Gene expression studies in Arabidopsis in response to South African Cassava Mosaic Virus infection utilizing microarrays

Student Number : 9610284H - MSc dissertation - School of Molecular and Cell Biology - Faculty of ScienceCassava Mosaic Disease is the most devastating disease affecting cassava (Manihot esculenta Crantz) crops worldwide. This disease is associated with eight species of geminiviruses, all belonging to the genus Begomovirus of the family Geminiviridae. In South Africa, in particular, CMD is caused by South African cassava mosaic virus (SACMV). Currently, there are no adequate methods for control of this disease as mechanisms within virus-host interactions are poorly understood. This brings about the need for development of virus-disease control strategies. This study was therefore conducted to identify the host’s response to an invading virus. The model plant, Arabidopsis was chosen as it is a well-characterized plant system, with expression databases readily available as its entire genome has been sequenced. This study was conducted, firstly, to phenotypically determine if Arabidopsis was resistant or susceptible to SACMV infection, and secondly, to identify the host’s response to pathogen infection on a molecular level through gene expression studies utilizing microarrays. Results from the symptomatology study revealed that Arabidopsis plants were fully symptomatic 28 days post-inoculation, displaying characteristic disease symptoms such as stunting, yellowing, and leaf deformation. This indicated that Arabidopsis was susceptible to SACMV infection. Microarray analyses revealed 86 differentially expressed genes, of which 48 showed up-regulation and 38 down-regulation. Relative quantification real-time PCR was performed on selected genes to confirm these results. Many up-regulated genes were shown to be primarily involved in a general stress response induced by the host, whereas those genes that were downregulated seemed to be involved in more specific responses to viral invasion, probably a consequence of suppression of host genes by SACMV to enhance its own replication. The majority of genes identified fell under the predominant functional categories involved in metabolism, transcription, and transport. To our knowledge, this is the first study in which a DNA geminivirus has been used in a host-pathogen interaction utilizing microarrays

A SMART decade: outcomes of an integrated, inclusive, first-year college-level STEM curricular innovation

In the early 2000s, our primarily undergraduate, white institution (PUI/PWI), began recruiting and enrolling higher numbers of students of color and first-generation college students. However, like many of our peer institutions, our established pedagogies and mindsets did not provide these students an educational experience to enable them to persist and thrive in STEM. Realizing the need to systematically address our lack of inclusivity in science majors, in 2012 faculty from multiple disciplines developed the Science, Math, and Research Training (SMART) program. Here, we describe an educational innovation, originally funded by a grant from the Howard Hughes Medical Institute, designed to support and retain students of color, first generation college students, and other students with marginalized identities in the sciences through a cohort-based, integrated, and inclusive first-year experience focused on community and sense of belonging. The SMART program engages first-year students with semester-long themed courses around “real world” problems of antibiotic resistance and viral infections while integrating the fields of Biology, Chemistry, Mathematics, and an optional Computer Science component. In the decade since its inception, 97% of SMART students have graduated or are on track to graduate, with 80.9% of these students earning a major in a STEM discipline. Here, we present additional student outcomes since the initiation of this program, results of the student self-evaluative surveys SALG and CURE, and lessons we have learned from a decade of this educational experience

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Transcriptome profiling in susceptible model and natural host systems in response to South African cassava mosaic virus

Geminiviruses causes diseases to many staple food and cash crops of great economic importance worldwide. Currently eight species of Begomoviruses belonging to the Geminivirus family exist, of which South African cassava mosaic virus SACMV-[ZA:99] is a member, and is known to cause cassava mosaic disease (CMD). Cassava (Manihot esculenta, Crantz) is considered to be an important food crop consumed in many tropical, sub-tropical and African countries, and is increasingly becoming well-known for its ethanol production on a global a scale. Various strategies to control CMD are currently being implemented, one of which is to elucidate mechanisms involved in host-virus interactions with the aim of identifying defence-related genes involved in the disease process. Many defence genes within the plant kingdom are evolutionary conserved, potentially providing methods of control not only to CMD but to other diseases as well. The research outlined in this thesis aimed to identify networks and pathways involved in disease susceptibility between the model plant host system, Arabidopsis thaliana and cassava T200 upon SACMV-[ZA:99] infection. Conclusions were also drawn from within host comparisons between susceptible cassava T200 and resistant cassava TME3 cultivars in order to explore if similarities, differences or common patterns of expression existed between genes governing resistance and susceptibility. Before transcriptomic profiling studies were carried out, it was important to improve South African cassava mosaic virus (SACMV-[ZA:99]) and African cassava mosaic virus (ACMV-[NG:Ogo:90]) infection efficiencies in recalcitrant crop systems such as cassava. Susceptible cassava cultivars T200, TMS60444, and SM14334 were tested for these purposes following infection with three different Agrobacterium strains (C58C1; AGL1; LBA4404). Results demonstrated that an overall increase in infection efficiency was achieved for each genotype and virus tested, although with varying infectivity levels, suggesting that although an improved method was established, basal levels of susceptibility differed between genotypes and therefore it was not possible to achieve 100% infection efficiencies for agroinfection methods. A 4 x 44k microarray whole genome study was then conducted to identify susceptible host genes involved in the interaction between the model plant system Arabidopsis thaliana and SACMV-[ZA:99]. An infectivity assay was carried out across three time points (14, 24, and 36 dpi), confirming that disease symptoms and virus infectivity levels correlated with an increase in differentially expressed transcripts across time points, with SACMV-[ZA:99] predominantly causing host-gene suppression. Many complex genes and pathways were disrupted and were shown to be involved in categories pertaining to stress and defence responses, phytohormone signalling pathways, cellular transport, metabolism and cell-cycle regulation strongly suggesting an attempt made by SACMV-[ZA:99] to affect homeostasis and antagonize host defence responses. This was the first geminivirus study identifying differentially expressed transcripts across 3 time points. Next generation sequencing (NGS) using the ABI Solid platform was then carried out on SACMV-[ZA:99] – infected susceptible cassava T200 cultivar at 3 time points (12, 32, and 67 dpi), comparing infection responses to mock-inoculated healthy controls. Similarly to the Arabidopsis microarray study, findings from this analysis also revealed a shift from up-regulated to down-regulated genes across time points, once again reflecting virus-specific suppression on host genes suggesting SACMV-[ZA:99] specific alterations were induced in the host, regardless of the host (Arabidopsis and cassava T200) or platform (microarray and NGS) used. Genes identified pertaining particularly to the susceptible cassava T200 - SACMV-[ZA:99] interaction such as the disease resistance protein families (TIR-NBS-LRR), RPP1, RPM1, and NHO1 were showing down-regulation demonstrating that SACMV-[ZA:99] pathogenicity proteins may be causing this suppression leading to inactivation of basal immunity. Comparisons between tolerant cassava TME3 and susceptible T200 showed similarities and differences in responses between the cultivars. Many similarities such as cell wall precursor proteins and glutathione-S-transferases were up-regulated in both cultivars, which may be due to the host attempting to mount appropriate defences. Opposite patterns of expression was observed for genes in categories involved in transcription and phytohormone signalling such as WRKY‘s, NAC, JAZ, and ERF where suppression was evident in susceptible cassava T200, confirming the suppressive nature of SACMV-[ZA:99] to establish a replication-competent environment. Findings in this study contributed to the little that is known about geminivirus disease progression within a previously uncharacterised susceptible host such as cassava

Effect of Antiplatelet Therapy on Survival and Organ Support–Free Days in Critically Ill Patients With COVID-19

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