ADP-dependent Conformational Changes Distinguish Mycobacterium tuberculosis SecA2 from SecA1

In bacteria, most secreted proteins are exported through the SecYEG translocon by the SecA ATPase motor via the general secretion or “Sec” pathway. The identification of an additional SecA protein, particularly in Gram-positive pathogens, has raised important questions about the role of SecA2 in both protein export and establishment of virulence. We previously showed in Mycobacterium tuberculosis, the causative agent of tuberculosis, the accessory SecA2 protein possesses ATPase activity that is required for bacterial survival in host macrophages, highlighting its importance in virulence. Here, we show that SecA2 binds ADP with much higher affinity than SecA1 and releases the nucleotide more slowly. Nucleotide binding also regulates movement of the precursor-binding domain in SecA2, unlike in SecA1 or conventional SecA proteins. This conformational change involving closure of the clamp in SecA2 may provide a mechanism for the cell to direct protein export through the conventional SecA1 pathway under normal growth conditions while preventing ordinary precursor proteins from interacting with the specialized SecA2 ATPase.     

Differences in metabolomic profiles of male db/db and s/s, leptin receptor mutant mice

Leptin, a protein hormone secreted by adipose tissue, plays an important role in regulating energy metabolism and the immune response. Despite similar extremes of adiposity, mutant mouse models, db/db, carrying spontaneous deletion of the active form of the leptin receptor (LEPR-B) intracellular signaling domain, and the s/s, carrying a specific point mutation leading to a dysfunctional LEPR-B-STAT3 signaling pathway, have been shown to have robust differences in glucose homeostasis. This suggests specific effects of leptin, mediated by non-STAT3 LEPR-B pathways. Differences in the LEPR-B signaling pathways in these two LEPR-B mutant mice models are expected to lead to differences in metabolism. In the current study, the hypothesized differences in metabolism were investigated using the metabolomics approach. Proton nuclear magnetic resonance spectroscopy ((1)HNMR) was conducted on 24 h urine samples in deuterium oxide using a 500 MHz instrument at 25°C. Principle Component Analysis showed clear separation of urine NMR spectra between the groups (P < 0.05). The CHENOMX metabolite database was used to identify several metabolites that differed between the two mouse models. Significant differences (P < 0.05) in metabolites associated with the glycine, serine, and homocysteine metabolism were observed. The results demonstrate that the metabolomic profile of db/db and s/s mice are fundamentally different and provide insight into the unique metabolic effects of leptin exerted through non-STAT3 LEPR-B pathways.     

Illuminating and mitigating the evolving impacts of COVID-19 on ethnocultural communities: a participatory action mixed-methods study

BACKGROUND:The COVID-19 pandemic has exacerbated disparities in poverty and illness for people in vulnerable circumstances in ethnocultural communities. We sought to understand the evolving impacts of COVID-19 on ethnocultural communities to inform intersectoral advocacy and community action.METHODS:The Illuminate Project used participatory action research, with cultural health brokers as peer researchers, from Sept. 21 to Dec. 31, 2020, in Edmonton, Alberta. Twenty-one peer researchers collected narratives from members of ethnocultural communities and self-interpreted them as they entered the narratives into the SenseMaker platform, a mixed-method data collection tool. The entire research team analyzed real-time, aggregate, quantitative and qualitative data to identify emerging thematic domains, then visualized these domains with social network analysis.RESULTS:Brokers serving diverse communities collected 773 narratives. Identified domains illuminate the evolving and entangled impacts of COVID-19 including the following: COVID-19 prevention and management; care of acute, chronic and serious illnesses other than COVID-19; maternal care; mental health and triggers of past trauma; financial insecurity; impact on children and youth and seniors; and legal concerns. We identified that community social capital and cultural brokering are key assets that facilitate access to formal health and social system supports.INTERPRETATION:The Illuminate Project has illustrated the entangled, systemic issues that result in poor health among vulnerable members of ethnocultural communities, and the exacerbating effects of COVID-19, which also increased barriers to mitigation. Cultural brokering and community social capital are key supports for people during the COVID-19 pandemic. These findings can inform policy to reduce harm and support community resiliency.

Mahatma Gandhi observed that “the true measure of any society can be found in how it treats its most vulnerable members.” Ethnocultural communities, defined by their unique shared characteristics (e.g., cultural traditions, language, country of origin),¹ face greater challenges and have higher rates of poverty and illness than the general Canadian population. Migration results in conditions that affect all social determinants of health and disproportionally affect health outcomes, herein referred to as vulnerable circumstances.^2,3 The emergence of major outbreaks of SARS-CoV-2 infections in ethnocultural communities highlights both the vulnerable circumstances of these communities and the disparities they face in accessing high-quality, culturally appropriate information and support.^4–7 Studies have shown substantial variation in deaths attributed to COVID-19 based on factors such as age, sex, ethnicity, length of time in Canada, income and education.^8–11 However, given the well-known gap in reporting comprehensive COVID-19 data in relation to race and ethnicity, efforts to measure its impact are hampered.^8–12 There is an urgent need to understand the evolving challenges of COVID-19 to inform action and public policy that can mitigate these challenges.To understand evolving situations of complexity and crisis, sensemaking, defined as “a continuous process to establish situational awareness,”¹³ is a crucial undertaking.¹⁴ Using participatory action research,^15–18 we sought to understand the evolving impacts of COVID-19 on ethnocultural communities to inform broader national efforts to migitate the impacts of COVID-19. Particularly, we sought to understand how the challenges of COVID-19 are entangled with contextual factors at multiple levels, how families and communities are leveraging strengths and social capital to adapt, and the role of cultural brokers in managing the crisis.     

Microfluidic models of the human circulatory system: versatile platforms for exploring mechanobiology and disease modeling

The human circulatory system is a marvelous fluidic system, which is very sensitive to biophysical and biochemical cues. The current animal and cell culture models do not recapitulate the functional properties of the human circulatory system, limiting our ability to fully understand the complex biological processes underlying the dysfunction of this multifaceted system. In this review, we discuss the unique ability of microfluidic systems to recapitulate the biophysical, biochemical, and functional properties of the human circulatory system. We also describe the remarkable capacity of microfluidic technologies for exploring the complex mechanobiology of the cardiovascular system, mechanistic studying of cardiovascular diseases, and screening cardiovascular drugs with the additional benefit of reducing the need for animal models. We also discuss opportunities for further advancement in this exciting field.     

Carnitine is a pharmacological allosteric chaperone of the human lysosomal α-glucosidase

Pompe disease is an inherited metabolic disorder due to the deficiency of the lysosomal acid α-glucosidase (GAA). The only approved treatment is enzyme replacement therapy with the recombinant enzyme (rhGAA). Further approaches like pharmacological chaperone therapy, based on the stabilising effect induced by small molecules on the target enzyme, could be a promising strategy. However, most known chaperones could be limited by their potential inhibitory effects on patient’s enzymes. Here we report on the discovery of novel chaperones for rhGAA, L- and D-carnitine, and the related compound acetyl-D-carnitine. These drugs stabilise the enzyme at pH and temperature without inhibiting the activity and acted synergistically with active-site directed pharmacological chaperones. Remarkably, they enhanced by 4-fold the acid α-glucosidase activity in fibroblasts from three Pompe patients with added rhGAA. This synergistic effect of L-carnitine and rhGAA has the potential to be translated into improved therapeutic efficacy of ERT in Pompe disease.     

Exosome-mediated mRNA delivery in vivo is safe and can be used to induce SARS-CoV-2 immunity

Functional delivery of mRNA has high clinical potential. Previous studies established that mRNAs can be delivered to cells in vitro and in vivo via RNA-loaded lipid nanoparticles (LNPs). Here we describe an alternative approach using exosomes, the only biologically normal nanovesicle. In contrast to LNPs, which elicited pronounced cellular toxicity, exosomes had no adverse effects in vitro or in vivo at any dose tested. Moreover, mRNA-loaded exosomes were characterized by efficient mRNA encapsulation (∼90%), high mRNA content, consistent size, and a polydispersity index under 0.2. Using an mRNA encoding the red light-emitting luciferase Antares2, we observed that mRNA-loaded exosomes were superior to mRNA-loaded LNPs at delivering functional mRNA into human cells in vitro. Injection of Antares2 mRNA-loaded exosomes also led to strong light emission following injection into the vitreous fluid of the eye or into the tissue of skeletal muscle in mice. Furthermore, we show that repeated injection of Antares2 mRNA-loaded exosomes drove sustained luciferase expression across six injections spanning at least 10 weeks, without evidence of signal attenuation or adverse injection site responses. Consistent with these findings, we observed that exosomes loaded with mRNAs encoding immunogenic forms of the SARS-CoV-2 Spike and Nucleocapsid proteins induced long-lasting cellular and humoral responses to both. Taken together, these results demonstrate that exosomes can be used to deliver functional mRNA to and into cells in vivo.     

Polycyclic Aromatic Hydrocarbons (PAHs) in Surface Sediments of Lake Manzala,Egypt

This investigation represents the first extensive study of the spatial distribution, sources, and potential effects of polycyclic aromatic hydrocarbons (PAHs) in sediments from Lake Manzala, the largest of Egypt's Mediterranean coastal lakes. The concentrations of PAHs (Σ39 components) ranged from 246 to 9910 ng g^?1 dry wt., the highest values corresponding to urban hotspots with high anthropogenic input coming from wastewater discharges and combustion activities and decreasing offshore. The levels of PAHs were significantly lower compared to values reported in several coastal/estuarine areas (e.g., in Spain, Italy, USA, and Egypt) receiving substantial anthropogenic inputs from urban and industrial activities. Source ratios indicated that the PAHs were mainly from petrogenic sources in near-shore urban hotspots, with higher contributions of pyrolytic sources in coastal and offshore areas which are little influenced by human activities. Sediment quality guidelines (SQGs) showed that except at one station heavily impacted by sewage discharge, the total and individual PAH concentrations were below effect range low (ERL) concentrations that are not likely to adversely affect benthic biota.     

Development of stability indicating HPLC method for the separation and validation of enantiomers of miconazole

A selective and sensitive stability indicting HPLC method was developed for the analysis of enantiomers of miconazole. For this purpose, six different polysaccharide‐based chiral columns were evaluated. Optimization was performed using several polar organic and alcohol‐hydrocarbon mobile phases. As a result of optimization studies, the analysis was carried out using Lux Cellulose‐3, methanol as a mobile phase at a flow rate of 1 mL·min^?1, and the detection wavelength was arranged to 230 nm. Developed method has been fully validated according to International Council on Harmonization guidelines. Method was found linear in the concentration range of 1 to 200 μg·mL^?1. Coefficient of determination (R²) was calculated as 0.9996, intraday precision of the method was found with the RSD% of 0.56, and the recovery of the method was calculated close to 100%. Furthermore, some other validation parameters like specificity, selectivity, LOD, and LOQ were also investigated. Stability indicating capability of this method was shown by forced degradation studies, and the run time for each analysis was less than 6 minutes. As a result, simple, fast, reliable HPLC method was developed for the separation and determination of the enantiomers of miconazole. Applicability of the developed method was shown with the application of marketed pharmaceutical preparations.     

Phenotypic and functional characterization of mesenchymal stromal cells isolated from pediatric patients with severe idiopathic nephrotic syndrome

Background

Idiopathic nephrotic syndrome (INS) is one of the most common renal diseases in the pediatric population; considering the role of the immune system in its pathogenesis, corticosteroids are used as first-line immunosuppressive treatment. Due to its chronic nature and tendency to relapse, a significant proportion of children experience co-morbidity due to prolonged exposure to corticosteroids and concomitant immunosuppression with second-line, steroid-sparing agents. Mesenchymal stromal cells (MSCs) are multipotent cells that represent a key component of the bone marrow (BM) microenvironment; given their unique immunoregulatory properties, their clinical use may be exploited as an alternative therapeutic approach in INS treatment.