Building momentum through networks: Bioimaging across the Americas.

In September 2023, the two largest bioimaging networks in the Americas, Latin America Bioimaging (LABI) and BioImaging North America (BINA), came together during a 1-week meeting in Mexico. This meeting provided opportunities for participants to interact closely with decision-makers from imaging core facilities across the Americas. The meeting was held in a hybrid format and attended in-person by imaging scientists from across the Americas, including Canada, the United States, Mexico, Colombia, Peru, Argentina, Chile, Brazil and Uruguay. The aims of the meeting were to discuss progress achieved over the past year, to foster networking and collaborative efforts among members of both communities, to bring together key members of the international imaging community to promote the exchange of experience and expertise, to engage with industry partners, and to establish future directions within each individual network, as well as common goals. This meeting report summarises the discussions exchanged, the achievements shared, and the goals set during the LABIxBINA2023: Bioimaging across the Americas meeting.

Modeling hypercholesterolemia and vascular lipid accumulation in LDL receptor mutant zebrafish.

Elevated plasma LDL cholesterol is the dominant risk factor for the development of atherosclerosis and cardiovascular disease. Deficiency in the LDL receptor (LDLR) is a major cause of familial hypercholesterolemia in humans, and the LDLR knockout mouse is a major animal model of atherosclerosis. Here we report the generation and characterization of an ldlr mutant zebrafish as a new animal model to study hypercholesterolemia and vascular lipid accumulation, an early event in the development of human atherosclerosis. The ldlr mutant zebrafish were characterized by activated SREBP-2 pathway and developed moderate hypercholesterolemia when fed a normal diet. However, a short-term, 5-day feeding of ldlr mutant larvae with a high-cholesterol diet (HCD) resulted in exacerbated hypercholesterolemia and accumulation of vascular lipid deposits. Lomitapide, an inhibitor of apoB lipoprotein secretion, but not the antioxidant probucol, significantly reduced accumulation of vascular lipid deposits in HCD-fed ldlr mutant larvae. Furthermore, ldlr mutants were defective in hepatic clearance of lipopolysaccharides, resulting in reduced survival. Taken together, our data suggest that the ldlr knockout zebra-fish is a versatile model for studying the function of the LDL receptor, hypercholesterolemia, and related vascular pathology in the context of early atherosclerosis.

Betaglycan knock-down causes embryonic angiogenesis defects in zebrafish.

Angiogenesis is an essential requirement for embryonic development and adult homeostasis. Its deregulation is a key feature of numerous pathologies and many studies have shown that members of the transforming growth factor beta (TGF-ß) family of proteins play important roles in angiogenesis during development and disease. Betaglycan (BG), also known as TGF-ß receptor type III, is a TGF-ß coreceptor essential for mice embryonic development but its role in angiogenesis has not been described. We have cloned the cDNA encoding zebrafish BG, a TGF-ß-binding membrane proteoglycan that showed a dynamic expression pattern in zebrafish embryos, including the notochord and cells adjacent to developing vessels. Injection of antisense morpholinos decreased BG protein levels and morphant embryos exhibited impaired angiogenesis that was rescued by coinjection with rat BG mRNA. In vivo time-lapse microscopy revealed that BG deficiency differentially affected arterial and venous angiogenesis: morphants showed impaired pathfinding of intersegmental vessels migrating from dorsal aorta, while endothelial cells originating from the caudal vein displayed sprouting and migration defects. Our results reveal a new role for BG during embryonic angiogenesis in zebrafish, which has not been described in mammals and pose interesting questions about the molecular machinery regulating angiogenesis in different vertebrates. genesis 53:583-603, 2015. © 2015 Wiley Periodicals, Inc.

Label-free fluorescence microscopy: revisiting the opportunities with autofluorescent molecules and harmonic generations as biosensors and biomarkers for quantitative biology.

Over the past decade, the utilization of advanced fluorescence microscopy technologies has presented numerous opportunities to study or re-investigate autofluorescent molecules and harmonic generation signals as molecular biomarkers and biosensors for in vivo cell and tissue studies. The label-free approaches benefit from the endogenous fluorescent molecules within the cell and take advantage of their spectroscopy properties to address biological questions. Harmonic generation can be used as a tool to identify the occurrence of fibrillar or lipid deposits in tissues, by using second and third-harmonic generation microscopy. Combining autofluorescence with novel techniques and tools such as fluorescence lifetime imaging microscopy (FLIM) and hyperspectral imaging (HSI) with model-free analysis of phasor plots has revolutionized the understanding of molecular processes such as cellular metabolism. These tools provide quantitative information that is often hidden under classical intensity-based microscopy. In this short review, we aim to illustrate how some of these technologies and techniques may enable investigation without the need to add a foreign fluorescence molecule that can modify or affect the results. We address some of the most important autofluorescence molecules and their spectroscopic properties to illustrate the potential of these combined tools. We discuss using them as biomarkers and biosensors and, under the lens of this new technology, identify some of the challenges and potentials for future advances in the field.

A nitroalkene derivative of salicylate alleviates diet-induced obesity by activating creatine metabolism and non-shivering thermogenesis.

Obesity-related type II diabetes (diabesity) has increased global morbidity and mortality dramatically. Previously, the ancient drug salicylate demonstrated promise for the treatment of type II diabetes, but its clinical use was precluded due to high dose requirements. In this study, we present a nitroalkene derivative of salicylate, 5-(2-nitroethenyl)salicylic acid (SANA), a molecule with unprecedented beneficial effects in diet-induced obesity (DIO). SANA reduces DIO, liver steatosis and insulin resistance at doses up to 40 times lower than salicylate. Mechanistically, SANA stimulated mitochondrial respiration and increased creatine-dependent energy expenditure in adipose tissue. Indeed, depletion of creatine resulted in the loss of SANA action. Moreover, we found that SANA binds to creatine kinases CKMT1/2, and downregulation CKMT1 interferes with the effect of SANA in vivo. Together, these data demonstrate that SANA is a first-in-class activator of creatine-dependent energy expenditure and thermogenesis in adipose tissue and emerges as a candidate for the treatment of diabesity.

Id gene regulation and function in the prosensory domains of the chicken inner ear: a link between Bmp signaling and Atoh1.

Bone morphogenetic proteins (Bmps) regulate the expression of the proneural gene Atoh1 and the generation of hair cells in the developing inner ear. The present work explored the role of Inhibitor of Differentiation genes (Id1-3) in this process. The results show that Id genes are expressed in the prosensory domains of the otic vesicle, along with Bmp4 and Bmp7. Those domains exhibit high levels of the phosphorylated form of Bmp-responding R-Smads (P-Smad1,5,8), and of Bmp-dependent Smad transcriptional activity as shown by the BRE-tk-EGFP reporter. Increased Bmp signaling induces the expression of Id1-3 along with the inhibition of Atoh1. Conversely, the Bmp antagonist Noggin or the Bmp-receptor inhibitor Dorsomorphin elicit opposite effects, indicating that Bmp signaling is necessary for Id expression and Atoh1 regulation in the otocyst. The forced expression of Id3 is sufficient to reduce Atoh1 expression and to prevent the expression of hair cell differentiation markers. Together, these results suggest that Ids are part of the machinery that mediates the regulation of hair cell differentiation exerted by Bmps. In agreement with that, during hair cell differentiation Bmp4 expression, P-Smad1,5,8 levels and Id expression are downregulated from hair cells. However, Ids are also downregulated from the supporting cells which contrarily to hair cells exhibit high levels of Bmp4 expression, P-Smad1,5,8, and BRE-tk-EGFP activity, suggesting that in these cells Ids escape from Bmp/Smad signaling. The differential regulation of Ids in time and space may underlie the multiple functions of Bmp signaling during sensory organ development.

Phasor-based hyperspectral snapshot microscopy allows fast imaging of live, three-dimensional tissues for biomedical applications.

Hyperspectral imaging is highly sought after in many fields including mineralogy and geology, environment and agriculture, astronomy and, importantly, biomedical imaging and biological fluorescence. We developed ultrafast phasor-based hyperspectral snapshot microscopy based on sine/cosine interference filters for biomedical imaging not feasible with conventional hyperspectral detection methods. Current approaches rely on slow spatial or spectral scanning limiting their application in living biological tissues, while faster snapshot methods such as image mapping spectrometry and multispectral interferometry are limited in spatial and/or spectral resolution, are computationally demanding, and imaging devices are very expensive to manufacture. Leveraging light sheet microscopy, phasor-based hyperspectral snapshot microscopy improved imaging speed 10-100 fold which, combined with minimal light exposure and high detection efficiency, enabled hyperspectral metabolic imaging of live, three-dimensional mouse tissues not feasible with other methods. As a fit-free method that does not require any a priori information often unavailable in complex and evolving biological systems, the rule of linear combinations of the phasor could spectrally resolve subtle differences between cell types in the developing zebrafish retina and spectrally separate and track multiple organelles in 3D cultured cells over time. The sine/cosine snapshot method is adaptable to any microscope or imaging device thus making hyperspectral imaging and fit-free analysis based on linear combinations broadly available to researchers and the public.

A novel nitroalkene vitamin E analogue inhibits the NLRP3 inflammasome and protects against inflammation and glucose intolerance triggered by obesity.

Chronic metabolic diseases, like obesity, type II diabetes and atherosclerosis often involve a low-grade and sterile systemic inflammatory state, in which activation of the pro-inflammatory transcription factor NF-kB and the NLRP3 inflammasome play a major role. It is well established that genetic inhibition of the NLRP3 inflammasome ameliorates acute and chronic inflammation. Indeed, accumulating experimental evidences in murine models and also in humans suggest that inhibition of the NLRP3 inflammasome might be a suitable approach to tackle the deleterious effects of chronic metabolic diseases. In this work, we explored our previously synthesized nitroalkene-Trolox™ derivative named NATx0, as a non-conventional anti-inflammatory strategy to treat chronic inflammatory diseases, such as obesity-induced glucose intolerance. We found that NATx0 inhibited NF-kB nuclear translocation and pro-inflammatory gene expression in macrophages in vitro. In addition, treatment with NATx0 prevented NLRP3 inflammasome activation after LPS/ATP stimulation in macrophages in vitro. When tested acutely in vivo, NATx0 inhibited neutrophil recruitment in zebrafish larvae, and also diminished IL-1ß production after LPS challenge in mice. Finally, when NATx0 was administered chronically to diet-induced obese mice, it decreased muscle tissue inflammation and glucose intolerance, leading to improved glucose homeostasis. In conclusion, we propose that this novel nitroalkene-Trolox derivative is a suitable tool to tackle acute and chronic inflammation in vitro and in vivo mainly due to inhibition of NF-kB/NLRP3 activation.

A novel nitroalkene-α-tocopherol analogue inhibits inflammation and ameliorates atherosclerosis in Apo E knockout mice.

BACKGROUND AND PURPOSE: Atherosclerosis is characterized by chronic low-grade inflammation with concomitant lipid accumulation in the arterial wall. Anti-inflammatory and anti-atherogenic properties have been described for a novel class of endogenous nitroalkenes (nitrated-unsaturated fatty acids), formed during inflammation and digestion/absorption processes. The lipid-associated antioxidant α-tocopherol is transported systemically by LDL particles including to the atheroma lesions. To capitalize on the overlapping and complementary salutary properties of endogenous nitroalkenes and α-tocopherol, we designed and synthesized a novel nitroalkene-α-tocopherol analogue (NATOH) to address chronic inflammation and atherosclerosis, particularly at the lesion sites. EXPERIMENTAL APPROACH: We synthesized NATOH, determined its electrophilicity and antioxidant capacity and studied its effects over pro-inflammatory and cytoprotective pathways in macrophages in vitro. Moreover, we demonstrated its incorporation into lipoproteins and tissue both in vitro and in vivo, and determined its effect on atherosclerosis and inflammatory responses in vivo using the Apo E knockout mice model. KEY RESULTS: NATOH exhibited similar antioxidant capacity to α-tocopherol and, due to the presence of the nitroalkenyl group, like endogenous nitroalkenes, it exerted electrophilic reactivity. NATOH was incorporated in vivo into the VLDL/LDL lipoproteins particles to reach the atheroma lesions. Furthermore, oral administration of NATOH down-regulated NF-κB-dependent expression of pro-inflammatory markers (including IL-1ß and adhesion molecules) and ameliorated atherosclerosis in Apo E knockout mice. CONCLUSIONS AND IMPLICATIONS: In toto, the data demonstrate a novel pharmacological strategy for the prevention of atherosclerosis based on a creative, natural and safe drug delivery system of a non-conventional anti-inflammatory compound (NATOH) with significant potential for clinical application.

Electrophilic nitroalkene-tocopherol derivatives: synthesis, physicochemical characterization and evaluation of anti-inflammatory signaling responses.

Inflammation plays a major role in the onset and development of chronic non-communicable diseases like obesity, cardiovascular diseases and cancer. Combined, these diseases represent the most common causes of death worldwide, thus development of novel pharmacological approaches is crucial. Electrophilic nitroalkenes derived from fatty acids are formed endogenously and exert anti-inflammatory actions by the modification of proteins involved in inflammation signaling cascades. We have developed novel nitroalkenes derived from α-tocopherol aiming to increase its salutary actions by adding anti-inflammatory properties to a well-known nutraceutical. We synthesized and characterized an α-tocopherol-nitroalkene (NATOH) and two hydrosoluble analogues derived from Trolox (NATxME and NATx0). We analyzed the kinetics of the Michael addition reaction of these compounds with thiols in micellar systems aiming to understand the effect of hydrophobic partition on the reactivity of nitroalkenes. We studied NATxME in vitro showing it exerts non-conventional anti-inflammatory responses by inducing Nrf2-Keap1-dependent gene expression and inhibiting the secretion of NF-κB dependent pro-inflammatory cytokines. NATxME was also effective in vivo, inhibiting neutrophil recruitment in a zebrafish model of inflammation. This work lays the foundation for the rational design of a new therapeutic strategy for the prevention and treatment of metabolic and inflammation-related diseases.