Zebrafish as a high throughput model for organ preservation and transplantation research.

Despite decades of effort, the preservation of complex organs for transplantation remains a significant barrier that exacerbates the organ shortage crisis. Progress in organ preservation research is significantly hindered by suboptimal research tools that force investigators to sacrifice translatability over throughput. For instance, simple model systems, such as single cell monolayers or co-cultures, lack native tissue structure and functional assessment, while mammalian whole organs are complex systems with confounding variables not compatible with high-throughput experimentation. In response, diverse fields and industries have bridged this experimental gap through the development of rich and robust resources for the use of zebrafish as a model organism. Through this study, we aim to demonstrate the value zebrafish pose for the fields of solid organ preservation and transplantation, especially with respect to experimental transplantation efforts. A wide array of methods were customized and validated for preservation-specific experimentation utilizing zebrafish, including the development of assays at multiple developmental stages (larvae and adult), methods for loading and unloading preservation agents, and the development of viability scores to quantify functional outcomes. Using this platform, the largest and most comprehensive screen of cryoprotectant agents (CPAs) was performed to determine their toxicity and efficiency at preserving complex organ systems using a high subzero approach called partial freezing (i.e., storage in the frozen state at -10°C). As a result, adult zebrafish cardiac function was successfully preserved after 5 days of partial freezing storage. In combination, the methods and techniques developed have the potential to drive and accelerate research in the fields of solid organ preservation and transplantation.

Zebrafish Models of Cardiac Disease: From Fortuitous Mutants to Precision Medicine.

Heart disease is the leading cause of death worldwide. Despite decades of research, most heart pathologies have limited treatments, and often the only curative approach is heart transplantation. Thus, there is an urgent need to develop new therapeutic approaches for treating cardiac diseases. Animal models that reproduce the human pathophysiology are essential to uncovering the biology of diseases and discovering therapies. Traditionally, mammals have been used as models of cardiac disease, but the cost of generating and maintaining new models is exorbitant, and the studies have very low throughput. In the last decade, the zebrafish has emerged as a tractable model for cardiac diseases, owing to several characteristics that made this animal popular among developmental biologists. Zebrafish fertilization and development are external; embryos can be obtained in high numbers, are cheap and easy to maintain, and can be manipulated to create new genetic models. Moreover, zebrafish exhibit an exceptional ability to regenerate their heart after injury. This review summarizes 25 years of research using the zebrafish to study the heart, from the classical forward screenings to the contemporary methods to model mutations found in patients with cardiac disease. We discuss the advantages and limitations of this model organism and introduce the experimental approaches exploited in zebrafish, including forward and reverse genetics and chemical screenings. Last, we review the models used to induce cardiac injury and essential ideas derived from studying natural regeneration. Studies using zebrafish have the potential to accelerate the discovery of new strategies to treat cardiac diseases.

lncExACT1 and DCHS2 Regulate Physiological and Pathological Cardiac Growth.

BACKGROUND: The heart grows in response to pathological and physiological stimuli. The former often precedes cardiomyocyte loss and heart failure; the latter paradoxically protects the heart and enhances cardiomyogenesis. The mechanisms underlying these differences remain incompletely understood. Although long noncoding RNAs (lncRNAs) are important in cardiac development and disease, less is known about their roles in physiological hypertrophy or cardiomyogenesis. METHODS: RNA sequencing was applied to hearts from mice after 8 weeks of voluntary exercise-induced physiological hypertrophy and cardiomyogenesis or transverse aortic constriction for 2 or 8 weeks to induce pathological hypertrophy or heart failure. The top lncRNA candidate was overexpressed in hearts with adeno-associated virus vectors and inhibited with antisense locked nucleic acid-GapmeRs to examine its function. Downstream effectors were identified through promoter analyses and binding assays. The functional roles of a novel downstream effector, dachsous cadherin-related 2 (DCHS2), were examined through transgenic overexpression in zebrafish and cardiac-specific deletion in Cas9-knockin mice. RESULTS: We identified exercise-regulated cardiac lncRNAs, called lncExACTs. lncExACT1 was evolutionarily conserved and decreased in exercised hearts but increased in human and experimental heart failure. Cardiac lncExACT1 overexpression caused pathological hypertrophy and heart failure; lncExACT1 inhibition induced physiological hypertrophy and cardiomyogenesis, protecting against cardiac fibrosis and dysfunction. lncExACT1 functioned by regulating microRNA-222, calcineurin signaling, and Hippo/Yap1 signaling through DCHS2. Cardiomyocyte DCHS2 overexpression in zebrafish induced pathological hypertrophy and impaired cardiac regeneration, promoting scarring after injury. In contrast, murine DCHS2 deletion induced physiological hypertrophy and promoted cardiomyogenesis. CONCLUSIONS: These studies identify lncExACT1-DCHS2 as a novel pathway regulating cardiac hypertrophy and cardiomyogenesis. lncExACT1-DCHS2 acts as a master switch toggling the heart between physiological and pathological growth to determine functional outcomes, providing a potentially tractable therapeutic target for harnessing the beneficial effects of exercise.

Risk factors determining adherence to tyrosine kinase inhibitors in chronic myeloid leukaemia.

OBJECTIVE: Tyrosine kinase inhibitors (TKIs) have successfully changed the natural course of chronic myeloid leukaemia (CML). Although they are highly effective drugs, their clinical benefit is conditioned by adherence. This study aims to analyse the adherence of CML patients treated with TKIs and to identify the main factors influencing their adherence to TKIs treatment. MATERIAL AND METHODS: An 8-month prospective, observational, multicentre study which included patients diagnosed with CML on treatment with TKIs attending the outpatient departments (OPD) of the Pharmacy Services of the participating hospitals. Adherence was assessed using two methods: the Simplified Medication Adherence Questionnaire (SMAQ) and the register of treatment dispensations from the OPDs. To analyse the predictors of adherence, a questionnaire was developed to report demographic and socio-economic information on the patients. RESULTS: A total of 130 patients enrolled in this study. Adherence rate was 56.9% (n = 74) among individuals, not conditioned by the type of drug used: imatinib (54.8%), nilotinib (63.6%) or dasatinib (54.3%) (p = 0.67). The patient educational level (p = 0.047) and employment status (p = 0.028) were predictors of non-adherence to treatment. CONCLUSIONS: Adherence is one of the most relevant parameters affecting the effectiveness of highly effective chronic treatments. Approximately half of our patients showed inadequate adherence to treatment with TKIs, with employment status and the individual's level of education emerging as the determining factors.

Actin dynamics and the Bmp pathway drive apical extrusion of proepicardial cells.

The epicardium, the outer mesothelial layer enclosing the myocardium, plays key roles in heart development and regeneration. During embryogenesis, the epicardium arises from the proepicardium (PE), a cell cluster that appears in the dorsal pericardium (DP) close to the venous pole of the heart. Little is known about how the PE emerges from the pericardial mesothelium. Using a zebrafish model and a combination of genetic tools, pharmacological agents and quantitative in vivo imaging, we reveal that a coordinated collective movement of DP cells drives PE formation. We found that Bmp signaling and the actomyosin cytoskeleton promote constriction of the DP, which enables PE cells to extrude apically. We provide evidence that cell extrusion, which has been described in the elimination of unfit cells from epithelia and the emergence of hematopoietic stem cells, is also a mechanism for PE cells to exit an organized mesothelium and fulfil their developmental fate to form a new tissue layer, the epicardium.

Effect of chronic lithium on mechanical sensitivity and trabecular bone loss induced by type-1 diabetes mellitus in mice.

Type-1 diabetes mellitus (T1DM) is a chronic condition characterized by long-term hyperglycemia that results in several complications such as painful peripheral neuropathy, bone deterioration, and increased risk of bone fractures. Lithium, a first-line therapy for bipolar disorder, has become an attractive agent for attenuating peripheral neuropathy and menopause-induced bone loss. Therefore, our aim was to determine the effect of chronic lithium treatment on mechanical hypersensitivity and trabecular bone loss induced by T1DM in mice. T1DM was induced in male C57BL/6J mice by intraperitoneal injection of streptozotocin (STZ, 50 mg/kg/day, for 5 consecutive days). 12 weeks after T1DM-induction, mice received a daily intraperitoneal injection of vehicle, 30 or 60 mg/kg lithium (as LiCl) for 6 weeks. Throughout the treatment period, blood glucose levels and mechanical sensitivity were evaluated every 2 weeks. After lithium treatment, the femur and L5 vertebra were harvested for microcomputed tomography (microCT) analysis. T1DM mice showed significant hyperglycemia, mechanical hypersensitivity, and significant trabecular bone loss as compared with the control group. Chronic lithium treatment did not revert the hindpaw mechanical hypersensitivity nor hyperglycemia associated to T1DM induced by STZ. In contrast, microCT analysis revealed that lithium reverted, in a dose-dependent manner, the loss of trabecular bone associated to T1DM induced by STZ at both the distal femur and L5 vertebra. Lithium treatment by itself did not affect any trabecular bone parameter in non-diabetic mice.

Active indole-3-acetic acid biosynthesis by the bacterium Azospirillum brasilense cultured under a biogas atmosphere enables its beneficial association with microalgae.

AIMS: This study assessed, at the physiological and molecular levels, the effect of biogas on indole-3-acetic acid (IAA) biosynthesis by Azospirillum brasilense as well as the impact of this bacterium during CO2 fixation from biogas by Chlorella vulgaris and Scenedesmus obliquus. METHODS AND RESULTS: IpdC gene expression, IAA production and the growth of A. brasilense cultured under air (control) and biogas (treatment) were evaluated. The results demonstrated that A. brasilense had a better growth capacity and IAA production (105.7 ± 10.3 µg ml-1 ) when cultured under biogas composed of 25% CO2  + 75% methane (CH4 ) with respect to the control (72.4 ± 7.9 µg ml-1 ), although the ipdC gene expression level was low under the stressful condition generated by biogas. Moreover, this bacterium was able to induce a higher cell density and CO2 fixation rate from biogas by C. vulgaris (0.27 ± 0.08 g l-1 d-1 ) and S. obliquus (0.22 ± 0.08 g l-1 d-1 ). CONCLUSIONS: This study demonstrated that A. brasilense has the capacity to grow and actively maintain its main microalgal growth-promoting mechanism when cultured under biogas and positively influence CO2 fixation from the biogas of C. vulgaris and S. obliquus. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings broaden research in the field of Azospirillum-microalga interactions and the prevalence of Azospirillum in environmental and ecological topics in addition to supporting the uses of plant growth-promoting bacteria to enhance biotechnological strategies for biogas upgrading.

Nucleus Accumbens Stimulation Modulates Inhibitory Control by Right Prefrontal Cortex Activation in Obsessive-Compulsive Disorder.

Inhibitory control is considered a compromised cognitive function in obsessive-compulsive (OCD) patients and likely linked to corticostriatal circuitry disturbances. Here, 9 refractory OCD patients treated with deep brain stimulation (DBS) were evaluated to address the dynamic modulations of large-scale cortical network activity involved in inhibitory control after nucleus accumbens (NAc) stimulation and their relationship with cortical thickness. A comparison of DBS "On/Off" states showed that patients committed fewer errors and exhibited increased intraindividual reaction time variability, resulting in improved goal maintenance abilities and proactive inhibitory control. Visual P3 event-related potentials showed increased amplitudes during Go/NoGo performance. Go and NoGo responses increased cortical activation mainly over the right inferior frontal gyrus and medial frontal gyrus, respectively. Moreover, increased cortical activation in these areas was equally associated with a higher cortical thickness within the prefrontal cortex. These results highlight the critical role of NAc DBS for preferentially modulating the neuronal activity underlying sustained speed responses and inhibitory control in OCD patients and show that it is triggered by reorganizing brain functions to the right prefrontal regions, which may depend on the underlying cortical thinning. Our findings provide updated structural and functional evidence that supports critical dopaminergic-mediated frontal-striatal network interactions in OCD.

Serum retinol levels are associated with cognitive function among community-dwelling older Mexican adults.

BACKGROUND: Vitamin A (VA) provides neuroprotection against oxidative stress and brain inflammation. VA deficiency (VAD) increases the risk of neurodegeneration in animal models, but results are inconclusive in humans: particularly in the older adult (OA) population which is at higher risk for micronutrient deficiencies and cognitive impairment. OBJECTIVE: To estimate the association between serum retinol levels and cognitive function (CF) in older Mexican adults. METHODS: Cross-sectional study with 803 adults aged ≥60 years with fasting blood sample from the southern region of Mexico, collected in summer of 2015. Low serum retinol (LSR) was defined if serum retinol ≤20 µg/dl. CF was evaluated using Semantic Verbal Fluency Test (SVFT). Mild cognitive impairment (MCI) was defined using normative values for SVFT. Linear and logistic regression models were used to estimate the association of LSR with CF and MCI, respectively. RESULTS: Prevalence of MCI was 9.35% and LSR 3.36%. OA with LSR evoked less words in the SVFT (ß = -2.8, CI95% -4.6, -0.9) and had higher probability of MCI (OR = 2.7, CI95% 0.9, 7.7). Associations remained significant when considered IL-6. CONCLUSION: Frequency of LSR in older Mexican adults was low, but strongly associated with MCI. This result suggests that VA plays a role in maintaining CF in the elderly population. Since VAD is a reversible condition, further studies are needed in order to identify the main causes of LSR and prevent MCI in populations which are at higher risk for malnutrition.Trial registration: ClinicalTrials.gov identifier: NCT04820465.

Is there an association between inflammatory markers and lower physical performance in older adults?

BACKGROUND: Maintenance of physical performance is essential for achievement of healthy aging. A few studies have explored the association between inflammatory markers and physical performance in older adults with inconclusive results. Our aim was to analyze the association of tumor necrosis factor-alpha (TNF-α), Interleukin-10 (IL-10), and C-reactive protein (CRP) with physical performance in a sample of older adults in rural settings of Mexico. METHODS: Our study comprised 307 community-dwelling older men and women who participated in the third wave of the Rural Frailty Study. We assessed the physical performance with the Short Physical Performance Battery (SPPB) and classified older adults as low performance if SPPB scored ≤8. Inflammatory markers were ascertained using serum by immunodetection methods. Logistic regression models were used to estimate the associations between inflammatory markers and physical performance. RESULTS: In comparison with the normal physical performance group, low physical performance individuals mainly were female (P <  0.01), older (P <  0.01), more illiterate (P = 0.02), more hypertensive (P < 0.01), fewer smokers (P = 0.02), and had higher CRP levels (P < 0.01). The logistic model results showed a significant association between the 3rd tertile of CRP and low physical performance (OR = 2.23; P = 0.03). IL-10 and TNF-α levels did not show a significant association. CONCLUSIONS: The results of this study were mixed, with a significant association of physical performance with higher CRP levels but nonsignificant with IL-10 and TNF-α. Further studies with improved designs are needed by incorporating a broader set of inflammatory markers.

Association between physical activity and physical and functional performance in non-institutionalized Mexican older adults: a cohort study.

BACKGROUND: Aging is an independent risk factor for deterioration in functional capacity. Some studies have reported that physical activity (PA) improves functional capacity and physical performance among older adults (OA). Thus the objective of the present study was to assess the longitudinal association between PA and functional and physical performance in non-institutionalized OA. METHODS: A longitudinal analysis using data from the Frailty, Dynapenia and Sarcopenia in Mexican adults (FRADYSMEX, by its Spanish acronym) cohort study was conducted. PA was assessed through the Community Healthy Activities Model Program for Seniors (CHAMPS) instrument. Functionality was measured with the Barthel index and the Lawton and Brody scale, while physical performance was measured with the Short Physical Performance Battery (SPPB). To evaluate the association between the level of PA and physical and functional performance as a continuous variable, a linear regression of mixed effects was performed. To assess PA and dependence in basic activities of the daily life (BADL), instrumental activities of the daily life (IADL), and low physical performance (PP), generalized estimation equation models [to compute odds ratios (OR) and 95% confidence intervals (95%CI)] were computed. RESULTS: Older people who performed moderate to vigorous-intensity PA had a lower risk of dependence in IADL (OR = 0.17; 95%CI: 0.10, 0.80) and lower risk of low PP (OR = 0.18; 95%CI: 0.11, 0.58) compared to those in lower categories of PA. CONCLUSIONS: Older adults living in the community who perform PA of moderate to vigorous intensity have a lower risk of dependence in BADL and IADL and have a lower risk of low PP.

RECK Variants are Associated with Clinicopathological Features and Decreased Susceptibility in Mexican Patients with Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of death worldwide. Down-regulation of the cysteine-rich reversion-inducing protein with Kazal motifs (RECK) has been confirmed in numerous human cancers and is clinically associated with metastasis. This study aims to explore, for the first time, the possible association of the RECK variants rs11788747 and rs10972727 with CRC susceptibility and clinicopathological features. DNA from 130 CRC patients and 130 healthy blood donors was analyzed. Identification of genetic variants was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology. Association was calculated using the odds ratio (OR) test and P values were adjusted using the Bonferroni test. Individuals carrying the G/G genotype for the rs11788747 variant showed a lower risk of colorectal cancer (OR 0.33; 95% CI 0.16-0.70; P = 0.006). Patients older than 50 years who carry the G/G genotype have a lower risk of CRC (OR 0.26; 95% CI 0.09-0.73; P = 0.019) and of developing advanced tumor-nodule-metastasis (TNM) stages (OR 0.23; 95% CI 0.09-0.54; P = 0.001). Individuals carrying the A/A genotype of the rs10972727 variant also showed decreased risk of CRC (OR 0.38; 95% CI 0.19-0.77; P = 0.011), and were associated with age (over 50 years), sex, advanced TNM stages, and tumor location in the colon. Our results suggest that the RECK variants studied here (rs11788747 and rs10972727) are associated with decreased CRC risk, TNM stages and tumor location.

Nanohybrid of Thymol and 2D Simonkolleite Enhances Inhibition of Bacterial Growth, Biofilm Formation, and Free Radicals.

Due to the current concerns against opportunistic pathogens and the challenge of antimicrobial resistance worldwide, alternatives to control pathogen growth are required. In this sense, this work offers a new nanohybrid composed of zinc-layered hydroxide salt (Simonkolleite) and thymol for preventing bacterial growth. Materials were characterized with XRD diffraction, FTIR and UV-Vis spectra, SEM microscopy, and dynamic light scattering. It was confirmed that the Simonkolleite structure was obtained, and thymol was adsorbed on the hydroxide in a web-like manner, with a concentration of 0.863 mg thymol/mg of ZnLHS. Absorption kinetics was described with non-linear models, and a pseudo-second-order equation was the best fit. The antibacterial test was conducted against Escherichia coli O157:H7 and Staphylococcus aureus strains, producing inhibition halos of 21 and 24 mm, respectively, with a 10 mg/mL solution of thymol-ZnLHS. Moreover, biofilm formation of Pseudomonas aeruginosa inhibition was tested, with over 90% inhibition. Nanohybrids exhibited antioxidant activity with ABTS and DPPH evaluations, confirming the presence of the biomolecule in the inorganic matrix. These results can be used to develop a thymol protection vehicle for applications in food, pharmaceutics, odontology, or biomedical industries.

Optimization of 5-CQA Extraction Conditions from Green Coffee By-Product (Coffea arabica) Using a Response-Surface Design and the Study of Its Extraction Kinetics.

To take advantage of the residues generated in the production of products from green coffee and due to the special interest in the compounds contained in the bean, a by-product obtained after the extraction of the oil was studied. The physical characterization of the green-coffee-bean by-product was carried out. Subsequently, the extraction of compound 5-CQA was carried out via leaching using central composition design 24 and evaluating factors such as temperature, time, solid/solvent ratio, and ethanol percentage, and its yield was quantified using HPLC. In addition, the response-surface methodology was used to maximize the efficiency of 5-CQA extraction and to perform the kinetic study. Yields of 59 ± 2 mg of 5-CQA/g from the by-product were obtained, and by selecting the best leaching conditions, the kinetic study was performed at 45, 60, and 75 °C, increasing the yield to a total of 61.8 ± 3 mg of 5-CQA/g. By applying the kinetic model of mass transfer, a fit of R2 > 0.97 was obtained, with KLa values between 0.266 and 0.320 min−1. This study showed an approach to optimize the 5-CQA extraction conditions, resulting in a simple, fast, reproducible, accurate, and low-cost method.

Hepatic Deficiency of Augmenter of Liver Regeneration Predisposes to Nonalcoholic Steatohepatitis and Fibrosis.

BACKGROUND AND AIMS: The augmenter of liver regeneration (ALR) protein is critical for lipid homeostasis and mitochondrial function. We investigated high-fat/high-carbohydrate (HF/HC) diet-induced nonalcoholic fatty liver disease (NAFLD) in wild-type (WT), hepatocyte-specific ALR-knockout (ALR-H-KO), and ALR-heterozygous (ALR-H-HET) mice. ALR was measured in serum of human nonalcoholic steatohepatitis (NASH) and NASH-induced cirrhosis (serum and liver). APPROACH AND RESULTS: HF/HC feeding decreased ALR expression in all groups of mice. The otherwise normal ALR-H-HET mice gained more weight and steatosis than WT mice when challenged metabolically with the HF/HC diet; ALR-H-KO mice gained the least weight and had the least steatosis. These findings were consistent with correspondingly increased triglycerides and cholesterol and altered expression of carnitine palmitoyltransferase 1a, sterol regulatory element-binding protein, acetyl coenzyme A carboxylase, and fatty acid synthase. All HF/HC-fed mice developed insulin resistance, the magnitude being lower in ALR-H-KO mice. HF/HC-fed ALR-H-HET mice were more resistant to glucose challenge than WT or ALR-H-KO mice. The frequency of tumor necrosis factor alpha-producing, interleukin 6 (IL6)-producing, and IL17-producing cells was greater in ALR-H-KO than ALR-H-HET and lowest in WT mice. HF/HC feeding did not increase their number in ALR-H-KO mice, and the increase in ALR-H-HET was greater than that in WT mice except for IL17 cells. Cluster of differentiation 25-positive (CD25+ ) forkhead box P3-positive CD4+ regulatory T-cell frequency was lower in ALR-H-HET than WT mice and further reduced in ALR-H-KO mice; HF/HC reduced regulatory T-cell frequency only in WT mice. HF/HC-fed ALR-H-HET, but not WT, mice developed fibrosis; and ALR-H-KO mice progressed to cirrhosis. White adipose tissue of HF/HC-fed ALR-deficient mice developed strong inflammation, indicating bidirectional interactions with the liver. Hepatic and serum ALR levels were significantly reduced in patients with NASH-cirrhosis. Serum ALR was also significantly lower in patients with NASH. CONCLUSIONS: Hepatic ALR deficiency may be a critical predisposing factor for aggressive NAFLD progression.

The systemic administration of neural stem cells expressing an inducible and soluble form of growth arrest specific 1 inhibits mammary gland tumor growth and the formation of metastases.

BACKGROUND AIMS: Metastasis to different organs is the major cause of death in breast cancer patients. The poor clinical prognosis and lack of successful treatments for metastatic breast cancer patients demand the development of new tumor-selective therapies. Thus, it is necessary to develop treatments capable of releasing therapeutic agents to both primary tumors and metastases that avoid toxic side effects in normal tissue, and neural stem cells are an attractive vehicle for tracking tumor cells and delivering anti-cancer agents. The authorspreviously demonstrated that a soluble form of growth arrest specific 1 (GAS1) inhibits the growth of triple-negative breast tumors and glioblastoma. METHODS: In this study, the authors engineered ReNcell CX (EMD Millipore, Temecula, CA, USA) neural progenitor cells to express truncated GAS1 (tGAS1) under a tetracycline/on inducible system using lentiviral vectors. RESULTS: Here the authors show that treatment with ReNcell-tGAS1 in combination with tetracycline decreased primary tumor growth and inhibited the formation of metastases in tumor-bearing mice by diminishing the phosphorylation of AKT and ERK1/2 in orthotopic mammary gland tumors. Moreover, the authors observed that ReNcell-tGAS1 prolonged the survival of 4T1 tumor-bearing mice. CONCLUSIONS: These data suggest that the delivery of tGAS1 by ReNcell cells could be an effective adjuvant for the treatment of triple-negative breast cancer.

A Diet High in Fat and Fructose Induces Early Hepatic Mitochondrial Aging.

ABSTRACT: To investigate the effect of high fructose diet on ultrastructure and content of hepatic mitochondria, we randomized 6-8 weeks old male C57Bl6/J mice to ad lib chow or high-fat-high-fructose (HF2) diet for 32 weeks. HF2-fed mice gained more weight, had higher plasma alanine aminotransferase, and fasting glucose levels and increased hepatic triglyceride content at all time points compared to chow-fed mice. HF2-fed mice had lower mitochondrial to nuclear DNA ratio compared to chow-fed mice. HF2-fed mice had lower average mitochondrial surface area and the number of mitochondria compared to chow-fed mice. HF2-fed mice had higher expression of the hepatic endoplasmic reticulum stress marker Chop, compared to chow-fed mice. A diet high in fat and fructose leads to enhanced hepatic mitochondrial aging, depletion, and dysfunction, which may be important determinants of nonalcoholic steatohepatitis pathogenesis.

Transient fibrosis resolves via fibroblast inactivation in the regenerating zebrafish heart.

In the zebrafish (Danio rerio), regeneration and fibrosis after cardiac injury are not mutually exclusive responses. Upon cardiac cryoinjury, collagen and other extracellular matrix (ECM) proteins accumulate at the injury site. However, in contrast to the situation in mammals, fibrosis is transient in zebrafish and its regression is concomitant with regrowth of the myocardial wall. Little is known about the cells producing this fibrotic tissue or how it resolves. Using novel genetic tools to mark periostin b- and collagen 1alpha2 (col1a2)-expressing cells in combination with transcriptome analysis, we explored the sources of activated fibroblasts and traced their fate. We describe that during fibrosis regression, fibroblasts are not fully eliminated but become inactivated. Unexpectedly, limiting the fibrotic response by genetic ablation of col1a2-expressing cells impaired cardiomyocyte proliferation. We conclude that ECM-producing cells are key players in the regenerative process and suggest that antifibrotic therapies might be less efficient than strategies targeting fibroblast inactivation.

Validation of an Analytical Method for the Simultaneous Determination of Hyaluronic Acid Concentration and Molecular Weight by Size-Exclusion Chromatography.

The hyaluronic acid (HA) global market growth can be attributed to its use in medical, cosmetic, and pharmaceutical applications; thus, it is important to have validated, analytical methods to ensure confidence and security of its use (and to save time and resources). In this work, a size-exclusion chromatography method (HPLC-SEC) was validated to determine the concentration and molecular distribution of HA simultaneously. Analytical curves were developed for concentration and molecular weight in the ranges of 100-1000 mg/L and 0.011-2.200 MDa, respectively. The HPLC-SEC method showed repeatability and reproducibility greater than 98% and limits of detection and quantification of 12 and 42 mg/L, respectively, and was successfully applied to the analysis of HA from a bacterial culture, as well as cosmetic, and pharmaceutical products.

Notch and Bmp signaling pathways act coordinately during the formation of the proepicardium.

BACKGROUND: The epicardium is the outer mesothelial layer of the heart. It encloses the myocardium and plays key roles in heart development and regeneration. It derives from the proepicardium (PE), cell clusters that appear in the dorsal pericardium (DP) close to the atrioventricular canal and the venous pole of the heart, and are released into the pericardial cavity. PE cells are advected around the beating heart until they attach to the myocardium. Bmp and Notch signaling influence PE formation, but it is unclear how both signaling pathways interact during this process in the zebrafish. RESULTS: Here, we show that the developing PE is influenced by Notch signaling derived from the endothelium. Overexpression of the intracellular receptor of notch in the endothelium enhances bmp expression, increases the number of pSmad1/5 positive cells in the DP and PE, and enhances PE formation. On the contrary, pharmacological inhibition of Notch1 impairs PE formation. bmp2b overexpression can rescue loss of PE formation in the presence of a Notch1 inhibitor, but Notch gain-of-function could not recover PE formation in the absence of Bmp signaling. CONCLUSIONS: Endothelial Notch signaling activates bmp expression in the heart tube, which in turn induces PE cluster formation from the DP layer.