Decoding functional proteome information in model organisms using protein language models.

Protein language models have been tested and proved to be reliable when used on curated datasets but have not yet been applied to full proteomes. Accordingly, we tested how two different machine learning-based methods performed when decoding functional information from the proteomes of selected model organisms. We found that protein language models are more precise and informative than deep learning methods for all the species tested and across the three gene ontologies studied, and that they better recover functional information from transcriptomic experiments. The results obtained indicate that these language models are likely to be suitable for large-scale annotation and downstream analyses, and we recommend a guide for their use.

Amidation of arabinoglucuronoxylans to modulate their flow behavior.

Arabinoglucuronoxylans obtained from the exudate of Cercidium praecox (Brea gum) were subjected to an amidation reaction to modulate their flow behavior to obtain a product with similar behavior to gum Arabic. The amidation reaction of the uronic acids present in this exudate was studied using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) system with the aim of maximizing product yield and minimizing by-product. An analysis of the significant factors involved in the reaction was carried out and a response surface methodology was conducted to optimize the stoichiometry of the reagents used. It was possible to obtain models for predicting the degree of amidation (DA) of arabinoglucuronoxylans and the formation of by-products. The formation of a secondary product derived from the amino acid ß-alanine which has not been reported previously in the reaction with polysaccharides, was described. The flow behavior of an amidated product (DA = 52 %) was determined, showing a pseudoplastic behavior and a decreased Newtonian viscosity (η0 = 36.2 Pa s) at the lowest shear rate range with respect to native product solution (η0 = 115 Pa s). Amidated arabinoglucuronoxylans had a flow behavior more similar to that of gum Arabic.

A hotspot for posttranslational modifications on the androgen receptor dimer interface drives pathology and anti-androgen resistance.

Mutations of the androgen receptor (AR) associated with prostate cancer and androgen insensitivity syndrome may profoundly influence its structure, protein interaction network, and binding to chromatin, resulting in altered transcription signatures and drug responses. Current structural information fails to explain the effect of pathological mutations on AR structure-function relationship. Here, we have thoroughly studied the effects of selected mutations that span the complete dimer interface of AR ligand-binding domain (AR-LBD) using x-ray crystallography in combination with in vitro, in silico, and cell-based assays. We show that these variants alter AR-dependent transcription and responses to anti-androgens by inducing a previously undescribed allosteric switch in the AR-LBD that increases exposure of a major methylation target, Arg761. We also corroborate the relevance of residues Arg761 and Tyr764 for AR dimerization and function. Together, our results reveal allosteric coupling of AR dimerization and posttranslational modifications as a disease mechanism with implications for precision medicine.

Chemoinformatic modelling of the antioxidant activity of phenolic compounds.

BACKGROUND: Antioxidants are chemicals used to protect foods from deterioration by neutralizing free radicals and inhibiting the oxidative process. One approach to investigate the antioxidant activity is to develop quantitative structure-activity relationships (QSARs). RESULTS: A curated database of 165 structurally heterogeneous phenolic compounds with the Trolox equivalent antioxidant capacity (TEAC) was developed. Molecular geometries were optimized by means of the GFN2-xTB semiempirical method and diverse molecular descriptors were obtained afterwards. For model development, V-WSP unsupervised variable reduction was used before performing the genetic algorithms-variable subset selection (GAs-VSS) to construct the best five-descriptor multiple linear regression model. The coefficient of determination and the root mean square error were used to measure the performance in calibration (R2 = 0.789 and RMSEC = 0.381), and test set prediction (Q2 = 0.748 and RMSEP = 0.416), along several cross-validation criteria. To thoroughly understand the TEAC prediction, a fully explained mechanism of action of the descriptors is provided. In addition, the applicability domain of the model defined a theoretical chemical space for reliable predictions of new phenolic compounds. CONCLUSION: This in silico model conforms to the five principles stated by the Organisation for Economic Co-operation and Development. The model might be useful for virtual screening of the antioxidant chemical space and for identifying the most potent molecules related to an experimental measurement of TEAC activity. In addition, the model could assist chemists working on computer-aided drug design for the synthesis of new targets with improved activity and potential uses in food science. © 2023 Society of Chemical Industry.

Antioxidant pectins from eggplant (Solanum melongena) fruit exocarp, calyx and flesh isolated through high-power ultrasound and sodium carbonate.

Dried and milled eggplant fruit peel and calyces (PC) and mesocarp, placenta and core (Mes) were utilized as natural sources of valuable chemicals. Pectins were extracted with 0.1 M Na2CO3 (1 h; 23 °C). A high-power ultrasound (US) pretreatment (10 min net time; 12.76 W/cm2 power intensity) in 10:200 (g/mL) powder:water ratio led to the lowest solvent and energy consumptions after the subsequent 0.1 M Na2CO3 stirring, permitting the highest recoveries of uronic acid (UA) from PC and Mes (80.25 and 93.8 %, respectively). Homogalacturonans (>65 % w/w UA) of low degree of methylesterification, of acetylation, and 90,214-138,184 Da molecular weights with low polydispersity (≈1.32-1.40) were obtained. They included released ferulate (≈3.5 mg/100 g) esterified pectins. Antioxidants (caffeoylquinic acid, putrescine and spermidine derivatives, ß-carotene, lutein) gave additional technological value to their thickening effect as pectins protected tryptophan, tyrosine, alkyl side chains and sulfhydryl of skim milk proteins from UV-C photo-oxidation.

The multivalency of the glucocorticoid receptor ligand-binding domain explains its manifold physiological activities.

The glucocorticoid receptor (GR) is a ubiquitously expressed transcription factor that controls metabolic and homeostatic processes essential for life. Although numerous crystal structures of the GR ligand-binding domain (GR-LBD) have been reported, the functional oligomeric state of the full-length receptor, which is essential for its transcriptional activity, remains disputed. Here we present five new crystal structures of agonist-bound GR-LBD, along with a thorough analysis of previous structural work. We identify four distinct homodimerization interfaces on the GR-LBD surface, which can associate into 20 topologically different homodimers. Biologically relevant homodimers were identified by studying a battery of GR point mutants including crosslinking assays in solution, quantitative fluorescence microscopy in living cells, and transcriptomic analyses. Our results highlight the relevance of non-canonical dimerization modes for GR, especially of contacts made by loop L1-3 residues such as Tyr545. Our work illustrates the unique flexibility of GR's LBD and suggests different dimeric conformations within cells. In addition, we unveil pathophysiologically relevant quaternary assemblies of the receptor with important implications for glucocorticoid action and drug design.

Genotoxic risk in humans and acute toxicity in rats of a novel oral high-dose coenzyme Q10 oleogel.

Coenzyme Q10 (CoQ10) supplementation has demonstrated to be safe and effective in primary and secondary CoQ10 deficiencies. Previously, we have designed a high-dose CoQ10 oleogel (1 g/disk) with excipients used in quantities that do not represent any toxic risk. However, it was necessary to demonstrate their safety in the final formulation. Following this purpose, an acute toxicity study of the oleogel in rats was performed. Furthermore, the genotoxic risk was evaluated in human volunteers after CoQ10 supplementation with oleogel and compared to the solid form (1 g/three 00-size-capsules). In addition, the general health status and possible biochemical changes of the participants were determined using serum parameters. Results suggested the absence of adverse effects caused by the interaction of the components in the oleogel formulation. Therefore, we conclude that the designed novel high-dose CoQ10 oleogel was safe for oral consumption.

Mutation of SPINOPHILIN (PPP1R9B) found in human tumors promotes the tumorigenic and stemness properties of cells.

Rationale: SPINOPHILIN (SPN, PPP1R9B) is an important tumor suppressor involved in the progression and malignancy of different tumors depending on its association with protein phosphatase 1 (PP1) and the ability of the PP1-SPN holoenzyme to dephosphorylate retinoblastoma (pRB). Methods: We performed a mutational analysis of SPN in human tumors, focusing on the region of interaction with PP1 and pRB. We explored the effect of the SPN-A566V mutation in an immortalized non-tumorigenic cell line of epithelial breast tissue, MCF10A, and in two different p53-mutated breast cancer cells lines, T47D and MDA-MB-468. Results: We characterized an oncogenic mutation of SPN found in human tumor samples, SPN-A566V, that affects both the SPN-PP1 interaction and its phosphatase activity. The SPN-A566V mutation does not affect the interaction of the PP1-SPN holoenzyme with pocket proteins pRB, p107 and p130, but it affects its ability to dephosphorylate them during G0/G1 and G1, indicating that the PP1-SPN holoenzyme regulates cell cycle progression. SPN-A566V also promoted stemness, establishing a connection between the cell cycle and stem cell biology via pocket proteins and PP1-SPN regulation. However, only cells with both SPN-A566V and mutant p53 have increased tumorigenic and stemness properties. Conclusions: SPN-A566V, or other equivalent mutations, could be late events that promote tumor progression by increasing the CSC pool and, eventually, the malignant behavior of the tumor.

Perennial halophyte Salicornia neei Lag.: Cell wall composition and functional properties of its biopolymers.

Salicornia neei halophyte extends in Argentina seashores. To envisage potential applications, cell wall sequential extraction performed on dry plant yielded 1.1, 2.4, 0.3 and 0.9% of pectin fractions respectively extracted by room temperature water, 90 °C-water, CDTA and Na2CO3. They contained 21-33% uronic acids (UA) with low degree of methylation and 0.5-1.2 M ratios of neutral sugars to UA. High arabinose level suggests that long arabinan side-chains maintain cell wall flexibility in water deficit. Fractions also contained 10-36% of proteins. The KOH-soluble fractions (4.3%) were mainly arabinoxylans. At 2.0% w/v, pectin fractions developed "weak gel"-type networks with Ca2+, while arabinoxylans generated "dilute solutions". Cellulose (28%) and lignin (45.1%) were the main biopolymers in the final residue, which showed low water swelling capacity (3.6 mL/g) due to lignin, increasing when arabinoxylans were also present. Phenolics (9.8%) were mainly water-extractable. Salicornia is a source of biopolymers and antioxidants potentially useful for food applications.

Sunflower (Helianthus annuus L.) Seed Hull Waste: Composition, Antioxidant Activity, and Filler Performance in Pectin-Based Film Composites.

Helianthus annuus L. seed hull is an abundant waste of the edible oil industry. To envisage potential applications of this waste, here, we aimed to analyze the chemical composition of milled sunflower hulls (SP), constituted mainly by 210 µm (51.4%) and 420 µm (27.6%) average mesh particle sizes. SP contained almost 30% of cellulose, 26.4% of lignin, 38.5% of neutral sugars, mainly hemicelluloses, and only 1.3% of proteins. The important lignin content and low pectin content (4.0% of uronic acids) present in SP were both ascribed to its low hydrophilic behavior and hydration capacity. Phenolic compounds were mostly proanthocyanidins (168 mg/100 g SP), with lower amounts of extractable (31.4 mg/100 g SP) phenolics (O-caffeoylquinic acid), all of them associated with the DPPH radical scavenging capacity (95 mg ascorbic acid equiv./100 g) and ferric reducing power (FRAP: 152 mg ascorbic acid equiv./100 g) shown by SP. Esterified ferulic acid (52.9 mg/100 g SP) was also found, mostly as monomers and trimers. SP of 53 µm particle size was then assayed as a filler (0, 5, 8, and 12% concentrations) in calcium low methoxyl pectin-based films, which showed antioxidant capacity (DPPH and FRAP assays) in an SP-concentration-dependent manner. SP showed homogeneous dispersion in composite films equilibrated at 57.7% relative humidity. Water content decreased while film thickness increased with SP concentration. When loaded at a 12% level, the presence of 53-µm SP decreased the water vapor permeability and increased the normal stress at film fracture. Sunflower hulls can then be applied to the development of active materials like 12% SP film, which can be proposed as a food slice antioxidant separator to be investigated in a future work.

Randomised trial of external-beam radiotherapy alone or with high-dose-rate brachytherapy for prostate cancer: Mature 12-year results.

BACKGROUND AND PURPOSE: A randomised phase-III trial compared external beam radiotherapy (EBRT) alone with EBRT combined with high-dose-rate brachytherapy boost (HDR-BTb) in localised prostate adenocarcinoma. Previous analysis, at median follow up of 85 months, demonstrated improved relapse free survival (RFS) with EBRT + HDR-BTb. This data has now been updated with a median follow up of 131 months. MATERIALS AND METHODS: From December 1997 to August 2005, patients were assigned either to EBRT alone delivering 55 Gy in 20 fractions over 4 weeks or EBRT followed by a temporary high-dose-rate implant delivering 2 × 8·5 Gy over 24 h. The primary endpoint was RFS defined by a PSA rise ≥2.0 µg/l above nadir, clinical progression or death. Actuarial survival rates and Hazard Ratios (HRs) were calculated using the Kaplan-Meier method and Cox's Proportional Hazard Model, respectively. Secondary endpoints were overall survival (OS), urinary and bowel toxicity. RESULTS: One hundred and six patients received EBRT alone and 110 EBRT + HDR-BTb. Median time to relapse was 137 months in the HDR-BTb arm compared to 82 months for EBRT alone (p = 0·01). A 27% risk of recurrence with EBRT alone was observed (p = 0·001), resulting in a 21% improvement in RFS at 12 years with EBRT + HDR-BTb. In multivariate analysis treatment arm, risk category and no androgen deprivation therapy were significant covariates for risk of relapse. Differences in overall survival were not significant. CONCLUSION: At 12 years there remains a significant improvement in RFS after EBRT + HDR-BTb; both treatments were equitoxic for severe late urinary and bowel events and urethral strictures.

Bioavailability of coenzyme Q10 loaded in an oleogel formulation for oral therapy: Comparison with a commercial-grade solid formulation.

Coenzyme Q10 (CoQ10) is essential in mitochondrial bioenergetics and is a potent endogenous antioxidant. Low CoQ10 levels are associated with neurodegenerative, metabolic, muscular and cardiovascular disorders. Early treatment with high doses (5-50 mg/kg/day) demonstrated to limit the onset and progression of neuropathology. Recently, we developed an oleogel matrix able to support a high dose of oil-dissolved CoQ10, easy to swallow by CoQ10-deficient patients who suffer from secondary dysphagia. In the present study, we evaluated the bioavailability of oleogel-dissolved CoQ10 and plasma antioxidant status in healthy adults in single-dose and repeated-dose studies. The single-dose study demonstrated that, in terms of CoQ10 bioavailability, 1 g CoQ10/5g oleogel-disk was equivalent to the solid form (1 g CoQ10/three 00-size-capsules), whereas the repeated-dose study (14-days-administration) demonstrated a significantly higher increase in plasma CoQ10 when administered through the oleogel, which could be compatible with the levels necessary to achieve an adequate therapeutic response. Also, a trend to a higher plasma apparent half-life (greater than24 h) was observed for the oleogel-loaded-CoQ10. In conclusion, the oleogel matrix does not compromise the oil-dissolved CoQ10 bioavailability and can prevent the non-adherence to this vital supplementation in patients with high CoQ10 requirements. No significant variation in the plasma antioxidant status (vitamins A, E and C, glutathione and TBARs) was observed.

Origin and Evolution of Polycyclic Triterpene Synthesis.

Polycyclic triterpenes are members of the terpene family produced by the cyclization of squalene. The most representative polycyclic triterpenes are hopanoids and sterols, the former are mostly found in bacteria, whereas the latter are largely limited to eukaryotes, albeit with a growing number of bacterial exceptions. Given their important role and omnipresence in most eukaryotes, contrasting with their scant representation in bacteria, sterol biosynthesis was long thought to be a eukaryotic innovation. Thus, their presence in some bacteria was deemed to be the result of lateral gene transfer from eukaryotes. Elucidating the origin and evolution of the polycyclic triterpene synthetic pathways is important to understand the role of these compounds in eukaryogenesis and their geobiological value as biomarkers in fossil records. Here, we have revisited the phylogenies of the main enzymes involved in triterpene synthesis, performing gene neighborhood analysis and phylogenetic profiling. Squalene can be biosynthesized by two different pathways containing the HpnCDE or Sqs proteins. Our results suggest that the HpnCDE enzymes are derived from carotenoid biosynthesis ones and that they assembled in an ancestral squalene pathway in bacteria, while remaining metabolically versatile. Conversely, the Sqs enzyme is prone to be involved in lateral gene transfer, and its emergence is possibly related to the specialization of squalene biosynthesis. The biosynthesis of hopanoids seems to be ancestral in the Bacteria domain. Moreover, no triterpene cyclases are found in Archaea, invoking a potential scenario in which eukaryotic genes for sterol biosynthesis assembled from ancestral bacterial contributions in early eukaryotic lineages.

Rheology of partially and totally oxidized red seaweed galactans.

Agarose and κ-carrageenan were oxidized using (2,2,6,6-tetramethylpiperidinyl)oxy (TEMPO) in the presence of NaOCl and NaBr. Products with several degrees of oxidation were structurally characterized. The mechanical spectra were determined: derivatives with a medium to high degree of oxidation give rise to polysaccharides that behave like dilute solutions in water, whereas those with a degree of oxidation close to 20 % keep the gelling properties with a different thermo-rheological response towards pH (6.5 or 4.0) and counterions (K+ or Ca2+) in comparison with the native polysaccharides. For instance, they showed a marked dependence on the presence of calcium ions, observed in the increase of thermal stability and dynamic elastic component (G') value, due to the known interaction of this divalent cation with the carboxylate groups. In this sense, these derivatives with low oxidation degrees have proven to be not only thermosensitive, like the native polysaccharide, but also pH- and calcium-sensitive.

Chemical structure and rheological studies of arabinoglucuronoxylans from the Cercidium praecox exudate brea gum.

The structure of the arabinoglucuronoxylans from brea gum was elucidated through an chemical and NMR spectroscopical analysis. They are composed of xylose, arabinose, glucuronic acid and 4-O-methylglucuronic acid in a molar ratio 1:0.44:0.16:0.22. The structure consists of a central chain of (1→4)-ß-d-xylopyranose of which ca.70% are susbstituted in C2 with single stubs of others sugars (ß-d-Xylp, α-d-GlcpA and 4-O-Me-α-d-GlcpA), with disaccharides (α-l-Arap-(1→2)-4-O-Me-α-d-GlcpA-(1→, α-l-Arap-(1→2)-α-d-GlcpA-(1→, ß-l-Araf-(1→3)-α-l-Araf-(1→ and α-l-Araf-(1→3)-α-l-Araf-(1→5), and possibly with trisaccharides of xylose. The determination of the location of the acetyl groups and their quantification in these arabinoglucuronoxylans has been achieved for the first time. Brea gum presents a higher thickening effect than gum arabic in 5% aqueous solution, demonstrating its potential usefulness for food and pharmaceutical applications.

VRK1 functional insufficiency due to alterations in protein stability or kinase activity of human VRK1 pathogenic variants implicated in neuromotor syndromes.

Very rare polymorphisms in the human VRK1 (vaccinia-related kinase 1) gene have been identified in complex neuromotor phenotypes associated to spinal muscular atrophy (SMA), pontocerebellar hypoplasia (PCH), microcephaly, amyotrophic lateral sclerosis (ALS) and distal motor neuron dysfunctions. The mechanisms by which these VRK1 variant proteins contribute to the pathogenesis of these neurological syndromes are unknown. The syndromes are manifested when both of these rare VRK1 polymorphic alleles are implicated, either in homozygosis or compound heterozygosis. In this report, to identify the common underlying pathogenic mechanism of VRK1 polymorphisms, we have studied all human VRK1 variants identified in these neurological phenotypes from a biochemical point of view by molecular modeling, protein stability and kinase activity assays. Molecular modelling predicted that VRK1 variant proteins are either unstable or have an altered kinase activity. The stability and kinase activity of VRK1 pathogenic variants detected two groups. One composed by variants with a reduced protein stability: R133C, R358X, L195V, G135R and R321C. The other group includes VRK1variants with a reduced kinase activity tested on several substrates: histones H3 and H2AX, p53, c-Jun, coilin and 53BP1, a DNA repair protein. VRK1 variants with reduced kinase activity are H119R, R133C, G135R, V236M, R321C and R358X. The common underlying effect of VRK1 pathogenic variants with reduced protein stability or kinase activity is a functional insufficiency of VRK1 in patients with neuromotor developmental syndromes. The G135 variant cause a defective formation of 53BP1 foci in response to DNA damage, and loss Cajal bodies assembled on coilin.

Non-canonical dimerization of the androgen receptor and other nuclear receptors: implications for human disease.

Nuclear receptors are transcription factors that play critical roles in development, homeostasis and metabolism in all multicellular organisms. An important family of nuclear receptors comprises those members that respond to steroid hormones, and which is subdivided in turn into estrogen receptor (ER) isoforms α and ß (NR3A1 and A2, respectively), and a second subfamily of so-called oxosteroid receptors. The latter includes the androgen receptor (AR/NR3C4), the glucocorticoid receptor (GR/NR3C1), the mineralocorticoid receptor (MR/NR3C2) and the progesterone receptor (PR/NR3C3). Here we review recent advances in our understanding of the structure-and-function relationship of steroid nuclear receptors and discuss their implications for the etiology of human diseases. We focus in particular on the role played by AR dysregulation in both prostate cancer (PCa) and androgen insensitivity syndromes (AIS), but also discuss conditions linked to mutations of the GR gene as well as those in a non-steroidal receptor, the thyroid hormone receptor (TR). Finally, we explore how these recent results might be exploited for the development of novel and selective therapeutic strategies.

Antioxidant pectin enriched fractions obtained from discarded carrots (Daucus carota L.) by ultrasound-enzyme assisted extraction.

Carrot residues were upgraded as pectin-enriched fractions (PEFs) useful for functional food formulation due to co-extracted antioxidants (α- and ß-carotenes, lutein, α-tocopherol), and gelling effect. High power ultrasound (US)-enzyme assisted extraction was applied for efficiency and sustainability. Carrot powder (CP) in citrate-buffer (pH 5.20) was submitted to US-pretreatment (12.27 W/cm2: 20 kHz, 80% amplitude, 20 min) and a subsequent digestion (5 h-40 °C) without or with hemicellulase or cellulase. US-hemicellulase led to the highest PEF yield (27.1%), and extracted almost the whole pectin content of CP. US-pretreatment increased the extraction yield of all PEFs, but the existence of an additional positive effect of the following step depended on the enzyme used. PEFs contained 40-47% of UA with low DM (24-49.9%), and co-extracted antioxidants. US decreased the antioxidant contents, DM, and molecular weight, but allowed obtaining calcium crosslinked true gels, also with higher elastic modulus than non-US-extracted PEFs, being promising as food additives.

Husks of Zea mays as a potential source of biopolymers for food additives and materials' development.

Maize husks, an agricultural and industrial residue generated in a large volume, were investigated as a potential source of useful biopolymers. Thus, their chemical composition was firstly studied, after which two biopolymer products were obtained and characterized. Maize husks were dried and milled, obtaining a 210 µm-main particle size powder (MHP). It contained carotenes (4 mg/100 g), and exhibited antioxidant capacity (≈195 mg ascorbic acid/100 g MHP) coming also from extractable coumaric and cinnamic acids-derivatives (14 mg/100 g). A 31% of the MPH was water-soluble at room temperature, mainly constituted by fructose, glucose, and sorbitol of mesophylls' intracellular origin. The water insoluble fiber (WIF, ≈70%), which showed antioxidant capacity (≈25-33 mg ascorbic acid/100 g WIF), was almost entirely constituted by the cell wall biopolymers or alcohol insoluble residue (AIR) of the MPH, mostly arabinoxylans (≈26%) crosslinked by ferulic residues (18.6 mg/100 g MPH), and cellulose (26%). Low levels of pectins (5.5%) and lignin (7%) were found. Hence, a 1.25%-sulfur nanocellulose (NCC) was directly obtained with sulfuric acid (-15 mV Zeta-potential; 147 °C onset of thermal-degradation) without the necessity of previous delignification. On the other hand, a water soluble arabinoxylan enriched fraction (AX-EF) with pseudoplastic behavior in water and sensibility to calcium ions (≈3 Pa⋅s initial Newtonian-viscosity) was isolated by alkaline hydrolysis of diferulate bridges. Despite a 56% of crystallinity, NCC showed the highest water absorption capacity when compared to that of the AX-EF and AIR. Maize husks constitute an important source of biopolymers for development of materials and food additives/ingredients with relevant hydration and antioxidant properties.

miARma-Seq, a comprehensive pipeline for the simultaneous study and integration of miRNA and mRNA expression data.

Messenger RNAs (mRNAs) fulfil specific biological roles in cells and, thus, their expression may be adapted to suit specific circumstances. This is in part achieved through selective gene transcription and post-transcriptional events, the regulation of which must be tightly integrated and controlled. To comprehensively study the coordinated effects of transcriptional and post-transcriptional regulatory elements, and to obtain coherent results, it is advisable to use different methodologies. Adequately integrating the data derived from these distinct methodologies then becomes critical to elucidating the relationships between the coordinated cellular effects assayed, particularly when applied to normal and disease states. Such integrated studies are likely to be particularly useful to identify markers suitable for early detection of diseases and to devise strategies for therapeutic interventions. Throughout this chapter, we will focus on the methods currently available to analyse mRNA and microRNA (miRNA) expression, paying special attention to the influence of miRNAs on mRNA metabolism. We will introduce miARma-Seq, a comprehensive pipeline that facilitates the simultaneous integration of mRNA and miRNA expression data. For illustrative purposes, we include a case study that incorporates data from RNASeq and small-RNASeq, detailing all the steps necessary to define the differential expression of both mRNA- and miRNA-encoding genes. Finally, we explore the possible regulatory relationships that drive significant and potentially relevant changes in mRNA and miRNA gene expression.