Category: Nevin Manimala

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R2228.

ABSTRACT

Fat embolism induced in rats by intravenous injection of triolein(T) causes pulmonary inflammation, fibrosis, and vasculitis after 48 hours ort 10 weeks. Administration of Aliskiren, Captopril and Losartan drugs, which interfere with the renin angiotensin aldosterone system, ameliorates the pulmonary damage. Macrophages and mast cell number decreased with drugs administration. Hearts do not exhibit inflammation or vasculitis although significant emboli are detected at 48 hours. Pulmonary expression of renin, prorenin(R/P) positive cells are also increased after T injection. This study investigates whether the presence of R/P cells correlates with macrophages and mast cell invasion in the hearts and lungs.

METHODOLOGY: 24 Sprague Dawley rats were divided into four groups and treated with 0.2 ml of T or Saline. Lungs and hearts from 3 animals from each group that were sacrificed 48 hours or 10 weeks, fixed in formalin and stained with Hematoxylin-Eosin, Trichrome or immunostained with antibodies to CD117, CD68 to identify mast cells and macrophages. Sections of the heart and the lung were evaluated at 400X magnification. Cells were counted, histopathology examined and statistical significance evaluated using GraphPad software.

RESULTS: While the lungs showed the previously reported, histopathological damage caused by the inflammation, vasculitis, fibrosis and cell necrosis, the hearts did not show such damage, thus confirming previous observations. Contrary to the lungs where the number of CD117, CD68 and R/P positive cells was significantly increased at both interval times, no difference in CD117, CD68, R/P positive cell numbers were observed in the heart. In the lungs, two distinct types of macrophages were identified, one large and the second one of normal size. Such histopathological changes were not observed in the hearts. Hearts exhibited distinct locations of CD117 and CD68 positive cells, with multiple cells located primarily near the myocardium and few in the adventitia of the coronary arteries, in contrast to lungs where the cells were mostly located around the arteries.

CONCLUSION: The study confirms the previously reported histopathological difference of the T induced damage in the lungs compared to that of the hearts in this model of fat embolization. This finding highlights the variation in cellular response of diverse organs to fat embolism. This difference is not only related to the number of the inflammatory cells but also to their location in different organs as evidenced by the difference in the distribution and location of R/P cells in hearts and lungs. Future studies will investigate the underlying mechanisms that lead to pulmonary susceptibility while preserving cardiac integrity in response to a FE induced inflammatory response in order to better design a therapeutic intervention for the fat embolism syndrome.

PMID:35557437 | DOI:10.1096/fasebj.2022.36.S1.R2228

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.L6410.

ABSTRACT

BACKGROUND: Pesticides are important biological agents that deter or kill pests. Globally, about 2 million tons of pesticides are being utilized each year which highlights their importance. Previous studies have suggested that high levels of pesticide exposure are associated with increased risk of chronic diseases, including cancers, cardiotoxicity, Parkinson’s disease, diabetes mellitus, neurological deficits, birth defects, and reproductive disorders. Prometryn is a commonly used selective triazine herbicide, which is currently used as an alternative to atrazine-based products. It is widely applied to agriculture as it has lower acute toxicity than atrazine herbicides, stable chemical properties, and a long period of effectiveness. Although past studies have shown that triazine herbicides have carcinogenic potential in humans, the cytotoxic effects of prometryn on cardiac and hepatic systems are not well investigated or understood. The aim of this study was to determine the effects of prometryn on the heart and liver.

METHODS: H9c2 cardiac and HepG2 liver carcinoma cells were used to study the in-vitro effects of prometryn. Cells were treated with varying concentrations of prometryn for 24 h. Cell viability, mitochondrial membrane potential, and reactive oxygen species (ROS) levels were assessed. For animal studies, male mice (10 weeks old) (n=24) were randomized to two treatment groups (control and prometryn). The control group received corn oil while the prometryn treated group were exposed to 185mg/kg b.w. prometryn dissolved in corn oil every 48 hours for 7 days. After a total of 4 doses, mice were euthanized, and the heart and liver tissues were collected. Proteasome and immunoproteasome activity were measured in the heart and liver. Protein expression levels of markers for oxidative stress, apoptosis, and proteasomal degradation such as GSTT2, GSTA1/2, Hsp70, Hsp27, NADPH oxidase, PARP-1, p53, PSMD12, LMP2, DNP, VU-1 were detected using immunoblotting. Protein oxidation and polyubiquitination were detected as an indicator of oxidative stress. A paired t-test was used to assess group differences for protein levels and p values <0.05 were deemed statistically significant.

RESULT: We found that prometryn had a concentration-dependent effects on H9c2 and HepG2 cell lines when we assessed cell viability, mitochondria function, and total intracellular reactive oxygen species formation. Increasing the concentration of prometryn increased the ROS formation, decreased cell viability and decreased mitochondrial membrane potential in these cell lines. Furthermore, in animal studies we observed changes in the proteasome and immunoproteasome activities in the liver but not in the heart. Changes in protein expression levels of oxidative stress and proteasomal degradation markers were only detected in the liver.

CONCLUSION: These results indicate that H9c2 and HepG2 cells are sensitive to prometryn in-vitro. However, prometryn effects on the heart and liver tissue in male mice is significantly different. Overall, our data supports the deduction that prometryn affected mitochondrial function, induced oxidative stress in cells, but also alters the ubiquitin-proteasome system and increases oxidative stress in mice liver. These results help towards elucidation of the mechanism by which prometryn could cause diseases.

PMID:35557455 | DOI:10.1096/fasebj.2022.36.S1.L6410

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R3602.

ABSTRACT

OBJECTIVE: The goal of this study was to analyze possible associations between fibroblast growth factors (FGFs) and hemorrhagic conversion (HC) in patients with acute ischemic stroke that underwent mechanical thrombectomy (MT).

BACKGROUND: Emergent Large Vessel Occlusion (ELVO), often treated with MT, can lead to further complications such as HC. Little is known about predicting the risk and extent of HC. FGF-21 and 23 are angiogenic proteins of the endocrine subfamily and are independent factors for overall risk of stroke and intracranial hemorrhage (PMID: 33967812). Comorbidities such as diabetes, obesity, hypertension, and hypercholesterolemia are also known to increase risk of HC and affect FGF levels.

METHODS: The Center for Advanced Translational Stroke Science at the University of Kentucky utilizes the Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (www.

CLINICALTRIALS: gov NCT03153683) for collection of systemic (i.e. proximal to thrombus) arterial blood during a MT procedure. Blood samples collected from 61 patients were sent to Olink Proteomics for proteomic analysis. The European Cooperative Acute Stroke Study (ECASS II) grading system was used to differentiate between the types of HT: Hemorrhagic Infarction (HI 1 and 2) and Parenchymal Hemorrhagic Infarction (PH 1 and 2). Logistic regressions were used to determine the association of FGF with the subtypes of hemorrhagic conversion. Data analyses were performed using IBM SPSS Statistics.

RESULTS: FGF-21 and FGF-23 were significantly higher in patients with HI versus PH-type HC (p=0.043 and p=0.004 respectively). Logistic regression yielded a significant model for only FGF-21 (χ² = 5.196, p=0.023). Increasing levels of FGF-21 were associated with a decreased likelihood of having PH-type HC. Most patients receiving TPA had decreased levels of FGF-21 (p=0.04). Various comorbidities were tested but were not found to be significant.

DISCUSSION: The data provides an indication that lower levels of FGF-21 increase the odds for a worse type of hemorrhagic transformation. Lower levels of FGF-21 are reported in patients with hypercholesterolemia and hypertension. Our study found TPA correlated with lower levels of FGF-21. Taken together, patients with comorbidities that decrease FGF-21 levels, such as hypertension and hypercholesterolemia, and those who receive TPA should be monitored more carefully following thrombectomy. FGF-21 should be evaluated further as a potential biomarker for hemorrhagic transformation to administer adjunctive therapies to MT.

PMID:35557434 | DOI:10.1096/fasebj.2022.36.S1.R3602

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R6006.

ABSTRACT

BACKGROUND: Animal studies suggest that maternal obesity and high fat diet during pregnancy program physical activity (PA) patterns in the offspring. However, such nexus has not been established in humans. The purpose of this study was to evaluate the association of maternal adiposity measured early in pregnancy with offspring PA at age 2 years while taking into consideration confounding and modifying factors.

METHODS: Women (n=152) were enrolled early in pregnancy (<10 weeks). At enrollment, maternal adiposity [air displacement plethysmography, fat mass index (FMI, kg/m² ] was measured, and maternal dietary quality (Healthy Eating Index), age, race, and education level were assessed. Gestational weight gain was measured at the research facility. Offspring birthweight and sex were self-reported. At age 2 years, parental feeding practices (child feeding questionnaire) were assessed whereas anthropometrics (length and weight) and physical activity (ankle worn accelerometers) were objectively measured. Offspring body mass index z-scores were calculated. Generalized linear regression analysis with a natural log link was done to model the association of maternal FMI and offspring PA. The interaction between maternal FMI and covariates (i.e., all variables listed above) was assessed. Our final model was created using principles of purposeful selection. The best subset of these terms was identified by observation of minimum model AIC statistic.

RESULTS: There was interaction between maternal FMI and offspring sex in association with offspring total PA [average activity counts (AC)/day] at 2 years of age. Specifically, PA decreased with increasing maternal FMI but only in girls (β=-0.02, p=0.0470). In addition, total PA was higher in children born to women with college education compared to children born to women without college education (β=0.09, p=0.0369). Minutes spent in sedentary time increased (β= 0.01, p=0.0422) while minutes spent in light PA tended to decrease (β=-0.01, p=0.0536) with increasing maternal FMI in girls compared to boys.

CONCLUSIONS: There is sexual dimorphism in the association of maternal adiposity measured early in pregnancy with offspring PA at age 2 years. PA levels in 2-year-old girls was negatively associated with increasing maternal adiposity.

PMID:35557425 | DOI:10.1096/fasebj.2022.36.S1.R6006

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R4960.

ABSTRACT

OBJECTIVE: Bladder cancer is the most common malignancy involving the urinary system. As with all types of cancer, accurate monitoring of stage progression is critical in determining proper treatment strategies and predicting clinical outcomes. Collagen fibers are a major component of tumor microenvironment, and their post-translational modifications during the course of tumor progression are well-documented in a number of malignancies including bladder cancer. In the present feasibility study we sought to determine whether quantitative morphological features extracted from collagen fibers can be used to distinguish between urothelial carcinoma tumors pre-muscle-invasion (stage pT1) and post-muscle-invasion (stage pT2).

METHODS: PicroSirius Red (PSR) staining was used to detect fibrillar collagen in 8 tissue microarray (TMA) cores acquired from Biomax (4 stage pT1, 4 stage pT2). We applied auto-fluorescence imaging techniques to detect the PSR signal, and employed the CT-Fire freeware to automatically extract individual collagen fibers. Five morphological features (including length, width, straightness, orientation, and anisotropy) were computed for each detected fiber. Individual features were then combined within each slide to form five corresponding histograms per TMA core. Finally, for each histogram we computed two measures of central tendency (i.e., mean and median), two measures of spread (i.e., range and standard deviation), and two measures of symmetry (i.e., skewness and kurtosis).

RESULTS: To identify any statistically-significant differences between the pT1 and pT2 groups, we employed the student’s t-test (run in Python 3.8.5) to compare the two groups with respect to each of the five morphological features and across each of the six distributional measures mentioned in the Methods section (30 comparisons in total). Of these 30 comparisons, all but one (i.e., median) of the six straightness measures resulted in a p-value < 0.05. Further, each of the two measures of spread (i.e., range and standard deviation) of straightness returned a p-value < 0.001. We are currently working on expanding the size of our dataset to further validate these results.

CONCLUSION: Our preliminary findings are in line with the hypothesis that collagen remodeling is strongly associated with muscle-invasive bladder cancer, and suggest that quantitative features measuring the straightness of individual fibers may be used to improve tumor staging. This type of quantitative approach is particularly helpful in distinguishing between stage pT1 and stage pT2 of bladder cancer, a task which can be challenging at times even for experienced pathologists.

PMID:35557422 | DOI:10.1096/fasebj.2022.36.S1.R4960

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.L7744.

ABSTRACT

The lungs are constantly exposed to a diversity of microbes. On average, the human inhales between 0.7 and 7000 bacterial colony forming units (CFU) every minute. The airway epithelium and the airway surface liquid (ASL) which lines the luminal surface, play a vital role in the defence against these inhaled organisms. Glucose concentration in the ASL is much lower than that of blood (approximately 12.5 times lower). It was proposed that low glucose concentration in the ASL contributes to innate protection against the growth of pathogenic organisms which can utilise glucose for growth. Previous research demonstrated that a sustained increase in blood glucose concentration (such as diabetes) led to increased glucose concentration found in the ASL in both human and animals. We therefore hypothesised that the microbial population of the lung would change in the diabetic lung. Seven-week-old female db/db (BKS.Cg-+Leprdb/+Leprdb/OlaHsd) and non-diabetic littermates (BKS.Cg-(Lean)/OlaHsd) db/db mice and non-diabetic littermate controls were purchased from Envigo (UK). Mice were maintained in standard animal housing in a 12h light/dark cycle; water and standard rodent chow available ad libitumand allowed to acclimatise for three weeks before lung microbiome collection. Mice were terminated with an overdose of pentobarbital (0.2ml of 100mg/ml i.p.). Blood was collected for glucose measurement. Bronchoalveolar lavage was performed and 1 mL of solution was used to extract bacterial DNA using QIAamp DNA Microbiome Kit (Qiagen). The V3-V4-region of the 16S rRNA gene was amplified and sequenced using 300 bp paired-end reads on the Illumina MiSeq platform. Bioinformatic analysis was performed using Mothur v1.39.5 as per the MiSeq SOP pipeline. After removing of contaminant sequence reads, downstream statistical analyses were performed using R statistical software. The bacterial diversity in BAL samples was highly variable within and between diabetic and non-diabetic mice. Hyperglycaemia did not affect the a-diversity of the lung microbiome (Inverse Simpson rating). However, hyperglycaemia had a significant effect on the b-diversity of lung microbiome (analysed with AMOVA, p=0.011, n=9) with the microbiome from diabetic mice clustering together. At the genus level, bacteria of genus Staphylococcus were more abundant in the normoglycaemic mice (n=9, p=0.019). The genus Pseudomonas were more abundant in diabetic mice (n=9, p=0.028) and Corynebacterium (n=9, p=0.0018), which are frequently found in the lung microbiome as commensal organisms, were decreased. Taken together, these data indicate that sustained hyperglycaemia modifies the lung microbiome, decreasing the abundance of commensal bacteria and promoting the growth of glucose-utilising bacteria such as Pseudomonas which may include potential pathogenic species such as P. aeruginosa.

PMID:35557398 | DOI:10.1096/fasebj.2022.36.S1.L7744

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.L8001.

ABSTRACT

The external respiration system is studied in various areas of human physiology and ecology in order to both identify the patterns of functioning of this system and to assess the nature and extent of the influence of environmental factors on respiratory function. Indicators of the external respiration system are considered as individual indicators of the body’s adaptation to the environment. The result of the influence of unfavorable environmental factors is most pronounced in the period of intrauterine development and largely determines the functional capabilities of the organism in the later periods of ontogenesis. The aim of our work was to study the relationship between birth weight and expiratory flow rates. 500 children aged 4 to 7 years were examined. With age, there is an increase in the volumetric expiratory flow rate, which is associated with the processes of general development and growth of the body. The results of the analysis of variance also indicate that this relationship is statistically significant. The value of the Fisher coefficient (F) is 6.73 at a significance level of 0.00021. The greatest increase in expiratory volume flow rates is observed between four and five years (0.54 l/s or 54%), between six and seven years (0.36 l/s or 36%), and the increase between five and six years is insignificant (p>0.05). In the course of our research, the dependence of the indicators of the volumetric expiratory velocity on the floor was also established. In boys, this figure was 1.34 l/s, in girls – 1.19 l/s. This difference was also significant after taking into account age (p<0.05). To identify the nature of the influence of anthropometric parameters at birth on the value of the volumetric expiratory flow rate, a multiple regression analysis was carried out, taking into account birth weight, birth length and age. To clarify the nature of the relationship under study, partial correlation coefficients were calculated, which showed that age and birth weight have the greatest influence on expiratory PTM. Body length at birth does not have a significant effect on external respiration. The partial correlation coefficients are as follows: body weight at birth – 0.244451; age – 0.569063; body length at birth – -0.156432. Since we found that there are certain differences in the expiratory flow rate between boys and girls, we performed a regression analysis for each group. Thus, we can conclude that the indicators of external respiration increase with age due to the processes of growth and general development of the body. The results of the regression analysis of the influence of anthropometric parameters on the volumetric expiratory flow rate indicate the greatest influence on PTM of age and body weight at birth, both in boys and girls.

PMID:35557412 | DOI:10.1096/fasebj.2022.36.S1.L8001

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.L7435.

ABSTRACT

The aim of the study is to investigate the effect of silencing of NILCO by siRNA on the MDR1 gene, which plays a role in chemoresistance. Cells of the human colon cancer lines Caco-2 and HT-29 were cultured at 37˚C in DMEM supplemented with 5% FBS in a humid incubator with 5% CO2. Exponentially growing untreated Caco-2 and HT-29 cells were collected and plated (4 x 10⁵ / well in 2 mL) 24 hours before transfection. Plated cells were treated with either double-stranded siRNA targeting Notch1 mRNA, leptin mRNA, IL-1 β mRNA, control siRNA, or Paclitaxel (PTX) alone, Notch1 siRNA+PTX, Leptin siRNA+PTX, IL-1β siRNA+PTX. The siRNAs of Notch1, IL-1β and Leptin, were applied to the cells at a final concentration of 30 nM, and the cells were transfected with siRNA for 48 hours. After transfection, 100 nM Paclitaxel chemotherapeutic agent for HT-29 and 130 nM for Caco-2 was applied to the cells. Cells were harvested for PCR analysis after treatment with PTX for 24 hours. According to our findings, MDR1 gene expression was significantly increased in the paclitaxel group compared to the control group (p<0.05). Silencing of Notch, leptin and IL-1β decreased MDR1 gene expression compared with the control group, but it was not statistically significant. (p>0.05). However, our results indicated that Notch1 siRNA+PTX, IL-1β siRNA+PTX and Leptin siRNA+PTX downregulated MDR gene expression in the Caco-2 cell line compared with PTX alone treated group (p<0.01). In addition, MDR1 gene expression levels were significantly decreased in the Leptin siRNA+PTX and IL-1β siRNA+PTX groups compared with PTX alone group in the HT-29 cell line, but the decrease in the Notch1 siRNA+PTX group was not statistically significant. (p<0.01, p<0.05, p>0.05). In conclusion, knockdown of NILCO genes is effective in regulating MDR1 expression, which is a hallmark in chemoresistance. The present study was supported by Eskisehir Osmangazi University Scientific Research Projects (BAP) Coordination Unit (grant no. 202011D11).

PMID:35557372 | DOI:10.1096/fasebj.2022.36.S1.L7435

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R3123.

ABSTRACT

Cancer stem cells (CSCs) and associated tumor microenvironment (TM) have been proposed to mediate cancer progression, and therapeutic resistance rendering them valuable therapeutic targets in head and neck squamous cell carcinomas (HNSCC). Oral CSCs drive their tumorigenic effect via stromal fibroblasts. Phytochemicals like allantoin are extensively used in wound healing modulating fibroblast activity. The present study aimed to evaluate whether allantoin can modulate CSCs and/or TM and subsequently contribute to sensitization of cancer cells to chemotherapeutic agents. Hep2, OECs, DPSC cell lines were used to test the cytotoxicity of allantoin on cancer, oral epithelial cells, and normal dental pulp stem cells, respectively. The effect of allantoin and/or 5-fluorouracil (5FU) on cell cycle, apoptosis and expression of stem cell and apoptotic markers was tested in vitro. Further validation was performed using in vivo 4-nitroquinoline 1-oxide model of tongue squamous cell carcinoma model with assessment of cytotoxic effect of allantoin by flow cytometry, immunohistochemical staining, western-blot and RT-PCR. Allantoin was safe on both normal oral epithelial cells and dental pulp stem cells. Allantoin showed a statistically significant and dose dependent increase in 5FU apoptotic effect via reduction of cancer stem cell markers CD133 and CD44. Regarding cell cycle arrest, there was no statistically significant difference with addition of allantoin to 5FU. In vivo, allantoin down-regulated SOX2, NANOG, ALDH and OCT4 gene expression in tongue squamous cell carcinoma. Additionally, allantoin reduced the expression IL-6, TGF-Β, PDGF, α- SMA and CD163. Our results proposed that high concentration of allantoin drives its antitumorigenic effect. This was mediated by modulating stromal component of TM via down-regulation of trans-differentiation of cancer associated fibroblasts and tumor associated macrophages. These findings shed the light on potential use of allantoin as cytotoxic drug either alone or combined with other chemotherapeutic drugs.

PMID:35557345 | DOI:10.1096/fasebj.2022.36.S1.R3123

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.0R581.

ABSTRACT

INTRODUCTION: Regular physical activity is essential for sedentary older adults (aged 65 years and above) who are at the risk for multiple comorbidities. By performing physical activity, older adults can maintain or improve their physical function and mobility that may help them to be independent for a long time and delay the occurrence of disability in the future.

OBJECTIVE: To assess the effect of the eccentric exercises prior to tai chi and yoga performance in the activities of daily living (ADL), balance and gait, and muscle strength in older adults.

METHODS: Nine sedentary independent older adults aged between 60 and 90 were recruited. The study included the medical pre-protocol assessment, the eccentric (ECC) exercise protocol with and without tai chi and yoga, subjective (Health activities of daily living (ADL) difficulty scale), and objective (Tinetti balance and gait, muscle strength) assessments, and medical post-protocol assessment.

RESULTS: The functional limitations, Tinetti balance and gait score, manual muscle strength testing, and active range of motion (AROM) were improved in the medical post-protocol assessment. Muscle strength showed statistically significant differences on the shoulder abductors (P=0.035), the elbow extensors (P=0.046), the hip abductors (P=0.028) and the hip extensors (P=0.03).

CONCLUSIONS: Our findings show low perceived exercises (ECC) prior to tai chi and yoga training are well suited to the sedentary independent older adults with chronic conditions.

PMID:35557356 | DOI:10.1096/fasebj.2022.36.S1.0R581