Aphids display “cyclic parthenogenesis,” in which parthenogenetically and sexually reproducing morphs seasonally alternate in the aphid annual life cycles. There are various characteristics that differ between asexual viviparous and sexual oviparous females. In oviparous females, swollen cuticular structures (~ 10 μm in diameter), called “scent plaques,” are scattered on the surface of hind tibias, and secrete monoterpenoid sex pheromones. However, the developmental processes of the pheromone glands and the biosynthetic pathways of monoterpenoid pheromones have yet to be elucidated.Comparisons of the developmental processes that form hind tibias between sexual and parthenogenetic females revealed that, in sexual females, the epithelial tissues in proximal parts of hind tibias become columnar in fourth instar nymphs, and circular pheromone glands with Class 1 gland cells appear in adults, although they do not appear in parthenogenetic females. Furthermore, by comparing the expression levels of genes involved in the mevalonate pathway, which is required for monoterpenoid synthesis, we show that genes that encode the downstream enzymes in the pathway are highly expressed in hind tibias of sexual females.Glandular tissues of scent plaque are differentiated from the fourth instar in sexual females, while parthenogenetic females lack the glandular cells. Only the downstream steps of the mevalonate pathway appear to occur in scent plaques on hind tibias of sexual females, although the upstream steps may occur somewhere in other body parts.The online version of this article (10.1186/s40851-018-0092-0) contains supplementary material, which is available to authorized users. Most aphid species show cyclic parthenogenesis, in which both sexual and asexual reproduction are performed depending on season –4 Fig. . From spAcyrthosiphon pisum, two monoterpenoids, -nepetalactone and -nepetalactol, have been identified as sex pheromones [Sexual aphid females are known to secrete sex pheromones to attract males –12. In meromones –17. The eromones , 18, 19.Generally, terpenoids can be synthesized by several biosynthetic pathways. The mevalonate pathway is the most well-known, and is essential to isoprenoid synthesis in eukaryotes, in which acetyl-CoA is converted to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) through several enzymatic reactions . In somehttp://www.genome.jp/kegg/pathway.html), seven major enzymes were chosen: acetoacetyl-CoA thiolase (AACT), HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) synthetase (HMGS), HMG-CoA reductase (HMGR), mevalonate kinase (MevK), phosphomevalonate kinase (MevPK), mevalonate 5-diphosphate decarboxylase (MevPPD), and isopentenyl pyrophosphate isomerase (IPPI) [Ips pini [In the present study, hind-tibia structure and development were compared between oviparous and parthenogenetic females, and detailed scent plaque structures were histologically observed to examine whether the scent plaque possesses characteristics of a pheromone gland. In addition, although the place of secretion is thought to be scent plaques, the places of reactions in the mevalonate pathway may not necessarily be scent plaques. In order to examine from which step of the mevalonate pathway was localized in hind tibias of oviparous aphids, expression levels of genes that encode enzymes in the mevalonate pathway were compared by realtime quantitative PCR (qPCR). Based on the KEGG pathway database (e (IPPI) –25. In aIps pini , 24, 26.Acrthosiphon pisum, which was suitable to the induction of sexual generation, was used in this study. This strain was originally obtained from Sapporo, Hokkaido (referred to as Sap05Ms2 in [A. pisum genome was sequenced and is publicly available [Vicia faba) was placed on wet vermiculite. The tubes were kept under long-day and room-temperature conditions  [The ApL strain of the pea aphid, 05Ms2 in ) and use05Ms2 in . The A. vailable . During ; 20 °C) .The induction of viviparous and oviparous females in the ApL strain was performed following the protocols previously described . BrieflyTo examine the detailed morphological features of hind legs in third, fourth, and fifth (adult) instars of female aphids, the surface structures were observed by SEM. Specimens for SEM observations were prepared as previously described . BrieflyTo determine inner morphological features of hind legs, female legs of third, fourth, and fifth instars were observed using paraffin sections. As described previously , aphid lTo determine inner ultrastructure of epidermal tissue from hind tibias of fourth and fifth instars of female aphids, TEM observations were performed. Specimens were prepared following the protocols described in . Aphid hA. pisum genome; http://www.ncbi.nlm.nih.gov/) using protein sequences of orthologous genes in Drosophila melanogaster or Tribolium castaneum as queries for Blast search. To confirm the putative orthologous genes in aphids, neighbor-joining trees of protein sequences were constructed using MEGA7 [http://www.megasoftware.net). To estimate node confidence, 1000 bootstrap replicates were performed. The confirmed enzyme genes and their gene IDs are listed in Table To compare expression levels of genes that encode enzymes involved in monoterpenoid synthesis, AACT, HMGS, HMGR, MevK, MevPK, MevPPD, IPPI, and FPPS were searched for in the pea aphid genome  with recombinant DNase1 (RNase-free)  from more than 40 hind tibias  and three whole bodies of adult aphids. For biological replication, three replicated samples were prepared for each of the following four categories: (1) hind tibias of adult oviparous females, (2) hind tibias of adult parthenogenetic females, (3) whole bodies of adult oviparous females, and (4) whole bodies of adult parthenogenetic females. For each sample, extracted total RNA was reverse-transcribed with the High Capacity cDNA Reverse Transcription Kit based on the manufacturer’s instructions .AACT1 and AACT2, the primers were designed to cover the conserved region shared by the two paralogs, because the DNA sequences of the two genes are too similar to design primers specific to each paralog.Primers for the target enzyme genes were designed using Primer Express software v3.0.1 (Applied Biosystems) , beta-actin, elongation factor 1 alpha (EF1a), and ribosomal protein L32 (rpL32), by three statistical software programs, geNorm [GAPDH was shown to be the most stable so that it was used as the reference gene.Quantitative RT-PCR (qRT-PCR) was performed using a SYBR Green I Chemistry System and Sequence Detection System ABI PRISM 7500 . To determine the endogenous control of constitutive expression, the suitability of different putative reference genes was evaluated among 4 candidate reference genes, , geNorm , BestKee, geNorm , and Nor, geNorm . As the p < 0.01) were performed.Data acquisition and analyses were performed by ABI Prism 7500 v2.0.1 . Baselines and Ct values (threshold cycle) were set automatically. The relative standard curve method was used for qRT-PCR quantification, as described in User Bulletin 2 for the ABI Prism 7700 Sequence Detection System . To evaluate the significance of expression differences among the categories, Student’s t-tests , no structural differences were found between the two morphs; i.e., scent plaques were not found on any leg part in either morph Fig. . The tibThe internal histological structures of hind tibias were first compared among adult individuals, and especially focused on the proximal part of hind tibias Fig. . In partTo understand the associated developmental processes, nymphal hind tibias were also observed. In the third-instar nymphs of both morphs, epithelial cell shape in hind tibias was columnar, and a newly formed cuticle was observed between the outer cuticle and epithelial cell layer Fig.  and f. TUltrastructural observation by transmission electron microscope (TEM) revealed that, in adult parthenogenetic females, each epithelial cell in the hind tibia possessed a small nucleus and numerous vacuoles Fig. . These eIn the fourth-instar nymphs of oviparous females, the epithelial tissues of hind tibias did not possess secretory vesicles, although epicuticular pores were already formed on newly formed cuticle Fig. . In partAACT, HMGS, HMGR, MevK, MevPPD, IPPI, and FPPS) were confirmed from the aphid gene database , MevPK, MevPPD1, IPPI1, and FPPS showed dramatically higher expression levels in oviparous females   Fig. . The expAACT1&2, HMGS1, and MevPPD did not show significant expression differences, but MevPK, IPPI1, and FPPS showed significantly higher expression levels in oviparous females   were not detected, probably due to very low levels of expression.In whole bodies, The present study describes the internal structures of the scent plaques associated with epidermal gland cells in the pea aphid. Epidermal gland cells are generally classified into three types, as follows: (1) Class 1 epidermal gland cells are monocellular and contact the glandular cuticle; (2) Class 2 epidermal gland cells do not contact the glandular cuticle, and transport secretory materials into adjacent Class 1 cells; and (3) Class 3 epidermal gland cells are multicellular, and consist of secretory cells and duct cells , 38. In In general, the epidermis of adult insects is flattened, as adult insects generally do not synthesize new cuticles , 43. HinI. pini, Dendroctonus jeffreyi, Phaedon cochleariae, and Gastrophysa viridula) used the mevalonate pathway to synthesize monoterpenes [Myzus persicae, was reported to convert the final products of mevalonate pathway to GPP [, which is consistent with the fact that only oviparous females possess Class 1 gland cells on hind tibias. These results indicate that oviparous females synthesize monoterpenoid sex pheromones by up-regulating gene expression of downstream enzyme genes in the Class 1 epidermal gland cells of hind tibias. In contrast, the expression levels of all paralogous genes of AACT, HMGR, and MevK were not detected from hind tibias. However, the expression of AACT were detected in whole bodies of both morphs  biosynthesis in insects, which suggests that expression levels in whole bodies may be caused by JH biosynthesis in the corpora allata. Juvenile hormone is reported to play important roles in the production of parthenogenetic females , and somParthenogenesis is thought to have evolved in aphid life cycles about 200 million years ago –57. In aThis study reveals that glandular tissues of scent plaque are differentiated from the fourth instar in sexual oviparous females, while parthenogenetic females lack the glandular cells. Furthermore, it was shown that the downstream steps of the mevalonate pathway are up-regulated in scent plaques on hind tibias of sexual females, although the upstream steps might occur somewhere in other body parts. In the aphid life cycle, oviparous sexual females only appear once a year, while parthenogenetic females occur throughout the rest of the annual life cycle –4. ThereAdditional file 1:Table S1. List of primer sequences for qRT-PCR. (PDF 23 kb)Additional file 2:Figure S1. Thickness comparison of hind-tibial cuticle between adult parthenogenetic and oviparous females. Posterior parts of hind tibias were measured. Asterisks indicate significant difference . Figure S2. Phylogenetic trees of the mevalonate-pathway genes with orthologs from other insects. A:Acetoacetyl-CoA thiolase (AACT). B:HMG-CoA synthetase ; C:HMG-CoA reductase (HMGR). D:Mevalonate kinase (MevK). E:Phosphomevalonate kinase (MevPK). F:Mevalonate 5-diphosphate decarboxylase (MevPPD). G:isopentenyl pyrophosphate isomelase (IPPI). H:Farbesyl diphosphate synthetase (FPPS). Figure S3. Relative expression levels of the enzyme genes in the whole body of parthenogenetic (PF) and oviparous females (OF). Vertical axes indicate relative expression levels. Asterisks indicate significant differences . AACT: acetoacetyl-CoA thiolase. HMGS: HMG-CoA synthetase (3-hydroxy-3-methylglutaryl-coenzyme A synthase). MevPK: phosphomevalonate kinase. MevPPD: mevalonate 5-diphosphate decarboxylase. IPPI: isopentenyl pyrophosphate isomelase. FPPS: farnesyl diphosphate synthetase. (PDF 507 kb)


EBUS-TBNA has revolutionized the diagnostic approach to thoracic diseases from a surgical to minimally invasive procedure. In non small-cell lung cancer (NCSLC) patients, EBUS-TBNA is able to dictate the consecutive therapy both for early and advanced stages, providing pathological diagnosis, mediastinal staging, and even adequate specimens for molecular analysis. This study reports on the ability of EBUS-TBNA to make different diagnoses and dictates the consecutive therapy in a large cohort of patients presenting different thoracic diseases.  All procedures performed from January 2012 to September 2016 were reviewed. Five groups of patients were created according to the main indications for the procedure. Group 1: lung cancer staging; Group 2: pathological diagnosis in advanced stage lung cancer; Group 3: lymphadenopathy in previous malignancies; Group 4: pulmonary lesions; Group 5: unknown origin lymphadenopathy. In each group, the diagnostic yield of the procedure was analysed. Non malignant diagnosis at EBUS-TBNA was confirmed by a surgical procedure or clinical and radiological follow-up.  1891 patients were included in the analysis. Sensitivity, negative predictive value, and diagnostic accuracy in each group were 90.7%, 79.4%, and 93.1% in Group 1; 98.5%, 50%, and 98.5% in Group 2; 92.4%, 85.1%, and 94.7% in Group 3; 90.9%, 51.0%, and 91.7% in Group 4; and 25%, 83.3%, and 84.2% in Group 5. Overall sensitivity, negative predictive value, and accuracy were 91.7%, 78.5%, and 93.6%, respectively.  EBUS-TBNA is the best approach for invasive mediastinal investigation, confirming its strategic role and high accuracy in thoracic oncology. Mediastinal adenopathy has always been assessed by radiological imaging such as computed tomography (CT) and positron emission tomography (PET), with high sensitivity but a low diagnostic accuracy for the purposes of correct clinical decision-making . To dateIn the early 2000's, a minimally invasive convex probe endobronchial ultrasound (EBUS) procedure able to perform real-time transbronchial needle aspiration (TBNA) was described with high accuracy for mediastinal and hilar lymph node staging . Since tIn thoracic oncology, EBUS-TBNA has revolutionized the diagnostic approach to lung cancer and other neoplasms from a surgical to minimally invasive procedure. Especially in non small-cell lung cancer (NCSLC), EBUS-TBNA is able to dictate the consecutive therapy both for early and advanced stages, providing pathological diagnosis, mediastinal staging, and even adequate specimens for molecular analysis . In addiThis study reports the largest published experience in the use of EBUS-TBNA in a high-volume thoracic oncology institution. We aimed to describe the utility and diagnostic yield of EBUS-TBNA in different clinical scenarios in thoracic diseases, dividing our series into five different groups according to the main indication for the procedure  and reporting our results in terms of sensitivity, negative predictive value, and diagnostic accuracy.This single-centre retrospective study with a prospective follow-up was approved by the Institutional Review Board and the individual consent was obtained.From January 2012 to September 2016, 1958 EBUS-TBNA procedures were performed at our institution. The indications for EBUS-TBNA, lymph node stations, number of lymph nodes sampled, cytological results, and cancer cell type were obtained for the analysis. To better standardize the series, different groups of patients were defined according to the indication for the procedure.Group 1 included patients referred for EBUS-TBNA for mediastinal staging in potentially operable lung cancer and patients with mediastinal involvement but no evidence of distant metastasis. Patients with proven or suspected NSCLC were included, and pathological cell type was performed in the same procedure. According to our institutional protocol, suspect lymph nodes were defined as lymph nodes with a pathological PET scan uptake and/or enlarged lymph nodes with more than 1 cm in the short axis at the CT scan.Group 2 included patients with metastatic and/or bulky mediastinal disease referred for EBUS-TBNA for pathological diagnosis and molecular mutational analysis for targeted therapy; Group 3 included patients with a previous (thoracic or extrathoracic) malignancy who developed mediastinal and hilar lymphadenopathies suspected for recurrence; Group 4 included patients who underwent EBUS-TBNA for primary tissue sampling in pulmonary lesions ; and Group 5 included patients with mediastinal and hilar lymphadenopathy of unknown origin with no history of malignancy. Diagnostic sensitivity, accuracy, and negative predictive value were calculated according to standard definitions. Sensitivity was calculated for the diagnosis of malignancy.EBUS-TBNA samples were considered diagnostic when a definitive diagnosis was obtained. Lymph node samples negative for malignancy underwent surgical confirmation (mediastinoscopy or VATS) or clinical and radiological follow-up (at least 12 months of follow-up). Patients with loss of follow-up were excluded.EBUS-TBNA samples were considered false negative when surgery changed the final diagnosis, or there was clinical and radiological progression of the disease during follow-up. EBUS-TBNA samples insufficient or inadequate for diagnosis were considered false negative in the calculation of diagnostic accuracy. Patients without a specific diagnosis at EBUS-TBNA  but a definitive non-malignant diagnosis at mediastinoscopy were considered false negative.During EBUS-TBNA procedures, all lymphadenopathies with increased PET scan pathological uptake were sampled in patients with a non-malignant diagnosis at rapid on-site evaluation (ROSE), and at least one mediastinal and two hilar stations were sampled when ROSE was negative for tumour cells and suspected for lymphadenitis granulomatosis.Patients with negative EBUS-TBNA samples were referred for confirmatory surgical procedures after a multidisciplinary team discussion with thoracic surgeons, pulmonologists, radiologists, oncologists, and radiotherapists when lymphadenopathies were considered highly suspicious for recurrence based on CT and/or PET scan characteristics.EBUS-TBNA procedures were performed under local anaesthesia (1% lidocaine), and moderate sedation was provided by an anaesthesiologist with spontaneous ventilation. The same team of interventional pulmonologists using a convex probe  and a dedicated ultrasound processor  performed all procedures. All EBUS-TBNA specimens were collected with a 22 gauge dedicated needle .A very small amount of the aspirated material was pushed out by the internal stylet and smeared onto glass slides for immediate on-site evaluation (ROSE). Mirror slides were alcohol-fixed for posterior evaluation. Air-dried smears were immediately stained with a modified May-Grünwald Giemsa stain  and evaluated by the cytopathologist to confirm adequate tumour cells and/or lymph node material. The remaining aspirate and other needle passages (at least three needle passages each station) were fixed in a formalin solution for cell block processing and histological evaluation. Alcohol-fixed smears were stained with Papanicolaou and haematoxylin-eosin stains.Out of 1958 patients, 1891 were included in the study. Sixty-seven patients were excluded from statistical analysis due to loss of follow-up with the impossibility to confirm the negativity of lymph node samples. Patients' mean age was 65 years (range: 20–92), 1197 (63.3%) were men.The leading indication for EBUS-TBNA at our institution was lung cancer staging in 728 (38.5%) patients followed by pathological diagnosis and mutational status in advanced stage lung cancer in 401 (21.2%) patients, lymphadenopathy in previous malignancies in 320 (17%) cases, diagnosis of pulmonary lesions in 290 (15.3%), and diagnosis of unknown origin lymphadenopathy in 152 (8%) patients. Patient characteristics and indications for the procedures are reported in A total of 2298 lymph node stations were sampled: 1003 superior mediastinal: 2 high upper station #1, 56 station #2R, 8 station #2L, 740 station #4R, and 197 station #4L; 789 subcarinal station #7; 506 N1 nodes: 57 hilar (37 station #10R and 20 station #10L), 365 interlobar (191 station #11R and 174 station #11L), 83 lobar (53 station #12R and 30 station #12L), and 1 segmental station #13L. Lymph node sampled locations and number of lymph nodes are listed in Cytological diagnoses were adenocarcinoma in 692 patients, squamous cell carcinoma in 216, neuroendocrine tumours in 143 , 19 large-cell neuroendocrine tumours, 9 mixed large- and small-cell neuroendocrine tumours, 10 typical carcinoids, 3 atypical carcinoids), 154 metastases from extrathoracic malignancy, 65 poorly differentiated NSCLC, 24 lymphoproliferative disorders, 34 other malignancies , 100 lymphadenitis granulomatosis (97 sarcoid-like reaction and 3 tuberculosis), 316 normal/reactive lymph nodes, and 28 other non-neoplastic diagnoses .Overall sensitivity, negative predictive value, and diagnostic accuracy were 91.7% (95% CI: 90.1–93.0), 78.5% (95% CI: 74.9–81.7), and 93.6% (95% CI: 92.4–94.6), respectively.Mycobacterium tuberculosis.Out of 728 (60.2%) patients, 438 underwent EBUS-TBNA for mediastinal staging and the diagnosis of the cell-type tumour in the same procedure, and 244 (33.5%) had a previous pathological diagnosis of NSCLC, and EBUS-TBNA was performed for complete mediastinal staging, and in 46 (6.3%) patients, EBUS-TBNA was performed for restaging the mediastinum after induction chemotherapy for stage IIIA (pN2) NSCLC. In this group, EBUS-TBNA was diagnostic for malignancy in 485 (66.6%) patients, and a mean of 1, 5 lymph nodes per patient was biopsied. In 187 (25.7%) cases, EBUS-TBNA revealed normal or reactive lymph node samples, and in six (0.8%), EBUS-TBNA revealed a granulomatous reaction in five cases of sarcoid-like reaction compatible with sarcoidosis and one case of necrotizing granulomatosis with a positive culture for Malignant diagnoses included 278 adenocarcinoma, 102 squamous cell carcinoma, 55 neuroendocrine, 27 poorly differentiated nonsmall-cell lung cancer, and 23 other malignancies .Fourty-six patients underwent EBUS-TBNA for restaging the mediastinum after neoadjuvant chemotherapy. There were 16 out of 46 (34.8%) positive for malignancy and 6 (13%) false negative cases. From false negative cases, four patients received chemotherapy and mediastinal radiotherapy and two patients' neoadjuvant chemotherapy. Sensitivity, negative predictive value, and diagnostic accuracy in this subgroup of patients were 66.7% (95% CI: 45.4–82.8), 78.1% (95% CI: 61.2–89), and 84.8% (95% CI: 71.8–92.4), respectively.Among 193 patients with a nonmalignant diagnosis at EBUS-TBNA, 146 underwent surgical procedures for primary tumours confirming the negativity of lymph node samples, and 47 patients were excluded from surgery for clinical reasons and underwent a mean of 17.9 month's clinical and radiological follow-up. In this group, there were 50 (6.9%) false-negative samples. Sensitivity, negative predictive value, and diagnostic accuracy in the staging group were 90.7% (95% CI: 87.9–92.8), 79.4% (95% CI: 73.9–84.0), and 93.1% (95% CI: 91.1–94.8), respectively.This group comprised 401 patients. In 344 (85.8%) patients, EBUS-TBNA was performed for the first cell type diagnosis in stages IIIB and IV lung cancer, and in 57 (14.2%) patients, the procedure was performed after an initial established pathological diagnosis for tissue sampling for molecular analysis including the epidermal growth factor (EGFR) mutational profile, anaplastic lymphoma kinase (ALK) fusion genes, and mesenchymal-epithelial transition (MET) protooncogene amplification. Sensitivity, negative predictive value, and diagnostic accuracy in this group were 98.5% (95% CI: 96.7–99.3), 50% (95% CI: 25.4–74.6), and 98.5% (95% CI: 96.8–99.3), respectively.Out of 320 (59.4%) patients, 190 had a diagnosis of a recurrence of a previous tumour and 113 (35.3%) had a different diagnosis from the previous tumour. There were 17 (5.3%) false negative cases. In 97 patients, EBUS-TBNA revealed a definitive nonmalignant diagnosis, and 35 patients had evidence of a granulomatosis disease. Twenty-two out of 114 (19.3%) patients with nonmalignant diagnoses at EBUS-TBNA underwent mediastinoscopy or other surgical procedures to confirm the negativity of EBUS-TBNA diagnosis, and 92 (80.7%) patients underwent clinical and radiological median follow-up of 17 months. Sensitivity, negative predictive value, and diagnostic accuracy were 92.4% (95% CI: 88.1–95.2), 85.1% (95% CI: 77.4–90.5), and 94.7% (95% CI: 91.7–96.7), respectively.EBUS-TBNA was diagnostic for malignancy in 241 out of 290 (83.1%) cases. Cytological diagnoses were 10 lymphoproliferative disorders, 95 adenocarcinomas, 55 squamous cell carcinoma, 25 neuroendocrine tumours, 29 metastasis from other neoplasms, 14 poorly differentiated nonsmall-cell lung cancer, and 13 other malignancies . EBUS-TBNA revealed a nonmalignant diagnosis in 25 cases: 5 hamartomas, 4 fibrotic lesions after radiotherapy, 6 bronchogenic cyst, 1 pericardial cyst, 1 benign leiomyoma, 5 pneumonias, 1 mycetoma and 2 cases of granulomatosis (1sarcoidosis and 1 tuberculosis). In this group of patients, sensitivity, negative predictive value, and diagnostic accuracy were 90.9% (95% CI: 86.9–93.8), 51.0% (95% CI: 37.5–64.4), and 91.7% (95% CI: 88.0–94.4), respectively.This group included 152 patients: 59 granulomatosis lymphadenitis, 60 reactive lymph nodes, 3 lymphoproliferative disorders, 2 adenocarcinomas, 2 metastasis from other sites (thyroid and breast), 1 small-cell lung cancer, 1 ectopic thymus tissue, and 24 false negative samples. Sensitivity, negative predicted value, and diagnostic accuracy were 25% (95% CI: 13.3–42.1), 83.3% (95% CI: 76.4–88.5), and 84.2% (95% CI: 77.6–89.2), respectively. The diagnostic performance of EBUS-TBNA in the different groups of patients according to clinical indication for the procedure is shown in Many literature studies have reported on EBUS-TBNA experience for mediastinal staging in lung cancer patients and some have described the utility of EBUS-TBNA in other pathologies with mediastinal lymphadenopathies such as sarcoidosis, tuberculosis, and lymphoma , 11.During the last decade, EBUS-TBNA has become an essential diagnostic procedure in thoracic disease, revolutionizing the approach to diagnosis and treatment and guiding the best treatment option in a large number of patients. To date, the procedure has been widely used to perform mediastinal staging and has been included in almost all guidelines as the preferred first approach for invasive mediastinal staging in lung cancer patients , 12.A recent meta-analysis showed a sensitivity of EBUS-TBNA for mediastinal staging ranging from 81 to 95% . LikewisConsidering our Group 2 , EBUS-TBNA sensitivity and diagnostic accuracy were both 98.5%. EBUS-TBNA also provided adequate material for molecular analysis whenever requested in advanced stage adenocarcinomas in 98% of cases. This datum confirms our previous published experience of molecular analysis with EBUS-TBNA specimens where we showed a sensitivity of 96.9% . Our preDue to the low invasive and the feasibility of repeated procedures, EBUS-TBNA is the ideal procedure to establish a cell-type diagnosis and to provide adequate specimens for molecular assay in advanced lung cancer patients. The procedure's high accuracy and low risk of complications make it suitable for low performance status patients who could still benefit from targeted therapy .In Group 3 , EBUS-TBNA showed a sensitivity and diagnostic accuracy of 92.4% and 94.7%, respectively, in agreement with a previous similar study published by Navani et al. and also with our previous reported experience in this subset of patients , 9. In tIn the diagnosis of Group 4 patients (pulmonary lesions), EBUS-TBNA demonstrated a sensitivity and accuracy of 90.9% and 91.7%, respectively, in line with Nakajima et al.'s published data  . ClinicaIn the investigation of Group 5 (unknown origin lymphadenopathy), EBUS-TBNA presented a diagnostic accuracy of 84.2%. Due to the very low prevalence of malignancy in this group of patients, sensitivity was not surprisingly very low (25%). The vast majority of patients in this group had an inflammatory disease with the most frequent diagnosis of granulomatous lymphadenitis compatible with sarcoidosis.Our results refer to the largest published series in EBUS-TBNA, in a high-volume thoracic oncology institution, demonstrating the utility and high accuracy of EBUS-TBNA in different clinical scenarios in daily clinical practice.Some additional findings have also emerged from this study. First, our study showed a wide use of EBUS-TBNA in elderly patients (732 patients over 70 years old and 120 over 80 years old), with a very low rate (0.74%) of postprocedure minor complications and no major events. Other interesting findings included the possibility to repeat biopsies easily and safely, obtaining tumour specimens for genetic assessment in stage IV NSCLC patients and in other tumour recurrences without the need for further more invasive procedures. These aspects highlight the importance of EBUS-TBNA as a diagnostic approach in different thoracic oncology scenarios, underpinning modern oncological strategy also for fragile patients and thereby avoiding more invasive and demanding procedures.An essential aspect to achieve optimal results is the specimen handling in EBUS-TBNA . The useOur study has some limitations. The first is its retrospective nature, which probably limits and influences all of the variables included in the analysis. Another bias is that not all patients underwent a surgical procedure (mediastinoscopy or VATS) to confirm negative EBUS-TBNA samples but all patients had a clinical and radiological follow-up longer than 15 months.In our experience, EBUS-TBNA is not just a technique, but a philosophy for the investigation of thoracic disease. EBUS-TBNA feasibility and optimal results largely depend on the cytopathology and pulmonologists' experience but also the way the procedure is performed. Adequate sedation and ROSE are crucial and should always be used. In experienced hands, EBUS-TBNA offers everything chest physicians need for the best medical practice without further invasive procedures. Surgical procedures are still required in highly suspicious NSCLC cases when EBUS-TBNA is negative for malignancy and in some cases for lymphoma subtyping.In conclusion, EBUS-TBNA represents the best diagnostic approach in many different clinical scenarios in high-volume thoracic oncology centres and dictates the consecutive treatment option in the vast majority of patients. EBUS-TBNA is useful and accurate in a high percentage of cases underpinning modern oncological therapy, guiding the best treatment options in lung cancer and avoiding useless and invasive procedures in case of benign disease.Based on our previous expertise, we are developing new applications for EBUS-TBNA such as a new protocol for EBUS-TBNA primary lung cancer cell culture and microRNA profile in stage IIIA (pN2) NSCLC patients, testing the feasibility of a microRNA profile obtained from EBUS-TBNA  and the


ARS), providing an indication of how animals view complex resource landscapes, and also the resolutions at which the analysis should proceed; and (2) identifying places which the animal revisits multiple times and performs ARS; for these, we extract two scale‐dependent statistical measures—the mean visit duration and the number of revisits in each place. The association between these measures is suggested as a signature of how animals utilize different habitats or resource types. The framework is validated through computer simulations combining different movement strategies and resource maps. We suggest that the framework provides information that is especially relevant when interpreting movement data in light of optimal behavior models, and which would have remained uncovered by either coarser or finer analyses.The residence time is the amount of time spent within a predefined circle surrounding each point along the movement path of an animal, reflecting its response to resource availability/quality. Two main residence time‐based methods exist in the literature: (1) The variance of residence times along the path plotted against the radius of the circle was suggested to indicate the scale at which the animal perceives its resources; and (2) segments of the path with homogeneous residence times were suggested to indicate distinct behavioral modes, at a certain scale. Here, we modify and integrate these two methods to one framework with two steps of analysis: (1) identifying several distinct, nested scales of area‐restricted search ( A peakd Tveraa , statingHere, we introduce several improvements to the variance‐scale curve method. Fauchald and Tveraa  examinedAnother issue raised by Fauchald and Tveraa  is that backward + FPTforward, but also additional forward and backward path segments found within the circle, as long as the time outside the circle between these segments is shorter than some predefined threshold. These homogeneous movement bouts can then be correlated with environmental data providing insight regarding the response of the animal to its environment and resources.Following the variance‐scale method, Barraquand and Benhamou  suggesteBarraquand and Benhamou  did not 33.1r and as the coefficient of variation in RT against r. For each curve, we measured the number of peaks, the strength of the peak signature, and the radii of ARS. The latter was compared with direct estimations measured from the visualized paths. This was possible as the simulations, albeit stochastic in nature, created ARS within one or two distinct scale domains, which could be distinguished visually  locations of the animal.The algorithm has four steps illustrated in Fig. en route and recorded path segments of the latter are filtered out. As a filtering criterion, it is possible to use the mean duration per visit or the total duration spent around recorded locations, as both should distinguish the long ARS revisits from the fast occasional ones; here, we examined and compared both. As a filtering threshold, we suggest the mean or median (whichever is largest—as these distributions are strongly left‐skewed) of the distribution of the criterion chosen.In step (2), revisited ARS places are distinguished from other places the animal crossed more than once, while In step (3), spatially distinct ARS places are identified by considering the remaining locations as the nodes of a graph, in which connections (edges) exist if two locations are found within the circles of each other. ARS places are defined as connected components of this graph; that is, subgroups in which any two nodes are indirectly connected to each other see Fig. .Step (4) estimates the exact location of each place, its mean visit duration, and number of revisits. The mean visit duration and number of revisits to recorded locations that are assigned to the same ARS place constitute various portions of the full revisit history of this place. Therefore, one of the locations in each ARS place should be chosen to represent the central location of the place and its revisit history. Choosing this location was done using the same two criteria used for filtering—we examined both the location with the longest mean duration per visit and the location with the longest total duration spent within its circle Fig. .We used the simulation of simple ARS for patchy resources to evaluate the performance of the algorithm including the examination of filtering and choosing criteria. As the simulated resource maps and movement modes are simple and well defined, we could identify ARS places visually from each realized resource map and path. We could therefore compare the ARS places identified by the algorithm to those identified visually. This allowed us to spot failures and erroneous identifications of the algorithm: places that were erroneously united because of their proximity, stopping places, which are not ARS places (these have long visit duration and were therefore filtered in when the mean visit duration was used for filtering and choosing locations), and places with occasional, multiple, and short revisits . The simulation was run 30 times to ensure that the algorithm identifies most of the ARS places successfully, and to properly estimate error rates.3.3Simulations were run for 60,000 time steps on resource maps of 1,000 × 1,000 cells. Three spatial structures of food items were created as follows: scattered, simple patchiness, and two‐level hierarchical patchiness  to 1 (complete attraction). The sign before p is minus for attraction, or plus for repulsion. Different combinations of vb and p yield a variety of movement behaviors: from directed movement toward the target to concentrated ARS around the target to widely dispersed exploration behavior  yielded low and constant variance‐scale curve, peaking only at s Figure . When ARs Figure . Using vs Figure . The var4.2The algorithm performed slightly different when using the mean visit duration versus total accumulated time as a criterion for filtering out non‐ARS path segments and choosing the recorded location representing each ARS place. Using the mean visit duration as a filtering and choosing criterion, the algorithm identified most of the food patches in which ARS were performed in the simulated paths Figure . This er55.1The variance‐scale curve indicates the scales at which animals respond to their environment. Considering resources as the only movement driver in our simulations, the curves discriminated between distinct scale domains formed in the path when the animal responded to resources organized in a spatially hierarchical manner  The differences between the two criteria for filtering out locations and choosing locations to represent ARS places are important. If the goal is to identify ARS places, the mean visit duration should be the favored criterion. But in some cases, places that were relatively briefly revisited multiple times are meaningful, so the goals of the specific study should direct the choice of a criterion. Alternatively, the analysis may be performed twice using both criteria, and the results can be compared; (2) adjacent, distinct ARS places may be erroneously identified as one, especially when patches are very close to each other. To overcome this problem, after applying the analysis, the connected components should be visualized, and components which appear to have a radius twice larger than the characteristic radius of ARS places  should be split manually. The problem becomes crucial for radii, which are large relative to the whole occurrence area of the animal, making the method inappropriate for such cases, for example, for studying home‐range behavior. For such purposes, utilization distributions are more appropriate; (3) performing the analysis using characteristic radii smaller than the actual radius of ARS bears a serious problem of erroneously counting several in–out movements as several revisits. This problem may be overcome by summing into the RT additional time segments within the circle, up to some threshold of time outside (Barraquand & Benhamou, 6While the variance‐scale curve gives a holistic view of the spatial behavior, the visit durations and number of revisits are scale‐ and habitat‐specific, and provide information regarding the detailed space use. This information is remains hidden if only resource selection functions Manly,  or otherNevertheless, our framework assumes that animals respond to resource abundance and distribution in a way that maximizes time within profitable places. Different associations between resources and movement could manifest themselves differently in terms of the RT. For example, an animal having a good knowledge of the location of food items could perform short and straight in–out movements into a food patch, such that peaks in the variance‐scale curve would not indicate ARS size or patch size. Choosing the proper scale using the variance‐scale method in such a case is problematic, but the revisit analysis would still be indicative on its own. Such associations between movement and resources should emerge when resources interact with other factors causing movement—reproduction prospects, conspecific attraction, and predator avoidance (Creel & Winnie, Matlab analysis code is uploaded as the online supporting information. Sample data will be archived on Dryad upon acceptance.The authors declare no conflict of interest.Dror Kapota, Amit Dolev, and David Saltz developed and integrated the methodological approach; Dror Kapota and David Saltz wrote the manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Drosophila transcription factors, the classic EMT inducer Snail and the Drosophila orthologue of hGATAs4/6, Serpent, which drives an alternative mechanism of EMT; both Snail and GATA are specifically expressed in a number of human cancers, particularly at the invasive front and in metastasis. Thus, we recreated conditions of Snail and of Serpent high expression in the fly imaginal wing disc and analysed their effect. While either Snail or Serpent induced a profound loss of epithelial polarity and tissue organisation, Serpent but not Snail also induced an increase in the size of wing discs. Furthermore, the Serpent-induced tumour-like tissues were able to grow extensively when transplanted into the abdomen of adult hosts. We found the differences between Snail and Serpent to correlate with the genetic program they elicit; while activation of either results in an increase in the expression of Yorki target genes, Serpent additionally activates the Ras signalling pathway. These results provide insight into how transcription factors that induce EMT can also promote primary tumour growth, and how in some cases such as GATA factors a ‘multi hit’ effect may be achieved through the aberrant activation of just a single gene.Several transcription factors have been identified that activate an epithelial-to-mesenchymal transition (EMT), which endows cells with the capacity to break through basement membranes and migrate away from their site of origin. A key program in development, in recent years it has been shown to be a crucial driver of tumour invasion and metastasis. However, several of these EMT-inducing transcription factors are often expressed long before the initiation of the invasion-metastasis cascade as well as in non-invasive tumours. Increasing evidence suggests that they may promote primary tumour growth, but their precise role in this process remains to be elucidated. To investigate this issue we have focused our studies on two  Drosophila EMT-inducers Snail and Serpent are both capable of driving overproliferation. However, Snail overproliferation is accompanied by a decrease in cell size as well as cell death, and consequently the tissue does not increase in size. Serpent also drives cell proliferation but this occurs together with an increase in cell size, but not cell death, thus having a profound effect on the overall size of the tissue. We show that both Snail and Serpent trigger activation of the Yorki pathway and in addition Serpent, but not Snail, also triggers activation of the Ras pathway. These results provide insight into how activation of some EMT-inducing genes can also promote primary tumour growth.Many cancer cells acquire abnormal motility behaviour leading to metastasis, the main cause of cancer related deaths. In many cancers, transcription factors capable of inducing motile migratory cell behaviours, so-called EMT transcription factors, are found highly expressed. However, the expression of these genes is not restricted to metastatic invasive cancers; they are often found in benign tumours, or in tumours long before they show any sign of metastasis. This observation motivated us to ask if they may play a role in driving primary tumour growth. Our results show that the  Epithelial cells display a remarkable plasticity and while this property is vital for morphogenesis during normal embryonic development, there is now strong evidence supporting its critical role in tumour progression (reviewed in –3). SeveActivating an EMT program promotes tumour invasion and metastasis, as it endows cells with the ability to break through basement membranes and migrate away from their site of origin. However, as well as promoting tumour dissemination, there is also evidence implicating EMT transcription factors in promoting primary tumour growth (reviewed in ). For exDrosophila orthologue of hGATAs4/6, and modelled the effects of creating gain-of-function conditions in the wing imaginal discs of the fly. Due to the work of many laboratories, wing discs are now a well-established system for modelling epithelial tumours , the urs (see ), where Drosophila primordia of adult wing structures, the wing imaginal discs, are epithelial monolayers that actively proliferate during larval development to give rise to a 1,000-fold increase in the number of cells and tissue size. They are now widely used to study and dissect the basic mechanisms underlying different stages of epithelial tumour progression . We theview see  and usedme discs ; or nub-ng pouch . Regionaitecture . F-actinitecture  and cellitecture . In addiThe effects of Sna and Srp on tissue organisation vary notably, depending on the region of the wing disc, with the effects of Sna relatively restricted to the peripodial membrane , arrow aDrosophila embryo through the direct transcriptional repression of the key cell polarity regulator Crumbs (Crb)  and assembled into figures using both Fiji and the Adobe Photoshop software.The following antibodies used were: rabbit anti-aPKC ; rat anti-crb ; mouse anti-Dlg ; rabbit anti-dpERK ; goat anti-GFP ; rabbit anti-RFP ; Phalloidin TRITC ; mouse anti-Patched ; rat anti-Srp (1:500 made in the Casanova lab). The TUNEL assay was performed using the nub-Gal4, UAS-srcGFP; tubulinGal80ts alone, or crossed to UAS-sna or UAS-srp were dissected from third instar larvae that had been kept at 18 °C until late in L2 and shifted to 29 °C for 48 hours, and transferred to cold PBS. Discs were fragmented into small pieces using Vannas scissors (World precision Instruments) and implanted into the abdomens of virgin females, as described in [Wing discs with ribed in . Fly hosnub-Gal4, UAS-srcGFP; tubulinGal80ts was crossed to either UAS-sna or UAS-srp. Crosses were kept at 18 °C until late in L2 when larvae were shifted to 29 °C for 48 hours and dissected. The size of the GFP expressing area was then measured using Fiji software. For Anterior Posterior size ratios, the maximal width of the anterior compartment was divided by that of the posterior, again measured using Fiji software.For measuring the effects on the wing disc proper, S1 FigTS discs  and in nub-Gal4, UAS-Srp; tub-Gal80TS discs  48 hours after shifting to 29°C, the permissive temperature. Staining for Crb (A-C) shows that Crb is lost from the nub expressing region upon ectopic Sna or Srp expression . D, E Staining for F-Act and Dlg show that cells towards the edges of the Sna expressing regions lose polarity . F, G Srp overexpression drives a dramatic loss of cell polarity throughout the nub expressing region, as seen by staining for F-Act and Dlg  Scale bars—100μm.A-G Staining for polarity markers in control (A), nub-Gal4, UAS-Sna; tub-Gal80(TIF)Click here for additional data file.S2 FigTS and the discs are fixed 48 hours after shifting to 29°C. Scale bars—100μm. (C) Histogram plotting the A/P width ratio of wing primordia expressing either GFP alone (wt), Sna alone (sna), Sna together with GFP  or Sna together with both GFP and p35 . Data are presented as mean ± SD. *P<0.005; paired t-test. There is no significant difference between wt and sna, GFP, p35.A, B, 3rd instar wing discs from either individuals expressing GFP to mark anterior the anterior compartment together with Sna (A) or together with both Sna and p35, which blocks cell death (B). Discs are stained for GFP (green) and F-Act (red). Transgenes are under the control of the ci-Gal4 driver and tub-Gal80(TIF)Click here for additional data file.S3 FigTS and the discs are fixed 48 hours after shifting to 29°C. A Staining for Dcp1 to visualise cell death. D. Histogram plotting the A/P width ratio of wing primordia expressing either GFP alone (wt), Srp alone (srp) or Srp together with EGFRRNAi. Data are presented as mean ± SD. *P<0.005; paired t-test.A-C 3rd instar wing discs from either individuals expressing GFP to mark anterior the anterior compartment together with Srp  and RasRNAi (A); or just Srp (B). Transgenes are under the control of the ci-Gal4 driver and tub-Gal80(TIF)Click here for additional data file.S4 Figsrp were generated in an otherwise wild type background. Clones were induced by a 30 min heat shock and fixed after 24 hours. Staining for dpERK shows that Ras signalling is activated both inside (arrow) and outside (arrowhead) clones.(A) Clones of UAS-(TIF)Click here for additional data file.


Falls among persons over 60 present significant risks for serious injury or debility. Falls place burdens on Emergency Medical Services (EMS), hospitals, and the adults themselves. Recognizing a need to provide interventions to minimize risk, Orange County Emergency Services (OCES), the Orange County Department on Aging (OCDoA), and the University of North Carolina at Chapel Hill (UNC) partnered to create the Stay Up and Active Program (SUAA). The purpose of this study was to determine if SUAA was a feasible program to implement in the community.A streamlined workflow algorithm between the OCES and OCDoA was created and employed to provide falls risk assessment and necessary services. Qualitative techniques were used to assess the need for such a program and its potential impact. A subset of individuals was interviewed 3 months after the intervention to assess the impact of the intervention on their fall risk. Formal stakeholder interviews were not conducted, but anecdotal information from EMS providers was obtained and reported.n = 16) reported no recurrent fall.In the first 7 months, 478 instances of individuals who called OCES screened positive for falls risk. Of the 478 positive screenings, 55 individuals were identified as having received more than one positive fall screen due to multiple calls. The maximum number of positive screenings by one individual was 14. More women (61.3%) than men screened positive for fall risk. Individuals 88 years of age (6.9%) represented the highest number of individuals with positive screens. Nineteen (4.0%) people who called OCES and received the intervention completed a 3-month follow-up survey. Of the 19, 86% (The number of individuals who screened positive supports the need for early identification and intervention through SUAA. This program identified several challenges connecting older adults with services already available to keep them independent, which provided insight to all stakeholders regarding factors that inhibit the program’s success. The program evaluation should continue to provide suggestions for improvement and ensure sustainability. Falls in older adults comprise a significant portion of health-care expenditures and resource use in the United States. One of every three older adults falls annually resulting in a total of 12 million falls  of the Orange County Emergency Services (OCES) is the sole provider of Advanced Life Support (ALS) services in the county. EMS consists of 75 full-time and 20 part-time employees, and staffs 5–9 ambulances any given day. OCES began tracking EMS calls classified as falls-related in 2010. Between 2010 and 2013, the EMS Division averaged 10,384 calls per year . Of thesThe Orange County Department on Aging (OCDoA) encounters over 190,000 participants per year at its hosted events . The OCDEmergency Medical Services has frequent contact with older adults who would benefit from OCDoA services to minimize their risk of falling. The opportunity to leverage the “first responder” relationship and connect older, at-risk adults with the resources in the community was the inspiration for the Stay Up and Active Program (SUAA). SUAA was designed to be a fall risk identification and management program implemented by EMS to connect at-risk older adults with the services they need. The intent of connecting these adults with services is to reduce the number of falls by older adults in their homes. This program represents the first time EMS formally collaborated with the Department on Aging to meet a need in the community.Initial discussions between the OCDoA and EMS supported that an EMS-centric model would be optimal for a community falls prevention program. As EMS providers are frequently the first caregivers in any fall, OCES was well positioned to link the at-risk population with the services and resources provided by the OCDoA. EMS providers have the opportunity to obtain accurate and complete histories from the older adults and possible bystanders on scene, and can assess the older adult’s safety in their environment. EMS and OCDoA agreed to initiate SUAA with EMS as the first point of contact with potential at-risk individuals. EMS would then schedule an in-home visit to further assess the older adult and communicate with OCDoA to connect the older adults with community resources.The purpose of this study was to determine if SUAA was a feasible program to implement in the community. Specifically, it was necessary to know if the perceived need for the program was accurate, if the workflow developed to implement SUAA for EMS staff was efficient and effective, if older adults who called EMS for a falls-related issue would be receptive to a second home visit, and if the system designed to facilitate communication between OCES and OCDoA achieved the goals of the project. The information from this study will help inform future steps to this collaborative project to address the problem of falls in the community.The OCDoA provides services to all county residents aged 60 years and older. Therefore, the SUAA program included any adult who is 60 years of age or older in Orange County who received EMS support resulting from a call for service. An algorithm and workflow were developed by both organizations to identify the level of risk and appropriate intervention Figure .•Are you worried that you are going to fall?•Have you fallen in the past year?•Are you unsteady when walking or standing? All adults over the age of 60 years, who called EMS, were screened for fall risk. During the course of an EMS intervention, providers would ask the following screening questions from the American Geriatrics Society (AGS) Clinical Practice Guidelines questions:A positive screen was based on a “Yes” response to any of the questions. Those that screened positive were asked if they would like to receive a follow-up phone call and additional home safety services from EMS. Those who agreed signed a form allowing EMS to access their name and phone number for further follow-up. Any EMS provider, regardless of their certification level, was able to conduct a fall risk screen. This screening was designed to supplement the standard patient assessment, and was incorporated to be as streamlined as possible for field EMS staff.The older adults who agreed to follow-up were entered into the EMS WebEOC (online emergency incident management technology) database to track their status. The purpose of WebEOC was to notify the SUAA team that an older adult screened positive for the program, to track their status and to communicate outcomes between agencies. Seventy-two hours following the initial EMS service call, a follow-up telephone call by EMS was initiated to schedule a home visit. If no contact was made after three telephone call attempts, the patient record was closed. If contact was made, a home visit from EMS personnel would be scheduled.The home visit consisted of a translation of the Centers for Disease Control’s STEADI  toolkit, which is an evidence-based fall risk management algorithm for clinicians . The STESubsequent to a home visit from EMS, and with approval from the older adult, a notification was sent to OCDoA from the WebEOC database. The goal was for OCDoA to make an assessment and connect the older adult with the appropriate resources in the community. At the OCDoA follow-up, appropriate referrals for occupational therapy, physical therapy, counseling, caregiver support group, in-home health-care services, and others were made.In an effort to streamline communication and share findings, the assessments and referrals were recorded in WebEOC by both EMS and by OCDoA and used for participant monitoring. Following the completion of a WebEOC ticket, the initial EMS crews were notified of the outcome of OCES and OCDoA follow-up with the older adults.Prior to implementation, SUAA was reviewed by the UNC Office of Human Research Ethics Institutional Review Board as Study 13-2942 and was granted exempt status from further review as the submission was considered a quality improvement program and did not constitute human subjects research under 45 CFR 46.102 (d or f) and 21 CFR 56.102(c)(e)(l). Additionally, neither special funding was allocated nor was grant funding obtained to implement this program. All resources were obtained from preexisting sources within county offices. Any materials given to adult participants were free of charge and donated by relevant organizations.The SUAA program was evaluated by a team based at the UNC Chapel Hill Center for Aging and Health to determine the feasibility of implementing the project. The first part of the evaluation determined the SUAA program met a need in the community. Implementation based on the established workflow and related IRB status enabled the data collection and analysis effort. The second part of the SUAA program evaluation consisted of interviews with a subset of individuals who received the home visit to assess their response to the program. The evaluation work was funded by a grant received by the University of North Carolina.Formal interviews of EMS providers were not conducted; however, anecdotal information obtained by providers was obtained when reviewing cases with these providers. The primary method of obtaining information about patients and their condition was by reviewing the patient care reports submitted by EMS providers after the initial 9-1-1 call. These reports provided valuable information about the patient’s health and current social environment.Between September 1, 2013 and March 31, 2014, there were a total of 704 EMS calls for a fall and 37 EMS calls for “Lift Assistance” . There wOf patients who screened positive, accurate phone numbers were only recorded in 31% (148 instances) of the patient care reports. Of the 148 instances of positive screening for falls risk and accurate phone number collected, 54 participants agreed to a home visit by EMS. Of those patients who received a home visit by EMS, 20 participants agreed to a follow-up visit from the OCDoA. Nineteen of the participants who received follow-up from the OCDoA agreed to further follow-up from UNC in the form of a 3-month follow-up survey conducted by phone interview.Of the 19 participants who completed the 3-month follow-up survey, 86% did not report a recurrent fall at 3-month follow-up. A total of 74% were very satisfied, and 26% were satisfied with the home visit from EMS. When asked about the value of the program, 5% found it not helpful, 16% found it somewhat helpful, 21% found it helpful, and 42% very helpful; 10 respondents answered other. As a result of the home visit, 16% felt somewhat confident, 16% felt confident, and 32% felt very confident that he or she could take actions to reduce risk of falling. Ten participants remarked other and commented, “They have already done everything they could to prevent falls” and, “The visit helped to heighten their awareness that they had to do something to prevent falls.” In the survey, 95% would recommend the EMS home visit program to a friend who may need help to stay independent in their home and 5% responded maybe.Older adult falls, and older adults who fall more than once, are a public health problem in Orange County, NC, USA. With more than 11% of all EMS calls being fall-related, coupled with a rapidly expanding aging population within the community, there was a definite need for a local structured falls prevention program. An efficient workflow incorporating evidence-based assessments was constructed and adopted by OCES and OCDoA. The WebEOC system allowed for transparent and timely exchanges of information between providers. The program recruited only 54 older adults in a 7-month pilot period, a lower than expected number. Of the 19 participants who completed the 3-month follow-up survey, 86% did not report a recurrent fall and the overall satisfaction rate was positive.During the 7-month trial period, there were a total of 704 EMS calls for a fall, and 37 EMS calls for “Lift Assistance.” Based on this information alone, the EMS unit hour utilization and ED bed time use expected as a result of these calls causes a significant burden to health-care resources. Further studies are warranted to investigate if SUAA has any impact on decreasing the number of annual falls related EMS calls. First responders should continue to be utilized as they offer a unique and innovative way to access older, at-risk adults who would otherwise be left underserved by their community resources.There were a total of 741 falls related EMS calls during the study period, but only 478 instances of positive screenings. As the falls risk assessment could be performed on any patient aged 60 years or greater no matter what the nature of the call , it was expected that at least as many positive screenings would be recorded. Since this is not the case, further investigation is needed to determine and quantify whether or not all fall victims were screened or if they screened negative. If they are simply were not being screened, then further training and emphasis will need to be placed on the necessity for asking the three falls risk assessment questions with field EMS staff. If the patients are screening negative, then evaluation of EMS recording and other potential areas of outreach need to be explored with this program.In examining the demographics of the patients who screened positive, more women screened positive than men, consistent with the national data that shows women over 60 falls more frequently than men . The OraThere were several barriers and limitations discovered during implementation of this program. Barriers fell into two categories: system-based change and older adults. Initially, information sharing to track participants who agreed to follow-up was difficult. In response, the WebEOC boards were reviewed, modified, and republished to ensure ease of access and use for all agencies. A second barrier encountered was the poor phone number collection by field EMS staff. Without an accurate phone number, patients could not be contacted for follow-up which was reflected by low participant rates.There was significant difficulty getting participants’ agreement to a home visit by EMS based on the EMS telephone contact effort. Several factors that contributed to this were failure to self-identify as at-risk, currently receiving care at the time of phone call , unable to contact, and no interest in speaking with a representative from EMS. It was also difficult to find one time frame  that could be applied to all patients to call to schedule a follow-up. To address these barriers, patients are now to be asked at the time of the field assessment if they would like a follow-up and contact information will be obtained for both the patient and their primary caregiver (if possible). This process amendment will hopefully reduce the difficulty in trying to explain the program over the phone and make it easier to schedule a follow-up visit.The 3-month follow-up survey revealed areas of success and room for improvement. One participant remarked that the, “EMS made suggestions to get the wheelchair through the doorway” while a family member of another participant commented, “The older adult won’t comply with the recommendations.” Some of the additional comments included: (1) “Daughter was very frustrated – she has been spending considerable time caring for her mother and needs help. The daughter has minimal transportation and hasn’t been able to go to work due to caring for her mother. Her mother had been taken to the hospital in the morning with a mini-stroke. The daughter repeatedly said that her mother needs a wheelchair,” (2) “Talked with son-in-law of patient. He was present at the EMS home visit and very enthusiastically supports it.”The idea of utilizing EMS to provide population health services is not novel; programs have been established for the EMS staff to augment immunization and fall prevention services provided in the rural areas of upstate New York . SUAA suThe study shows SUAA addresses a need within the community, but adjustments are needed to improve processes to ensure sustainability.Elderly, frail patients with multi-morbidity require greater time and resources to maintain independent living. In an effort to intercept the unique health-care concerns of a rapidly expanding aging population, the SUAA offers a potential solution by targeting at-risk individuals and providing assessment and resources. The goal is to not only decrease the number of EMS calls for falls but also the overall community morbidity as a result of preventable falls in older adults. This program represents a tremendous effort put forth by UNC, OCDoA, and OCES. The historical data and results from the pilot phase of Stay Up and Active demonstrate the need in Orange County for more than simple emergency response to injury and illness. Orange County EMS is in a prime position to provide the falls assessment questions as an integrated part of their services, and must continue implementation of this program as well as address the barriers identified in this report. Furthermore, SUAA represents a national trend for EMS systems to address community needs of their patients and begin to shift resources toward population health as a means to alleviate the burdens they face. However, with a large aging population, both local and national attention should be given to help individuals safely age in place as a way to help offset future health-care costs.SL is the primary author for this work. LE served as content and conceptual review for the work throughout the process. TS was the program implementation lead. KK made iterative changes and periodic updates to the work throughout the process. ER and JB-W provided subject-matter expertise to the research and evaluation oversight.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The PRAME tumour antigen is expressed in several tumour types but in few normal adult tissues. A dose-escalation phase I/II study (NCT01149343) assessed the safety, immunogenicity and clinical activity of the PRAME immunotherapeutic (recombinant PRAME protein (recPRAME) with the AS15 immunostimulant) in patients with advanced melanoma. Here, we report the phase I dose-escalation study segment.Patients with stage IV PRAME-positive melanoma were enrolled to 3 consecutive cohorts to receive up to 24 intramuscular injections of the PRAME immunotherapeutic. The RecPRAME dose was 20, 100 or 500 µg in cohorts 1, 2 and 3, respectively, with a fixed dose of AS15. Adverse events (AEs), including predefined dose-limiting toxicity (DLT) and the anti-PRAME humoral response (ELISA), were coprimary end points. Cellular immune responses were evaluated using in vitro assays.66 patients were treated . AEs considered by the investigator to be causally related were mostly grade 1 or 2 injection site symptoms, fatigue, chills, fever and headache. Two DLTs (grade 3 brain oedema and proteinuria) were recorded in two patients in two cohorts (cohorts 2 and 3). All patients had detectable anti-PRAME antibodies after four immunisations. Percentages of patients with predefined PRAME-specific-CD4+T-cell responses after four immunisations were similar in each cohort. No CD8+ T-cell responses were detected.The PRAME immunotherapeutic had an acceptable safety profile and induced similar anti-PRAME-specific humoral and cellular immune responses in all cohorts. As per protocol, the phase II study segment was initiated to further evaluate the 500 µg PRAME immunotherapeutic dose.NCT01149343, Results. Checkpoint-inhibitors are successfully used for the treatment of metastatic melanoma, but more specific, tumour antigen-targeted immunotherapies would be desirable to increase specificity and decrease side effects of immunotherapeutic approaches.The human tumour antigen PReferentially expressed Antigen of MElanoma (PRAME) is a potential candidate because it is expressed by several tumour types, including melanoma. T-cell immune responses have been induced in previous studies, with no safety issues raised.This phase I/II dose-escalation study evaluated the PRAME cancer immunotherapeutic (three dosages of recPRAME+AS15 immunostimulant) in patients with advanced malignant melanoma.Two weeks post-treatment 4, the safety profile was clinically acceptable at all three dosages investigated.Treatment-induced robust humoral immune responses in all patients.Specific PRAME-reactive CD4+ T-cells responses were observed at all three dosages, although CD8+ T-cell immunogenicity was barely detectable and CD8+ T-cells responses were absent.As per protocol, the highest dose was selected for assessment of clinical activity in the phase II segment of the study.This study provides evidence that PRAME is a possible target for specific immunotherapy in melanoma. Initial implication for clinical practice will be assessed in the phase II study segment.Cutaneous melanoma is the most aggressive form of skin cancer and patients with metastatic disease have a poor prognosis.PRAME is expressed in low levels in a normal ovary, endometrium, kidney and adrenal tissues,4In the context of these advances, the aim of specific immunotherapies is to induce clinically significant, long-lasting responses with moderate toxicity. The human tumour antigen PReferentially expressed Antigen of MElanoma (PRAME) was originally identified as the target antigen of a cytolytic T-lymphocyte clone derived from a patient with melanoma.PRAME is a potential candidate for cancer immunotherapy because it is expressed by a variety of tumours and can induce T-cell immune responses.In a dose-escalation phase I study, we sought to determine an adequate dose of a recombinant PRAME protein  administered with GSK's proprietary immunostimulant AS15, through evaluation of the safety and immunogenicity of the PRAME immunotherapeutic in patients with PRAME-positive metastatic melanoma. Here we present safety and immunogenicity data two weeks after dose 4 that led to dose selection according to protocol-defined rules. A phase II study segment is ongoing and will assess clinical activity of the selected dose of recPRAME. Clinical activity observed in phase I will be described at the time of the final analysis.http://www.clinical trials.gov NCT01149343) study protocol was approved by institutional review boards at each participating centre. Written informed consent was obtained from each patient prior to the performance of any study-specific procedures, including PRAME screening.The open-label, phase I dose-escalation study  and the anti-PRAME humoral immune response. Secondary objectives included evaluation of additional indicators of safety and immunogenicity in terms of antigen-specific cell-mediated immune (CMI) responses.Patients were ≥18 years of age with histologically proven cutaneous PRAME antigen-positive melanoma. Eligible patients had stage IV M1b-c melanoma, including completely resected stage IV patients except those with IV M1c disease with serum lactate dehydrogenase >1.5 times the upper limit of normal, or with active involvement of the central nervous system. See the 10.1136/esmoopen-2016-000068.supp1supplementary dataThe PRAME immunotherapeutic (recPRAME+AS15) was administered intramuscularly into the deltoid or thigh. The composition of the PRAME immunotherapeutic is provided in the online Escalating doses of recPRAME  combined with a fixed dose of AS15 were evaluated in three consecutive cohorts. A maximum of 24 doses of PRAME immunotherapeutic could be administered. The treatment schedule is provided in the online A DLT was defined as any of the following AEs considered related or possibly related to administration of the PRAME immunotherapeutic: (1) ≥grade 3 AE ; (2) ≥grade 2 allergic reaction occurring within 24 hours postinjection of the PRAME immunotherapeutic; (3) any decrease in renal function with a creatinine clearance <40 mL/min considered related or possibly related to the PRAME immunotherapeutic; or (4) any symptomatic and confirmed adrenal insufficiency related or possibly related to the PRAME immunotherapeutic.Anti-PRAME IgG antibodies were measured by ELISA prior to administration of dose 1, 2 weeks postdose 2 and 2 weeks postdose 4, as described in the online CMI was measured prior to the first dose and 2 weeks postdose 4 as described in the online The dose was selected based on safety and immunogenicity data. A dose was considered adequate if ≤two cases of DLT were reported at any time among the 15 patients in each cohort; and if the dose showed ≥70% (≥11/15) anti-PRAME antibody responses after four immunisations. If more than one dose level satisfied safety and humoral immune response criteria, the selection of the dose would also take into account CMI responses. If the best immunological dose could not be determined by applying these criteria, the highest dose with acceptable safety and immunogenicity was selected.The study was descriptive and no comparative tests were performed. See the online PRAME expression and 66 were enrolled , 66 patients had received a total of 468 doses of the PRAME immunotherapeutic by the data lock point (DLP). The main reason for treatment discontinuation was disease progression . Two patients withdrew due to AEs not considered related to treatment: one patient in cohort 1 developed atrial fibrillation 5 days postdose 2 and one patient in cohort 2 developed dehydration on the day of dose 4. Two patients withdrew consent (not due to an AE) during treatment cycle 2. Until the DLP, two patients died from melanoma progression during treatment.DLTs were reported by two patients. All were categorised as treatment-related grade 3 AEs. A patient in cohort 2 with small pre-existing residual local brain oedema after surgery and cerebral radiotherapy for brain metastases developed worsening of the focal brain oedema  5 days postdose 6. The patient recovered and continues the study treatment and remains disease-free. One patient in cohort 3 had microalbuminuria on the day of dose 8 and proteinuria 22 days postdose 8 . The microalbuminuria resolved and the patient was withdrawn due to progression of melanoma.There were nine SAEs in six patients, including the DLT case of focal brain oedema, of which eight were considered as treatment unrelated. One potential immune-mediated disease was reported in a patient who developed grade 1 vitiligo on the day of dose 3, considered to be treatment-related. This patient demonstrated a humoral response but did not have a clinical response to treatment, nor CD4+ or CD8+ responses.The majority of AEs reported from dose 1 were grade 1 or 2. There were 17 grade 3 AEs reported by 11 patients distributed across all three study cohorts and encompassing a wide range of conditions  and one patient in cohort 2 (maximum severity grade 2) on two occasions . These events were considered unrelated to the PRAME immunotherapeutic.There were no grade 4 laboratory abnormalities. Grade 3 laboratory abnormalities occurred in seven patients  and included anaemia (2 patients), increased γ-glutamyl transpeptidase , increased lymphocyte count (1 patient).One patient (cohort 3) was reported to have grade 1 adrenal insufficiency concomitantly with decreased blood cortisol levels at dose 5. This finding was not reported as an AE.All patients were seronegative for anti-PRAME IgG antibodies at baseline. At least 73% of patients in each cohort were seropositive after two doses. All patients had a humoral response (seroconversion) after 4 doses. Anti-PRAME antibody concentrations were higher after dose 4 than after dose 2 in all cohorts .After receiving dose 4, the number of patients with PRAME-specific CD4+ T-cell (TNF-α+/IFN-γ+) immunogenicity scores ≥cut-off was 7/9 in cohort 1, 7/11 in cohort 2 and 11/15 in cohort 3 . No patiOut of all doses fulfilling the predefined criteria for dose selection, the 500 µg dose was selected for the phase II segment of the study as per protocol.PRAME expression in 84.6% of patients, which is in the range reported in the literature for metastatic melanoma.3This dose-escalation study evaluated the PRAME immunotherapeutic in patients with advanced malignant melanoma. We observed Antigen-specific cancer immunotherapeutics typically induce less toxicity than cytotoxic agents and non-specific immunotherapeutic treatments targeting T-cell checkpoints.No patients had pre-existing anti-PRAME antibodies and the PRAME immunotherapeutic induced a humoral immune response in all patients. Spontaneous anti-PRAME antibodies were reported in a small proportion of patients with NSCLC expressing PRAME, as was also the case for NSCLC and melanoma expressing MAGE-A3.As observed in the parallel study in NSCLC (adjuvant setting), CD8+ T-cell immunogenicity was barely detectable or undetectable and CD8+ T-cells responses were absent. These results in patients with solid tumours contrast with studies in other clinical settings (patients with leukaemia where contact with circulating T-cells occurs) in which CD8+ T-cell responses to PRAME have been observed.MAGE-A3 expression in patients with melanoma,PRAME antigen expression into all progression/relapse lesions evaluated illustrates that a specific antigen loss variant does not seem to be the main mechanism of absence of clinical response to the PRAME immunotherapeutic.As observed with In this phase I dose-escalation study, the PRAME immunotherapeutic 500 μg dose showed a clinically acceptable safety profile and was immunogenic, with humoral and specific CD4+ responses observed in the majority of patients. The highest dose was selected for assessment of clinical activity in the phase II segment of the study in melanoma.


Emerging literature suggests that mobility and cognition are linked. Epidemiological data support a negative association between cognition and falls among cognitively intact older adults. A small number of intervention studies found that regimented cognitive training (CT) improves mobility among this population, suggesting that CT may be an under-explored approach toward reducing falls. To date, no studies have examined the impact of CT on balance among those who are cognitively impaired. The purpose of this study was to assess the feasibility of implementing a CT program among cognitively impaired older adults and examine whether there are potential improvements in balance following CT.A single group repeated measures design was used to identify change in balance, depressive symptoms, and global cognition. A mixed method approach was employed to evaluate the feasibility of a CT intervention among a cohort of cognitively impaired older adults. CT was delivered in a group 2 days/week over 10 weeks using an online brain exercise program, Posit Science Brain HQ (20 h). All participants completed a one-on-one data collection interview at baseline and post-program.N = 20) were on average 80.5 years old and had mild to moderate cognitive impairment. Following the 10-week CT intervention, mean scores on 4 of the 5 balance measures improved among CT participants. Although none of the balance improvements reached significance, these findings are promising given the small sample size. Depressive symptoms significantly improved between baseline and 10 weeks (p = 0.021). Mean global cognition also improved across the study period, but neither of these improvements were statistically significant. Based on participant responses, the CT program was feasible for this population.Participants (This study provides support for the feasibility of implementing a CT program among cognitively impaired older adults in an adult day setting. Our findings also add to emerging literature that CT may be a novel and innovative approach to fall prevention among older adults. Each year approximately one-third of community-dwelling older adults fall . Falls iA 2012 Cochrane Review of 159 fall prevention interventions implemented in community settings concluded that the most promising strategies for reducing falls risk are multifactorial and include multiple-component exercise programs, home safety assessment and modification, cardiac pacemakers when medically justified, and reduction of psychotropic medications . Of the Cognitive processing specifically, executive function (EF), is linked to balance, gait, and falls –10. EF iAs individuals progress into older age, fluid cognitive abilities, including EF, typically decline. Despite the decline in cognitive processing associated with increasing age, a growing body of evidence supports that the aging brain has exceptional neuroplasticity . CognitiN = 51) who were older adult independent living (IL) residents with a history of falls. Participants were randomly assigned to a computer-based CT intervention (Posit Science) that met 3 days/week for 60 min over 10 weeks or to a no-contact control group that received CDC pamphlets on fall prevention. Individuals randomly assigned to the CT intervention demonstrated significantly better balance compared to controls (N = 45). Participants were randomly assigned to CT or a no-contact control, but this time CT classes were held at Chicago Senior Centers over 10 weeks. Compared to controls, intervention participants improved significantly in balance and gait speed. Li et al. also used a randomized trial among older adults to show that CT was associated with significant improvements in body sway and dynamic balance compared to controls , and behavior. Alzheimer’s disease, a common form of dementia, is estimated to affect over 5 million adults in the United States and rise to be between 11 and 16 million by the year 2050 . AlzheimA single group repeated measures design was used to identify change in balance, depressive symptoms, and global cognition. A mixed method approach was employed to evaluate the feasibility of a CT intervention among a cohort of cognitively impaired older adults. This evaluation was carried out in accordance with the University of Illinois Chicago, Office of the Protection of Research Subjects as an exempt status. All participant information was secondary data and de-identified before evaluation.Twenty participants were recruited from the Easter Seals adult day care program to participate in an online brain exercise program. All participants provided consent to participate. To be included in the study participants must have (1) been admitted to Easter Seals day services ≥1 month prior to program onset, (2) been fluent in English, (3) been able to engage in computerized cognitive tasks, (4) been willing to commit to the time commitments required by the program, (5) been cognitively impaired as indicated based on a score <27 on the mini mental state exam (MMSE), and (6) self-reported a diagnosis of mild cognitive impairment or early stage Alzheimer’s disease. Individuals were excluded if they (1) reported a physical impairment that would prohibit them from using a computer mouse or keyboard, (2) were unable to perform the neuropsychological evaluations, or (3) could not comprehend study instructions or were incapable of providing written informed consent.Individual- and site-level data were gathered at baseline and post-intervention. CT was delivered in a group 2 days/week over 10 weeks using an online brain exercise program, Posit Science Brain HQ (20 h). Participants worked individually on desktop computers during weeks 2 through 9 to complete the CT. The Posit Science program targets EF through selective and divided attention, visuospatial WM, speed of processing, and dual-task processing – cognitive processes that are linked to balance and gait , 33. TheAll participants completed a one-on-one data collection interview at baseline and post-program, week 10. Balance was measured by the 4-position balance stand and timed up and go (TUG). The 4-position balance stand consists of the most sensitive balance stands taken from the Berg Balance scale, a valid, reliable, and clinically relevant measure of balance in older adults. The TUG is a brief physical performance test that measures time required to stand up from chair, walk 3 m, turn around, and sit back down. The TUG is a valid and reliable measure of balance –36. DeprIn order to capture program feasibility, qualitative data were collected using key informant interviews with program staff to examine facilitators and barriers to program implementation and maintenance using the RE-AIM model.t-tests were used to assess whether there were significant differences between the two time points. Given the pilot nature of this study, potential confounding factors were not controlled for in the analysis. All analyses were conducted using the IBM SPSS Statistics v 24.0 software.Repeated measures paired Twenty cognitively impaired older adults were enrolled into this study. Participants had a mean age of 80.5 years and were categorized as having mild to moderate dementia as indicated by the mean baseline MMSE score of 21.4 (Table t-tests exhibited a significant improvement in depressive symptoms between baseline and 10 weeks   = 1.63, p = 0.122); (MMSE t(15) = −0.24, p = 0.81)] , although this improvement did not reach significance  enjoyed the program” and noted that when participants “improved confidence in one area, the confidence trickles into other (behaviors)”. The staff delivering the intervention also reported that improvements in participants’ confidence were apparent over the course of the 10 weeks. The program was embedded within the structure of the adult day program and required few additional resources to implement. However, one challenge identified by key informants was facility space. Due to limited space availability, they expressed that it would likely be difficult to identify a quiet space to conduct this intervention on an ongoing basis.To our knowledge, this is the first study to examine whether cognitively impaired older adults experience improvements in balance following CT. Participants improved in 4 of the 5 balance measures over the study period. Although none of the balance improvements reached significance, these findings are promising given the small sample size.The primary purpose of this study was to examine the feasibility of conducting a group-based CT intervention among a cohort of cognitively impaired older adults. The program was embedded within an Easter Seals Adult Day Program. Program staff received training to implement the CT program and to collect study measures. Key informant interviews at the conclusion of the program with the program director and staff revealed that with the exception of limited space availability, the program implementation was both feasible and enthusiastically embraced by both staff and participants. Based on the successful implementation of the program within this environment and among this cohort, we conclude that it is feasible to implement a CT program within an adult day program.Our repeated measures analysis found that balance, depressive symptoms, and global cognition improved in the hypothesized direction following the 10-week CT program; however, depressive symptoms was the only outcome to significantly improve among our cohort of cognitively impaired older adults. This may be the first CT intervention to examine whether cognitively impaired older adults experience improvements in depressive symptoms. During post-program participant debriefing sessions, many expressed having a positive experience with the program and, following an initial period of staff guidance, increased confidence in their ability to complete the CT independently resulting in a sense of cognitive control. Staff also observed an improvement in participant confidence over the 10-week training period. It is possible that the self-reported improved sense of independence and control following the program mediated the significant reduction in depressive symptoms.We found that mean balance performance on TUG and three of the four balance stands improved across the 10-week training period; however, the post-program improvement in balance was not significant. To date, six studies, including two by Smith-Ray, have shown that CT improves motor tasks, such as balance and walking in older adults , 37, 38.This study is not without limitations. First, limited resources were available to conduct this study, and as such, we were unable to collect and analyze data on important covariates, such as polypharmacy/medication regimen, which is known to impact balance. Because we were unable to control for confounding factors, we cannot say conclusively that the significant improvements in depressive symptoms were due to CT rather than another explanatory factor, such as change in social engagement. We were also unable to conduct a cost effectiveness analysis of the intervention; such evidence would provide further support to justify or refute the feasibility of this intervention. Another limitation of the study was the use of a pre-post, within-subjects design with a small number of participants. Larger randomized studies among this population are needed to confirm these findings.Our findings support the feasibility of implementing a CT intervention to cognitively impaired older adults within an adult day setting. We found a significant improvement in depressive symptoms post-program in addition to improvements in global cognition that did not reach significance. We also found that balance improved within participants following 10 weeks of CT. These results are in line with findings reported by similar studies and support the hypothesis that CT may be a novel approach to improve balance among older adults. This study not only adds to emerging literature that CT may be a novel and innovative approach to fall prevention among older adults, but is the first to demonstrate this relationship among a cohort of cognitively impaired older adults.RS-R and CI co-led this project as investigators and together developed the research method, analytic plan, identified research site, and trained staff on data collection. Ms. KB assisted with data analysis and manuscript writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.