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Private Christian schools in North America endeavour to provide an educational experience that integrates religious and academic training for their preK-12 students. The integration of faith and ac...

Drug resistance remains the major driving factor behind the clinical failure of targeted therapeutics. Current oncology precision medicine approaches rely on targeting known acquired resistance mutations, such as EGFR T790M or ALK/ROS mutations in NSCLC with 2nd and 3rd generation molecules designed to overcome or prevent resistance. These next generation targeted therapeutic approaches have increasingly long and complex drug development timelines and burdensome toxicities from off target effects (e.g. wild-type receptor targeting) or drug-drug interactions (DDI). The toxicities limit tolerability, compliance and combinability of different targeted therapeutics. RNA-based immunotherapy approaches offer an increasingly attractive alternative to next generation small molecule targeted therapeutics approaches: (1) RNA-based approaches only require a known acquired resistance sequence, (2) drug development timelines, cost and complexity can be meaningfully condensed, and (3) multiple acquired resistance mutations can be targeted with the same candidate. RBI-1000 is a candidate using a novel type of self-replicating RNA (srRNA) to generate robust immunity directed against acquired resistance mutations that develop in ER+ breast cancer (ER+ BC) in response to endocrine therapy. RBI-1000 includes on-target mutations within the estrogen receptor ligand binding domain, and bypass mutations either in the form of activating mutations in the PI3K kinase domain or amplifications of HER2/HER3. Here, we demonstrate that this srRNA encapsulated in a lipid nanoparticle primes polyfunctional CD4 and CD8 T cells leading to tumor growth inhibition and improved survival in a mouse model expressing the targeted acquired resistance mutation. Priming of T cells against acquired mutations is also confirmed in human HLA-transgenic mice. The immune cell-mediated elimination of clones expressing the acquired resistance mutations is predicted to prolong endocrine control of ER+BC, in an analogous manner to small molecule or monoclonal antibody targeted therapies, but with a more favorable dosing and adverse event profile due to precise immunologic targeting and no DDI. Citation Format: Zelanna Goldberg, Christian Maine, Gabrielle P. Dailey, Christine Domingo, Gaelle Picarda, Hunter Little, Annie Chou, Jessica Sparks, Darina Spasova, Shigeki Miyake-Stoner, Zachary C. Hartman, Christopher A. Rabiola, Erika J. Crosby, Herbert K. Lyerly, Nathaniel Wang, Parinaz Aliahmad. A self-replicating RNA precision medicine approach to overcoming resistance to endocrine therapy in ER+BC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6403.

Christian educators must regularly articulate the ways in which they add value to the learning experiences of students, recognizing that pedagogy and purpose are inextricably linked. This study inv...

There continues to be a debate as to the role and value of educator preparation programs throughout the world. This paper examines self-report data of the instructional language learning methods of Nigerien English as a Foreign Language (EFL) teachers. This study sought to understand what instructional methods EFL teachers are using in their classrooms and if there is any connection between instructional methods and teacher training. All EFL teachers in Niger were surveyed to answer these questions. Teachers used a variety of instructional methods based on their preservice training; however, these differences were contained to teachers in their first five years of teaching. The findings support that teacher training is associated with the instructional decisions of teachers.

• PURPOSE: To determine whether ocular phenotypic features of keratoconjunctivitis sicca (KCS) and/or participant-reported symptoms of dry eye disease are associated with depression in women participants enrolled in the Sjögren’s International Collaborative Clinical Alliance (SICCA). • DESIGN: Cross-sectional study. • METHODS: Women enrolled in the SICCA registry from 9 international research sites. Participants met at least 1 of 5 inclusion criteria for registry enrollment (including complaints of dry eyes or dry mouth, a previous diagnosis of Sjögren syndrome (SS), abnormal serology (positive anti-Sjögren syndrome antigen A and/or B [anti-SSA and/or anti-SSB]), or elevated antinuclear antibody and rheumatoid factor), bilateral parotid gland enlargement, or multiple dental caries). At baseline, participants had oral, ocular, and rheumatologic examination; blood and saliva collection; and a labial salivary gland biopsy (LSGB). They also completed an interview and questionnaires including assessment of depression with the Patient Health Questionnaire 9 (PHQ-9). Univariate logistic regression was used to assess the association between depression and demographic characteristics, participant-reported health, phenotypic features of Sjögren syndrome, and participant-reported symptoms. Mixed-effects modeling was performed to determine if phenotypic features of KCS and/or participant-reported symptoms of dry eye disease were associated with depression, controlling for health, age, country or residence, and sex and allowing for nonindependence within geographic site. • RESULTS: Dry eye complaints produced a 1.82-fold (95% confidence interval [CI] 1.38–2.40) higher odds of having depression compared to being symptom-free (P < .001). Additionally, complaints of specific ocular sensations were associated with a higher odds of depression including burning sensation (odds ratio 2.25, 95% CI 1.87–2.72, P < .001) compared to those without complaints. In both women with and without SS, the presence of symptoms of dry eyes and/or dry mouth rather than SS itself resulted in higher odds of depression. One particular ocular phenotypic feature of SS, a positive ocular staining score, was inversely correlated with depression. • CONCLUSIONS: Participant-reported eye symptoms, particularly specific ocular sensations such as burning, were found to be positively associated with individual American College of Rheumatology/EUropean League Against Rheumatism (ACR/EULAR) SS criteria items.

Objective To examine health-related quality of life (HRQoL) and depression among participants in an international Sjögren’s syndrome (SS) registry, comparing those with and without SS. Methods Cross-sectional study of participants in the Sjögren’s International Collaborative Clinical Alliance (SICCA) registry. The 2016 American College of Rheumatology/European League Against Rheumatism SS classification criteria were used to determine disease status. HRQoL was assessed using the Short Form 12, version 2 Health Survey to derive scores for physical component summary (PCS) and mental component summary (MCS). Depression was assessed using the 9-Item Patient Health Questionnaire. Multivariate linear and logistic regression analyses were performed to identify predictors of HRQoL and depression while controlling for potential confounders. Results Among 2401 SICCA participants who had symptoms of dry eyes and dry mouth, 1051 had SS (44%) and 1350 did not (56%). After controlling for confounders, when compared with non-SS participants, those with SS had better PCS (p

Summary Although environmental stress likely plays a significant role in promoting aging, the relationship remains poorly understood. To characterize this interaction in a more comprehensive manner, we examined the stress response profiles for 46 long-lived yeast mutant strains across four different stress conditions (oxidative, ER, DNA damage, and thermal), grouping genes based on their associated stress response profiles. Unexpectedly, cells lacking the mitochondrial AAA protease gene AFG3 clustered strongly with long-lived strains lacking cytosolic ribosomal proteins of the large subunit. Similar to these ribosomal protein mutants, afg3Δ cells show reduced cytoplasmic mRNA translation, enhanced resistance to tunicamycin that is independent of the ER unfolded protein response, and Sir2-independent but Gcn4-dependent lifespan extension. These data demonstrate an unexpected link between a mitochondrial protease, cytoplasmic mRNA translation, and aging.

Objective Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are responsible for several craniosynostosis and chondrodysplasia syndromes as well as some human cancers, including bladder and cervical carcinoma. Despite a high frequency in some benign skin disorders, FGFR3 mutations have not been reported in cutaneous malignancies. Actinic cheilitis (AC) is a sun-induced premalignancy affecting the lower lip that frequently progresses to squamous cell carcinoma (SCC). The objective of this study was to determine if FGFR3 gene mutations are present in AC and SCC of the lip. Study design DNA was extracted and purified from microdissected, formalin-fixed, paraffin-embedded tissue sections of 20 cases of AC and SCC arising in AC. Exons 7, 15, and 17 were PCR amplified and direct sequenced. Results Four novel somatic mutations in the FGFR3 gene were identified: exon 7 mutation 742C→T (amino acid change R248C), exon 15 mutations 1850A→G (D617G) and 1888G→A (V630M), and exon 17 mutation 2056G→A (E686K). Grade of dysplasia did not correlate with presence of mutations. Conclusion The frequency of FGFR3 receptor mutations suggests a functional role for the FGFR3 receptor in the development of epithelial disorders, and perhaps this change may contribute to the pathogenesis of some AC and SCC.

SummaryMany genes that affect replicative lifespan (RLS) in the budding yeast Saccharomyces cerevisiae also affect aging in other organisms such as C. elegans and M. musculus. We performed a systematic analysis of yeast RLS in a set of 4,698 viable single-gene deletion strains. Multiple functional gene clusters were identified, and full genome-to-genome comparison demonstrated a significant conservation in longevity pathways between yeast and C. elegans. Among the mechanisms of aging identified, deletion of tRNA exporter LOS1 robustly extended lifespan. Dietary restriction (DR) and inhibition of mechanistic Target of Rapamycin (mTOR) exclude Los1 from the nucleus in a Rad53-dependent manner. Moreover, lifespan extension from deletion of LOS1 is nonadditive with DR or mTOR inhibition, and results in Gcn4 transcription factor activation. Thus, the DNA damage response and mTOR converge on Los1-mediated nuclear tRNA export to regulate Gcn4 activity and aging.

Therapeutic regulatory T cells (Tregs) can reverse pre-established autoimmune pathology. In this study, using a mouse model of autoimmune diabetes, we aimed to determine the means by which therapeutic Tregs control islet inflammation. Islet Ag-specific Tregs infiltrated inflamed islets soon after infusion into prediabetic mice, which was quickly followed by a selective reduction of mRNA associated with effector T cells in the islets. This change was partially due to decreased CD8+ T cell accumulation in the tissue. CD8+ T cells that remained in the islets after Treg treatment were able to engage dendritic cells in a manner similar to that found in untreated mice, consistent with the retention of an activated phenotype by islet dendritic cells shortly after Treg treatment. Nonetheless, Treg treatment abrogated IFN-γ production by intraislet CD8+ and CD4+ T cells at the protein level with minimal effect on IFN-γ mRNA. Sustained expression of IFN-γ protein by effector T cells was dependent on common γ-chain cytokine activation of the mTOR pathway, which was suppressed in islet CD8+ T cells in vivo after Treg treatment. These multifaceted mechanisms underlie the efficacy of therapeutic Treg subversion of effector T cell functions at the site of inflammation to restore normal tissue homeostasis.

Combining two or more longevity-altering interventions and determining the resulting effect on lifespan is a common method for examining the relationship between such interventions. An important subset of this type of analysis occurs when one of the factors under study promotes longevity, such as daf-16 in Caenorhabditis elegans or SIR2 in Saccharomyces cerevisiae. For both of these genes, several studies have combined a lifespan shortening null allele with an intervention that extends lifespan. A resulting lifespan similar to that of the short-lived single mutant has generally been interpreted as suggesting that the factors act in the same pathway. In contrast, an intervention extending the lifespan of the short-lived mutant has been interpreted as suggesting that the factors act in genetically distinct pathways. Specific examples of this type of comparison are studies in which DR fails to extend lifespan in yeast (Lin et al. 2000), invertebrates (Rogina & Helfand 2004; Wang & Tissenbaum 2006), and mice (Li et al. 2008) when Sir2-orthologs are mutated. These data have been, and continue to be, interpreted by some to support a model in which DR promotes longevity and healthspan through activation of sirtuins (Baur et al. 2010). It has been previously reported that deletion of SIR2 blocks RLS extension from DR by reduction of glucose and in strains lacking GPA2 or HXK2, two genetic mimics of DR, but not in a strain lacking the rDNA replication fork block protein, FOB1 (Kaeberlein et al. 2004). In order to examine the influence of deleting SIR2 on RLS extension more generally, we generated 30 additional double mutant strains in which a RLS extending deletion was combined with deletion of SIR2. We also tested three additional methods of DR involving growth on alternative carbon sources (ethanol, glycerol, or raffinose). Strikingly, none of these interventions resulted in a significant RLS extension relative to sir2Δ cells (Figure 1; Figure S2; Table S1). Figure 1 Single-gene deletions that extend RLS in wild-type cells do not extend RLS of sir2Δ cells One possible interpretation of these data is that each of the RLS-extending interventions acts upstream of Sir2, perhaps by promoting Sir2 activity. Two observations are inconsistent with this model. First, at least eight single-gene deletions that increase wild type RLS, and all four forms of DR, significantly extend the RLS of sir2Δ fob1Δ cells (Figure S1A; Figure S2; Table S1), demonstrating that SIR2 is not absolutely required for RLS extension in these cases. Second, at least five long-lived deletion mutants show no indication of enhanced Sir2 activity in vivo, as measured by rDNA recombination or rDNA silencing (Figure S3). A similar lack of increased Sir2 activity has been previously reported in cells subjected to DR (Kaeberlein et al. 2005; Riesen & Morgan 2009; Smith et al. 2009). Interestingly, deletion of TOR1 caused a significant decrease in rDNA recombination, but this effect was independent of SIR2 (Figure S3A). An alternative explanation for these data is that loss of SIR2 alters aging such that molecular processes that do not limit RLS in wild-type cells become limiting in sir2Δ cells. Sir2 has multiple functions, including repression of extrachromosomal rDNA circle formation (Kaeberlein et al. 1999), enhancing global rDNA stability and silencing (Gottlieb & Esposito 1989; Smith & Boeke 1997), promoting asymmetric inheritance of damaged proteins (Aguilaniu et al. 2003), and maintaining telomeric chromatin during aging (Dang et al. 2009). Our observation that only deletion of FOB1 is sufficient to suppress the short RLS of sir2Δ cells suggests that (1) the primary RLS-limiting defect in sir2Δ cells is likely related to rDNA instability and (2) none of the 32 deletions tested that slow aging in wild-type cells is able to overcome this defect. One prior study reported that overexpression of Hsp104 could also suppress the short RLS of sir2Δ cells (Erjavec et al. 2007), raising the possibility that accumulation of damaged proteins in sir2Δ mother cells may also contribute to the reduced longevity. While it is likely that many of the genes examined in this study do not require Sir2 for their effect on RLS, we do not believe that all of the 32 long-lived single gene deletion mutants examined here necessarily act via Sir2-independent mechanisms. For example, deletion of SAS2, a histone acetyltransferase known to antagonize Sir2 effects on chromatin (Dang et al. 2009), extends wild-type RLS but fails to extend the RLS of sir2Δ fob1Δ cells (FigureS2b). Thus, both functional and genetic evidence suggest that Sas1 likely acts in the same longevity pathway as Sir2. This study provides a clear demonstration of the challenges associated with interpreting longevity epistasis data. In particular, the failure of a longevity-intervention to extend lifespan in a short-lived background may not be informative regarding the mechanism of lifespan extension in the wild-type context. In the absence of strong evidence indicating that the lifespan shortening is caused by acceleration of the wild-type aging process, caution is warranted when interpreting these types of data.

Chronological aging of budding yeast cells results in a reduction in subsequent replicative life span through unknown mechanisms. Here we show that dietary restriction during chronological aging delays the reduction in subsequent replicative life span up to at least 23 days of chronological age. We further show that among the viable portion of the control population aged 26 days, individual cells with the lowest mitochondrial membrane potential have the longest subsequent replicative lifespan. These observations demonstrate that dietary restriction modulates a common molecular mechanism linking chronological and replicative aging in yeast and indicate a critical role for mitochondrial function in this process.

There is growing evidence that stochastic events play an important role in determining individual longevity. Studies in model organisms have demonstrated that genetically identical populations maintained under apparently equivalent environmental conditions display individual variation in life span that can be modeled by the Gompertz–Makeham law of mortality. Here, we report that within genetically identical haploid and diploid wild-type populations, shorter-lived cells tend to arrest in a budded state, while cells that arrest in an unbudded state are significantly longer-lived. This relationship is particularly notable in diploid BY4743 cells, where mother cells that arrest in a budded state have a shorter mean life span (25.6 vs. 35.6) and larger coefficient of variance with respect to individual life span (0.42 vs. 0.32) than cells that arrest in an unbudded state. Mutations that cause genomic instability tend to shorten life span and increase the proportion of the population that arrest in a budded state. These observations suggest that randomly occurring damage may contribute to stochasticity during replicative aging by causing a subset of the population to terminally arrest prematurely in the S or G2 phase of the cell cycle.

Chronological and replicative aging have been studied in yeast as alternative paradigms for post-mitotic and mitotic aging, respectively. It has been known for more than a decade that cells of the S288C background aged chronologically in rich medium have reduced replicative lifespan relative to chronologically young cells. Here we report replication of this observation in the diploid BY4743 strain background. We further show that the reduction in replicative lifespan from chronological aging is accelerated when cells are chronologically aged under standard conditions in synthetic complete medium rather than rich medium. The loss of replicative potential with chronological age is attenuated by buffering the pH of the chronological aging medium to 6.0, an intervention that we have previously shown can extend chronological lifespan. These data demonstrate that extracellular acidification of the culture medium can cause intracellular damage in the chronologically aging population that is asymmetrically segregated by the mother cell to limit subsequent replicative lifespan.

Activation of Sir2-orthologs is proposed to increase lifespan downstream of dietary restriction (DR). Here we describe an examination of the effect of 32 different lifespan-extending mutations and four methods of dietary restriction on replicative lifespan (RLS) in the short-lived sir2Δ yeast strain. In every case, deletion of SIR2 prevented RLS extension; however, RLS extension was restored when both SIR2 and FOB1 were deleted in several cases, demonstrating that SIR2 is not directly required for RLS extension. These findings indicate that suppression of the sir2Δ lifespan defect is a rare phenotype among longevity interventions and suggest that sir2Δ cells senesce rapidly by a mechanism distinct from that of wild-type cells. They also demonstrate that failure to observe life span extension in a short-lived background, such as cells or animals lacking sirtuins, should be interpreted with caution.

The Ras/mitogen-activated protein kinase (MAPK) pathway is considered to be a positive regulator of tumor initiation, progression, and maintenance. This study reports an opposite finding: we have found strong evidence that the MAPK pathway is inhibited in a subset of adenoid cystic carcinomas (ACCs) of the salivary glands. ACC tumors consistently overexpress the receptor tyrosine kinase (RTK) c-Kit, which has been considered a therapeutic target. We performed mutational analysis of the c-Kit gene (KIT in 17 cases of ACC and found that 2 cases of ACC had distinct missense mutations in KIT at both the genomic DNA and messenger RNA levels. These mutations caused G664R and R796G amino acid substitutions in the kinase domains. Surprisingly, the mutations were functionally inactive in cultured cells. We observed a significant reduction of MAPK (ERK1/2) activity in tumor cells, as assessed by immunohistochemistry. We performed further mutational analysis of the downstream effectors in the c-Kit pathway in the genes HRAS, KRAS, NRAS, BRAF, PIK3CA, and PTEN. This analysis revealed that two ACC tumors without KIT mutations had missense mutations in either KRAS or BRAF, causing S17N K-Ras and V590I B-Raf mutants, respectively. Our functional analysis showed that proteins with these mutations were also inactive in cultured cells. This is the first time that MAPK activity from the RTK signaling has been shown to be inhibited by gene mutations during tumor development. Because ACC seems to proliferate despite inactivation of the c-Kit signaling pathway, we suggest that selective inhibition of c-Kit is probably not a suitable treatment strategy for ACC.

This paper presents unprecedented accomplishments toward unidirectional guidance of cellular migration via durotaxis-based microtopography. In contrast to previous efforts, micropost arrays of varying anisotropy (μPVAs) optimize unidirectional control of cell migration through dual axis durotaxis cues which restrict movement in the lateral direction in addition to promoting migration in the direction of increasing micropost stiffness. Preliminary results show 79% of bovine aortic endothelial cells (BAECs) cultured on μPVA substrates migrated within ±60° of the direction of increasing micropost anisotropy. µPVAs offer a simple, yet powerful technique for enabling unidirectional control of cellular migration for a variety of applications in tissue engineering, biomaterials, and medical device implantation.

We examined whether mosquitofish (Gambusia affinis) exhibit any preferences for potential prey items on the basis of size or color. Using fishing lures to control for movement and other variables, we compared the number of initial foraging attempts (biting) exhibited towards lures based on size and based on color. Mosquitofish showed a non-significant trend (P = 0.14) toward preferring the largest item. Mosquitofish showed an overwhelming preference for a green lure compared to red, black and yellow, and black and green lures. These results suggest that mosquitofish may exhibit preferences for prey based on color, and to a lesser extent, on size.

Adenoid cystic carcinoma (ACC) is a malignant salivary gland tumor well-known for spreading in wide local area and invasion of vital structures of the head and neck. Unfortunately, conventional therapies have not improved longer-term survival, resulting in significant morbidity and a very low overall survival rate. Improved systemic therapies are clearly needed for this population. ACC tumors overexpress the receptor tyrosine kinase c-Kit, which is thought to be a therapeutic target. However, the role of c-Kit in the pathogenesis of ACC is not currently understood. We addressed this question through a functional genomic study with 17 sporadic ACC tumor specimens. We found heterogeneous missense mutations in three genes in the c-Kit signaling pathway, although mutations were identified at a low frequency. Two ACC tumors had distinct missense mutations in the gene KIT, resulting in G664R and R796G amino acid substitutions in the c-Kit kinase domain. Furthermore, two ACC tumors without KIT mutations had missense mutations in either KRAS or BRAF, causing S17N K-Ras and V590I B-Raf mutants. KRAS and BRAF are downstream effectors of c-Kit. We explored the functional consequences of these amino acid substitutions and found that all of c-Kit, K-Ras and B-Raf mutations in our ACC samples were inactive. These observations suggest that the c-Kit signaling pathway must be dispensable for maintaining established ACC. As a result, we believe that selective c-Kit inhibition is not a suitable treatment for the patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 351.

Background Bleeding following colonoscopic polypectomy is a common complication and has been reported to occur in up to 6.1% of patients. Several risk factors have been discussed but their overall contribution to post-polypectomy bleeding remains controversial. The aim of the study was to determine the rate of post polypectomy bleeding and to analyse the role of potential risk factors especially the role of aspirin. Methods We conducted a retrospective cohort study of all patients who underwent polypectomy at Dunedin Hospital, New Zealand between January 2007 and June 2009. Results During the study period, 514 patients underwent colonoscopy with polypectomy and a total of 1502 polyps were removed. From further analysis we excluded 21 patients; 15 patients had surgery immediately after colonoscopy for the diagnosis of colorectal carcinoma and 6 patients presented with symptoms of an acute lower gastrointestinal bleed prior to colonoscopy. Of the remaining 493 patients, 11 patients (2.2%) presented with post-polypectomy bleeding within 30 days of the investigation of which 8 were on aspirin. In total 145 patients were taking aspirin prior to colonoscopy and 348 patients were not taking aspirin. The use of aspirin was associated with an increased prevalence of post-polypectomy bleeding (OR=6.72, 95% C.I. 1.76 to 25.7). Interestingly, the use of non-steroidal anti-inflammatory drugs (NSAIDs) was not associated with risk of bleeding after polypectomy (OR=2.82, 95% C.I, 0.34 to 23.3). Conclusion Our study confirmed a significantly increased risk of lower gastrointestinal bleeding following polypectomy in patients taking aspirin. We would recommend approaching the patient on aspirin coming forward for a colonoscopy with potential polypectomy with caution.