Yeast Two Hybrid Screen of a Putative Toxoplasma gondii Cyclin, TGME49_266900

In this current research, protein-protein interactions with a putative Toxoplasma gondii cyclin, TGME49_266900 or Cyc6, were discovered via a yeast two hybrid screen. Several putative interacting protein partners were isolated and described from a cDNA library of an asynchronous tachyzoite transcriptome. After false positives were weaned from the study, two proteins were identified as Cyc6 interacting partners. These two proteins are described from the toxodb.org bioinformatic database as a DJ-1 family protein (TGME49_214290) and a ThiF protein (TGME49_314890). Interestingly, the interacting DJ-1 protein has been shown in previous research to play a role in T. gondii microneme secretion. Additionally, ThiF proteins share distinct traits with E1 enzymes at the start of the ubiquitin pathway in eukaryotes. After no evidence of an interacting CDK partner for Cyc6 was obtained, a direct experiment was conducted testing for an interaction between Cyc6 and a putative CDK with expression levels notably higher in bradyzoites instead of tachyzoites. The outcome of this experiment showed no interaction between Cyc6 and the putative CDK. Although no interacting CDK partner was evident from this yeast two hybrid screen, two proteins were found to display a strong and biologically relevant interaction with the putative cyclin of interest. Future studies regarding Cyc6 should explore potential noncanonical roles for this putative cyclin in not only tachyzoites, but also bradyzoites and the purpose of Cyc6\u27s interaction with the two proteins discovered from this screen

miRNA-Based Regulation of Alternative RNA Splicing in Metazoans

Alternative RNA splicing is an important regulatory process used by genes to increase their diversity. This process is mainly executed by specific classes of RNA binding proteins that act in a dosage-dependent manner to include or exclude selected exons in the final transcripts. While these processes are tightly regulated in cells and tissues, little is known on how the dosage of these factors is achieved and maintained. Several recent studies have suggested that alternative RNA splicing may be in part modulated by microRNAs (miRNAs), which are short, non-coding RNAs (~22 nt in length) that inhibit translation of specific mRNA transcripts. As evidenced in tissues and in diseases, such as cancer and neurological disorders, the dysregulation of miRNA pathways disrupts downstream alternative RNA splicing events by altering the dosage of splicing factors involved in RNA splicing. This attractive model suggests that miRNAs can not only influence the dosage of gene expression at the post-transcriptional level but also indirectly interfere in pre-mRNA splicing at the co-transcriptional level. The purpose of this review is to compile and analyze recent studies on miRNAs modulating alternative RNA splicing factors, and how these events contribute to transcript rearrangements in tissue development and disease

Identification and Prioritization of Canadian Society of Nephrology Clinical Practice Guideline Topics With Multidisciplinary Stakeholders and People Living With Kidney Disease: A Clinical Research Protocol

Background: Despite efforts to provide evidence-based care for people living with kidney disease, health care provider goals and priorities are often misaligned with those of individuals with lived experience of disease. Coupled with competing interests of time, resources, and an abundance of suitable guideline topics, identifying and prioritizing areas of focus for the Canadian nephrology community with a patient-oriented perspective is necessary and important. Similar priority-setting exercises have been undertaken to establish research priorities for kidney disease and to standardize outcomes for kidney disease research and clinical care; however, research priorities are distinct from priorities for guideline development. Inclusion of people living with health conditions in the selection and prioritization of guideline topics is suggested by patient engagement frameworks, though the process to operationalizing this is variable. We propose that the Canadian Society of Nephrology Clinical Practice Guideline Committee (CSN CPGC) takes the opportunity at this juncture to incorporate evidence-based prioritization exercises with involvement of people living with kidney disease and their caregivers to inform future guideline activities. In this protocol, we describe our planned research methods to address this. Objective: To establish consensus-based guideline topic priorities for the CSN CPGC using a modified Delphi survey with involvement of multidisciplinary stakeholders, including people living with kidney disease and their caregivers. Study design: Protocol for a Modified Delphi Survey. Setting: Pilot-tested surveys will be distributed via email and conducted using the online platform SurveyMonkey, in both French and English. Participants: We will establish a group of multidisciplinary clinical and research stakeholders (both within and outside CSN membership) from Canada, in addition to people living with kidney disease and/or their caregivers. Methods: A comprehensive literature search will be conducted to generate an initial list of guideline topics, which will be organized into three main categories: (1) International nephrology-focused guidelines that may require Canadian commentary, (2) Non-nephrology specific guidelines from Canada that may require CSN commentary, and (3) Novel topics for guideline development. Participants will engage in a multi-round Modified Delphi Survey to prioritize a set of “important guideline topics.” Measures: Consensus will be reached for an item based on both median score on the Likert-type scale (≥ 7) and the percentage agreement (≥ 75%); the Delphi process will be complete when consensus is reached on each item. Guideline topics will then be given a priority score calculated from the total Likert ratings across participants, adjusted for the number of participants. Limitations: Potential limitations include participant response rates and compliance to survey completion. Conclusions: We propose to incorporate evidence-based prioritization exercises with the engagement of people living with kidney disease and their caregivers to establish consensus-based guideline topics and inform future guidelines activities of the CSN CPGC

Obesity, inflammatory and thrombotic markers, and major clinical outcomes in critically ill patients with COVID‐19 in the US

Objective This study aimed to determine whether obesity is independently associated with major adverse clinical outcomes and inflammatory and thrombotic markers in critically ill patients with COVID‐19. Methods The primary outcome was in‐hospital mortality in adults with COVID‐19 admitted to intensive care units across the US. Secondary outcomes were acute respiratory distress syndrome (ARDS), acute kidney injury requiring renal replacement therapy (AKI‐RRT), thrombotic events, and seven blood markers of inflammation and thrombosis. Unadjusted and multivariable‐adjusted models were used. Results Among the 4,908 study patients, mean (SD) age was 60.9 (14.7) years, 3,095 (62.8%) were male, and 2,552 (52.0%) had obesity. In multivariable models, BMI was not associated with mortality. Higher BMI beginning at 25 kg/m2 was associated with a greater risk of ARDS and AKI‐RRT but not thrombosis. There was no clinically significant association between BMI and inflammatory or thrombotic markers. Conclusions In critically ill patients with COVID‐19, higher BMI was not associated with death or thrombotic events but was associated with a greater risk of ARDS and AKI‐RRT. The lack of an association between BMI and circulating biomarkers calls into question the paradigm that obesity contributes to poor outcomes in critically ill patients with COVID‐19 by upregulating systemic inflammatory and prothrombotic pathways