using-computer-vision-libraries-to-streamline-nuclei-quantification

Using Computer Vision Libraries to Streamline Nuclei Quantification

Live cell assays and image-based cell analyses require data normalization for accurate interpretation. A commonly used method is to stain and quantify ...

Texas Tech University

Muscle damage imposes stress on mitochondria resulting in mitochondrial fusion, fission, and mitophagy. Testosterone is a regulator of these processes. However, no study has examined the effect of sex-specific resistance exercise (RE)-induced hormonal response on mitochondrial dynamics and mitophagy after muscle damage in untrained men and women. Untrained men and women performed two sessions of 80 unilateral maximal eccentric knee extensions (ECC) followed by upper-body RE (ECC+RE) aimed to induce hormonal changes and maintain a similar lower body demands between conditions or 20 min seated rest (ECC+REST). Vastus lateralis samples were analyzed for gene and protein expression of OPA1, MFN1, DRP1, PINK1, and Parkin at baseline (BL), 12 and 24 h. Testosterone area under the curve was greater for ECC+RE than ECC+REST in men and was greater in men than women for both conditions. A significant time × sex × condition effect was found for Parkin protein expression. At 12 and 24 h, Parkin was lower for ECC + REST than ECC + RE for men; whereas, Parkin was increased at 24 h for women regardless of condition. A significant time effect was found for OPA1 protein expression increasing at 12 and 24 h. A significant time × sex × condition effects were found for MFN1, DRP1, and PINK1 gene expression with increases at 12 h in men for ECC + RE. A significant time × sex effect was found for OPA1 gene expression with a decrease at 12 h in men, and 12 h expression in men was lower than women. RE-induced hormonal changes promoted expression of fission, fusion, and mitophagy markers in men. With muscle damage, regardless of condition, expression of inner mitochondrial membrane fusion markers are promoted in both sexes; whereas, those for mitophagy were promoted in women but reduced in men.

Sex-specific mitochondrial dynamics and mitophagy response to muscle damage.

With diabetes, skeletal muscle mitochondrial quality (fusion, fission & mitophagy) and muscle mass are compromised. Geranylgeraniol (GGOH) can prevent mitochondrial damage, inflammation, and improve muscle health; however, the effect of GGOH on a diabetic model is not known. This study aimed to determine the effect of GGOH on mitochondrial quality and muscle mass in diabetic rats.

Geranylgeraniol Supplementation Mitigates Soleus Muscle Atrophy  Changes in Mitochondrial Quality in Diabetic Rats.

To assess hydration status, hydration markers [urine color, osmolality, and urine-specific gravity (USG)] are used. Urine color, osmolality, and USG have shown to be stable for 7, 7, and 3 days, respectively, at 4 °C. However, refrigeration could produce a dry environment which enhances evaporation and potentially affects urine hydration markers.

Storing urine samples with moisture preserves urine hydration marker stability up to 21 days.

Oxidative stress, regulated by SOD2 and mitochondrial dynamics, contributes to muscle atrophy in diabetes. Ginger root extract (GRE) reduces oxidative stress. However, its effect on oxidative stress, mitochondrial dynamics, and muscle atrophy is not known in the diabetic muscle. This study examined the effect of GRE on intramuscular oxidative stress, mitochondrial dynamics, and muscle size in diabetic rats.

Ginger Supplementation Attenuated Mitochondrial Fusion and Improved Skeletal Muscle Size in Type 2 Diabetic Rats.

Muscle-damaging exercise (e.g., downhill running [DHR]) or heat exposure bouts potentially reduce physiological and/or cellular stress during future exertional heat exposure; however, the true extent of their combined preconditioning effects is unknown. Therefore, this study investigated the effect of muscle-damaging exercise in the heat on reducing physiological and cellular stress during future exertional heat exposure. Ten healthy males (mean ± Standard Definition; age, 23 ± 3 years; body mass, 78.7 ± 11.5 kg; height, 176.9 ± 4.7 cm) completed this study. Participants were randomly assigned into two preconditioning groups: (a) DHR in the heat (ambient temperature [T], 35 °C; relative humidity [RH], 40%) and (b) DHR in thermoneutral (T, 20 °C; RH, 20%). Seven days following DHR, participants performed a 45-min flat run in the heat (Flat [T, 35 °C; RH, 40%]). During exercise, heart rate and rectal temperature (T) were recorded at baseline and every 5-min. Peripheral blood mononuclear cells were isolated to assess heat shock protein 72 (Hsp72) concentration between conditions at baseline, immediately post-DHR, and immediately pre-Flat and post-Flat. Mean T during Flat between hot (38.23 ± 0.38 °C) and thermoneutral DHR (38.26 ± 0.38 °C) was not significantly different (P = 0.68), with no mean heart rate differences during Flat between hot (172 ± 15 beats min) and thermoneutral conditions (174 ± 8 beats min; P = 0.58). Hsp72 concentration change from baseline to immediately pre-Flat was significantly lower in hot (-51.4%) compared to thermoneutral (+24.2%; P = 0.025) DHR, with Hsp72 change from baseline to immediately post-Flat also lower in hot (-52.6%) compared to thermoneutral conditions (+26.3%; P = 0.047). A bout of muscle-damaging exercise in the heat reduces cellular stress levels prior to and immediately following future exertional heat exposure.

Eccentric muscle-damaging exercise in the heat lowers cellular stress prior to and immediately following future exertional heat exposure.

Type 2 diabetes (T2D) is associated with increased inflammation and reactive oxygen species (ROS) in muscles, leading to basal satellite cell (SC) myogenic impairment (i.e., reduction in SC pool), which is critical for maintaining skeletal muscle mass. T2D may contribute to muscle atrophy, possibly due to reductions in the SC pool. Geranylgeraniol (GGOH) and ginger can reduce inflammation and enhance SC myogenesis in damaged muscles, thereby alleviating muscle atrophy; however, their effect on basal SC myogenic state and muscle mass in T2D rats is limited. Rats consumed a control diet (CON), high-fat diet with 35 mg/kg of streptozotocin (HFD), a HFD with 800 mg/kg body weight of GGOH (GG), or a HFD with 0.75% ginger root extract (GRE). In the eighth week, their soleus muscles were analyzed for Pax7, MyoD, and MSTN gene and protein expression, SC myogenic state, and muscle cross-sectional area (CSA). The HFD group had a significantly lower number of Pax7/MyoD and Pax7/MSTN cells, less Pax7 and MyoD gene expression, and less MyoD and MSTN protein expression, with a smaller CSA than the CON group. Compared to the GG and GRE groups, the HFD group had a significantly lower number of Pax7/MSTN cells, less MyoD protein expression, and smaller CSA. The GRE group also had a significantly lower number of Pax7/MyoD and greater MSTN protein expression than the HFD group. Nevertheless, the CON group had a significantly greater number of Pax7/MyoD than the GG and GRE groups, and a greater number of Pax7/MyoD cells than the GRE group with a larger CSA than the GG group. GGOH and ginger persevered muscle CSA, possibly through increased MyoD and the ability to maintain the SC pool in T2D rats.

The Effect of Geranylgeraniol and Ginger on Satellite Cells Myogenic State in Type 2 Diabetic Rats.

Dehydration-induced increased plasma osmolality (P) alters whole body fluid balance which could alter resistance exercise (RE) induced intramuscular (IM) fluid shift.

Passive dehydration reduces muscle thickness after resistance exercise.

Muscle recovery after damage is mediated by circulating factors and intracellular signaling pathways. Our previous studies have demonstrated that resistance exercise (RE)-induced circulating factors elicited sex-differential responses in damaged muscle. However, the global effects of these circulating factors on damaged muscle are largely understudied. We examined the differential effects of RE-induced circulating factors and sex on the damaged muscle proteome. Damaged vastus lateralis muscle from 3 men and 3 women from a parent study were analyzed. Participants completed 2 identical bouts of unilateral eccentric knee extensions immediately followed by either upper body RE to induce circulating factors (EXE) or 20-min seated rest (CON). Muscle biopsies collected from the damaged leg at 24 h were used. 900 proteins were identified by LC-MS/MS analysis. Ingenuity Pathway Analysis was used to detect activation prediction using z-scores for functional and pathway analyses. In men, 79 proteins were downregulated and 15 were upregulated in EXE versus CON. These differentially expressed proteins were associated with immunological and inflammatory signaling pathways. Biological functions of the differentially expressed proteins in EXE vs. CON in men include inactivating acute inflammatory signaling, neutrophil extracellular trap signaling, ROS production, and activating IL-12 signaling. These results underline that RE-induced circulating factors have a sex-specific effect on the damaged muscle proteome, where immune signaling is altered in men but not women. Given that the immune response is critical for recovery from muscle damage, these results highlight the potential role of RE-induced circulating factors that could be essential in mediating muscle recovery.

Resistance exercise-induced circulating factors influence the damaged skeletal muscle proteome in a sex-dependent manner.

Bacteriophage (phage) therapy, which uses lytic viruses as antimicrobials, is a potential strategy to address the antimicrobial resistance crisis. Cystic fibrosis, a disease complicated by recurrent Pseudomonas aeruginosa pulmonary infections, is an example of the clinical impact of antimicrobial resistance. Here, using a personalized phage therapy strategy that selects phages for a predicted evolutionary trade-off, nine adults with cystic fibrosis (eight women and one man) of median age 32 (range 22-46) years were treated with phages on a compassionate basis because their clinical course was complicated by multidrug-resistant or pan-drug-resistant Pseudomonas that was refractory to prior courses of standard antibiotics. The individuals received a nebulized cocktail or single-phage therapy without adverse events. Five to 18 days after phage therapy, sputum Pseudomonas decreased by a median of 10 CFU ml, or a mean difference of 10 CFU ml (P = 0.006, two-way analysis of variance with Dunnett's multiple-comparisons test), without altering sputum microbiome, and an analysis of sputum Pseudomonas showed evidence of trade-offs that decreased antibiotic resistance or bacterial virulence. In addition, an improvement of 6% (median) and 8% (mean) predicted FEV was observed 21-35 days after phage therapy (P = 0.004, Wilcoxon signed-rank t-test), which may reflect the combined effects of decreased bacterial sputum density and phage-driven trade-offs. These results show that a personalized, nebulized phage therapy trade-off strategy may affect clinical and microbiologic endpoints, which must be evaluated in larger clinical trials.

Casey R. Appell

Applied Exercise Physiology Laboratory,
Department of Kinesiology and Sport Management,
Applied Exercise Physiology Laboratory, Department of Kinesiology and Sport Management

Texas Tech University

Using Computer Vision Libraries to Streamline Nuclei Quantification

Casey R. Appell

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Applied Exercise Physiology Laboratory,Department of Kinesiology and Sport Management,Applied Exercise Physiology Laboratory, Department of Kinesiology and Sport Management

Texas Tech University

Using Computer Vision Libraries to Streamline Nuclei Quantification

Applied Exercise Physiology Laboratory,
Department of Kinesiology and Sport Management,
Applied Exercise Physiology Laboratory, Department of Kinesiology and Sport Management