 4.8 D. Figurez = 3.7 and z = 4.25, respectively. Until t’ = 25, the volume of V0 is unchanged and its height is around 0.18. Then its volume decreases during the merging process with V1. The front of V0 moves with a longitudinal velocity of 66.7 until t’ = 85. Furthermore, x = 0.18 (zU = 74). The front of V1 remains at the same place in the time interval. Two starting points,  and , are chosen for the discussion because they give rise to two kinds of motion.In t’ = 25  and finally passes V0 at t’ = 85, as explained in t’ = 85 the fluid element is at a transverse position much greater than the height of V0. Reaching V1, the fluid leaves the instability region .On the contrary, the fluid remains close to the instability layer if z>Uzroll C. It reaf V1 see  ∆Φ = 23 xo decreases from 0.1, the DSL reaches a higher transverse position during the fluid excursion, i.e., the fluid explores the diffusion layer more deeply, and it enters the vortex layer at a higher distance from the source position. This means that the high transverse amplitude of the trajectory and the large dispersion of the DSL allow a fluid exchange between different distant vortices in contrast to the periodic regime.As zo = 2.7 between V0/+ and V1/+ . This starting point is located inside the Ec spot close to the membrane, i.e., in the fluid film below V/−. If t’ < 80 (z < 3), the fluid trajectory is similar to the trajectory beginning at ox = 0.015, shown in t’ = 100 , the fluid element reaches the region where xU is small. However, the fluid element does not cross the boundary xU = 0 because the inter-vortex region expands. Therefore, the fluid element continues to move upward and leaves the inter-vortex region with an increasing longitudinal velocity, which becomes higher than t’ > 135, the DSL enters the V1/+ rear at t’ = 175 as explained previously and then the fluid trajectory makes a loop inside V1/+.Let us consider the first trajectory roll see C. As thexo = 0.025 (green trajectory), the fluid element is initially inside V/−. The vortex is isolated from the neighborhood. So, until t’ = 55, the fluid element remains inside V/− and rotates counter-clockwise two times. At t’ = 60, V/− disappears; therefore, until t’ = 125 . The inter-vortex region between V0/+ and V1/+ is so small that the rear of V0/+ passes the fluid element during its upward motion at t’ = 200 . The fluid consequently moves upstream inside V0/+ to the front  see . x = 0.1, z = 4.8), the electro-migration contribution becomes dominant with t’ = 95 ox increases from 0.035 to 0.085, the ions deviate from the ejection region as they leave the vortex. Consequently, the transverse amplitude of their trajectory decreases, and the cusps move upstream.Consider first the case g period A, the antion see C, the iozo = 4.55). Two families of trajectory can be observed. The first one is identical to the trajectories shown in ox lies between 0.035 and 0.055. However, the stagnation point is replaced by an elongated and tilted loop because the anions reach a transverse position lower than the rotation center one and move to the vortex front by diffusion xo, Therefore, the longitudinal Fickian contribution is of great importance to the Lagrangian anionic motion. The two last Figures also show that despite the electric force directed upward, the anions remain in the layer explored by the fluid DSL. At the outer-edge of this layer, the Fickian diffusion balances the electro-migration and keeps the anions leaving the region.zo = 3, In regards to the cations B, the dyν. Despite this observation, the present study was performed mainly with In this work, the analysis of electro-convective instabilities in a laminar boundary layer along a cation exchange membrane is presented. The periodic and aperiodic modes were studied. In particular, for the latter mode, vortex association/dissociation and fusion, as well as time evolution of cluster and counter-clockwise vortex, were investigated. The influence of the instability structure and dynamics on the fluid and ion transfer was also investigated by means of a Lagrangian approach. The aperiodic mode is sensitive to the value of the parameters The vortex structure and dynamics were analyzed by considering the spatial distribution of the longitudinal component of the electric and pressure force. This distribution is composed of a layer along the membrane inside the non-equilibrium EDL and of two spots on both sides of the vortex fronts and above the force layer. These two structures undergo a change of sign at the front and the rear of the vortices. The link between a spot and a layer of negative sign plays a key role in the vortex dynamics and in the birth of counter-clockwise vortices and clusters. This link bends the streamlines in the upward direction and induces a neck at the outer edge of the inter-vortex region where the fluid is pushed downward by the forced flow. The counter-clockwise vortices occur in the inter-vortex region over which the negative spot is located. This vortex is isolated from the neighboring clockwise vortices and the membrane by a thin fluid film coming from the neck. The clockwise vortices associate into a cluster when the neck closes, and a continuous path of negative longitudinal force occurs over the vortex group along the membrane. In this case, a thin film also wraps the vortex group, which is therefore separated from the membrane and the boundary layer. The counter-clockwise vortices are excluded from the clusters in contact with the membrane during the clustering process. The upstream fluid motion in the film along the membrane plays a similar role as the electro-osmotic boundary conditions.The real motion of the fluid, as well as the ions, must be computed by considering the unsteadiness of the velocity and flux fields. In the periodic mode, the fluid trajectories passing through a vortex, computed with the SSL, form a trochoid. The SSL vortices are fictitious. In the vortex reference frame, these DSLs form rolls whose rotation center is above the SSL vortex center and whose size is much greater. The real vortex layer is therefore in contact with the diffusion layer containing high ion concentration and it controls the mixing layer expansion. This is not the case for the inter-vortex region. Surprisingly, the anions follow the same kind of trajectory with roughly the same transverse amplitude. The Fickian diffusion plays a key role in the loop formation and in the transverse amplitude. The outer edge of the region explored by the anions corresponds to the balance between the electro-migration and the Fickian diffusion fluxes in the transverse direction. Further away from the instability threshold, the electric field is high enough for the anions to be ejected from the vortex layer.For the aperiodic mode, the real instability layer visualized by the DSL is also much larger than the SSL rolls. Its size increases with ∆Φ, imposing the upward displacement of the diffusion layer. In this case, fluid exchange is performed between distant SSL vortices. The transverse amplitude of the trajectories is high enough for the mean Lagrangian velocity to be much greater than the SSL roll one. The size increase allows the rolls to attract and trap the fluid elements. It is also observed that the inter-vortex regions contribute to the mixing process. The variation of the inter-vortex length destabilizes the fluid trajectory leading to the fluid ejection outside the SSL vortex layer and initiating a trochoid-like trajectory.


Sebaceous carcinoma (SC) is a rare aggressive cutaneous malignant tumour. It accounts for less than 1 % of all cutaneous malignant tumours. Sebaceous carcinomas are divided into ocular and extraocular constituting 75 % and 25 % respectively. The most common extraocular site is parotid gland. Chest wall is a rare site of this tumour.In line with SCARE criteria (Agha et al., 2018 [Sebaceous carcinoma is a rare and aggressive adnexal tumour. It is categorized as ocular and extraocular. Of these two, the more common is extraocular. The chest wall is a rare site for this tumour. The definitive diagnosis of this kind of tumour is done by tissue biopsy. In dealing with this tumour, surgical excision with wide margins is the standard treatment.Sebaceous carcinoma is a rare aggressive malignant tumour originating from sebaceous gland. Early diagnosis and wide excision with negative margins improves survival. It is p2The patient is a 45-year-old African male who presented with a two-year history of small lump on the right anterior chest wall. He reported that it was painless, firm and covered by hyper-pigmented skin. He sought for medical assistance after a year of noticing a gradual growth of the lesion. Subsequently, an incisional biopsy was done and histology results confirmed sebaceous adenocarcinoma. The patient absconded and presented a year later with a very large, pedunculated, mushrooming and fungating tumour. Its stalk was on the second intercostal space extending to the parasternal area  A. The rPostero-anterior (PA) and lateral chest x-rays (CXRs) were done, with a PA showing multi-lobulated soft tissue homogeneous opacity in the upper mediastinum, which extends to the right hemithorax A. A lateA non-contrast computed tomography (CT) scan showed anterior chest wall tumour with a calcified capsule without bones involvement A. A CT-sen bloc  stained sections showed ulcerated invasive sebaceous carcinoma evidenced by ulcerated skin with an invasive basaloid tumour A with se3Sebaceous carcinoma is an uncommon aggressive adnexal tumour. The great majority of SCs 75 %) occur in the periocular area, commonly, the eyelid  % occur . MTS is The morphological subtype in this case report is basaloid. This SC subtype can mimic different types of neoplasms, which makes correct diagnosis a challenge . The morThe clinical presentation is not a pathognomonic and is often non-specific. The tumour is usually firm; slow enlarging, yellowish to red-brown plaque, friable, crusted and often ulcerated. The size of extraocular tumour varies, with ranges of 6−10 cm on its greatest dimension. Judged against studies prior to this one, this tumour is the largest with the size of 180 × 140 × 30 mm to be reported.The definitive diagnosis of this tumor is by histology results. The presence of sebocytes characterized by multi-vacuolated cytoplasm and pleomorphic nuclei suggests the diagnosis of SCs on microscopy . HoweverAdjunct investigations include the CXR, CT-scan and MRI, as done in this case report. The MRI is an important modality not only for diagnosis, but also for planning of surgery. It gives adequate information about location, size and extent of invasion of adjacent structures by the tumour. Surgical excision with wide margins is the standard treatment . In caseOn both physical examination and radiological investigations, the patient did not have regional lymph node involvement. This implies a better diagnosis. The rate of metastasis in sebaceous tumour is about 14–25 %, which is similar for both extraocular and ocular types . The nod4Sebaceous carcinoma is a rare but aggressive tumour, which is notorious for mimicking other tumours. The earlier the diagnosis, the better the prognosis. In the case of this tumour, histological analysis is the gold standard for diagnosis. Surgical excision with wide margin is standard treatment. A recurrence is uncommon, hence follow-up is mandatory.None.Sefako Makgatho University Research Ethics Committee (SMUREC) approved the publication of this case report.SMUREC/M/287/2019INSTITUTIONAL REVIEW BOARD (IRB00010386).Written informed consent to publish this case report and images was obtained from the patient. The copy of written informed consent will be available for Editor-in- Chief on request.Dr. Sekgololo J.M. wrote the case report.Prof Chauke R.F. and Dr. Tshazi N., both critically revised the case report.Not applicable, because is a single case report.Dr. Sekgololo J.M.Not commissioned, externally peer-reviewed.None.


In the second paragraph of the Results, fifth sentence, the number 19.91 should have appeared as 1.91, and in the final sentence of that paragraph, P < .02 should have appeared as P = .02. In Table 3 in the second row of the Stroke column, the 95% CI for 0.78 should have appeared as 0.54-1.13. In Figure 2A, the older age group should have appeared as ≥65 and the P values for residence and drinking status both should have appeared as .56. In Figure 2B, the P value for body mass index should have appeared as .91. This article has been corrected.1In the Original Investigation titled “Association of Depressive Symptoms With Incident Cardiovascular Diseases in Middle-Aged and Older Chinese Adults,”


Potentially, monitoring tumour neo-antigen specific T cell profiles is a highly sensitive method for determining disease recurrence.Non-small cell lung cancer (NSCLC) causes 19% of all Australian cancer deaths, with a 5-year survival post-resection of around 60%. Post-operative recurrence is due to metastases that were undetectable pre-operatively, or growth of microscopic locoregional residual disease. However, post-operative imaging modalities typically only detect more advanced tumours; where PET-CT has a detection limit of 6–7 mm. Detection of small deposits of lung metastatic disease is of importance in order to facilitate early and potentially more effective treatment. In this study, in a murine model of lung metastatic disease, we explore whether neo-antigen specific T cells are a sensitive marker for the detection of lung cancer after primary tumour resection. We determine lung metastatic disease by histology, and then compare detection by PET-CT and neo-antigen specific T cell frequency. Detection of lung metastatic disease within the histology positive group by PET-CT and neo-antigen specific T cell frequency were 22.9% and 92.2%, respectively. Notably, neo-antigen specific T cells in the lung draining lymph node were indicative of metastatic disease (82.8 ± 12.9 spots/10 However, disease recurrence after surgery for many cancers is frequent, either due to the growth of metastases that were not detectable before the operation or due to progressive growth of microscopic residual disease4.Globally, more than 15 million cancer patients undergo resection of their tumour every year5. For example, in melanoma patients the efficacy of tremilimumab in treatment of stage III disease is 60%, compared to 17.7% in stage IV disease6. Similarly, in animal models, immunotherapy is most effective when tumour deposits are small7. Accordingly, earlier detection of metastatic disease may improve patient outcomes to immunotherapy.Immunotherapy is a breakthrough modality for the treatment of a number of cancers, and works best when tumour deposits are relatively small8. 5-year survival after resection is approximately 60%9, indicating that current curative-intent treatment has limited efficacy and that residual microscopic tumour deposits remain undetected. Imaging modalities are typically used to detect the presence of metastatic disease, although it is known to miss small deposits. PET-CT is one of the main such imaging modalities and is routinely used in pre- and post-operative staging. Depending upon anatomical location, PET scanning has a tumour detection limit of 6–7 mm, with detection limits for CT at 3–4 mm for malignant masses. Given the proportion of patients who go on to develop metastatic disease post-resection, current imaging modalities miss a large proportion of the microdeposits of tumour that are actually present10.In this study we focus on NSCLC, one of the most common lethal cancers. NSCLC has an incidence of around 43 cases per 100,000, and accounts for 19% of all cancer deaths12 and in fact, tumour specific T cell responses can be detected even when subcutaneous tumours are completely impalpable13. Therefore, tumour-specific CD8 T cells could be a sensitive marker for the presence of metastatic disease. On the other hand, any such T cells detected could simply be a memory population, driven by the original tumour that was resected and not reflective of the presence of metastases at all.Unlike imaging, immune recognition of tumours can occur when there is no detectable lesion present. In murine models, neo-antigen specific anti-tumour CD8 T cells are generated in response to very low levels of tumour antigen17. We reason that the most sensitive method of detection of the presence of metastatic disease may be a detectable shift in neo-antigen specific CD8 T cell frequency. This could allow for greater benefit from early adjuvant immunotherapy18 before tumour is even detectable by imaging.It is now considered most likely that the main targets for immune recognition are tumour neo-antigens (tumour-specific mutations). These neo-antigens, and the T cell (specific) response to them, can be identified in animals and patients by using advanced sequencing, bioinformatics and T cell assays14. Uqcrc2 is a protein important in oxidative phosphorylation, Unc45a is a protein involved in the regulation of the actomyosin system. In vivo immune responses to mutated Uq2 and Unc45a are detected in AB1-HA tumours and their draining lymph nodes. In this study we compared neo-antigen specific T cell responses with PET-CT imaging to determine if the former was indeed more sensitive to the presence of metastatic disease than imaging.Our AB1-HA cell line is an ideal probe to study this question. AB-HA cells express the model viral neo-antigen haemagglutinin (HA). It also carries 2 natural carcinogen-induced mouse neo-antigens in Uqcrc2 and Unc45a, discovered using whole exome sequencing (WES) and RNAseqWe show that increased T cell responses to neo-antigen are indeed a sensitive marker of early metastatic lung disease, and that responses to a combination of several tumour specific neo-antigen T cell responses performed even better than single neo-antigen responses as an sensitive method of detection of metastatic lung disease compared to PET-CT.6 AB1-HA luciferase expressing (AB1-HA_LUC) cells intravenously  imaging . Figure 3) and neo-antigen specific T cell frequency in the subcutaneous tumour draining lymph node using the Spearman Rho . These data indicate that frequency of neo-antigen specific T cells increased with lung metastatic disease onset.Next, we combined the frequency of HA, Uq2 and Unc45 neo-antigen specific T cells and compared metastatic lung disease and healthy lungs Fig. . CombineWe next determined if total lung tumour burden from metastatic disease correlated with neo-antigen specific T cell frequency. Increased neo-antigen specific T cells in the LDLN positively correlated with lung metastatic disease. The Spearman Rho correlation coefficient was 0.642, 0.805, 0.813 and 0.809 comparing tumour size and HA, Uq2 or Unc45 or combined neo-antigen specific T cells, all with significance p = 0.000. Fig. .Further, next examined the neo-antigen specific T cell response in the spleen. Neo-antigen specific T cell responses by IFΝγ ELISPOT assay were not significantly increased for either individual or combined HA, Uq2 and Unc45 compared to the naïve group  to 20%6.Treatment for the early stages of NSCLC consists of surgical resection followed by adjuvant chemotherapy, immunotherapy, and/or radiotherapy. Unfortunately, the relapse rate from recurrence or onset of metastatic disease after primary lung tumour resection remains high. For example, the Stage IA/B or IIA/B the 5-year survival rates are 49/45% and 30/31%, respectively5, so it is thought that early detection of metastatic disease, before tumour deposits become large, will increase treatment efficacy. This study showed that monitoring of neo-antigen specific T cell responses may be an early indicator of disease, that is more sensitive to the presence of metastatic tumour relative to current imaging modalities.Crucially, immunotherapy is more effective when tumours are small21, in our animal model. Although PET-CT imaging detected metastatic lung disease in the range 3–4 mm (>30 mm3), it missed metastatic disease deposits. This is consistent with the clinical setting in which, the limits of PET-CT detection of tumour in patients are in the order 6–7 mm (>216 mm3). Although a useful diagnostic tool, the potential to miss small metastatic lesions, along with the known lack of sensitivity22 and potential for significant radiation exposure associated with sequential scans, highlight the practical limitations of PET-CT and the need for additional disease biomarkers to be investigated.In this study we tested the sensitivity of PET-CT, an imaging technique that combines functional and anatomical information in a non-invasive fashion26. One advantage of the tumour neo-antigen specific T cell response is that they are truly unique to the tumour tissue, and therefore will not have undergone prior clonal deletion or tolerance in the thymus16, in contrast to tumour associated antigens (TAAs), and germline cancer antigens (CGAs). The selective expression of neo-antigens on tumour cells strategically places neo-antigen specific T cells as a biological marker to reflect tumour burden. Limitations in the clinical application of personalised neo-antigen based therapies include limitations in the capacity of current genomics approaches to identify all bona-fide neo-antigens using current in silico prediction algorithms. Accordingly, development of new methods for the rapid and more accurate identification of neo-antigens on tumour cells are a current research focus in cancer27. It is also possible that immune responses against other tumour antigens  could offer an early diagnostic marker of metastasis, but this needs to be further tested.Due to advances in next generation sequencing, bioinformatics and peptide binding algorithms, it has recently become possible to track tumour neo-antigen specific T cell responses to cancer, i.e. responses to the mutations that are present within each tumour28. In humans, melanoma neo-antigen specific T cells delivered as adoptive cell therapy contract in number following complete tumour regression29.Neo-antigen specific T cells are not just memory cells but reflect the presence of tumour: We show that surgical resection of the primary subcutaneous tumour reduced the frequency of neo-antigen specific T cells specific for all three neo-antigens HA, Uq2 and Unc45 when examined in the primary tumour dLN 19 days post-surgery. We have previously shown, using T cell proliferation assays in the Lyons-parish assay, that HA-specific T cells in the draining lymph node steadily decline after surgical resection, reaching non-detectable levels by day 14 post-surgeryIn this study HA, Uq2 and Unc45 neo-antigen specific T cell response declined with tumour resection alone, however, when occult tumour cells were introduced on the day of surgery, residual neo-antigen specific T cells in the tumour draining lymph nodes remained. Taken together, the presence of tumour antigen might be driving the continuous generation of neo-antigen specific T cells in the tumour draining lymph nodes, and as such removal of the primary tumour leads to a reduction in neo-antigen specific T cells.11. We demonstrate here that neo-antigen specific T cell responses in the LDLN of mice can be detected with metastasis as small as 0.8 mm3. This is substantially more sensitive than PET-CT imaging in terms of the limits of detection.In this paper we further demonstrate lung draining lymph node neo-antigen specific T cell response post-surgery correlates with onset of metastatic disease. Tumour specific CD8 T cells are activated (‘cross-primed’) in dLNs in a process that is sensitive to low amounts of total tumour antigen, around 200 pget al., unpublished data]. Other studies have also shown that neo-antigen specific T cell responses correlate more closely with tumour volume according to neo-antigen type or antigen expression levels on the tumour surface11. Our data suggest that monitoring of more than one neo-antigen specific T cell response may be necessary for optimal, reliable detection of metastatic disease. This notion takes on extra importance in the knowledge that neo-antigen response are dynamic in tumour evolution. For example, tracking of neo-antigen T cell responses in melanoma patients indicates T cell driven immune-editing of tumour29 and in an ovarian cancer patient, detection of T cell response to a neo-antigen HSDL1L25Vwas seen at first recurrence but not in primary or secondary recurrence tumour tissue15. This indicates that changes in the neo-antigen T cell response and expressed tumour neo-antigens can occur during metastasis and/or recurrence. Studies also indicate tumour cell heterogeneity may be found when comparing the primary tumour to metastatic disease sites15. This means that the same neo-antigen may be expressed on some, but not all, cancer cells. Further tumour cells may suppress their expression of immunologically targeted neo-antigens, or neo-antigen specific T cells may become tolerogenic or exhausted30, though recent studies in melanoma have shown that neo-antigen specific T cell populations remained similar in the primary tumour compared to metastatic disease sites31. Furthermore, two neo-antigen specific T cell populations were responsible for killing 90% of autologous melanoma cells31. Here, we tracked 3 tumour specific neo-antigens T cell response that were individually predictive of metastatic disease. Importantly, in this study the combination HA, Uq2 and Unc45 neo-antigen specific T cell responses was most strongly predictive of lung metastatic disease. This suggests that patients will require careful examination of the neoantigen load in the primary resected tumour and a number of such neoantigens used to monitor the patient for recurrence.Further, combinations of neo-antigen reactivities are more sensitive to the presence of tumour than responses to single neo-antigens. In this study we observed variations in HA, Uq2 and Unc45 immunoreactivity between individual mice, and in many cases a particular neo-antigen dominated the response to metastatic disease. This is a common finding in individual animals, even when the strain and tumour are identical [Ma S In this study model, we determine disease recurrence via neo-antigen specific T cells generated to primary tumours by the immune system. A better model may be orthotopically inoculated primary tumour however lung tumour resection in mice is not always feasible. In addition, orthotopic models and/or spontaneous metastases models often have poor reproducibility. Importantly, we have been able to establish a murine metastatic disease model that stimulates neo-antigen specific immune response to primary tumour, in order to determine relevance to metastatic disease.Finally, given that we focussed our attention on the dLN, which cannot be easily examined when following up patients, we also examined neoantigen responses in the spleen, as large volume blood samples are impossible to obtain in mice. We did not find the same close correlation with metastasis that we found in the dLNs. This does not mean that sequential bloods, which are the easiest to obtain, will not provide the relevant early information in metastatic disease, because the spleen is a secondary lymphoid organ, not the blood. Only sequential studies in patients’ post-surgery will accurately test the implications of our animal study. We currently have patient studies in progress wherein we collect lymph node and blood samples, for subsequent analysis of neoantigen responses to correlate with PET-CT imaging, after lung cancer surgery (RGS0000001516). Although blood sampling will be studied, sequential lymph node sampling via endobronchial ultrasound (EBUS) sampling for neo-antigen specific T cell fluctuations may also be a powerful tool for early diagnosis of metastasis.In summary, we show increased neo-antigen specific T cells in response to very small deposits of tumour growth in the lung. Indeed, low burden lung metastatic disease showed a strong correlation with HA, Uq2 and Unc45 neo-antigen specific T cell frequency in the LDLNs. The specific neo-antigen specific T cells responses to the same tumour neo-antigen can vary between individuals but combined multi-neo-antigen specific T cell responses were most strongly predictive of metastatic disease. These observations have major translational implications. If verified in humans, sequential tracking of neoantigen responses may enable clinicians to begin adjuvant therapy much earlier than otherwise possible, with the prospect of improving the survival rate in patients.th edition, 2013 and the University of Western Australia animal ethics and guidelines protocols .Eight-week old female BALB/c mice were obtained from the Animal Resource Centre  and housed at the Harry Perkins Bioresources Facility . All mice were maintained under standard specific pathogen free housing conditions and animal experiments were conducted in accordance with the Australian code for the care and use of animals for scientific purposes, 8in vivo and in vitro until a stable, clonal cell line was obtained32. The AB1 cell line was then transfected with influenza hemagglutinin (HA) from the Mt Sinai strain of PR8/34/H1N1 influenza virus to generate the well characterised AB1-HA cell line13, which was further transfected to express the luciferase expressing cell line AB-HA_LUC.The murine mesothelioma cell line AB1-HA cell line was generated by inoculating crocidolite asbestos intraperitoneally (i.p.) in BALB/c mice, collecting the peritoneal exudate and passaging cells 5 AB1-HA cells on day 0, tumour growth was monitored with callipers, and tumour surgically resected on day 14. On the day of surgery mice received intravenous injection of AB1-HA_LUC cells. Lung tumour growth was determined by IVIS, PET-CT, and/or histology at the indicated time points.Mice received 5 × 1033.Animals were administered analgesic buprenorphine s.c. (0.1 mg/kg) at 30 min prior to surgery. Anaesthesia in mice was inducted with 4% isofluorane in 100% oxygen and maintained on at 2% isofluorane, at a flow rate of 2 L/min for the duration of surgery. Resections were performed by elliptical incisions over subcutaneous tumours spanning twice the length of the lesion, with at least 3 mm lateral margins. Tumours were dissected clear of the adjacent fascia, and wounds were closed with 7 mm wound clips  or uninterrupted sutures . After surgery mice received buprenorphine at 12 and 24 hMice were injected i.p. with 100 µl Luciferin , according to the manufacturer’s instructions. Mice were imaged for peak bioluminescence to 25 min in a Lumina II Imager .Mice were fasted for at least 6 hours, prior to anaesthesia and intravenous injection 15-FDG (7–15 MBq) and remained under 2% isofluorane for an uptake period of 60 min prior to scanning on the Bioscan BioPet/CT 105 camera, with respiratory monitoring.All animal imaging was performed at the Australian Cancer Research Foundation, Cancer Imaging Facility at the Harry Perkins Institute of Medical Research, Perth, Australia. A Bioscan, hybrid PET/CT preclinical scanner was used to obtain images after intraperitoneal injection of mice with 18-Fluorine fluorodeoxyglucose (FDG). A one hour uptake period was performed under 2% isoflurane in 100% oxygen at a flow rate of 2 L/min.PET/CT images were taken, following the 1 hour uptake phase, consisting of 1 emission bed position with the chest in the centre of the FOV. A 15 minute emission scan was acquired with a 250–700 KeV energy window followed by a CT, 40 kV tube voltage and 139.5 uA tube current, covering the PET FOV. PET images were reconstructed using 2D and 3D OSEM algorithms. 3D OSEM using 1 iteration and 80 subsets and 2D OSEM using 2 iterations and 16 subsets both with random and scatter correction. SUV semi-quantitative analysis was performed.Lungs from experimental mice were taken at day 20 and fixed in 10% formalin, and processed as paraffin embedded blocks. Lungs were serially sectioned every 750 microns onto glass slides and stained with Hematoxylin and Eosin (H&E). Slides were viewed using Aperio Scanscope XT 20x imaging , and ImageScope software was used for image analysis. Tumour volume was determined in serial sections for each mouse using FIJI software and a 1000 micron grid.34. In brief, inguinal lymph node (ILN), axillary lymph node (AxLN), lung draining lymph nodes  were subject to digestion with type IV collagenase (1/5 mg/mL); Worthington Biochemical, Lakewood, NJ) with type I DNase . Once digested, samples were centrifuged, washed, filtered through 100 µm filters and resuspended in PBS with 2% FCS.Single cell suspensions were prepared from draining lymph node (dLN) upon harvest as described14 on fresh single cells. In brief, 1 × 105 cells were incubated in a microtitre plate pre-coated with 1 µg/ml IFNγ , with 1 µg/ml peptide , 1 µg/mL CD3 , or media alone. Cells were incubated 20 h at 37 °C and cytokine secretion detected with 1 µg/ml biotinylated IFNγ antibody . Plates were counted on the Autoimmune Diagnostic AID ELISPOT reader system with count algorithm.3.2.x. The mean background SFU/105 cells was subtracted from the test wells.ELISPOT assays were performed as previousResults are mean and standard error of the mean (±SEM) unless otherwise stated. ONE-way ANOVA, with Sidaks correction for multiple testing was used to determine significance between groups. Unpaired, two-tailed, T-test was used to measure significance between two groups. Linear regression coefficient and significance were determined using Spearman rho. All analysis was performed using GraphPad Prism version 7 software  or SPSS* statistics version 22 .Supplementary Figure 1