Swallow is a complex behavior that consists of three coordinated phases: oral, pharyngeal, and esophageal. Esophageal distension (EDist) has been shown to elicit pharyngeal swallow, but the ...physiologic characteristics of EDist-induced pharyngeal swallow have not been specifically described. We examined the effect of rapid EDist on oropharyngeal swallow, with and without an oral water stimulus, in spontaneously breathing, sodium pentobarbital anesthetized cats (n = 5). Electromyograms (EMGs) of activity of 8 muscles were used to evaluate swallow: mylohyoid (MyHy), geniohyoid (GeHy), thyrohyoid (ThHy), thyropharyngeus (ThPh), thyroarytenoid (ThAr), cricopharyngeus (upper esophageal sphincter: UES), parasternal (PS), and costal diaphragm (Dia). Swallow was defined as quiescence of the UES with overlapping upper airway activity, and it was analyzed across three stimulus conditions: 1) oropharyngeal water infusion only, 2) rapid esophageal distension (EDist) only, and 3) combined stimuli. Results show a significant effect of stimulus condition on swallow EMG amplitude of the mylohyoid, geniohyoid, thyroarytenoid, diaphragm, and UES muscles. Collectively, we found that, compared to rapid cervical esophageal distension alone, the stimulus condition of rapid distension combined with water infusion is correlated with increased laryngeal adductor and diaphragm swallow-related EMG activity (schluckatmung), and post-swallow UES recruitment. We hypothesize that these effects of upper esophageal distension activate the brainstem swallow network, and function to protect the airway through initiation and/or modulation of a pharyngeal swallow response.
•Mitochondrial volume density is higher in type I and IIa than type IIx/IIb fibers.•Mitochondria in type I and IIa fibers are more filamentous than type IIx/IIb fibers.•SDHmax per unitary ...mitochondrial volume is higher in type I and IIa than type IIx/IIb fibers.
In diaphragm muscle (DIAm), type I and IIa fibers are recruited to accomplish breathing, while type IIx/IIb fibers are recruited only during expulsive/straining behaviors. Thus, type I and IIa DIAm fibers are much more active (duty cycle of ∼40 %) than type IIx/IIb fibers (duty cycle of <1%), which we hypothesized underlies intrinsic differences in mitochondrial structure and function. MitoTracker Green labeled mitochondria were imaged in 3-D using confocal microscopy. Mitochondrial volume density (MVD, per muscle fiber volume) was higher, and mitochondria were more filamentous in type I and IIa DIAm compared to type IIx/IIb fibers. The maximum velocity of the succinate dehydrogenase reaction (SDHmax), measured using a quantitative histochemical technique was found to be higher in type I and IIa DIAm fibers compared to type IIx/IIb fibers with and without normalizing for MVD. These results are consistent with fiber type differences in the intrinsic structural and functional properties of DIAm fibers and closely match differences in energetic demands.
Given the potential for unintended pregnancy and exposure to sexually transmitted infections, both of which can have long-term deleterious health consequences, the identification of predictors of ...adolescent risky sexual behavior remains an important line of inquiry. Although prior research has identified a variety of family and individual factors that are associated with risky sexual behavior, few studies have examined the role of family economic stress. The current study utilized three waves of data from a community sample of African American families with adolescents (N = 778, 54% girls, average age = 10.4 years old at Wave 1, 12.3 years old at Wave 2, 15.6 years old at Wave 3) to test the family stress model as an explanation of adolescent risky sexual behavior. Multi-group analyses examined gender differences in the family processes expected to link economic stress and risky sexual behavior. Unlike most studies utilizing this theoretical perspective, family structure was also taken into account. The results supported the propositions of the family stress model for boys and girls for both two-caregiver and single-mother households. Further, in single-mother households, maternal psychological distress continued to have a positive effect on adolescent risky sex even after taking into account the impact of parenting behaviors. Overall, the results suggest that economic stress ripples through the family system, increasing adolescent risky sexual behavior through its negative impact on family processes, highlighting the need for systemic policy changes rather than individual-level intervention/prevention efforts.
Phrenic motor neuron (PhMN) development in early onset hypertonia is poorly understood. Respiratory disorders are one of the leading causes of morbidity and mortality in individuals with early onset ...hypertonia, such as cerebral palsy (CP), but they are largely overshadowed by a focus on physical function in this condition. Furthermore, while the brain is the focus of CP research, motor neurons, via the motor unit and neurotransmitter signaling, are the targets in clinical interventions for hypertonia. Furthermore, critical periods of spinal cord and motor unit development also coincide with the timing that the supposed brain injury occurs in CP. Using an animal model of early-onset spasticity (
mouse B6.Cg-Glrbspa/J with a glycine receptor mutation), we hypothesized that removal of effective glycinergic neurotransmitter inputs to PhMNs during development will result in fewer PhMNs and reduced PhMN somal size at maturity. Adult
(Glrb-/-), and wild-type (Glrb+/+) mice underwent unilateral retrograde labeling of PhMNs via phrenic nerve dip in tetramethylrhodamine. After three days, mice were euthanized, perfused with 4% paraformaldehyde, and the spinal cord excised and processed for confocal imaging.
mice had ~30% fewer PhMNs (
= 0.005), disproportionately affecting larger PhMNs. Additionally, a ~22% reduction in PhMN somal surface area (
= 0.019), an 18% increase in primary dendrites (
< 0.0001), and 24% decrease in dendritic surface area (
= 0.014) were observed. Thus, there are fewer larger PhMNs in
mice. Fewer and smaller PhMNs may contribute to impaired diaphragm neuromotor control and contribute to respiratory morbidity and mortality in conditions of early onset hypertonia.
Phrenic motor neuron (PhMN) development in early-onset hypertonia is poorly understood. Yet, respiratory disorders are a common cause of morbidity and mortality. In
mice, an animal model of early-onset hypertonia, we found ~30% fewer PhMNs, compared with controls. This PhMN loss disproportionately affected larger PhMNs. Thus, the number and heterogeneity of the PhMN pool are decreased in
mice, likely contributing to the hypertonia, impaired neuromotor control, and respiratory disorders.
Abstract
Background
Encapsulation of follicles within a biomatrix is one approach to maintaining 3-D follicle architecture during culture. Hyaluronan is one component of the natural extracellular ...matrix (ECM) that provides support to cells in vivo. This report describes the application of a novel tyramine-linked hyaluronan for 3-D
in vitro
follicle culture and the production of developmentally competent metaphase II oocytes.
Materials and Methods
Enzymatically isolated mouse preantral follicles or follicle clusters (FL-C) from fresh or vitrified ovaries were encapsulated in 3 mg/ml of hyaluronan gel (HA). Follicle growth, antrum formation and meiotic maturation to metaphase II oocytes was monitored. Chromatin staining was used to assess GV oocyte progression towards meiotic competence. Functional competence of
in vitro
matured (IVM) oocytes was evaluated by
in vitro
fertilization and ability to develop to blastocyst. Modifying the HA gel by inclusion of laminin (HA-LM), mouse sarcoma extracellular matrix (Matrigel;HA-MG) or placental extracellular matrix (HA-PM) was also tested to see if this might further enhance IVM outcomes.
Results
A total of 402 preantral follicles were cultured in HA gel. After hCG trigger, 314 oocyte-cumulus complexes ovulated from the embedded follicles. Meiotic maturation rate to the metaphase II stage was 73% (228/314). After insemination 83% (188/228) of IVM oocytes fertilized with a subsequent blastulation rate of 46% (87/188). A pilot transfer study with 3 recipient mice resulted in the birth of a single pup. HA gel supported individually isolated follicles as well ovarian tissue fragments containing clusters of 6–8 preantral follicles. Meiotic maturation was lower with FL-clusters from vitrified versus fresh ovaries (34% and 55%, respectively; p < 0.007). Modification of the HA gel with ECMs or laminin affected antrum formation and follicle retention. Maturation rates to the metaphase II stage were however not significantly different: 74% for HA gel alone as compared to HA-LM (67%), HA-MG (56%) and HA-PM (58%).
Conclusion
Hyaluronan gel is an effective and versatile extracellular matrix based biomaterial for 3-D culture of ovarian follicles. This culture model allowed ovulation of functionally competent metaphase II oocytes, capable of fertilization, genomic activation and blastulation. Future testing with human follicles that require longer
in vitro
culture times should be considered.
Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease globally. NAFLD is a consequence of fat accumulation in the liver leading to lipotoxicity. Increasing evidence ...has demonstrated the critical role of autophagy in NAFLD. This study uncovers the unexpected role of immune surveillance protein DDX58/Rig-1 (DExD/H box helicase 58) in activating macroautophagy/autophagy and protecting from lipotoxicity associated with NAFLD. Here we show for the first time that DDX58 protein is significantly reduced in nonalcoholic steatohepatitis (NASH) mouse model, an aggressive form of NAFLD characterized by inflammation and fibrosis of the liver. In addition to decreased expression of DDX58, we found that DDX58 activity can be attenuated by treatments with palmitic acid (PA), a saturated fatty acid. To investigate whether PA inhibition of DDX58 is harmful to the cell, we characterized DDX58 function in hepatocytes when exposed to high doses of PA in the presence and/or absence of DDX58. We show that siRNA knockdown of DDX58 promotes apoptosis. Importantly, we show that stable overexpression of DDX58 is protective against toxic levels of PA and stimulates autophagy. This study begins to demonstrate the regulation of the autophagy receptor protein SQSTM1/p62 through DDX58. DDX58 expression directly influences SQSTM1 mRNA and protein levels. This work proposes a model in which activating DDX58 increases an autophagic response and this aids in clearing toxic lipid inclusion bodies, which leads to inflammation and apoptosis. Activating a DDX58-induced autophagy response may be a strategy for treating NAFLD.
Abbreviations:5ʹpppdsRNA: 5ʹ triphosphate double-stranded RNA; CDAHFD: choline-deficient, L-amino acid defined high-fat diet; CEBPB: CCAAT/enhancer binding protein (C/EBP), beta; CQ: chloroquine; DDX58/retinoic acid inducible gene 1/Rig-1: DExD/H box helicase 58; h: hours; IFIH1/MDA5: interferon induced with helicase C domain 1; IFNB/IFN-β: interferon beta 1, fibroblast; KO: knockout; MAVS: mitochondrial antiviral signaling protein; NAFLD: nonalcoholic fatty liver disease; NASH: nonalcoholic steatohepatitis; NFKB/NF-κB: nuclear factor of kappa light polypeptide gene enhancer in B cells; PA: palmitic acid; poly:IC: polyinosinic:polycytidylic acid; PRR: pattern recognition receptors; PSR: picrosirus red; RAP: rapamycin; RLR: RIG-I-like receptor; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK-binding kinase 1.
MicroRNAs are potent regulators of gene expression that have been widely implicated in reproduction and embryo development. Recent studies have demonstrated that miR-21, a microRNA extensively ...studied in the context of disease, is important in multiple facets of reproductive biology including folliculogenesis, ovulation, oocyte maturation and early mammalian development. Surprisingly, little is known about the mechanisms that regulate miR-21 and no studies have characterized these regulatory pathways in cumulus-oocyte complexes (COCs). We therefore investigated miR-21 in an in vitro model of bovine oocyte maturation. Levels of the primary transcript of miR-21 (pri-miR-21) and mature miR-21 increased markedly in COCs over the maturation period. Cloning of the bovine pri-miR-21 gene and promoter by 5'3'RACE (rapid amplification of cDNA ends) revealed a highly conserved region immediately upstream of the transcription start site and two alternatively-spliced variants of pri-miR-21. The promoter region contained several putative transcription factor binding sites, including two for signal transducer and activator of transcription 3 (STAT3). Mutation of these sites significantly decreased both the intrinsic activity of pri-miR-21 promoter-luciferase constructs and the response to leukemia inhibitory factor (LIF) (a STAT3 activator) in cultured MCF7 cells. In COCs, treatment with a STAT3 pathway inhibitor markedly decreased pri-miR-21 expression and prevented cumulus expansion. Pri-miR-21 expression was also inhibited by the protein synthesis inhibitor cycloheximide, suggesting that a protein ligand or signaling cofactor synthesized during maturation is necessary for transcription. Together these studies represent the first investigation of signaling pathways that directly influence miR-21 expression in bovine oocytes and cumulus cells.
The tongue is a muscular hydrostat, with lingual movements occurring during breathing, chewing, swallowing, vocalization, vomiting, coughing and grooming/sexual activities. In the elderly, reduced ...lingual dysfunction and weakness contribute to increased risks of obstructive sleep apnea and aspiration pneumonia. In Fischer 344 (F344) rats, a validated model of aging, hypoglossal motor neuron death is apparent, although there is no information regarding tongue strength. The intrinsic tongue muscles, the superior and inferior longitudinal, transversalis and verticalis exist in an interdigitated state. Recently, we established a method to measure the specific force of individual intrinsic tongue muscle, accounting for the tissue bulk that is not in the direction of uniaxial force. In the longitudinal muscles of 6- (n = 10), 18- (n = 9) and 24-month-old (n = 12) female and male F344 rats, we assessed specific force, fatigability, fiber type dependent cross-sectional area (CSA) and overall CSA. Muscle force and fatigue was assessed ex vivo using platinum plate simulation electrodes. Tongue muscles were frozen in melting isopentane, and transverse sections cut at 10 µm. Muscle fiber type was classified based on immunoreactivity to myosin heavy chain (MyHC) isoform antibodies. In H&E stained muscle, CSA and uniaxial muscle contributions to total tongue bulk was assessed. We observed a robust ∼30% loss of longitudinal specific force, with reductions in overall longitudinal muscle fiber CSA and specific atrophy of type IIx/IIb fibers. It will be important to investigate the mechanistic underpinnings of hypoglossal motor neuron death and tongue muscle weakness to eventually provide therapies for age-associated lingual dysfunctions.
•Tongue dysfunction and weakness is implicated in the increased age-associated risks of OSA and aspiration pneumonia.•We assessed uniaxial specific force, fatigability and muscle fiber CSA of the superior and inferior longitudinal muscles.•In old age reduced specific force and muscle fiber atrophy is evident, which may underpin age-related lingual dysfunctions.
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