Isolation of bacterial consortia with probiotic potential from the rumen of tropical calves.

Probiotics are live microorganisms that confer health benefits to their animal host by balancing the composition of its gastrointestinal microbiota and modulating its immune response. In this work, we studied bacterial consortia isolated from the rumen of 28- and 42-day-old calves to select those showing probiotic capacity. Consortia were characterized and their growth dynamics were determined in several growth media. The number of viable bacteria was larger in the Man, Rogosa and Sharpe broth (MRS) than in nutritive medium A (MNA) and the largest was for A3D42. Antibiotic susceptibility of bacterial consortia in MRS was higher than in MNA and the most susceptible samples were A1D28 and A3D42. In turn, A3D42 showed the highest tolerance to bile salts in MRS and MNA. Moreover, all bacterial consortia showed optimal growth at pH 5, 5.5, 6 and 7 in both media, while their temperature tolerance was higher in MRS. The antagonistic activity of bacterial consortia in MNA was higher than in MRS with A2D42 showing the best antagonistic activity for Pseudomona aureginosa (ATCC 9027) and Staphylococcus aureus (ATCC 6538) in MNA. Additionally, A1D42 and A2D42 in MRS and A3D42 in MNA had significant adhesion to mucins, and A1D42 in MRS had the highest. Regarding their species composition, all bacterial consortia in MRS belonged to the phylum Firmicutes, and the class Bacilli and bacterial consortia in MNA belonged to three phyla; Proteobacteria, Firmicutes, and Bacteroidetes. Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus fermentum, and Lactobacillus johnsonii were identified in all bacterial consortia in MRS broth. Based on these results, A1D42 and A3D42 grown in MRS showed the best potential as probiotics for calves, which could result in health benefits and improve their production.

Study on manipulation of ruminal fermentation using a bioelectrochemical system.

The purpose of this work was to develop a two-chamber bioelectrochemical cell to modify the metabolic activity of rumen microorganisms by applying an electric potential to the ruminal liquid. Carbohydrate fermentation changes were evaluated along with a molecular characterization by DNA sequencing of the ruminal microbial community. We observed that an electrochemical stimulation potential of 0.75 V enhanced basal acetate, propionate, and butyrate production by 71%, 86%, and 63%, respectively, with no detectable effects on grass substrate disappearance. The applied electric potential also led to changes in the volatile fatty acids production but not on the core microbiome.

Insulin resistance rewires the metabolic gene program and glucose utilization in human white adipocytes.

BACKGROUND: In obesity, adipose tissue dysfunction resulting from excessive fat accumulation leads to systemic insulin resistance (IR), the underlying alteration of Type 2 Diabetes. The specific pathways dysregulated in dysfunctional adipocytes and the extent to which it affects adipose metabolic functions remain incompletely characterized. METHODS: We interrogated the transcriptional adaptation to increased adiposity in association with insulin resistance in visceral white adipose tissue from lean men, or men presenting overweight/obesity (BMI from 19 to 33) and discordant for insulin sensitivity. In human adipocytes in vitro, we investigated the direct contribution of IR in altering metabolic gene programming and glucose utilization using 13C-isotopic glucose tracing. RESULTS: We found that gene expression associated with impaired glucose and lipid metabolism and inflammation represented the strongest association with systemic insulin resistance, independently of BMI. In addition, we showed that inducing IR in mature human white adipocytes was sufficient to reprogram the transcriptional profile of genes involved in important metabolic functions such as glycolysis, the pentose phosphate pathway and de novo lipogenesis. Finally, we found that IR induced a rewiring of glucose metabolism, with higher incorporation of glucose into citrate, but not into downstream metabolites within the TCA cycle. CONCLUSIONS: Collectively, our data highlight the importance of obesity-derived insulin resistance in impacting the expression of key metabolic genes and impairing the metabolic processes of glucose utilization, and reveal a role for metabolic adaptation in adipose dysfunction in humans.

Vasoinhibin Suppresses the Neurotrophic Effects of VEGF and NGF in Newborn Rat Primary Sensory Neurons.

BACKGROUND/AIMS: Studies on the biological actions of vasoinhibins have focused mainly on endothelial cells. However, there is incipient knowledge about how vasoinhibins affect the nervous system, even if the target cells and mechanisms of action involved in these effects are unknown. METHODS: In order to determine if neurons are direct targets of vasoinhibins, we examined cellular outcomes and the intracellular pathways involved in the neuronal actions of vasoinhibins using newborn rat dorsal root ganglion (DRG) neurons as a model system. RESULTS: Vascular endothelial growth factor (VEGF) or nerve growth factor (NGF) treatment for 48 h resulted in neurite outgrowth stimulation in both DRG cultured explants and isolated primary sensory neurons. Interestingly, a recombinant vasoinhibin containing the first 123 amino acids of human prolactin antagonized the VEGF- and NGF-induced stimulation of neurite outgrowth. Vasoinhibin significantly reduced the density of neurites in DRG explants and obliterated neuritogenesis in isolated DRG neurons in primary culture, supporting a direct neuronal effect of vasoinhibin. In cultures of isolated DRG cells, virtually all ß3-tubulin-labeled cells express TrkA, and the majority of these cells also express VEGFR2. Short-term VEGF or NGF treatment of DRG explants resulted in increased ERK1/2 and AKT phosphorylation, whereas incubation of DRG with the combination of either VEGF or NGF together with vasoinhibin resulted in blunted VEGF- or NGF-induced phosphorylation of both ERK1/2 and AKT. CONCLUSION: Our results show that primary sensory neurons are direct targets of vasoinhibin, and suggest that vasoinhibin inhibition of neurite outgrowth involves the disruption of ERK and AKT phosphorylation cascades.

Shock Wave-Induced Damage and Poration in Eukaryotic Cell Membranes.

Shock waves are known to permeabilize eukaryotic cell membranes, which may be a powerful tool for a variety of drug delivery applications. However, the mechanisms involved in shock wave-mediated membrane permeabilization are still poorly understood. In this study, the effects on both the permeability and the ultrastructural features of two human cell lineages were investigated after the application of underwater shock waves in vitro. Scanning Electron Microscopy of cells derived from a human embryo kidney (HEK)-293 and Michigan Cancer Foundation (MCF)-7 cells, an immortalized culture derived from human breast adenocarcinoma, showed a small amount of microvilli (as compared to control cells), the presence of hole-like structures, and a decrease in cell size after shock wave exposure. Interestingly, these effects were accompanied by the permeabilization of acid and macromolecular dyes and gene transfection. Trypan blue exclusion assays indicated that cell membranes were porated during shock wave treatment but resealed after a few seconds. Deformations of the cell membrane lasted for at least 5 min, allowing their observation in fixed cells. For each cell line, different shock wave parameters were needed to achieve cell membrane poration. This difference was correlated to successful gene transfection by shock waves. Our results demonstrate, for the first time, that shock waves induce transient micro- and submicrosized deformations at the cell membrane, leading to cell transfection and cell survival. They also indicate that ultrastructural analyses of cell surfaces may constitute a useful way to match the use of shock waves to different cells and settings.

PPAR Agonists Promote the Differentiation of Porcine Bone Marrow Mesenchymal Stem Cells into the Adipogenic and Myogenic Lineages.

PURPOSE: The aim of this work was to evaluate the effect of PPAR agonists on the differentiation and metabolic features of porcine mesenchymal stem cells induced to the adipogenic or myogenic lineages. METHODS: Bone marrow MSCs from neonate pigs were isolated and identified by cell proliferation, cell surface markers or the gene expression of stem cells (CD44, CD90, CD105 or Oct4 and Nanog, respectively). Cells were differentiated into adipose or muscle cells and treated with the PPAR agonists; adipogenic and myogenic differentiation was promoted by adding these compounds. The expression of PPARγ (an adipose marker) and MyoD1 and MyHC (muscle markers), metabolic changes and expression levels of metabolic enzymes involved in glycolysis, lipogenesis, lipolysis and the pentose phosphate pathway were tested by qPCR. RESULTS: MSCs from neonate pigs exhibited high proliferation and were positive for CD44, CD90 and CD105 markers and Oct4 and Nanog expression. The treatment that promoted the highest expression of PPARγ was 50 µM of conjugated linoleic acid (CLA) c9 t11 (6.44 ± 0.69-fold, p ≤ 0.0001) in the adipose differentiation, and upregulation of HX2, ACCAα, ATGL, LPL and G6DP (p ≤ 0.0001) and downregulation of PFK and ACCAß (p ≤ 0.0001) were found. For muscle differentiation, the best treatment was 50 µM of CLA c10 t12 (59.72 ± 4.72-fold, p ≤ 0.0001), and metabolic changes were upregulation of PFK, ACCAß, G6DP, CPT1 and PPARß/δ (p ≤ 0.0001), but no effect was observed with HX2 and ACCAα (p ≥ 0.05). CONCLUSIONS: Our results suggest that differentiated cells exhibit a typical cell lineage metabolism and higher efficiencies both in anabolism and catabolism.

Bovine (Bos taurus) Bone Marrow Mesenchymal Cell Differentiation to Adipogenic and Myogenic Lineages.

PURPOSE: We evaluated the effect of peroxisome proliferator-activated receptor (PPAR) agonists on the differentiation and metabolic features of bovine bone marrow-derived mesenchymal cells induced to adipogenic or myogenic lineages. METHODS: Cells isolated from 7-day-old calves were cultured in basal medium (BM). For adipogenic differentiation, cells were cultured for one passage in BM and then transferred to a medium supplemented with either rosiglitazone, telmisartan, sirtinol or conjugated c-9, t-11 linoleic acid; for myogenic differentiation, third-passage cells were added with either bezafibrate, telmisartan or sirtinol. The expression of PPARx03B3; (an adipogenic differentiation marker), myosin heavy chain (MyHC; a myogenic differentiation marker) and genes related to energy metabolism were measured by quantitative real-time PCR in a completely randomized design. RESULTS: For adipogenic differentiation, 20 µM telmisartan showed the highest PPARx03B3; expression (15.58 ± 0.62-fold, p < 0.0001), and differences in the expression of energy metabolism-related genes were found for hexokinase II, phosphofructokinase, adipose triglyceride lipase, acetyl-CoA carboxylase α(ACACα) and fatty acid synthase (p < 0.001), but not for ACACß (p = 0.4275). For myogenic differentiation, 200 µM bezafibrate showed the highest MyHC expression (73.98 ± 11.79-fold), and differences in the expression of all energy metabolism-related genes were found (p < 0.05). CONCLUSIONS: Adipocyte and myocyte differentiation are enhanced with telmisartan and bezafibrate, respectively, and energy uptake, storage and mobilization are improved with both.

Recovery from experimental parkinsonism by semaphorin-guided axonal growth of grafted dopamine neurons.

Cell therapy in animal models of Parkinson's disease (PD) is effective after intrastriatal grafting of dopamine (DA) neurons, whereas intranigral transplantation of dopaminergic cells does not cause consistent behavioral recovery. One strategy to promote axonal growth of dopaminergic neurons from the substantia nigra (SN) to the striatum is degradation of inhibitory components such as chondroitin sulphate proteoglycans (CSPG). An alternative is the guidance of DA axons by chemotropic agents. Semaphorins 3A and 3C enhance axonal growth of embryonic stem (ES) cell-derived dopaminergic neurons in vitro, while Semaphorin 3C also attracts them. We asked whether intranigral transplantation of DA neurons, combined with either degradation of CSPG or with grafts of Semaphorin 3-expressing cells, towards the striatum, is effective in establishing a new nigrostriatal dopaminergic pathway in rats with unilateral depletion of DA neurons. We found depolarization-induced DA release in dorsal striatum, DA axonal projections from SN to striatum, and concomitant behavioral improvement in Semaphorin 3-treated animals. These effects were absent in animals that received intranigral transplants combined with Chondroitinase ABC treatment, although partial degradation of CSPG was observed. These results are evidence that Semaphorin 3-directed long-distance axonal growth of dopaminergic neurons, resulting in behavioral improvement, is possible in adult diseased brains.

The neurovascular unit of capillary blood vessels in the rat nervous system. A rapid-Golgi electron microscopy study.

We describe a pericapillary organ in the rat forebrain and cerebellar cortex. It consists of a series of tripartite synapses with synaptic extensions enveloped by astrocytic endfeet that are linked to the capillary wall by synaptic extensions. Reciprocal specializations of the pericyte-capillary blood vessel (CBV) with such specialized synapses suggest a mechanoreceptor role. In Golgi-impregnated and 3D reconstructions of the cerebral cortex and thalamus, a series of TSs appear to be sequentially ordered in a common dendrite, paralleled by synaptic outgrowths termed golf club synaptic extensions (GCE) opposed to a longitudinal crest (LC) from the capillary basal lamina (BL). Our results show that, in the cerebellar cortex, afferent fibers and interneurons display microanatomical structures that strongly suggest an interaction with the capillary wall. Afferent mossy fiber (MF) rosettes and ascending granule cell axons and their dendrites define the pericapillary passage interactions that are entangled by endfeet. The presence of mRNA of the mechanosensitive channel Piezo1 in the MF rosettes, together with the surrounding end-feet and the capillary wall form mechanosensory units. The ubiquity of such units to modulate synaptic transmission is also supported by Piezo1 mRNA expressing pyramidal isocortical and thalamic neurons. This scenario suggests that ascending impulses to the cerebellar and cortical targets are presynaptically modulated by the reciprocal interaction with the mechanosensory pericapillary organ that ultimately modulates the vasomotor response.

Microbial colonization dynamics of the postnatal digestive tract of Bos indicus calves.

The rumen and the jejunum of calves have distinct functional roles; the former is in the storage and fermentation of feed, and the latter is in transporting digesta to the ileum. It is unknown how nutrition changes the evolution of the microbiome of these organs after birth. We sequenced and characterized the entire microbiome of the rumen and the jejunum from Bos indicus calves of the Mexican Tropics to study their dynamics at Days 0, 7, 28, and 42 after birth. Operational taxonomic units (OTUs) belonging to 185 and 222 genera from 15 phylum were observed in the organs, respectively. The most abundant OTUs were Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. We observed that proteobacterial species were outcompeted after the first week of life by Bacteroidetes and Firmicutes in the rumen and the jejunum, respectively. Moreover, Prevotella species were found to predominate in the rumen (36% of total OTUs), while the jejunum microbiome is composed of small proportions of several genera. Presumably, their high relative abundance assists in specialized functions and is more likely in fermentation since they are anaerobes. In summary, the rumen and the jejunum microbiomes were outcompeted by new microbiomes in a dynamic process that begins at birth.

In vitro conversion of ß-carotene to retinal in bovine rumen fluid by a recombinant ß-carotene- 15, 15'-monooxygenase.

Pasture-fed cattle yield carcasses with yellow fat; consumers often reject the resulting meat products because they assume they come from old and/or culled animals. Recombinant bacteria expressing beta-carotene 15, 15'-monooxygenase, introduced into the rumen of the animal, might help to reduce the coloration since this enzyme converts carotene to retinal, thereby eliminating the source of yellowness. The goal of this work was to evaluate the effect of a recombinant beta-carotene 15, 15'-monooxygenase (BCMO1) from Gallus gallus, expressed in Escherichia coli. The genetically modified microbe was introduced into ruminal fluid, and carotene conversion to retinal was measured. Under optimum conditions the enzyme produced 6.8 nmol of retinal per 1 mg of protein in 1 hour at 37 °C. The data on in vitro digestibility in ruminal fluid showed no differences in beta-carotene breakdown or in retinal production (p > 0.1) between E. coli with pBAD vector alone and E. coli with pBAD/BCMO1. The pBAD/BCMO1 plasmid was stable in E. coli for 750 generations. These results indicate that the protein did not break beta-carotene into retinal in ruminal fluid, perhaps due to its location in the periplasmic space in E. coli. Future research must consider strategies to release the enzyme into the rumen environment.

Fructose promotes more than glucose the adipocytic differentiation of pig mesenchymal stem cells.

The goal of this study was to evaluate how glucose and fructose affected the adipose differentiation of pig newborn mesenchymal stem cells (MSCs). Cells were grown with or without inosine in 7.5 mM glucose (substituted with 1.5 or 6 mM fructose). MSCs displayed adipose morphology after 70 days of differentiation. Fructose stimulated the highest levels of PPARγ and C/EBPß. Fructose at 6 mM, but not glucose at 7.5 mM or fructose at 1.5 mM, promotes differentiation of MSCs into adipocytes and increases 11-hydroxysteroid dehydrogenase (11ß-HSD1) and NADPH oxidase 4 (NOX4) mRNA in the absence of hepatic effects (as simulated by the inosine). Fructose and glucose increased xanthine oxide-reductase (XOR) catalytic activity almost 10-fold and elevated their products: intracellular reactive oxygen species (ROS) pool, extracellular H2 O2 pool by 4 orders of magnitude, and uric acid by a factor of 10. Therefore, in our experimental model, differentiation of MSCs into adipocytes occurs exclusively at the blood concentration of fructose detected after ingestion by people on a high fructose diet. PRACTICAL APPLICATIONS: The results of this study provide new evidence for fructose's adipogenic potential in mesenchymal stem cells, a model in which its effects on XOR activity had not been studied. The increased expression of genes such as C/EBPß, PPARγ, and NOX4, as well as the increased XOR activity and high production of ROS during the differentiation process in the presence of fructose, coincides in pointing to this hexose as an important factor in the development of adipogenesis in young animals, which could have a great impact on the development of future obesity.

A new strategy for the decellularization of whole organs by hydrostatic pressure.

Tissue engineering has been able to develop novel decellularization-recellularization techniques, which facilitates the research for the generation of functional organs. This is based in the initial obtention of the organ's extracellular matrix (ECM). Therefore, any improvement in the decellularization process would have a positive impact in the results of the recellularization process. Nevertheless, commonly the methods and equipment employed for this process are expensive and thus limit the access of this technique to various research groups globally. To develop a decellularization technique with the exclusive use of hydrostatic pressure of detergent solutions, to have an easily accessible and low-cost technique that meets the basic requirements of acellularity and functionality of the ECM. This experimental study was performed in 10 male Wistar rats, obtaining the liver to carry out serial washes, with 1%, 2%, and 3% Triton X-100 solutions and 0.1% SDS. The washes were performed by using a gravity perfusion system (GPS), which assured us a continuous hydrostatic pressure of 7.5 mmHg. The obtained ECM was processed using stains and immunostaining to determine the residual cell content and preservation of its components. The staining showed a removal of cellular and nuclear components of approximately 97% of the acellular ECM, with an adequate three-dimensional pattern of collagen and proteoglycans. Furthermore, the acellular ECM allowed the viability of a primary hepatocyte culture. The use of the GPS decellularization technique allowed us to obtain an acellular and functional ECM, drastically reducing experimentation costs.

Profiling of circulating chromosome 21-encoded microRNAs, miR-155, and let-7c, in down syndrome.

BACKGROUND: Down syndrome (DS) is the most common chromosomal survival aneuploidy. The increase in DS life expectancy further heightens the risk of dementia, principally early-onset Alzheimer's disease (AD). AD risk in DS is higher, considering that this population may also develop metabolic diseases such as obesity, dyslipidemias, and diabetes mellitus. The extra genetic material that characterizes DS causes an imbalance in the genetic dosage, including over-expression of AD's key pathophysiological molecules and the gene expression regulators, the microRNAs (miRNAs). Two miRNAs, chromosome 21-encoded, miR-155, and let-7c, are associated with cognitive impairment and dementia in adults; but, expression dynamics and relationship with clinical variables during the DS's lifespan had remained hitherto unexplored. METHODS: The anthropometric, clinical, biochemical, and profile expression of circulating miR-155 and let-7c were analyzed in a population of 52 control and 50 DS subjects divided into the young group (Aged ≤20 years) and the adult group (Aged ≥21 years). RESULTS: The expression changes for miR-155 were not significant; nevertheless, a negative correlation with HDL-Cholesterol concentrations was observed. Notably, let-7c was over-expressed in DS from young and old ages. CONCLUSION: Overall, our results suggest that let-7c plays a role from the early stages of DS's cognitive impairment while overexpression of miR-155 may be related to lipid metabolism changes. Further studies of both miRNAs will shed light on their potential as therapeutic targets to prevent or delay DS's cognitive impairment.

LIM-homeobox gene Lhx5 is required for normal development of Cajal-Retzius cells.

Cajal-Retzius (C-R) cells play important roles in the lamination of the mammalian cortex via reelin secretion. The genetic mechanisms underlying the development of these neurons have just begun to be unraveled. Here, we show that two closely related LIM-homeobox genes Lhx1 and Lhx5 are expressed in reelin+ cells in various regions in the mouse telencephalon at or adjacent to sites where the C-R cells are generated, including the cortical hem, the mantle region of the septal/retrobulbar area, and the ventral pallium. Whereas Lhx5 is expressed in all of these reelin-expressing domains, Lhx1 is preferentially expressed in the septal area and in a continuous domain spanning from lateral olfactory region to caudomedial territories. Genetic ablation of Lhx5 results in decreased reelin+ and p73+ cells in the neocortical anlage, in the cortical hem, and in the septal, olfactory, and caudomedial telencephalic regions. The overall reduction in number of C-R cells in Lhx5 mutants is accompanied by formation of ectopic reelin+ cell clusters at the caudal telencephalon. Based on differential expression of molecular markers and by fluorescent cell tracing in cultured embryos, we located the origin of reelin+ ectopic cell clusters at the caudomedial telencephalic region. We also confirmed the existence of a normal migration stream of reelin+ cells from the caudomedial area to telencephalic olfactory territories in wild-type embryos. These results reveal a complex role for Lhx5 in regulating the development and normal distribution of C-R cells in the developing forebrain.

Slit-Robo signals regulate pioneer axon pathfinding of the tract of the postoptic commissure in the mammalian forebrain.

During early vertebrate forebrain development, pioneer axons establish a symmetrical scaffold descending longitudinally through the rostral forebrain, thus forming the tract of the postoptic commissure (TPOC). In mouse embryos, this tract begins to appear at embryonic day 9.5 (E9.5) as a bundle of axons tightly constrained at a specific dorsoventral level. We have characterized the participation of the Slit chemorepellants and their Robo receptors in the control of TPOC axon projection. In E9.5-E11.5 mouse embryos, Robo1 and Robo2 are expressed in the nucleus origin of the TPOC (nTPOC), and Slit expression domains flank the TPOC trajectory. These findings suggested that these proteins are important factors in the dorsoventral positioning of the TPOC axons. Consistently with this role, Slit2 inhibited TPOC axon growth in collagen gel cultures, and interfering with Robo function in cultured embryos induced projection errors in TPOC axons. Moreover, absence of both Slit1 and Slit2 or Robo1 and Robo2 in mutant mouse embryos revealed aberrant TPOC trajectories, resulting in abnormal spreading of the tract and misprojections into both ventral and dorsal tissues. These results reveal that Slit-Robo signaling regulates the dorsoventral position of this pioneer tract in the developing forebrain.

Delivery of chemotropic proteins and improvement of dopaminergic neuron outgrowth through a thixotropic hybrid nano-gel.

Chemotropic proteins guide neuronal projections to their final target during embryo development and are useful to guide axons of neurons used in transplantation therapies. Site-specific delivery of the proteins however is needed for their application in the brain to avoid degradation and pleiotropic affects. In the present study we report the use of Poly (ethylene glycol)-Silica (PEG-Si) nanocomposite gel with thixotropic properties that make it injectable and suitable for delivery of the chemotropic protein semaphorin 3A. PEG-Si gel forms a functional gradient of semaphorin that enhances axon outgrowth of dopaminergic neurons from rat embryos or differentiated from stem cells in culture. It is not cytotoxic and its properties allowed its injection into the striatum without inflammatory response in the short term. Long term implantation however led to an increase in macrophages and glial cells. The inflammatory response could have resulted from non-degraded silica particles, as observed in biodegradation assays.

Glioblastoma multiforme: a multi-omics analysis of driver genes and tumour heterogeneity.

Glioblastoma (GBM) is the most aggressive and common brain cancer in adults with the lowest life expectancy. The current neuro-oncology practice has incorporated genes involved in key molecular events that drive GBM tumorigenesis as biomarkers to guide diagnosis and design treatment. This study summarizes findings describing the significant heterogeneity of GBM at the transcriptional and genomic levels, emphasizing 18 driver genes with clinical relevance. A pattern was identified fitting the stem cell model for GBM ontogenesis, with an upregulation profile for MGMT and downregulation for ATRX, H3F3A, TP53 and EGFR in the mesenchymal subtype. We also detected overexpression of EGFR, NES, VIM and TP53 in the classical subtype and of MKi67 and OLIG2 genes in the proneural subtype. Furthermore, we found a combination of the four biomarkers EGFR, NES, OLIG2 and VIM with a remarkable differential expression pattern which confers them a strong potential to determine the GBM molecular subtype. A unique distribution of somatic mutations was found for the young and adult population, particularly for genes related to DNA repair and chromatin remodelling, highlighting ATRX, MGMT and IDH1. Our results also revealed that highly lesioned genes undergo differential regulation with particular biological pathways for young patients. This multi-omic analysis will help delineate future strategies related to the use of these molecular markers for clinical decision-making in the medical routine.

Evolutionary Perspective and Expression Analysis of Intronless Genes Highlight the Conservation of Their Regulatory Role.

The structure of eukaryotic genes is generally a combination of exons interrupted by intragenic non-coding DNA regions (introns) removed by RNA splicing to generate the mature mRNA. A fraction of genes, however, comprise a single coding exon with introns in their untranslated regions or are intronless genes (IGs), lacking introns entirely. The latter code for essential proteins involved in development, growth, and cell proliferation and their expression has been proposed to be highly specialized for neuro-specific functions and linked to cancer, neuropathies, and developmental disorders. The abundant presence of introns in eukaryotic genomes is pivotal for the precise control of gene expression. Notwithstanding, IGs exempting splicing events entail a higher transcriptional fidelity, making them even more valuable for regulatory roles. This work aimed to infer the functional role and evolutionary history of IGs centered on the mouse genome. IGs consist of a subgroup of genes with one exon including coding genes, non-coding genes, and pseudogenes, which conform approximately 6% of a total of 21,527 genes. To understand their prevalence, biological relevance, and evolution, we identified and studied 1,116 IG functional proteins validating their differential expression in transcriptomic data of embryonic mouse telencephalon. Our results showed that overall expression levels of IGs are lower than those of MEGs. However, strongly up-regulated IGs include transcription factors (TFs) such as the class 3 of POU (HMG Box), Neurog1, Olig1, and BHLHe22, BHLHe23, among other essential genes including the ß-cluster of protocadherins. Most striking was the finding that IG-encoded BHLH TFs fit the criteria to be classified as microproteins. Finally, predicted protein orthologs in other six genomes confirmed high conservation of IGs associated with regulating neural processes and with chromatin organization and epigenetic regulation in Vertebrata. Moreover, this study highlights that IGs are essential modulators of regulatory processes, such as the Wnt signaling pathway and biological processes as pivotal as sensory organ developing at a transcriptional and post-translational level. Overall, our results suggest that IG proteins have specialized, prevalent, and unique biological roles and that functional divergence between IGs and MEGs is likely to be the result of specific evolutionary constraints.