Biology subjects

Saleem, A.

Publications and source records attributed to Saleem, A..

25 records · Page 2Linked to original sources

bioRxiv2022

Profiling skeletal muscle-derived secretome with differentiation and acute contractile activity

Extracellular vesicles (EVs) released from all cells, are essential to cellular communication, and contain biomolecular cargo that can affect recipient cell function. Studies on the effects of contractile activity (exercise) on EVs usually rely on plasma/serum-based assessments, which contain EVs from many different cells. To specifically characterize skeletal muscle-derived vesicles and the effect of acute contractile activity, we used an in vitro model where C2C12 mouse myoblasts were differentiated to form myotubes. EVs were isolated from conditioned media from muscle cells, pre-differentiation (myoblasts) and post-differentiation (myotubes), as well as from acutely stimulated myotubes (1hr @ 14V, C-Pace EM, IonOptix) using total exosome isolation reagent (TEI, ThermoFisher, referred to as extracellular particles [EPs]) and differential ultracentrifugation (dUC; EVs). Myotube-EPs (~98 nm) were 41% smaller than myoblast-EPs (~167 nm, p<0.001, N=8-10). Two-way ANOVA showed a significant main effect for size distribution of myotube vs. myoblast-EPs (p<0.01, N=10-13). Myoblast-EPs displayed a bimodal size distribution profile with peaks at <200 nm and 400-600 nm, compared to myotube-EPs that were largely 50-300 nm in size. Total protein yield from myotube-EPs was nearly 15-fold higher than myoblast-EPs, (p<0.001 N=6-9). Similar biophysical characteristics were observed when EVs were isolated using dUC: myotube-EVs (~195 nm) remained 41% smaller in average size than myoblast-EVs (~330 nm, p=0.07, N=4-6) and had comparable size distribution profiles as EPs isolated via TEI. Myotube-EVs also had 4.7-fold higher protein yield vs. myoblast EVs (p<0.05, N=4-6). Myotube-EPs had significantly decreased expression of exosomal marker proteins TSG101, CD63, ALIX and CD81 compared to myoblast-EPs (p<0.05, N=7-12). Conversely, microvesicle marker ARF6, and lipoprotein marker APO-A1was only found in the myotube-EPs (p<0.05, N=4-12). There was no effect of acute stimulation on myotube-EP biophysical characteristics (N=7), nor on expression of TSG101, ARF6 or CD81 (N=5-6). Myoblasts treated with control or acute stimulation-derived EPs (13 g/well) for 48hrs and 72hrs showed no changes in mitochondrial mass (MitoTracker Red), cell viability or cell count (N=3-4). Myoblasts treated with EP-depleted media (72hrs) had ~90% lower cell counts (p<0.01, N=3). Our data show that EVs differ in size, distribution, protein yield and expression of subtype markers pre- vs. post-skeletal muscle differentiation. There was no effect of acute stimulation on biophysical profile or protein markers in EPs. Acute stimulation-derived EPs did not alter mitochondrial mass nor cell count/viability. Further investigation into the effects of chronic contractile activity on the biophysical characteristics and cargo of skeletal muscle-specific EVs are warranted.

physiology↗

bioRxiv2022

Novel Association of Lyme Disease, Age, and Atopic Dermatitis

Borrelia burgdorferi (B. burgdorferi) is a bacterial spirochete that can cause Lyme disease after infecting a susceptible host. Immune responses to the bacteria are highly variable and host specific. The murine substrain, C3H/HeJ, is a frequently utilized mouse model of Lyme disease. In this study, we sought to investigate the correlation of age with onset and severity of dermatitis, both in mice infected with B. burgdorferi as well as humans who have had a diagnosis of Lyme disease. Female C3H/HeJ mice aged 6-8 weeks, 1 year, or 2 years were infected intraperitoneally with 105 B. burgdorferi. Dermatitis of the tail was evaluated by gross examination and histology. Additional female C3H/HeJ and C57BL/6J mice aged 5 weeks were injected intradermally with 105 B. burgdorferi containing the luciferase reporter gene then analyzed under in vivo imaging. Human data via electronic health records of 342,499 Finnish individuals was tested and analyzed for associations between Lyme disease and atopic dermatitis. Dermatitis worsened over the course of untreated infection, with ulceration, hemorrhaging, flaking, hair loss, and dark lesions as well as spongiosis and acanthosis. These features of dermatitis were present in infected mice after 1 year of age. We further confirm the presence of B. burgdorferi in the tail through quantification of bioluminescence and immunohistochemistry of both C3H/HeJ and C57BL/6J mice. This relationship among Lyme disease, atopic dermatitis, and host age seen in the mouse model is consistent with a large pool (342,499) of human epidemiological data from Finland. We identified 5,248 individuals with Lyme disease and 17,233 with atopic dermatitis in FinnGen. Retrospective analysis shows Lyme disease is associated with atopic dermatitis (OR = 1.91 [1.68 -2.37], P < 2e-16). More visits due to Lyme disease complications (3 or more visits versus 1 visit) were associated with atopic dermatitis (OR = 2.19 [1.35-3.55], P = 0.0014) and risk of developing atopic dermatitis over time (HR=2.26 [1.54-3.95], P = 0.0017). Data from mice and humans reveal a novel relationship among Lyme disease, age, and atopic dermatitis. Through defined pathological scoring, we demonstrate the onset of murine atopic dermatitis with B. burgdorferi infection, which is further exacerbated by host age at time of infection. In humans, a diagnosis of Lyme disease in FinnGen was associated with atopic dermatitis and further research is warranted to establish causation.

immunology↗

bioRxiv2020

Extracellular vesicles as predictors of individual response to exercise training in youth living with obesity

Exercise is associated with various health benefits, including the prevention and management of obesity and cardiometabolic risk factors. However, a strong heterogeneity in the adaptive response to exercise training exists. The objective of this study was to evaluate if changes in extracellular vesicles (EVs) after acute aerobic exercise (AE) were associated with the responder phenotype following 6-weeks of resistance exercise training. This is a secondary analysis of plasma samples from the EXIT trial (clinical trial #02204670). Eleven sedentary youth with obesity (15.7{+/-}0.5 years, BMI [≥] 95th percentile) underwent an acute bout of AE (60% heart rate reserve, 45 min). Blood was collected before exercise [at time (AT) 0 min], during [AT15, 30, 45 min], and 75 min after exercise [AT120]. Afterward, youth participated in 6-week resistance training program, and were categorized into responders (RE) or non-responders (NRE) based on changes in insulin sensitivity as measured by the Matsuda Index. EVs were isolated using size exclusion chromatography (Izon(R)). The primary outcome variable was EV biophysical profile, which includes size, zeta potential, protein yield and expression of markers associated with EV subtypes. The variables were analyzed in a single-blind fashion. Overall, there was a general increase in EV production in both groups. Average EV size was larger in RE (~147 nm) vs. NRE (~124 nm; p<0.05). Average EV size at AT0 was associated with absolute change in Matsuda index following 6-weeks of resistance training (r=0.44, p=0.08). EV size distribution revealed RE preferentially expressed EVs between 150 - 250 nm in size, whereas NRE expressed EVs between 50 - 100 nm (p<0.05). At baseline, RE-EVs contained ~25% lower Tsg101 protein, ~85% higher MMP2 content, while CD63 levels remained unchanged between the groups. Total protein yield in RE-EVs was higher than NRE at AT15 (p<0.05). Our data suggest that youth with obesity that respond to exercise training produce larger EVs, with lower exosome- and higher microvesicle-specific protein expression. RE-EVs also had higher EV protein yield during AE. The relationship between larger EV subtypes and/or cargo, and the individual response to exercise has yet to be fully elucidated.

physiology↗

bioRxiv2020

Modeling Genetic Epileptic Encephalopathies using Brain Organoids

Epileptic encephalopathies (EEs) are a group of disorders associated with intractable seizures, brain development and functional abnormalities, and in some cases, premature death. Pathogenic human germline biallelic mutations in tumor suppressor WW domain-containing oxidoreductase (WWOX) are associated with a relatively mild autosomal-recessive spinocerebellar ataxia-12 (SCAR12) and a more severe early infantile WWOX-related epileptic encephalopathy (WOREE). In this study, we generated an in-vitro model for EEs, using the devastating WOREE syndrome as a prototype, by establishing brain organoids from CRISPR-engineered human ES cells and from patient-derived iPSCs. Using these models, we discovered dramatic cellular and molecular CNS abnormalities, including neural population changes, cortical differentiation malfunctions, and Wnt-pathway and DNA-damage response impairment. Furthermore, we provide a proof-of-concept that ectopic WWOX expression could potentially rescue these phenotypes. Our findings underscore the utility of modeling childhood epileptic encephalopathies using brain organoids and their use as a unique platform to test possible therapeutic intervention strategies.

neuroscience↗

bioRxiv2020

Hierarchical design of multi-scale protein complexes by combinatorial assembly of oligomeric helical bundle and repeat protein building blocks

A goal of de novo protein design is to develop a systematic and robust approach to generating complex nanomaterials from stable building blocks. Due to their structural regularity and simplicity, a wide range of monomeric repeat proteins and oligomeric helical bundle structures have been designed and characterized. Here we describe a stepwise hierarchical approach to building up multi-component symmetric protein assemblies using these structures. We first connect designed helical repeat proteins (DHRs) to designed helical bundle proteins (HBs) to generate a large library of heterodimeric and homooligomeric building blocks; the latter have cyclic symmetries ranging from C2 to C6. All of the building blocks have repeat proteins with accessible termini, which we take advantage of in a second round of architecture guided rigid helical fusion (WORMS) to generate larger symmetric assemblies including C3 and C5 cyclic and D2 dihedral rings, a tetrahedral cage, and a 120 subunit icosahedral cage. Characterization of the structures by small angle x-ray scattering, x-ray crystallography, and cryo-electron microscopy demonstrates that the hierarchical design approach can accurately and robustly generate a wide range of macromolecular assemblies; with a diameter of 43nm, the icosahedral nanocage is the largest structurally validated designed cage to date. The computational methods and building block sets described here provide a very general route to new de novo designed symmetric protein nanomaterials.

biochemistry↗

bioRxiv2019

Cell cycle regulation of mitochondrial protein import revealed by genome-scale pooled bimolecular fluorescence complementation screening

A central focus of systems biology is the functional mapping of protein-protein interactions under physiological conditions. Here we describe MaGiCaL-BiFC, a lentivirus-based bimolecular fluorescence protein-fragment complementation approach for the high-throughput, genome-scale identification of protein-protein interactions in mammalian cells. After developing and validating this methodology using known protein-protein interaction pairs, we constructed genome-scale pooled BiFC libraries using the human ORFeome cDNA collection. These pooled libraries, containing [~] 12,000 unique human cDNAs, were used to screen for candidate interaction partners of the mitochondrial transmembrane protein TOMM22. Following infection of cells with the TOMM22 bait and the pooled cDNA libraries, cells harboring candidate TOMM22 interacting proteins were isolated from the cell pool via fluorescence activated cell sorting, and identified via microarray analysis. This approach identified several known interaction partners of TOMM22, as well as novel physical and functional partners that link the mitochondrial network to proteins involved in diverse cellular processes. Notably, protein kinase CK2 was identified as a novel physical interaction partner of human TOMM22. We found that this association occurs preferentially during mitosis and involves direct phosphorylation of TOMM22, an event that may lead to attenuation of mitochondrial protein import. Together, this data contributes to the growing body of evidence suggesting eloquent coordination between cell cycle progression and mitochondrial physiology. Importantly, through high-throughput screening and focused validation, our study demonstrates the power of the MaGiCaL-BiFC approach to uncover novel functional protein-protein interactions, including those involving proteins with membrane-spanning domains, or of a transient nature, all within their native cellular environment.

systems biology↗

bioRxiv2019

Knowledge and awareness of cervical cancer in Southwestern Ethiopia is lacking: a descriptive analysis

PurposeCervical cancer remains the second most common cancer and cancer-related death among women in Ethiopia. This is the first study, to our knowledge, describing the demographic, and clinicopathologic characteristics of cervical cancer cases in a mainly rural, Southwestern Ethiopian population with a low literacy rate to provide data on the cervical cancer burden and help guide future prevention and intervention efforts.\n\nMethodsA descriptive analysis of 154 cervical cancer cases at the Jimma University Teaching Hospital in Southwestern Ethiopia from January 2008 - December 2010 was performed. Demographic and clinical characteristics were obtained from patient questionnaires and cervical punch biopsies were histologically examined.\n\nResultsOf the 154 participants with a histopathologic diagnosis of cervical cancer, 95.36% had not heard of cervical cancer and 89.6% were locally advanced at the time of diagnosis. Moreover, 86.4% of participants were illiterate, and 62% lived in a rural area.\n\nConclusionA majority of the 154 women with cervical cancer studied at the Jimma University Teaching Hospital in Southwestern Ethiopia were illiterate, had not heard of cervical cancer and had advanced disease at the time of diagnosis. Given the low rates of literacy and knowledge regarding cervical cancer in this population which has been shown to correlate with a decreased odds of undergoing screening, future interventions to address the cervical cancer burden here must include an effective educational component.

epidemiology↗