Mustn1 ablation in skeletal muscle results in functional alterations.

Mustn1, a gene expressed exclusively in the musculoskeletal system, was shown in previous in vitro studies to be a key regulator of myogenic differentiation and myofusion. Other studies also showed Mustn1 expression associated with skeletal muscle development and hypertrophy. However, its specific role in skeletal muscle function remains unclear. This study sought to investigate the effects of Mustn1 in a conditional knockout (KO) mouse model in Pax7 positive skeletal muscle satellite cells. Specifically, we investigated the potential effects of Mustn1 on myogenic gene expression, grip strength, alterations in gait, ex vivo investigations of isolated skeletal muscle isometric contractions, and potential changes in the composition of muscle fiber types. Results indicate that Mustn1 KO mice did not present any substantial phenotypic changes or significant variations in genes related to myogenic differentiation and fusion. However, an approximately 10% decrease in overall grip strength was observed in the 2-month-old KO mice in comparison to the control wild type (WT), but this decrease was not significant when normalized by weight. KO mice also generated approximately 8% higher vertical force than WT at 4 months in the hindlimb. Ex vivo experiments revealed decreases in about 20 to 50% in skeletal muscle contractions and about 10%-20% fatigue in soleus of both 2- and 4-month-old KO mice, respectively. Lastly, immunofluorescent analyses showed a persistent increase of Type IIb fibers up to 15-fold in the KO mice while Type I fibers decreased about 20% and 30% at both 2 and 4 months, respectively. These findings suggest a potential adaptive or compensatory mechanism following Mustn1 loss, as well as hinting at an association between Mustn1 and muscle fiber typing. Collectively, Mustn1's complex roles in skeletal muscle physiology requires further research, particularly in terms of understanding the potential role of Mustn1 in muscle repair and regeneration, as well as with influence of exercise. Collectively, these will offer valuable insights into Mustn1's key biological functions and regulatory pathways.

Low intensity mechanical signals promote proliferation in a cell-specific manner: Tailoring a non-drug strategy to enhance biomanufacturing yields.

Biomanufacturing relies on living cells to produce biotechnology-based therapeutics, tissue engineering constructs, vaccines, and a vast range of agricultural and industrial products. With the escalating demand for these bio-based products, any process that could improve yields and shorten outcome timelines by accelerating cell proliferation would have a significant impact across the discipline. While these goals are primarily achieved using biological or chemical strategies, harnessing cell mechanosensitivity represents a promising - albeit less studied - physical pathway to promote bioprocessing endpoints, yet identifying which mechanical parameters influence cell activities has remained elusive. We tested the hypothesis that mechanical signals, delivered non-invasively using low-intensity vibration (LIV; <1g, 10-500Hz), will enhance cell expansion, and determined that any unique signal configuration was not equally influential across a range of cell types. Varying frequency, intensity, duration, refractory period, and daily doses of LIV increased proliferation in CHO-adherent cells (+79% in 96h) using a particular set of LIV parameters (0.2g, 500Hz, 3x30 min/d, 2h refractory period), yet this same mechanical input suppressed proliferation in CHO-suspension cells (-13%). Exposing these same CHO-suspension cells to distinct LIV parameters (30Hz, 0.7g, 2x60 min/d, 2h refractory period) increased proliferation by 210%. Particle image velocimetry combined with finite element modeling showed high transmissibility of these signals across fluids (>90%), and LIV effectively scaled up to T75 flasks. Ultimately, when LIV is tailored to the target cell population, its highly efficient transmission across media represents a means to non-invasively augment biomanufacturing endpoints for both adherent and suspended cells, and holds immediate applications, ranging from small-scale, patient-specific personalized medicine to large-scale commercial bio-centric production challenges.

Mustn1 ablation in skeletal muscle results in increased glucose tolerance concomitant with upregulated GLUT expression in male mice.

Glucose homeostasis is closely regulated to maintain energy requirements of vital organs and skeletal muscle plays a crucial role in this process. Mustn1 is expressed during embryonic and postnatal skeletal muscle development and its function has been implicated in myogenic differentiation and myofusion. Whether Mustn1 plays a role in glucose homeostasis in anyway remains largely unknown. As such, we deleted Mustn1 in skeletal muscle using a conditional knockout (KO) mouse approach. KO mice did not reveal any specific gross phenotypic alterations in skeletal muscle. However, intraperitoneal glucose tolerance testing (IPGTT) revealed that 2-month-old male KO mice had significantly lower glycemia than their littermate wild type (WT) controls. These findings coincided with mRNA changes in genes known to be involved in glucose metabolism, tolerance, and insulin sensitivity; 2-month-old male KO mice had significantly higher expression of GLUT1 and GLUT10 transporters, MUP-1 while OSTN expression was lower. These differences in glycemia and gene expression were statistically insignificant after 4 months. Identical experiments in female KO and WT control mice did not indicate any differences at any age. Our results suggest a link between Mustn1 expression and glucose homeostasis during a restricted period of skeletal muscle development/maturation. While this is an observational study, Mustn1's relationship to glucose homeostasis appears to be more complex with a possible connection to other key proteins such as GLUTs, MUP-1, and OSTN. Additionally, our data indicate temporal and sex differences. Lastly, our findings strengthen the notion that Mustn1 plays a role in the metabolic capacity of skeletal muscle.

Identification of pathways that regulate circadian rhythms using a larval zebrafish small molecule screen.

The circadian clock ensures that behavioral and physiological processes occur at appropriate times during the 24-hour day/night cycle, and is regulated at both the cellular and organismal levels. To identify pathways acting on intact animals, we performed a small molecule screen using a luminescent reporter of molecular circadian rhythms in zebrafish larvae. We identified both known and novel pathways that affect circadian period, amplitude and phase. Several drugs identified in the screen did not affect circadian rhythms in cultured cells derived from luminescent reporter embryos or in established zebrafish and mammalian cell lines, suggesting they act via mechanisms absent in cell culture. Strikingly, using drugs that promote or inhibit inflammation, as well as a mutant that lacks microglia, we found that inflammatory state affects circadian amplitude. These results demonstrate a benefit of performing drug screens using intact animals and provide novel targets for treating circadian rhythm disorders.

Inherited and De Novo Genetic Risk for Autism Impacts Shared Networks.

We performed a comprehensive assessment of rare inherited variation in autism spectrum disorder (ASD) by analyzing whole-genome sequences of 2,308 individuals from families with multiple affected children. We implicate 69 genes in ASD risk, including 24 passing genome-wide Bonferroni correction and 16 new ASD risk genes, most supported by rare inherited variants, a substantial extension of previous findings. Biological pathways enriched for genes harboring inherited variants represent cytoskeletal organization and ion transport, which are distinct from pathways implicated in previous studies. Nevertheless, the de novo and inherited genes contribute to a common protein-protein interaction network. We also identified structural variants (SVs) affecting non-coding regions, implicating recurrent deletions in the promoters of DLG2 and NR3C2. Loss of nr3c2 function in zebrafish disrupts sleep and social function, overlapping with human ASD-related phenotypes. These data support the utility of studying multiplex families in ASD and are available through the Hartwell Autism Research and Technology portal.

Correlates of physical disability in the elderly population of Rural North India (Haryana).

BACKGROUND: With the increase in life expectancy, the number of older persons is constantly rising. Disability rates for elderly people are also on the rise with an increase in the burden of chronic diseases depriving them of independence and the performance of activities of daily living. The study aimed to estimate the prevalence of physical disability and determine its correlates among elderly population of rural Haryana. MATERIALS AND METHODS: A community-based cross-sectional study was conducted among 322 elderly participants aged 60 years and above in the rural area of Haryana, India. Data pertaining to sociodemographic profile, self-reported chronic diseases/ailments, and disability assessment by means of Barthel and Katz index of activities of daily living was collected and analyzed. RESULTS: Overall, 21.4% and 18% elderly people had some form of disability according to the Barthel index and Katz index, respectively. With aging, disability increased and 52.5% of the elderly aged 75 years and above were found to have disability according to the Barthel's index. Females (P = 0.014), those who were currently not married (P = 0.001), currently unemployed (P = 0.001), and those with chronic diseases/ailments (P = 0.002), had significantly higher disability rates. Binary logistic regression analysis revealed age 75 years and above, current unemployment, and the presence of three or more chronic diseases/ailments as significant factors related to physical disability. CONCLUSION: Disability is associated with increasing age and an increase in the burden of chronic health ailments in the elderly. Consequently, there is the need to prioritize preventive, promotive, curative, and rehabilitative services for the geriatric population.

Neuropeptide Y Regulates Sleep by Modulating Noradrenergic Signaling.

Sleep is an essential and evolutionarily conserved behavioral state whose regulation remains poorly understood. To identify genes that regulate vertebrate sleep, we recently performed a genetic screen in zebrafish, and here we report the identification of neuropeptide Y (NPY) as both necessary for normal daytime sleep duration and sufficient to promote sleep. We show that overexpression of NPY increases sleep, whereas mutation of npy or ablation of npy-expressing neurons decreases sleep. By analyzing sleep architecture, we show that NPY regulates sleep primarily by modulating the length of wake bouts. To determine how NPY regulates sleep, we tested for interactions with several systems known to regulate sleep, and provide anatomical, molecular, genetic, and pharmacological evidence that NPY promotes sleep by inhibiting noradrenergic signaling. These data establish NPY as an important vertebrate sleep/wake regulator and link NPY signaling to an established arousal-promoting system.

Light-Dependent Regulation of Sleep and Wake States by Prokineticin 2 in Zebrafish.

Light affects sleep and wake behaviors by providing an indirect cue that entrains circadian rhythms and also by inducing a direct and rapid regulation of behavior. While circadian entrainment by light is well characterized at the molecular level, mechanisms that underlie the direct effect of light on behavior are largely unknown. In zebrafish, a diurnal vertebrate, we found that both overexpression and mutation of the neuropeptide prokineticin 2 (Prok2) affect sleep and wake behaviors in a light-dependent but circadian-independent manner. In light, Prok2 overexpression increases sleep and induces expression of galanin (galn), a hypothalamic sleep-inducing peptide. We also found that light-dependent, Prok2-induced sedation requires prokineticin receptor 2 (prokr2) and is strongly suppressed in galn mutants. These results suggest that Prok2 antagonizes the direct wake-promoting effect of light in zebrafish, in part through the induction of galn expression in the hypothalamus.

Genetic Analysis of Histamine Signaling in Larval Zebrafish Sleep.

Pharmacological studies in mammals and zebrafish suggest that histamine plays an important role in promoting arousal. However, genetic studies using rodents with disrupted histamine synthesis or signaling have revealed only subtle or no sleep/wake phenotypes. Studies of histamine function in mammalian arousal are complicated by its production in cells of the immune system and its roles in humoral and cellular immunity, which can have profound effects on sleep/wake states. To avoid this potential confound, we used genetics to explore the role of histamine in regulating sleep in zebrafish, a diurnal vertebrate in which histamine production is restricted to neurons in the brain. Similar to rodent genetic studies, we found that zebrafish that lack histamine due to mutation of histidine decarboxylase (hdc) exhibit largely normal sleep/wake behaviors. Zebrafish containing predicted null mutations in several histamine receptors also lack robust sleep/wake phenotypes, although we are unable to verify that these mutants are completely nonfunctional. Consistent with some rodent studies, we found that arousal induced by overexpression of the neuropeptide hypocretin (Hcrt) or by stimulation of hcrt-expressing neurons is not blocked in hdc or hrh1 mutants. We also found that the number of hcrt-expressing or histaminergic neurons is unaffected in animals that lack histamine or Hcrt signaling, respectively. Thus, while acute pharmacological manipulation of histamine signaling has been shown to have profound effects on zebrafish and mammalian sleep, our results suggest that chronic loss of histamine signaling due to genetic mutations has only subtle effects on sleep in zebrafish, similar to rodents.

Porokeratotic Adnexal Ostial Nevus-Report of a Case With Unusual Clinical and Histologic Features.

An 11-year-old Tanzanian girl presented with diffuse verrucous lesions of varying morphology, scarring alopecia, and keloid scars over the face with a predilection for the ears. Physical examination revealed dark keratoderma and patches of hypopigmentation near the midline of the dorsal trunk (Figure 1a). Her forearms were densely covered by verrucous lesions with the exception of a clear linear patch on the dorsal aspect of the left forearm (Figure 1b). The perioral area was notable for white spires projecting from verrucous papules (Figure 1c) while the oral mucosa and teeth appeared normal on visual examination. The rest of her body, including the palms and soles, was covered by patchy, scaly lesions of varying severity.

Morphological and Physiological Interactions Between GnRH3 and Hypocretin/Orexin Neuronal Systems in Zebrafish (Danio rerio).

GnRH neurons integrate internal and external cues to control sexual maturation and fertility. Homeostasis of energy balance and food intake correlates strongly with the status of reproduction. Neuropeptides secreted by the hypothalamus involved in modulating energy balance and feeding may play additional roles in the regulation of reproduction. Hypocretin (Hcrt) (also known as orexin) is one such peptide, primarily controlling sleep/wakefulness, food intake, and reward processing. There is a growing body of evidence indicating that Hcrt/orexin (Hcrt) modulates reproduction through interacting with the hypothalamo-pituitary-gonadal axis in mammals. To explore potential morphological and functional interactions between the GnRH and Hcrt neuronal systems, we employed a variety of experimental approaches including confocal imaging, immunohistochemistry, and electrophysiology in transgenic zebrafish, in which fluorescent proteins are genetically expressed in GnRH3 and Hcrt neurons. Our imaging data revealed close apposition and direct connection between GnRH3 and Hcrt neuronal systems in the hypothalamus during larval development through adulthood. Furthermore, the Hcrt receptor (HcrtR) is expressed in GnRH3 neurons. Electrophysiological data revealed a reversible inhibitory effect of Hcrt on GnRH3 neuron electrical activity, which was blocked by the HcrtR antagonist almorexant. In addition, Hcrt had no effect on the electrical activity of GnRH3 neurons in the HcrtR null mutant zebrafish (HcrtR-/-). Our findings demonstrate a close anatomical and functional relationship between Hcrt and GnRH neuronal systems in zebrafish. It is the first demonstration of a link between neuronal circuits controlling sleeping/arousal/feeding and reproduction in zebrafish, an important animal model for investigating the molecular genetics of development.

A Zebrafish Genetic Screen Identifies Neuromedin U as a Regulator of Sleep/Wake States.

Neuromodulation of arousal states ensures that an animal appropriately responds to its environment and engages in behaviors necessary for survival. However, the molecular and circuit properties underlying neuromodulation of arousal states such as sleep and wakefulness remain unclear. To tackle this challenge in a systematic and unbiased manner, we performed a genetic overexpression screen to identify genes that affect larval zebrafish arousal. We found that the neuropeptide neuromedin U (Nmu) promotes hyperactivity and inhibits sleep in zebrafish larvae, whereas nmu mutant animals are hypoactive. We show that Nmu-induced arousal requires Nmu receptor 2 and signaling via corticotropin releasing hormone (Crh) receptor 1. In contrast to previously proposed models, we find that Nmu does not promote arousal via the hypothalamic-pituitary-adrenal axis, but rather probably acts via brainstem crh-expressing neurons. These results reveal an unexpected functional and anatomical interface between the Nmu system and brainstem arousal systems that represents a novel wake-promoting pathway.

Norepinephrine is required to promote wakefulness and for hypocretin-induced arousal in zebrafish.

Pharmacological studies in mammals suggest that norepinephrine (NE) plays an important role in promoting arousal. However, the role of endogenous NE is unclear, with contradicting reports concerning the sleep phenotypes of mice lacking NE due to mutation of dopamine ß-hydroxylase (dbh). To investigate NE function in an alternative vertebrate model, we generated dbh mutant zebrafish. In contrast to mice, these animals exhibit dramatically increased sleep. Surprisingly, despite an increase in sleep, dbh mutant zebrafish have a reduced arousal threshold. These phenotypes are also observed in zebrafish treated with small molecules that inhibit NE signaling, suggesting that they are caused by the lack of NE. Using genetic overexpression of hypocretin (Hcrt) and optogenetic activation of hcrt-expressing neurons, we also find that NE is important for Hcrt-induced arousal. These results establish a role for endogenous NE in promoting arousal and indicate that NE is a critical downstream effector of Hcrt neurons.

A high content assay to assess cellular fitness.

A universal process in experimental biology is the use of engineered cells; more often, stably or transiently transfected cells are generated for the purpose. Therefore, it is important that cell health assessment is conducted to check for stress mediated by induction of heat shock proteins (Hsps). For this purpose, we have developed an integrated platform that would enable a direct assessment of transfection efficiency (TE) combined with cellular toxicity and stress response. We make use of automated microscopy and high content analysis to extract from the same well a multiplexed readout to assess and determine optimal chemical transfection conditions. As a proof of concept, we investigated seven commercial reagents, in a matrix of dose and time, to study transfection of an EGFP DNA plasmid into HeLa cells and their consequences on health and fitness; where we scored for cellular proliferation, EGFP positive cells, and induction of Hsp10 and Hsp70 as makers of stress responses. FuGENE HD emerged as the most optimal reagent with no apparent side effects suitable for performing microtiter based miniaturized transfection for both chemical and RNAi screening. In summary, we report on a high content assay method to assess cellular overall fitness upon chemical transfection.

Cognitive abnormalities and hippocampal alterations in monoamine oxidase A and B knockout mice.

The monoamine oxidase isoenzymes (MAOs) A and B play important roles in the homeostasis of monoaminergic neurotransmitters. The combined deficiency of MAO A and B results in significantly elevated levels of serotonin (5-hydroxytryptamine), norepinephrine, dopamine, and ß-phenylethylamine; in humans and mice, these neurochemical changes are accompanied by neurodevelopmental perturbations as well as autistic-like responses. Ample evidence indicates that normal levels of monoamines in the hippocampus, amygdala, frontal cortex, and cerebellum are required for the integrity of learning and memory. Thus, in the present study, the cognitive status of MAO A/B knockout (KO) mice was examined with a wide array of behavioral tests. In comparison with male wild-type littermates, MAO A/B KO mice exhibited abnormally high and overgeneralized fear conditioning and enhanced eye-blink conditioning. These alterations were accompanied by significant increases in hippocampal long-term potentiation and alterations in the relative expression of NMDA glutamate receptor subunits. Our data suggest that chronic elevations of monoamines, because of the absence of MAO A and MAO B, cause functional alterations that are accompanied with changes in the cellular mechanisms underlying learning and memory. The characteristics exhibited by MAO A/B KO mice highlight the potential of these animals as a useful tool to provide further insight into the molecular bases of disorders associated with abnormal monoaminergic profiles.

Allopregnanolone restores hippocampal-dependent learning and memory and neural progenitor survival in aging 3xTgAD and nonTg mice.

We previously demonstrated that allopregnanolone (APα) increased proliferation of neural progenitor cells and reversed neurogenic and cognitive deficits prior to Alzheimer's disease (AD) pathology (Wang, J.M., Johnston, P.B., Ball, B.G., Brinton, R.D., 2005. The neurosteroid allopregnanolone promotes proliferation of rodent and human neural progenitor cells and regulates cell-cycle gene and protein expression. J. Neurosci. 25, 4706-4718; Wang, J.M., Singh, C., Liu, L., Irwin, R.W., Chen, S., Chung, E.J., Thompson, R.F., Brinton, R.D., 2010. Allopregnanolone reverses neurogenic and cognitive deficits in mouse model of Alzheimer's disease. Proc. Natl. Acad. Sci. U. S. A. 107, 6498-6503). Herein, we determined efficacy of APα to restore neural progenitor cell survival and associative learning and memory subsequent to AD pathology in male 3xTgAD mice and their nontransgenic (nonTg) counterparts. APα significantly increased survival of bromodeoxyuridine positive (BrdU+) cells and hippocampal-dependent associative learning and memory in 3xTgAD mice in the presence of intraneuronal amyloid beta (Aß) whereas APα was ineffective subsequent to development of extraneuronal Aß plaques. Restoration of hippocampal-dependent associative learning was maximal by the first day and sustained throughout behavioral training. Learning and memory function in APα-treated 3xTgAD mice was 100% greater than vehicle-treated and comparable to maximal normal nonTg performance. In aged 15-month-old nonTg mice, APα significantly increased survival of bromodeoxyuridine-positive cells and hippocampal-dependent associative learning and memory. Results provide preclinical evidence that APα promoted survival of newly generated cells and restored cognitive performance in the preplaque phase of AD pathology and in late-stage normal aging.

Allopregnanolone reverses neurogenic and cognitive deficits in mouse model of Alzheimer's disease.

Our previous analyses showed that allopregnanolone (APalpha) significantly increased proliferation of rodent and human neural progenitor cells in vitro. In this study, we investigated the efficacy of APalpha to promote neurogenesis in the hippocampal subgranular zone (SGZ), to reverse learning and memory deficits in 3-month-old male triple transgenic mouse model of Alzheimer's (3xTgAD) and the correlation between APalpha-induced neural progenitor cell survival and memory function in 3xTgAD mice. Neural progenitor cell proliferation was determined by unbiased stereological analysis of BrdU incorporation and survival determined by FACS for BrdU+ cells. Learning and memory function was assessed using the hippocampal-dependent trace eye-blink conditioning paradigm. At 3 months, basal level of BrdU+ cells in the SGZ of 3xTgAD mice was significantly lower relative to non-Tg mice, despite the lack of evident AD pathology. APalpha significantly increased, in a dose-dependent manner, BrdU+ cells in SGZ in 3xTgAD mice and restored SGZ proliferation to normal magnitude. As with the deficit in proliferation, 3xTgAD mice exhibited deficits in learning and memory. APalpha reversed the cognitive deficits to restore learning and memory performance to the level of normal non-Tg mice. In 3xTgAD mice, APalpha-induced survival of neural progenitors was significantly correlated with APalpha-induced memory performance. These findings suggest that early neurogenic deficits, which were evident before immunodetectable Abeta, may contribute to the cognitive phenotype of AD, and that APalpha could serve as a regenerative therapeutic to prevent or delay neurogenic and cognitive deficits associated with mild cognitive impairment and Alzheimer's disease.

The 69 kDa Escherichia coli maltodextrin glucosidase does not get encapsulated underneath GroES and folds through trans mechanism during GroEL/GroES-assisted folding.

Escherichia coli chaperonin GroEL and GroES assist in folding of a wide variety of substrate proteins in the molecular mass range of approximately 50 kDa, using cis mechanism, but limited information is available on how they assist in folding of larger proteins. Considering that the central cavity of GroEL can accommodate a non-native protein of approximately 60 kDa, it is important to study the GroEL-GroES-assisted folding of substrate proteins that are large enough for cis encapsulation. In this study, we have reported the mechanism of GroEL/GroES-assisted in vivo and in vitro folding of a 69 kDa monomeric E. coli protein maltodextrin glucosidase (MalZ). Coexpression of GroEL and GroES in E. coli causes a 2-fold enhancement of exogenous MalZ activity in vivo. In vitro, GroEL and GroES in the presence of ATP give rise to a 7-fold enhancement in MalZ refolding. Neither GroEL nor single ring GroEL (SR1) in the presence or absence of ATP could enhance the in vitro folding of MalZ. GroES could not encapsulate GroEL-bound MalZ. All these experimental findings suggested that GroEL/GroES-assisted folding of MalZ followed trans mechanism, whereas denatured MalZ and GroES bound to the opposite rings of a GroEL molecule.