Unraveling the evolutionary origin of the complex Nuclear Receptor Element (cNRE), a cis-regulatory module required for preferential expression in the atrial chamber.

Cardiac function requires appropriate proteins in each chamber. Atria requires slow myosin to act as reservoirs, while ventricles demand fast myosin for swift pumping. Myosins are thus under chamber-biased cis-regulation, with myosin gene expression imbalances leading to congenital heart dysfunction. To identify regulatory inputs leading to cardiac chamber-biased expression, we computationally and molecularly dissected the quail Slow Myosin Heavy Chain III (SMyHC III) promoter that drives preferential expression to the atria. We show that SMyHC III gene states are orchestrated by a complex Nuclear Receptor Element (cNRE) of 32 base pairs. Using transgenesis in zebrafish and mice, we demonstrate that preferential atrial expression is achieved by a combinatorial regulatory input composed of atrial activation motifs and ventricular repression motifs. Using comparative genomics, we show that the cNRE might have emerged from an endogenous viral element through infection of an ancestral host germline, revealing an evolutionary pathway to cardiac chamber-specific expression.

Arjunolic acid modulate pancreatic dysfunction by ameliorating pattern recognition receptor and canonical Wnt pathway activation in type 2 diabetic rats.

BACKGROUND: Arjunolic acid (AA) is a potent phytochemical with multiple therapeutics effects. In this study, AA is evaluated on type 2 diabetic (T2DM) rats to understand the mechanism of ß-cell linkage with Toll-like receptor 4 (TLR-4) and canonical Wnt signaling. However, its role in modulating TLR-4 and canonical Wnt/ß-catenin crosstalk on insulin signaling remains unclear during T2DM. Aim The current study is aimed to examine the potential role of AA on insulin signaling and TLR-4-Wnt crosstalk in the pancreas of type 2 diabetic rats. METHOD: Multiple methods were used to determine molecular cognizance of AA in T2DM rats, when treated with different dosage levels. Histopathological and histomorphometry analysis was conducted using masson trichrome and H&E stains. While, protein and mRNA expressions of TLR-4/Wnt and insulin signaling were assessed using automated Western blotting (jess), immunohistochemistry, and RT-PCR. RESULTS: Histopathological findings revealed that AA had reversed back the T2DM-induced apoptosis and necrosis caused to rats pancreas. Molecular findings exhibited prominent effects of AA in downregulating the elevated level of TLR-4, MyD88, NF-κB, p-JNK, and Wnt/ß-catenin by blocking TLR-4/MyD88 and canonical Wnt signaling in diabetic pancreas, while IRS-1, PI3K, and pAkt were all upregulated by altering the NF-κB and ß-catenin crosstalk during T2DM. CONCLUSION: Overall results, indicate that AA has potential to develop as an effective therapeutic in the treatment of T2DM associated meta-inflammation. However, future preclinical research at multiple dose level in a long-term chronic T2DM disease model is warranted to understand its clinical relevance in cardiometabolic disease.

Phytochemical and Safety Evaluations of Finger Lime, Mountain Pepper, and Tamarind in Zebrafish Embryos.

Plants play a pivotal role in drug discovery, constituting 50% of modern pharmacopeia. Many human diseases, including age-related degenerative diseases, converge onto common cellular oxidative stress pathways. This provides an opportunity to develop broad treatments to treat a wide range of diseases in the ageing population. Here, we characterize and assess the toxicological effects of finger lime (Citrus australasica), mountain pepper (Tasmannia lanceolata), and small-leaved tamarind (Diploglottis australis) extracts. The characterization demonstrates that these Australian native plants have antioxidant potential and, importantly, they have high concentrations of distinct combinations of different antioxidant classes. Using zebrafish larvae as a high-throughput pre-clinical in vivo toxicology screening model, our experiment effectively discriminates which of these extracts (and at what exposure levels) are suitable for development towards future therapies. The LC50-96h for finger lime and tamarind were >480 mg/L, and 1.70 mg/L for mountain pepper. Critically, this work shows that adverse effects are not correlated to the properties of these antioxidants, thus highlighting the need for combining characterization and in vivo screening to identify the most promising plant extracts for further development. Thus, we present a high-throughput pre-clinical screening that robustly tests natural plant products to utilize the diversity of antioxidant compounds for drug development.

Restoring the oxidative balance in age-related diseases - An approach in glaucoma.

As human life expectancy increases, age-related health issues including neurodegenerative diseases continue to rise. Regardless of genetic or environmental factors, many neurodegenerative conditions share common pathological mechanisms, such as oxidative stress, a hallmark of many age-related health burdens. In this review, we describe oxidative damage and mitochondrial dysfunction in glaucoma, an age-related neurodegenerative eye disease affecting 80 million people worldwide. We consider therapeutic approaches used to counteract oxidative stress in glaucoma, including untapped treatment options such as novel plant-derived antioxidant compounds that can reduce oxidative stress and prevent neuronal loss. We summarize the current pre-clinical models and clinical work exploring the therapeutic potential of a range of candidate plant-derived antioxidant compounds. Finally, we explore advances in drug delivery systems, particular those employing nanotechnology-based carriers which hold significant promise as a carrier for antioxidants to treat age-related disease, thus reviewing the key current state of all of the aspects required towards translation.

Arjunolic acid downregulates elevated blood sugar and pro-inflammatory cytokines in streptozotocin (STZ)-nicotinamide induced type 2 diabetic rats.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a worldwide health issue primarily due to failure of pancreatic ß-cells to release sufficient insulin. PURPOSE: The present work aimed to assess the antidiabetic potential of arjunolic acid (AA) isolated from Terminalia arjuna in type 2 diabetic rats. STUDY DESIGN: After extraction, isolation and purification, AA was orally administered to type 2 diabetic Sprague Dawley rats to investigate antidiabetic effect of AA. METHOD: T2DM was induced via single intraperitoneal injection of streptozotocin-nicotinamide (STZ-NIC) in adult male rats. After 10 days, fasting and random blood glucose (FBG and RBG), body weight (BW), food and water intake, serum C-peptide, insulin and glycated hemoglobin (HbA1c) was measured to confirm T2DM development. Dose dependent effects of orally administered AA (25 and 50 mg/kg/day) for 4 weeks was investigated by measuring BW variation, fasting and postprandial hyperglycemia, oral glucose tolerance test (OGTT), and levels of serum HbA1c, serum total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), high density lipoprotein (HDL), serum and pancreatic C-peptide, insulin, growth differentiation factor 15 (GDF-15), serum and pancreatic inflammatory cytokines. RESULTS: The oral administration of AA in preclinical model of T2DM significantly normalized FBG and RBG, restored BW, controlled polyphagia, polydipsia and glucose tolerance. In addition, AA notably reduced serum HbA1c, TC, TG, LDL with non-significant increase in HDL. On the other hand, significant increase in serum and pancreatic C-peptide and insulin was observed with AA treatment, while serum and pancreatic GDF-15 were non-significantly altered in AA treated diabetic rats. Moreover, AA showed dose dependent reduction in serum and pancreatic proinflammatory cytokines including TNF-α, IL-1ß and IL-6. CONCLUSION: For the first time our findings highlighted AA as a potential candidate in type 2 diabetic conditions.

Lauric acid ameliorates lipopolysaccharide (LPS)-induced liver inflammation by mediating TLR4/MyD88 pathway in Sprague Dawley (SD) rats.

BACKGROUND: Lipopolysaccharide (LPS) is an endotoxin that leads to inflammation in many organs, including liver. It binds to pattern recognition receptors, that generally recognise pathogen expressed molecules to transduce signals that result in a multifaceted network of intracellular responses ending up in inflammation. Aim In this study, we used lauric acid (LA), a constituent abundantly found in coconut oil to determine its anti-inflammatory role in LPS-induced liver inflammation in Sprague Dawley (SD) rats. METHOD: Male SD rats were divided into five groups (n = 8), injected with LPS and thereafter treated with LA (50 and 100 mg/kg) or vehicle orally for 14 days. After fourteen days of LA treatment, all the groups were humanely killed to investigate biochemical parameters followed by pro-inflammatory cytokine markers; tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1ß. Moreover, liver tissues were harvested for histopathological studies and evaluation of targeted protein expression with western blot and localisation through immunohistochemistry (IHC). RESULTS: The study results showed that treatment of LA 50 and 100 mg/kg for 14 days were able to reduce the elevated level of pro-inflammatory cytokines, liver inflammation, and downregulated the expression of TLR4/NF-κB mediating proteins in liver tissues. CONCLUSION: These findings suggest that treatment of LA has a protective role against LPS-induced liver inflammation in rats, thus, warrants further in-depth investigation through mechanistic approaches in different study models.

Clove Oil and AQUI-S Efficacy for Zebrafish Embryo, Larva, and Adult Anesthesia.

Since the use of the zebrafish Danio rerio genetic model organism within the scientific research community continues to grow rapidly, continued procedural refinement to support high-quality, reproducible research and improve animal welfare remains an important focus. As such, anesthesia remains one of the most frequent procedures conducted. Here, we compared the effectiveness of clove oil (active ingredient eugenol) and AQUI-S (active ingredient iso-eugenol) with the currently most commonly used tricaine/MS-222 (ethyl 3-aminobenzoate methanesulfonate) and benzocaine anesthesia. We focused on embryos (1 day postfertilization), larvae (5 days postfertilization), and adults (9-11 months) and for the first time used exposure times that are the most relevant in research settings by using zebrafish as a genetic model system. For each age, tricaine and benzocaine achieved the most reproducible, robust anesthesia with the quickest induction and recovery. For some experimental procedures, specific clove oil concentrations in embryos and larvae may represent suitable alternatives. Although different aquatic species at specific ages respond differentially to these agents, the systematic study of comparable effective dosages for procedures most commonly employed represent an important step toward refinement.

Developmental and adult characterization of secretagogin expressing amacrine cells in zebrafish retina.

Calcium binding proteins show stereotypical expression patterns within diverse neuron types across the central nervous system. Here, we provide a characterization of developmental and adult secretagogin-immunolabelled neurons in the zebrafish retina with an emphasis on co-expression of multiple calcium binding proteins. Secretagogin is a recently identified and cloned member of the F-hand family of calcium binding proteins, which labels distinct neuron populations in the retinas of mammalian vertebrates. Both the adult distribution of secretagogin labeled retinal neurons as well as the developmental expression indicative of the stage of neurogenesis during which this calcium binding protein is expressed was quantified. Secretagogin expression was confined to an amacrine interneuron population in the inner nuclear layer, with monostratified neurites in the center of the inner plexiform layer and a relatively regular soma distribution (regularity index > 2.5 across central-peripheral areas). However, only a subpopulation (~60%) co-labeled with gamma-aminobutyric acid as their neurotransmitter, suggesting that possibly two amacrine subtypes are secretagogin immunoreactive. Quantitative co-labeling analysis with other known amacrine subtype markers including the three main calcium binding proteins parvalbumin, calbindin and calretinin identifies secretagogin immunoreactive neurons as a distinct neuron population. The highest density of secretagogin cells of ~1800 cells / mm2 remained relatively evenly along the horizontal meridian, whilst the density dropped of to 125 cells / mm2 towards the dorsal and ventral periphery. Thus, secretagogin represents a new amacrine label within the zebrafish retina. The developmental expression suggests a possible role in late stage differentiation. This characterization forms the basis of functional studies assessing how the expression of distinct calcium binding proteins might be regulated to compensate for the loss of one of the others.

Fate bias during neural regeneration adjusts dynamically without recapitulating developmental fate progression.

BACKGROUND: Regeneration of neurons in the central nervous system is poor in humans. In other vertebrates neural regeneration does occur efficiently and involves reactivation of developmental processes. Within the neural retina of zebrafish, Müller glia are the main stem cell source and are capable of generating progenitors to replace lost neurons after injury. However, it remains largely unknown to what extent Müller glia and neuron differentiation mirror development. METHODS: Following neural ablation in the zebrafish retina, dividing cells were tracked using a prolonged labelling technique. We investigated to what extent extrinsic feedback influences fate choices in two injury models, and whether fate specification follows the histogenic order observed in development. RESULTS: By comparing two injury paradigms that affect different subpopulations of neurons, we found a dynamic adaptability of fate choices during regeneration. Both injuries followed a similar time course of cell death, and activated Müller glia proliferation. However, these newly generated cells were initially biased towards replacing specifically the ablated cell types, and subsequently generating all cell types as the appropriate neuron proportions became re-established. This dynamic behaviour has implications for shaping regenerative processes and ensuring restoration of appropriate proportions of neuron types regardless of injury or cell type lost. CONCLUSIONS: Our findings suggest that regenerative fate processes are more flexible than development processes. Compared to development fate specification we observed a disruption in stereotypical birth order of neurons during regeneration Understanding such feedback systems can allow us to direct regenerative fate specification in injury and diseases to regenerate specific neuron types in vivo.