Gill filament permeabilization: A novel approach to assess mitochondrial function in sheepshead minnows (Cyprinodon variegatus) following anthraquinone exposure

Measuring mitochondrial respiration in intact single muscle fibers

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00229.2011 ◽

2012 ◽

Vol 302 (6) ◽

pp. R712-R719 ◽

Cited By ~ 40

Author(s):

Rosemary A. Schuh ◽

Kathryn C. Jackson ◽

Ramzi J. Khairallah ◽

Christopher W. Ward ◽

Espen E. Spangenburg

Keyword(s):

Skeletal Muscle ◽

Oxygen Consumption ◽

Mitochondrial Function ◽

Muscle Fibers ◽

Peak Oxygen Consumption ◽

Mdx Mouse ◽

Mdx Mice ◽

Total Oxygen ◽

Novel Approach ◽

Consumption Rates

Measurement of mitochondrial function in skeletal muscle is a vital tool for understanding regulation of cellular bioenergetics. Currently, a number of different experimental approaches are employed to quantify mitochondrial function, with each involving either mechanically or chemically induced disruption of cellular membranes. Here, we describe a novel approach that allows for the quantification of substrate-induced mitochondria-driven oxygen consumption in intact single skeletal muscle fibers isolated from adult mice. Specifically, we isolated intact muscle fibers from the flexor digitorum brevis muscle and placed the fibers in culture conditions overnight. We then quantified oxygen consumption rates using a highly sensitive microplate format. Peak oxygen consumption rates were significantly increased by 3.4-fold and 2.9-fold by simultaneous stimulation with the uncoupling agent, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), and/or pyruvate or palmitate exposure, respectively. However, when calculating the total oxygen consumed over the entire treatment, palmitate exposure resulted in significantly more oxygen consumption compared with pyruvate. Further, as proof of principle for the procedure, we isolated fibers from the mdx mouse model, which has known mitochondrial deficits. We found significant reductions in initial and peak oxygen consumption of 51% and 61% compared with fibers isolated from the wild-type (WT) animals, respectively. In addition, we determined that fibers isolated from mdx mice exhibited less total oxygen consumption in response to the FCCP + pyruvate stimulation compared with the WT mice. This novel approach allows the user to make mitochondria-specific measures in a nondisrupted muscle fiber that has been isolated from a whole muscle.

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Copper-dependent ATP7B up-regulation drives the resistance of TMEM16A-overexpressing head-and-neck cancer models to platinum toxicity

Biochemical Journal ◽

10.1042/bcj20190591 ◽

2019 ◽

Vol 476 (24) ◽

pp. 3705-3719 ◽

Cited By ~ 2

Author(s):

Avani Vyas ◽

Umamaheswar Duvvuri ◽

Kirill Kiselyov

Keyword(s):

Oxidative Stress ◽

Head And Neck ◽

Transcriptional Activation ◽

Platinum Resistance ◽

Mrna Levels ◽

Strong Positive Correlation ◽

Platinum Compounds ◽

Copper Chelation ◽

Novel Approach ◽

Cancer Models

Platinum-containing drugs such as cisplatin and carboplatin are routinely used for the treatment of many solid tumors including squamous cell carcinoma of the head and neck (SCCHN). However, SCCHN resistance to platinum compounds is well documented. The resistance to platinum has been linked to the activity of divalent transporter ATP7B, which pumps platinum from the cytoplasm into lysosomes, decreasing its concentration in the cytoplasm. Several cancer models show increased expression of ATP7B; however, the reason for such an increase is not known. Here we show a strong positive correlation between mRNA levels of TMEM16A and ATP7B in human SCCHN tumors. TMEM16A overexpression and depletion in SCCHN cell lines caused parallel changes in the ATP7B mRNA levels. The ATP7B increase in TMEM16A-overexpressing cells was reversed by suppression of NADPH oxidase 2 (NOX2), by the antioxidant N-Acetyl-Cysteine (NAC) and by copper chelation using cuprizone and bathocuproine sulphonate (BCS). Pretreatment with either chelator significantly increased cisplatin's sensitivity, particularly in the context of TMEM16A overexpression. We propose that increased oxidative stress in TMEM16A-overexpressing cells liberates the chelated copper in the cytoplasm, leading to the transcriptional activation of ATP7B expression. This, in turn, decreases the efficacy of platinum compounds by promoting their vesicular sequestration. We think that such a new explanation of the mechanism of SCCHN tumors’ platinum resistance identifies novel approach to treating these tumors.

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Collecting Words: A Clinical Example of a Morphology-Focused Orthographic Intervention

Language Speech and Hearing Services in Schools ◽

10.1044/2020_lshss-19-00050 ◽

2020 ◽

Vol 51 (3) ◽

pp. 544-560 ◽

Cited By ~ 3

Author(s):

Kimberly A. Murphy ◽

Emily A. Diehm

Keyword(s):

Written Language ◽

Morphological Knowledge ◽

English Orthography ◽

Word Level ◽

Novel Approach ◽

Language Pathology ◽

The One ◽

Critical Intervention ◽

Reading And Spelling ◽

Reading And Spelling Difficulties

Purpose Morphological interventions promote gains in morphological knowledge and in other oral and written language skills (e.g., phonological awareness, vocabulary, reading, and spelling), yet we have a limited understanding of critical intervention features. In this clinical focus article, we describe a relatively novel approach to teaching morphology that considers its role as the key organizing principle of English orthography. We also present a clinical example of such an intervention delivered during a summer camp at a university speech and hearing clinic. Method Graduate speech-language pathology students provided a 6-week morphology-focused orthographic intervention to children in first through fourth grade ( n = 10) who demonstrated word-level reading and spelling difficulties. The intervention focused children's attention on morphological families, teaching how morphology is interrelated with phonology and etymology in English orthography. Results Comparing pre- and posttest scores, children demonstrated improvement in reading and/or spelling abilities, with the largest gains observed in spelling affixes within polymorphemic words. Children and their caregivers reacted positively to the intervention. Therefore, data from the camp offer preliminary support for teaching morphology within the context of written words, and the intervention appears to be a feasible approach for simultaneously increasing morphological knowledge, reading, and spelling. Conclusion Children with word-level reading and spelling difficulties may benefit from a morphology-focused orthographic intervention, such as the one described here. Research on the approach is warranted, and clinicians are encouraged to explore its possible effectiveness in their practice. Supplemental Material https://doi.org/10.23641/asha.12290687

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