Citrus Huanglongbing (HLB) is the most destructive citrus disease worldwide. HLB is associated with three species of the phloem-limited, gram-negative, fastidious α-proteobacteria: Candidatus Liberibacter asiaticus (Las), Ca. L. americanus (Lam), and Ca. L. africanus (Laf) with Las being the most widespread species. Las has not been cultured in artificial media, which has greatly hampered our efforts to understand its virulence mechanisms. Las contains a complete Sec-translocon, which has been suggested to transport Las proteins including virulence factors into the extracytoplasmic milieu. In this study, we characterized the Sec-translocon dependent, signal peptide containing extracytoplasmic proteins of Las. A total of 166 proteins of Las-psy62 strain were predicted to contain signal peptides targeting them out of the cell cytoplasm via the Sec-translocon using LipoP, SigalP 3.0, SignalP 4.1, and Phobius. We also predicated SP containing extracytoplasmic proteins for Las-gxpsy and Las-Ishi-1, Lam, Laf, Ca. L. solanacearum (Lso), and L. crescens (Lcr). For experimental validation of the predicted extracytoplasmic proteins, Escherichia coli based alkaline phosphatase (PhoA) gene fusion assays were conducted. A total of 86 out of the 166 predicted Las proteins were experimentally validated to contain signal peptides. Additionally, Las-psy62 lepB (CLIBASIA_04190), the gene encodes signal peptidase I, was able to partially complement the amber mutant of lepB of E. coli. This work will contribute to the identification of Sec-translocon dependent effector proteins of Las, which might be involved in virulence of Las.

Human β defensin DEFB103 acts as both a stimulant and an attenuator of chemokine and cytokine responses: a dichotomy that is not entirely understood. Our predicted results using an in silico simulation model of dendritic cells and our observed results in human myeloid dendritic cells, show that DEFB103 significantly (p < 0.05) enhanced 6 responses, attenuated 7 responses, and both enhanced/attenuated the CXCL1 and TNF responses to Porphyromonas gingivalis hemagglutinin B (HagB). In murine JAWSII dendritic cells, DEFB103 significantly attenuated, yet rarely enhanced, the Cxcl2, Il6, and Csf3 responses to HagB; and in C57/BL6 mice, DEFB103 significantly enhanced, yet rarely attenuated, the Cxcl1, Csf1, and Csf3 responses. Thus, DEFB103 influences pro-inflammatory activities with the concentration of DEFB103 and order of timing of DEFB103 exposure to dendritic cells, with respect to microbial antigen exposure to cells, being paramount in orchestrating the onset, magnitude, and composition of the chemokine and cytokine response.

Miltefosine is an important drug for the treatment of leishmaniasis; however, its mechanism of action is still poorly understood. In these studies, we tested the hypothesis that like in cancer cells, miltefosine's efficacy in leishmaniasis is due to its inhibition of Akt activation in host cells. We show using pharmacologic agents that block Akt activation by different mechanisms and also using an inducible knockdown approach that miltefosine loses its efficacy when its access to Akt1 is limited. Interestingly, limitation of Akt activation results in clearance of established Leishmania infections. We then show, using fluorophore-tagged probes that bind to phosphoinositides, that Leishmania parasitophorous vacuole membranes (LPVMs) display the relevant phosphoinositides to which Akt can be recruited and activated continuously. Taken together, we propose that the acquisition of PI(4) P and the display of PI (3,4)P2 on LPVMs initiate the machinery that supports continuous Akt activation and sensitivity to miltefosine.

Background: Lesbian, gay, bisexual, and transgender (LGBT) seniors are generally a medically underserved population that faces unique healthcare challenges. When compared to younger patients, LGBT seniors are at a greater risk for social isolation and have higher rates of smoking, disability, physical and mental distress, and lack of access to healthcare services. They are often reluctant to discuss their sexual orientations and gender identities with healthcare providers due to fear of discrimination and receiving inferior care based on prior unsatisfactory experiences with untrained or insensitive healthcare providers. Furthermore, recent research has revealed that only about 50% of primary care providers indicated confidence in providing culturally competent LGBT healthcare, highlighting the need for more LGBT proficiency training in medical school curricula. Objectives: The aim of this study was to provide early intervention training to first-year medical students regarding best practices for equitable healthcare for LGBT seniors through integrative, small group, case-based discussions. The impact of this activity on the knowledge and attitudes of medical students regarding LGBT healthcare was also assessed. Methods: First-year medical students participated in a two-hour small group, case-based discussion. Each group consisted of seven to eight students with one of seven facilitators who were invited members of the LGBT community. Students were provided with two clinical case scenarios related to treatment of LGBT senior patients. Students were given a pre/post-session knowledge and attitude survey to assess the impact of the session on their attitudes and understanding of the importance of providing equitable healthcare to LGBT patients. A rubric was also used by facilitators to evaluate level of student engagement and professionalism. Results: A total of 51 first-year medical students attended the session and 38 (74.5%) completed the pre/post surveys. There was diverse representation in our student demographic with 5.2% of respondents identifying as LGBT. Survey results showed a significant increase in knowledge confidence and attitudes following the session. Students’ attitudes regarding determinants of health status changed significantly for nine of the 13 (69%) survey items. In addition, their confidence in knowledge regarding healthcare barriers, health issues, and practices for LGBT culturally competent care significantly increased post-session. Data from our assessment rubrics also show that students were highly professional and engaged with the LGBT facilitators. Conclusion: Our study provides some evidence that case-based training of medical students regarding issues that affect health of LGBT seniors can improve attitudes and sensitize them to the unique needs of this population. Through this activity, the students indicated their desire to learn more about the topics covered and to receive further training in this field of study. While the study wa...

β-hemoglobinopathies like sickle cell disease (SCD) and β-thalassemia are characterized by differing mutations in the hemoglobin subunit beta gene (HBB). These disorders vary in phenotypic presentation and severity, with more severe manifestations leading to transfusion dependence along with associated complications such as infection and iron overload. β-hemoglobinopathies symptoms rapidly worsen after birth as the levels of fetal hemoglobin (HbF) begin to decline. To reverse this decline, current treatment plans typically involve the use of pharmacological agents such as hydroxyurea to raise expression levels of HbF. However, these treatments only result in transient effects and must be consistently administered. Gene editing technologies such as CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR associated protein) offer the opportunity to create novel treatments which can raise HbF expression with potential permanent effects. Two gene targets, B-cell lymphoma/leukemia 11A gene (BCL11A) and the promoter regions of gamma globin genes (HBG1/2), have been identified to significantly increase HbF protein expression. In order to differentiate the effectiveness of BCL11A and HBG1/2 editing, a meta-analysis was performed by first identifying 119 studies for inclusion based on the search terms terms “β-Thalassemia,” “beta-thal” “sickle cell disease,” “SCD,” and “CRISPR.” Following application of exclusion and inclusion criteria, we performed analysis on 8 peer-reviewed published studies from 2018 to 2021 were included in the study. Forest plots were generated using R (version 4.1.2). Primary comparative analysis shows HBG1/2 had a significantly (p &lt; 0.01)greater impact on induction of HbF expression compared to BCL11A. This analysis leads us to conclude that HBG1/2 merits further investigation as a possible gene editing target for treatment of SCD and β-thalassemia.

IntroductionLesbian, Gay, Bi-sexual, Transgender, Questioning, Intersex, and Asexual (LGBTQIA+) patients report experiences of discrimination within healthcare settings due to a lack of provider knowledge and biases of healthcare workers. There is an identified need among all health professions to provide more culturally competent healthcare for this community. Early interventions during healthcare profession training programs may be effective to address this need. The overall goal of this study was to assess the educational impact of an active learning session that was specifically designed to enhance LGBTQIA+ cultural competency awareness using an interprofessional setting.MethodsThis 2-year study involved students from 16 healthcare professional programs joining virtually to form interprofessional teams. A small group case-based learning approach was used and included pre/post-activity surveys to measure the change in student attitude and confidence, as well as the change in perception of the importance of the activity.ResultsResults indicate an increase in perception of importance (p &lt; 0.005) and in overall level of confidence (p &lt; 0.001) with respect to LGBTQIA+ issues post-session. Key themes established through the session represent an overall recognition of the importance of interprofessional education and awareness of LGBTQIA+ healthcare needs.DiscussionThe results demonstrate the effectiveness of a case-based approach for enhancing cultural competency awareness across different healthcare professions programs. This session also provided an interprofessional learning environment to allow multiple healthcare professions program students to interact and share perspectives. The positive impact of this intervention in a highly collaborative virtual learning environment also highlights that this immersive active learning approach that can be adopted across different programs and institutions.

e21211 Background: Cancer management using cytotoxic drugs is hampered by limited efficacy. Hence, a personalized treatment approach matching chemotherapy with appropriate patients remains a persistent and unmet need in the clinic. Genomic heterogeneity among patients creates an opportunity to discern key genomic aberrations and pathways that confer resistance and response to standard treatment options. Pemetrexed, an antifolate, primarily inhibits thymidylate synthase (TYMS) while platinum compounds (carboplatin/cisplatin) trigger DNA damage similar to alkylating agents. When the DNA damage exceeds repair, apoptosis results. We conducted a pilot study using CBM to novel genomic biomarkers of response and resistance to pemetrexed–platinum treatment. Methods: 25 patients who received pemetrexed–platinum therapy were selected from TCGA data. Mutation and copy number aberrations from each case served as input into the CBM (generated from PubMed and other online resources) to create a patient-specific protein network map. Disease-biomarkers unique to each patient were identified within protein network maps. Drug impact on the disease network was digitally simulated to determine treatment efficacy by measuring effect of chemotherapy on the cell growth score, i.e., a composite of cell proliferation, viability, apoptosis, metastasis, DNA damage and other cancer hallmarks. Effectively, the mechanism of action of each drug was mapped to each patient’s genome and biological consequences due to genomic abnormalities were correlated with response. Results: Among the 25 patients, 23 responders (R) and 2 non-responders (NR). The computer simulation correctly predicted response in 19/25 with 76% accuracy, 100% specificity and 73.91% sensitivity. CBM identified co-occurrence of deleted segments of chromosome 6q, 13q, 14q and 17q were responsible for pemetrexed-platinum response. The key genes governing response on these chromosomes included drug transporters and DNA repair pathways. ATP7B (13q) which exports platinum-based drugs was deleted in responders. The loss of REV3L (6q ), MBD1 (6q ), ERCC1 (6q), BRCA2 (13q), BRCA1 (17q), FANCM (14q), RAD51B (14q), XRCC3 (14q ) led to failure of DNA repair. RB1 (13q) del also led to failure of BRG1 mediated DNA repair . Combinations of these repair genes and transporters formed the major response criteria among the 23 responders while these characteristics were absent in non-responders. Conclusions: This pilot study highlights how CBM simulation platform can identify patients for therapy response prediction. Copy number changes impact responsiveness to chemotherapy and should be routinely assessed. We suggest that this approach should be validated prospectively in a larger patient cohort.

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