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Background: Acute myeloid leukemia (AML) is a hematological malignancy that remains a therapeutic challenge due to the high incidence of disease relapse. To better understand resistance mechanisms and identify novel therapies, robust preclinical models mimicking the bone marrow (BM) microenvironment are needed. This study aimed to achieve an automated fabrication process of a three-dimensional (3D) AML disease model that recapitulates the 3D spatial structure of the BM microenvironment and applies to drug screening and investigational studies., Methods: To build this model, we investigated a unique class of tetramer peptides with an innate ability to self-assemble into stable hydrogel. An automated robotic bioprinting process was established to fabricate a 3D BM (niche-like) multicellular AML disease model comprised of leukemia cells and the BM's stromal and endothelial cellular fractions. In addition, monoculture and dual-culture models were also fabricated. Leukemia cell compatibility, functionalities (in vitro and in vivo), and drug assessment studies using our model were performed. In addition, RNAseq and gene expression analysis using TaqMan arrays were also performed on 3D cultured stromal cells and primary leukemia cells., Results: The selected peptide hydrogel formed a highly porous network of nanofibers with mechanical properties similar to the BM extracellular matrix. The robotic bioprinter and the novel quadruple coaxial nozzle enabled the automated fabrication of a 3D BM niche-like AML disease model with controlled deposition of multiple cell types into the model. This model supported the viability and growth of primary leukemic, endothelial, and stromal cells and recapitulated cell-cell and cell-ECM interactions. In addition, AML cells in our model possessed quiescent characteristics with improved chemoresistance attributes, resembling more the native conditions as indicated by our in vivo results. Moreover, the whole transcriptome data demonstrated the effect of 3D culture on enhancing BM niche cell characteristics. We identified molecular pathways upregulated in AML cells in our 3D model that might contribute to AML drug resistance and disease relapse., Conclusions: Our results demonstrate the importance of developing 3D biomimicry models that closely recapitulate the in vivo conditions to gain deeper insights into drug resistance mechanisms and novel therapy development. These models can also improve personalized medicine by testing patient-specific treatments., (© 2023. The Author(s).)

During the last years, the application of next-generation sequencing (NGS) technologies to search for specific genetic markers has become a crucial method for the characterization of microbial communities. Illumina MiSeq, likely the most widespread NGS platform for metabarcoding experiments and taxonomic classification, allows processing shorter reads than the classical SANGER sequencing method and therefore requires specific primer pairs that produce shorter amplicons. Specifically, for the analysis of the commonly studied Prochlorococcus and Synechococcus communities, the petB marker gene has recently stood out as able to provide deep coverage to determine the microdiversity of the community. However, current petB primer set produce a 597 bp amplicon that is not suitable for MiSeq chemistry. Here, we designed and tested a petB primer pair that targets both Prochlorococcus and Synechococcus communities producing an appropriate amplicon to be used with state-of-the-art Illumina MiSeq. This new primer set allows the classification of both groups to a low taxonomic level and is therefore suitable for high throughput experiments using MiSeq technologies, therefore constituting a useful, novel tool to facilitate further studies on Prochlorococcus and Synechococcus communities. •This work describes the de novo design of a Prochlorococcus and Synechococcus -specific petB primer pair, allowing the characterization of both populations to a low taxonomic level.•This primer pair is suitable for widespread Illumina MiSeq sequencing technologies.• petB was confirmed as an adequate target for the characterization of both picocyanobacteria., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Published by Elsevier B.V.)

The introduction and establishment of exotic species often result in significant changes in recipient communities and their associated ecosystem services. However, usually the magnitude and direction of the changes are difficult to quantify because there is no pre-introduction data. Specifically, little is known about the effect of marine exotic macrophytes on organic carbon sequestration and storage. Here, we combine dating sediment cores ( 210 Pb) with sediment eDNA fingerprinting to reconstruct the chronology of pre- and post-arrival of the Red Sea seagrass Halophila stipulacea spreading into the Eastern Mediterranean native seagrass meadows. We then compare sediment organic carbon storage and burial rates before and after the arrival of H. stipulacea and between exotic (H. stipulacea) and native (C. nodosa and P. oceanica) meadows since the time of arrival following a Before-After-Control-Impact (BACI) approach. This analysis revealed that H. stipulacea arrived at the areas of study in Limassol (Cyprus) and West Crete (Greece) in the 1930s and 1970s, respectively. Average sediment organic carbon after the arrival of H. stipulacea to the sites increased in the exotic meadows twofold, from 8.4 ± 2.5 g C org  m -2  year -1 to 14.7 ± 3.6 g C org  m -2  year -1 , and, since then, burial rates in the exotic seagrass meadows were higher than in native ones of Cymodocea nodosa and Posidonia oceanica. Carbon isotopic data indicated a 50% increase of the seagrass contribution to the total sediment C org pool since the arrival of H. stipulacea. Our results demonstrate that the invasion of H. stipulacea may play an important role in maintaining the blue carbon sink capacity in the future warmer Mediterranean Sea, by developing new carbon sinks in bare sediments and colonizing areas previously occupied by the colder thermal affinity P. oceanica., (© 2021 John Wiley & Sons Ltd.)

The role of the microbiome in sustaining seagrasses has recently been highlighted. However, our understanding of the seagrass microbiome lacks behind that of other organisms. Here, we analyse the endophytic and total bacterial communities of leaves, rhizomes, and roots of six Red Sea seagrass species and their sediments. The structure of seagrass bacterial communities revealed that the 1% most abundant OTUs accounted for 87.9% and 74.8% of the total numbers of reads in sediment and plant tissue samples, respectively. We found taxonomically distinct bacterial communities in vegetated and bare sediments. Yet, our results suggest that lifestyle (i.e. free-living or host-association) is the main driver of bacterial community composition. Seagrass bacterial communities were tissue- and species-specific and differed from those of surrounding sediments. We identified OTUs belonging to genera related to N and S cycles in roots, and members of Actinobacteria, Bacteroidetes, and Firmicutes phyla as particularly enriched in root endosphere. The finding of highly similar OTUs in well-defined sub-clusters by network analysis suggests the co-occurrence of highly connected key members within Red Sea seagrass bacterial communities. These results provide key information towards the understanding of the role of microorganisms in seagrass ecosystem functioning framed under the seagrass holobiont concept., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Obese individuals without metabolic comorbidities are categorized as metabolically healthy obese (MHO). MicroRNAs (miRNAs) may be implicated in MHO. This cross-sectional study explores the link between circulating miRNAs and the main components of metabolic syndrome (MetS) in the context of obesity. We also examine oxidative stress biomarkers in MHO vs. metabolically unhealthy obesity (MUO). We analysed 3536 serum miRNAs in 20 middle-aged obese individuals: 10 MHO and 10 MUO. A total of 159 miRNAs were differentially expressed, of which, 72 miRNAs (45.2%) were higher and 87 miRNAs (54.7%) were lower in the MUO group. In addition, miRNAs related to insulin signalling and lipid metabolism pathways were upregulated in the MUO group. Among these miRNAs, hsa-miR-6796-5p and hsa-miR-4697-3p, which regulate oxidative stress, showed significant correlations with glucose, triglycerides, HbA1c and HDLc. Our results provide evidence of a pattern of differentially expressed miRNAs in obesity according to MetS, and identify those related to insulin resistance and lipid metabolism pathways.

Aeolian prokaryotic communities (APC) are important components of bioaerosols that are transported freely or attached to dust particles suspended in the atmosphere. Terrestrial and marine ecosystems are known to release and receive significant prokaryote loads into and from the surrounded atmospheric air. However, compared to terrestrial systems, there is a lack of microbial characterization of atmospheric dust over marine systems, such as the Red Sea, which receives significant terrestrial dust loads and is centrally located within the Global Dust Belt. Prokaryotic communities are likely to be particularly important in the Global Dust Belt, the area between the west coast of North Africa and Central Asia that supports the highest dust fluxes on the planet. Here we characterize the diversity and richness of the APC over the Red Sea ecosystem, the only sea fully contained within the Global Dust Belt. MiSeq sequencing was used to target 16S ribosomal DNA of two hundred and forty aeolian dust samples. These samples were collected at ∼7.5 m high above the sea level at coastal and offshore sampling sites over a 2-year period (2015-2017). The sequencing outcomes revealed that the APC in the atmospheric dust is dominated by Proteobacteria (42.69%), Firmicutes (41.11%), Actinobacteria, (7.69%), and Bacteroidetes (3.49%). The dust-associated prokaryotes were transported from different geographical sources and found to be more diverse than prokaryotic communities of the Red Sea surface water. Marine and soil originated prokaryotes were detected in APC. Hence, depending on the season, these groups may have traveled from other distant sources during storm events in the Red Sea region, where the APC structure is influenced by the origin and the concentration of aeolian dust particles. Accordingly, further studies of the impact of atmospheric organic aerosols on the recipient environments are required., (Copyright © 2020 Aalismail, Díaz-Rúa, Ngugi, Cusack and Duarte.)

Studies focusing on marine macrophyte metabarcoding from environmental samples are scarce, due to the lack of a universal barcode for these taxa, and to their poor representation in DNA databases. Here, we searched for a short barcode able to identify marine macrophytes from tissue samples; then, we created a DNA reference library which was used to identify macrophytes in eDNA from coastal sediments. Barcoding of seagrasses, mangroves and marine macroalgae (Chlorophyta, Rhodophyta and Phaeophyceae) was tested using 18 primer pairs from six barcoding genes: the plant barcodes rbcL, matK and trnL, plus the genes ITS2, COI and 18S. The 18S gene showed the highest universality among marine macrophytes, amplifying 95%-100% of samples; amplification performance of the other barcodes was limited. Taxonomy was assigned using a phylogeny-based approach to create an 18S DNA reference library. Macrophyte tissue sequences were accurately identified within their phyla (88%), order (76%), genus (71%) and species (23%). Nevertheless, out of 86 macrophytes tested, only 48% and 15% had a reference sequence at genus and at species level, respectively. Identification at these levels can be improved by more inclusive reference libraries. Using the 18S mini-barcode and the reference library, we recovered eDNA from 21 marine macrophytes in sediments, demonstrating the barcode's ability to trace primary producers that contribute to blue carbon. We expect this barcode to also be useful for other ecological questions, such as tracing macro primary producers in marine food webs., (© 2020 John Wiley & Sons Ltd.)

Atmospheric transport is a major vector for the long-range transport of microbial communities, maintaining connectivity among them and delivering functionally important microbes, such as pathogens. Though the taxonomic diversity of aeolian microorganisms is well characterized, the genomic functional traits underpinning their survival during atmospheric transport are poorly characterized. Here we use functional metagenomics of dust samples collected on the Global Dust Belt to initiate a Gene Catalogue of Aeolian Microbiome (GCAM) and explore microbial genetic traits enabling a successful aeolian lifestyle in Aeolian microbial communities. The GCAM reported here, derived from ten aeolian microbial metagenomes, includes a total of 2,370,956 non-redundant coding DNA sequences, corresponding to a yield of ~31 × 10 6 predicted genes per Tera base-pair of DNA sequenced for the aeolian samples sequenced. Two-thirds of the cataloged genes were assigned to bacteria, followed by eukaryotes (5.4%), archaea (1.1%), and viruses (0.69%). Genes encoding proteins involved in repairing UV-induced DNA damage and aerosolization of cells were ubiquitous across samples, and appear as fundamental requirements for the aeolian lifestyle, while genes coding for other important functions supporting the aeolian lifestyle (chemotaxis, aerotaxis, germination, thermal resistance, sporulation, and biofilm formation) varied among the communities sampled.

Exposure to polycyclic aromatic carbons (PAHs) poses a growing risk to coral reefs due to increasing shipping and petroleum extraction in tropical waters. Damaging effects of specific PAHs can be further enhanced by the presence of ultraviolet radiation, known as phototoxicity. We tested phototoxic effects of the PAHs anthracene and phenanthrene on larvae of the scleractinian coral Acropora tenuis in the presence and absence of UVA (320-400 nm). Activity of superoxide dismutase (SOD) enzyme was reduced by anthracene while phenanthrene and UVA exposure did not have any effect. Gene expression of MnSod remained constant across all treatments. The genes Catalase, Hsp70 and Hsp90 showed increased expression levels in larvae exposed to anthracene, but not phenanthrene. Gene expression of p53 was upregulated in the presence of UVA, but downregulated when exposed to PAHs. The influence on stress-related biochemical pathways and gene expresson in A. tenuis larvae was considerably greater for anthracene than phenanthrene, and UVA-induced phototoxicity was only evident for anthracene. The combined effects of UVA and PAH exposure on larval survival and metamorphosis paralleled the sub-lethal stress responses, clearly highlighting the interaction of UVA on anthracene toxicity and ultimately the coral's development., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Intake of high-protein (HP) diets has increased over the last years, mainly due to their popularity for body weight control. Liver is the main organ handling ingested macronutrients and it is associated with the beginning of different pathologies. We aimed to deepen our knowledge on molecular pathways affected by long-term intake of an HP diet. We performed a transcriptome analysis on liver of rats chronically fed with a casein-rich HP diet and analyzed molecular parameters related to liver injury. Chronic increase in the dietary protein/carbohydrate ratio up-regulated processes related with amino acid uptake/metabolism and lipid synthesis, promoting a molecular environment indicative of hepatic triacylglycerol (TG) deposition. Moreover, changes in expression of genes involved in acid-base maintenance and oxidative stress indicate alterations in the pH balance due to the high acid load of the diet, which has been linked to liver/health damage. Up-regulation of immune-related genes was also observed. In concordance with changes at gene expression level, we observed increased liver TG content and increased serum markers of hepatic injury/inflammation (aspartate transaminase, C-reactive protein and TNF-alpha). Moreover, the HP diet strongly increased hepatic mRNA and protein levels of HSP90, a marker of liver injury. Thus, we show for the first time that long-term consumption of an HP diet, resulting in a high acid load, results in a hepatic transcriptome signature reflecting increased TG deposition and increased signs of health risk (increased inflammation, alterations in the acid-base equilibrium and oxidative stress). Persistence of this altered metabolic status could have unhealthy consequences., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Scope: To analyse the usefulness of isolated human peripheral blood mononuclear cells (PBMC) to rapidly/easily reflect n-3 long-chain polyunsaturated fatty acid (LCPUFA) effects on lipid metabolism/inflammation gene profile, and evaluate if these effects are body mass index (BMI) dependent., Methods and Results: PBMC from normoweight (NW) and overweight/obese (OW/OB) subjects were incubated with physiological doses of docosahexaenoic (DHA), eicosapentaenoic acid (EPA), or their combination. PBMC reflected increased beta-oxidation-like capacity (CPT1A expression) in OW/OB but only after DHA treatment. However, insensitivity to n-3 LCPUFA was evident in OW/OB for lipogenic genes: both PUFA diminished FASN and SREBP1C expression in NW, but no effect was observed for DHA in PBMC from high-BMI subjects. This insensitivity was also evident for inflammation gene profile: all treatments inhibited key inflammatory genes in NW; nevertheless, no effect was observed in OW/OB after DHA treatment, and EPA effect was impaired. SLC27A2, IL6 and TNFα PBMC expression analysis resulted especially interesting to determine obesity-related n-3 LCPUFA insensitivity., Conclusion: A PBMC-based human in vitro system reflects n-3 LCPUFA effects on lipid metabolism/inflammation which is impaired in OW/OB. These results confirm the utility of PBMC ex vivo systems for bioactive-compound screening to promote functional food development and to establish appropriate dietary strategies for obese population., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Research on biomarkers that provide early information about the development of future metabolic alterations is an emerging discipline. Gene expression analysis in peripheral blood mononuclear cells (PBMC) is a promising tool to identify subjects at risk of developing diet-related diseases., Objective: We analysed PBMC expression of key energy homeostasis-related genes in a time-course analysis in order to find out early markers of metabolic alterations due to sustained intake of high-fat (HF) and high-protein (HP) diets., Design: We administered HF and HP diets (4 months) to adult Wistar rats in isocaloric conditions to a control diet, mainly to avoid overweight associated with the intake of hyperlipidic diets and, thus, to be able to characterise markers of metabolically obese normal-weight (MONW) syndrome. PBMC samples were collected at different time points of dietary treatment and expression of relevant energy homeostatic genes analysed by real-time reverse transcription-polymerase chain reaction. Serum parameters related with metabolic syndrome, as well as fat deposition in liver, were also analysed., Results: The most outstanding results were those obtained for the expression of the lipolytic gene carnitine palmitoyltransferase 1a ( Cpt1a ). Cpt1a expression in PBMC increased after only 1 month of exposure to both unbalanced diets, and this increased expression was maintained thereafter. Interestingly, in the case of the HF diet, Cpt1a expression was altered even in the absence of increased body weight but correlated with alterations such as higher insulin resistance, alteration of serum lipid profile and, particularly, increased fat deposition in liver, a feature characteristic of metabolic syndrome, which was even observed in animals fed with HP diet., Conclusions: We propose Cpt1a gene expression analysis in PBMC as an early biomarker of metabolic alterations associated with MONW phenotype due to the intake of isocaloric HF diets, as well as a marker of increased risk of metabolic diseases associated with the intake of HF or HP diets., Competing Interests: and funding The authors declare no conflict of interest.

Peripheral blood mononuclear cells (PBMCs) are accessible in humans, and their gene expression pattern was shown to reflect overall physiological response of the body to a specific stimulus, such as diet. We aimed to study the impact of sustained intake (4months) of diets with an unbalanced macronutrient proportion (rich in fat or protein) administered isocalorically to a balanced control diet, as physiological stressors on PBMC whole-genome gene expression in rats, to better understand the effects of these diets on metabolism and health and to identify biomarkers of nutritional imbalance. Dietary macronutrient composition (mainly increased protein content) altered PBMC gene expression, with genes involved in immune response being the most affected. Intake of a high-fat (HF) diet decreased the expression of genes related to antigen recognition/presentation, whereas the high-protein (HP) diet increased the expression of these genes and of genes involved in cytokine signaling and immune system maturation/activation. Key energy homeostasis genes (mainly related to lipid metabolism) were also affected, reflecting an adaptive response to the diets. Moreover, HF diet feeding impaired expression of genes involved in redox balance regulation. Finally, we identified a common gene expression signature of 7 genes whose expression changed in the same direction in response to the intake of both diets. These genes, individually or together, constitute a potential risk marker of diet macronutrient imbalance. In conclusion, we newly show that gene expression analysis in PBMCs allows for detection of diet-induced physiological deviations that distinguish from a diet with a proper and equilibrated macronutrient composition., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objectives: Peripheral blood mononuclear cells (PBMC) constitute an easily obtainable blood cell fraction useful in nutrition and obesity studies. Our aim was to study the potential use of PBMC to reflect metabolic recovery associated with weight loss in rats., Methods: By real-time PCR, the fasting response of key energy homeostatic genes in PBMC samples of control and cafeteria-obese rats and of rats fed a control diet after the intake of a cafeteria diet (post-cafeteria model) was analyzed., Results: Fasting caused decreased mRNA expression of lipogenic (Fasn and Srebp1a) and adipogenic (Pparγ) genes in PBMC, whereas it increased the expression of the key beta-oxidation gene Cpt1a and the orexigenic gene Npy. Fasting response of the genes studied was impaired in cafeteria-obese animals but was recovered in post-cafeteria rats, which showed a significant body weight decrease and normalization of adipose and metabolic parameters. Npy expression analyzed in PBMC has been revealed to be especially useful as a marker of fasting sensitivity, as its fasting response is not affected by the age of the animals and it is recovered even after shorter time of exposure to a balanced diet., Conclusions: PBMC reflect homeostatic balance recovery associated with weight loss in obese animals, when reverting from a hyperlipidic to a control balanced diet., (© 2014 The Obesity Society.)

We have investigated the effects of long term intake of two unbalanced diets (rich in fat -HF- or protein -HP-) administered under isocaloric conditions to a control balanced diet (pair-feeding) to adult rats. Isocaloric intake of a HF diet did not affect the body weight but increased adiposity, liver-fat deposition, and induced insulin resistance. Gene expression changes in the liver and adipose tissue (increased lipolytic and decreased lipogenic gene expression) could try to compensate for increased adiposity. The HP diet decreased caloric intake, the body weight, the size of subcutaneous adipocytes, and circulating cholesterol. Higher insulin levels apparently not related to insulin resistance were observed. Changes at the gene expression level reflected an adaptation to lower diet carbohydrate content and to the use of amino acids as the energy source. The kidney size increased in HP-fed animals but serum creatinine was not affected. Circulating TNF-alpha levels were higher in both dietary models. Thus, a long-term increase in dietary fat proportion produces alterations related to metabolic syndrome even in the absence of increased body weight, whereas an increase in diet protein content reduces the body weight but alters metabolic parameters and kidney size which could be linked to an increased risk of suffering from different pathologies.

The increased intake of fat-rich foods is one of the causes of the increasing incidence of obesity. However, there are controversial data on the reversibility of diet-induced obesity and its metabolic complications when adopting a control energy-balanced diet. Our aim was to evaluate the ability to reset not only body weight but also metabolic disorders caused by a highly palatable high fat diet, cafeteria diet, administered to adult rats, when replaced by a control diet (post-cafeteria model). Four-months of cafeteria diet-feeding produced important metabolic alterations in comparison to a commercial purified high fat diet: a rapid, drastic increase in body weight, adiposity and related complications such as insulin resistance, decreased glucose tolerance and development of hepatic steatosis. At gene level, decreased lipogenic and increased lipolytic gene expression in key energy homeostatic tissues as a physiological adaptation to increased fat intake was observed. In addition, fasting response of serum parameters and of key genes in lipid metabolism was impaired in cafeteria-fed animals. Contrary to what we have previously described if cafeteria diet is administered early in life, when administered to adult animals, its replacement with a balanced diet is able to restore body weight. Cafeteria diet withdrawal also allows recovery from metabolic damage, gene expression regulation and fasting response, the degree of which is dependent on the time of exposure to the cafeteria diet. In conclusion, adherence to an ad libitum intake of a balanced standard diet can enable the recovery of healthy status in animals which were previously exposed to an unhealthy cafeteria diet in adult age., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Adiponutrin/PNPLA3 is a protein highly produced in adipose tissue whose expression is under tight nutritional regulation. It possesses lipogenic/lipolytic capacity and, although adiponutrin polymorphisms are related to obesity, its physiological role is not clear. To help clarify its role, we studied the effect of acute cold exposure on adiponutrin mRNA expression in different adipose tissues of lean/obese Zucker rats subjected to feeding/fasting/refeeding. The effect of cold on the expression of key lipogenic enzymes and on uncoupling protein-1 (UCP1) was evaluated in selected adipose depots. Adiponutrin mRNA levels were also determined in the adipose tissue of isoprenaline-treated rats and in cultured adipocytes treated with noradrenaline, isoprenaline and a selective β3-adrenoceptor (AR) agonist. Adiponutrin expression was strongly down-regulated by cold in the different adipose depots in lean animals, while this down-regulation was impaired in obese rats. Adiponutrin pattern of expression in response to cold correlated positively with that of the lipogenic enzymes and negatively with UCP1 expression. Acute intraperitoneal administration of isoprenaline also produced a decrease in adiponutrin expression in adipose tissue. In vitro data suggest that adiponutrin's inhibitory effect could be mediated, at least in part, by the sympathetic system via β1/β2-AR. In addition, improvement in metabolic parameters related to obesity in cold-exposed animals was related to an improvement in adiponutrin nutritional regulation. Thus, cold inhibition of adiponutrin expression in adipose tissue (which correlates with the response of lipogenic enzymes) supports a physiological role for this protein in lipogenesis. Moreover, alterations in adiponutrin expression and regulation in adipose tissue are related to obesity.