A comprehensive study on the multifunctional properties of galectin-4 in red-lip mullet (Planiliza haematocheilus): Insights into molecular interactions, antimicrobial defense, and cell proliferation.

Galectin-4 belongs to the galactoside-binding protein family and is a type of tandem repeat galectin. Despite previous studies indicating its importance in fish immunology, a comprehensive investigation is necessary to fully understand its role in immunomodulatory functions and cellular dynamics. Therefore, this study aimed to explore the immunomodulatory functions of galectin-4 with a particular focus on its antimicrobial and cellular proliferative properties. The open reading frame of PhGal4 spans 1092 base pairs and encodes a soluble protein of 363 amino acids with a theoretical isoelectric point (IEP) of 6.39 and a molecular weight of 39.411 kDa. Spatial expression analysis under normal physiological conditions revealed ubiquitous expression of PhGal4 across all examined tissues, with the highest level observed in intestinal tissue. Upon stimulation with poly I:C, LPS, and L. garvieae, a significant increase (p < 0.05) in PhGal4 expression was observed in both blood and spleen tissues. Subsequent subcellular localization assay demonstrated that PhGal4 was predominantly localized in the cytoplasm. The recombinant PhGal4 (rPhGal4) exhibited specific binding capabilities to pathogen-associated molecular patterns (PAMPs), including LPS and peptidoglycan, but not poly I:C. The rPhGal4 negatively affected the bacterial growth kinetics. Additionally, rPhGal4 demonstrated complete hemagglutination of fish erythrocytes, which could be inhibited by the presence of D-galactose and α-lactose. The overexpression of PhGal4 in FHM epithelial cells demonstrated a significant suppression of viral replication during VHSV infection. Furthermore, the in vitro scratch assay and WST-1 assay demonstrated a wound healing effect of PhGal4 overexpression in FHM cells, potentially achieved through the promotion of cell proliferation by activating genes involved in cell cycle regulation. In conclusion, the responsive expression to immune stimuli, antimicrobial properties, and cell proliferation promotion of PhGal4 suggest that it plays a crucial role in immunomodulation and cellular dynamics of red-lip mullet. The findings in this study shed light on the multifunctional nature of galectin-4 in teleost fish.

Molecular features, antiviral activity, and immunological expression assessment of interferon-related developmental regulator 1 (IFRD1) in red-spotted grouper (Epinephelus akaara).

Interferon-related developmental regulator 1 (IFRD1) is a viral responsive gene associated with interferon-gamma. Herein, we identified the IFRD1 gene (EaIFRD1) from red-spotted grouper (Epinephelus akaara), evaluated its transcriptional responses, and investigated its functional features using various biological assays. EaIFRD1 encodes a protein comprising 428 amino acids with a molecular mass of 48.22 kDa. It features a substantial domain belonging to the interferon-related developmental regulator superfamily. Spatial mRNA expression of EaIFRD1 demonstrated the highest expression levels in the brain and the lowest in the skin. Furthermore, EaIFRD1 mRNA expression in grouper tissues exhibited significant modulation in response to immune stimulants, including poly (I:C), LPS, and nervous necrosis virus (NNV) infection. We analyzed downstream gene regulation by examining type Ⅰ interferon pathway genes following EaIFRD1 overexpression. The results demonstrated a significant upregulation in cells overexpressing EaIFRD1 compared to the control after infection with viral hemorrhagic septicemia virus (VHSV). A subcellular localization assay confirmed the nuclear location of the EaIFRD1 protein, consistent with its role as a transcriptional coactivator. Cells overexpressing EaIFRD1 exhibited increased migratory activity, enhancing wound-healing capabilities compared to the control. Additionally, under H2O2 exposure, EaIFRD1 overexpression protected cells against oxidative stress. Overexpression of EaIFRD1 also reduced poly (I:C)-mediated NO production in RAW267.4 macrophage cells. In FHM cells, EaIFRD1 overexpression significantly reduced VHSV virion replication. Collectively, these findings suggest that EaIFRD1 plays a crucial role in the antiviral immune response and immunological regulation in E. akaara.

Exploring morphological variation in bismuth ferrite nanostructures by pulsed laser deposition: synthesis, structural and electrochemical properties.

Structural and electrochemical properties of bismuth ferrite nanostructures produced by pulsed laser deposition with various morphologies are reported. The nanostructures are also explored as electrode materials for high-performance supercapacitors. Scanning electron microscopy images revealed that various bismuth ferrite morphologies were produced by varying the background pressure (10-6, 0.01, 0.10, 0.25, 0.50, 1.0, 2.0 and 4.0 Torr) in the deposition chamber and submitting them to a thermal treatment after deposition at 500◦C. The as-deposited bismuth ferrite nanostructures range from very compact thin-film (10-6, 0.01, 0.10 Torr), to clustered nanoparticles (0.25, 0.50, 1.0 Torr), to very dispersed arrangement of nanoparticles (2.0 and 4.0 Torr). The electrochemical characteristic of the electrodes was investigated through cyclic voltammetry process. The increase in the specific surface area of the nanostructures as background pressure in the chamber increases does not lead to an increase in interfacial capacitance. This is likely due to the wakening of electrical contact between nanoparticles with increasing porosity of the nanostructures. The thermal treatment increased the contact between nanoparticles, which caused an increase in the interfacial capacitance of the nanostructure deposited under high background pressure in the chamber.

Subgingival biofilm microbiome in individuals with asthma and periodontitis: Metagenomic analysis.

OBJECTIVE: This observational study aimed to explore the metagenomics of subgingival biofilms in individuals with varying degrees of asthma, from severe to none, to elucidate the association between the subgingival microbiome and asthma. MATERIALS AND METHODS: Subgingival biofilm samples were collected from thirty participants at the Asthma Control Program Outpatient Clinic in Bahia (ProAR). These samples were categorized into six groups based on the severity of asthma and the presence or absence of periodontitis. We employed next-generation sequencing (Illumina MiSeq), targeting the 16S rRNA gene, to characterize the microbial communities present. Our analysis included descriptive statistics and sequencing data, evaluated using multivariate statistical methods such as the Shannon index, principal coordinate analysis, and the Bray-Curtis dissimilarity. RESULTS: Our findings indicate a higher prevalence of periodontally detrimental bacterial genera in individuals with severe asthma and periodontitis. Additionally, individuals with asthma, but without periodontitis, exhibited a tendency toward dysbiosis, particularly in cases of severe asthma. CONCLUSION: This research provides new insights into the composition of the subgingival microbiome in individuals with varying severities of asthma and periodontitis. The genera identified in this study underscore the need for further investigations to build upon these findings.

Relating mutational signature exposures to clinical data in cancers via signeR 2.0.

BACKGROUND: Cancer is a collection of diseases caused by the deregulation of cell processes, which is triggered by somatic mutations. The search for patterns in somatic mutations, known as mutational signatures, is a growing field of study that has already become a useful tool in oncology. Several algorithms have been proposed to perform one or both the following two tasks: (1) de novo estimation of signatures and their exposures, (2) estimation of the exposures of each one of a set of pre-defined signatures. RESULTS: Our group developed signeR, a Bayesian approach to both of these tasks. Here we present a new version of the software, signeR 2.0, which extends the possibilities of previous analyses to explore the relation of signature exposures to other data of clinical relevance. signeR 2.0 includes a user-friendly interface developed using the R-Shiny framework and improvements in performance. This version allows the analysis of submitted data or public TCGA data, which is embedded in the package for easy access. CONCLUSION: signeR 2.0 is a valuable tool to generate and explore exposure data, both from de novo or fitting analyses and is an open-source R package available through the Bioconductor project at ( https://doi.org/10.18129/B9.bioc.signeR ).

A mixture model for determining SARS-Cov-2 variant composition in pooled samples.

MOTIVATION: Despite of the fast development of highly effective vaccines to control the current COVID-19 pandemics, the unequal distribution and availability of these vaccines worldwide and the number of people infected in the world lead to the continuous emergence of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) variants of concern. Therefore, it is likely that real-time genomic surveillance will be continuously needed as an unceasing monitoring tool, necessary to follow the spread of the disease and the evolution of the virus. In this context, new genomic variants of SARS-CoV-2, including variants refractory to current vaccines, makes genomic surveillance programs tools of utmost importance. Nevertheless, the lack of appropriate analytical tools to quickly and effectively access the viral composition in meta-transcriptomic sequencing data, including environmental surveillance, represent possible challenges that may impact the fast adoption of this approach to mitigate the spread and transmission of viruses. RESULTS: We propose a statistical model for the estimation of the relative frequencies of SARS-CoV-2 variants in pooled samples. This model is built by considering a previously defined selection of genomic polymorphisms that characterize SARS-CoV-2 variants. The methods described here support both raw sequencing reads for polymorphisms-based markers calling and predefined markers in the variant call format. Results obtained using simulated data show that our method is quite effective in recovering the correct variant proportions. Further, results obtained by considering longitudinal data from wastewater samples of two locations in Switzerland agree well with those describing the epidemiological evolution of COVID-19 variants in clinical samples of these locations. Our results show that the described method can be a valuable tool for tracking the proportions of SARS-CoV-2 variants in complex mixtures such as waste water and environmental samples. AVAILABILITY AND IMPLEMENTATION: http://github.com/rvalieris/LCS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Molecular features, antioxidant potential, and immunological expression assessment of thioredoxin-like protein 1 (TXNL1) in yellowtail clownfish (Amphiprion clarkii).

Thioredoxin-like protein 1 (TXNL1) is a redox-active protein belonging to the thioredoxin family, which mainly controls the redox status of cells. The TXNL1 gene from Amphiprion clarkii (AcTXNL1) was obtained from a pre-established transcriptome database. The AcTXNL1 is encoded with 289 amino acids and is predominantly localized in the cytoplasm and nucleus. The TXN domain of AcTXNL1 comprises a34CGPC37 motif with redox-reactive thiol (SH-) groups. The spatial distribution pattern of AcTXNL1 mRNA was examined in different tissues, and the muscle was identified as the highest expressed tissue. AcTXNL1 mRNA levels in the blood and gills were significantly increased in response to different immunostimulants. In vitro antioxidant capacity of the recombinant AcTXNL1 protein (rACTXNL1) was evaluated using the ABTS free radical-scavenging activity assay, cupric ion reducing antioxidant capacity assay, turbidimetric disulfide reduction assay, and DNA nicking protection assay. The potent antioxidant activity of rAcTXNL1 exhibited a concentration-dependent manner in all assays. Furthermore, in the cellular environment, overexpression of AcTXNL1 increased cell viability under H2O2 stress and reduced nitric oxide (NO) production induced by lipopolysaccharides (LPS). Collectively, the experimental results revealed that AcTXNL1 is an antioxidant and immunologically important gene in A. clarkii.

Toll-like Receptor 4 in Acute Kidney Injury.

Acute kidney injury (AKI) is a common and devastating pathologic condition, associated with considerable high morbidity and mortality. Although significant breakthroughs have been made in recent years, to this day no effective pharmacological therapies for its treatment exist. AKI is known to be connected with intrarenal and systemic inflammation. The innate immune system plays an important role as the first defense response mechanism to tissue injury. Toll-like receptor 4 (TLR4) is a well-characterized pattern recognition receptor, and increasing evidence has shown that TLR4 mediated inflammatory response, plays a pivotal role in the pathogenesis of acute kidney injury. Pathogen-associated molecular patterns (PAMPS), which are the conserved microbial motifs, are sensed by these receptors. Endogenous molecules generated during tissue injury, and labeled as damage-associated molecular pattern molecules (DAMPs), also activate pattern recognition receptors, thereby offering an understanding of sterile types of inflammation. Excessive, uncontrolled and/or sustained activation of TLR4, may lead to a chronic inflammatory state. In this review we describe the role of TLR4, its endogenous ligands and activation in the inflammatory response to ischemic/reperfusion-induced AKI and sepsis-associated AKI. The potential regeneration signaling patterns of TLR4 in acute kidney injury, are also discussed.

Effect of 4 weeks of plyometric training in the pre-competitive period on volleyball athletes' performance.

We aimed to evaluate the effect of 4 weeks of plyometric training (PT), performed in the pre-competitive period, on the vertical jump performance of professional volleyball athletes. We recruited 17 professional female volleyball players (age: 19 ± 3 years; weight: 67.2 ± 5.50 kg; height: 1.81 ± 0.22 m; body fat: 14.4 ± 2.12%; squat 1RM test: 75.5 ± 7.82 kg; training time experience: 6.2 ± 3.4 years) to participate in four weeks of training and assessments. They were divided into an experimental group (EG = 9) and a control group (CG = 8). Both groups were submitted to friendly matches, technical, tactical and resistance training (4 weeks/˜9 sessions per week), and internal load monitoring was carried out. The EG performed PT twice a week. At the beginning and end of the four weeks, jump tests were performed. The main findings are: 1) PT when incorporated into the pre-competitive period can induce greater improvements in jumping performance (EG = 28.93 ± 3.24 cm to 31.67 ± 3.39 cm; CG = 27.91 ± 4.64 cm to 28.97 ± 4.58 cm; when comparing the percentage delta, we found a difference between groups with ES of 1.04 and P = 0.02); 2) this result is observed when the training load is similar between groups and increases over the weeks, respecting the linear progression principle. Therefore, including plyometric training in the preparatory period for volleyball, with low monotony and training strain increment, is an effective strategy for further CMJ performance improvement.

Rqc1 and other yeast proteins containing highly positively charged sequences are not targets of the RQC complex.

Previous work has suggested that highly positively charged protein segments coded by rare codons or poly (A) stretches induce ribosome stalling and translational arrest through electrostatic interactions with the negatively charged ribosome exit tunnel, leading to inefficient elongation. This arrest leads to the activation of the Ribosome Quality Control (RQC) pathway and results in low expression of these reporter proteins. However, the only endogenous yeast proteins known to activate the RQC are Rqc1, a protein essential for RQC function, and Sdd1, a protein with unknown function, both of which contain polybasic sequences. To explore the generality of this phenomenon, we investigated whether the RQC complex controls the expression of other proteins with polybasic sequences. We showed by ribosome profiling data analysis and western blot that proteins containing polybasic sequences similar to, or even more positively charged than those of Rqc1 and Sdd1, were not targeted by the RQC complex. We also observed that the previously reported Ltn1-dependent regulation of Rqc1 is posttranslational, independent of the RQC activity. Taken together, our results suggest that RQC should not be regarded as a general regulatory pathway for the expression of highly positively charged proteins in yeast.

Evaluating the conformational space of the active site of D2 dopamine receptor. Scope and limitations of the standard docking methods.

We report here for the first time the potential energy surfaces (PES) of phenyletilamine (PEA) and meta-tyramine (m-OH-PEA) at the D2 dopamine receptor (D2DR) binding site. PESs not only allow us to observe all the critical points of the surface (minimums, maximums, and transition states), but also to note the ease or difficulty that each local minima have for their conformational inter-conversions and therefore know the conformational flexibility that these ligands have in their active sites. Taking advantage of possessing this valuable information, we analyze how accurate a standard docking study is in these cases. Our results indicate that although we have to be careful in how to carry out this type of study and to consider performing some extra-simulations, docking calculations can be satisfactory. In order to analyze in detail the different molecular interactions that are stabilizing the different ligand-receptor (L-R) complexes, we carried out quantum theory of atoms in molecules (QTAIM) computations and NMR shielding calculations. Although some of these techniques are a bit tedious and require more computational time, our results demonstrate the importance of performing computational simulations using different types of combined techniques (docking/MD/hybrid QM-MM/QTAIM and NMR shielding calculations) in order to obtain more accurate results. Our results allow us to understand in details the molecular interactions stabilizing and destabilizing the different L-R complexes reported here. Thus, the different activities observed for dopamine (DA), m-OH-PEA, and PEA can be clearly explained at molecular level.

Health care professionals' perceptions about atrial fibrillation care in the Brazilian public primary care system: a mixed-methods study.

BACKGROUND: Atrial fibrillation (AF) negatively impacts health systems worldwide. We aimed to capture perceptions of and barriers and facilitators for AF care in Brazilian primary care units (PCUs) from the perspective of healthcare professionals (HCPs). METHODS: This mixed-methods, cross-sectional study utilised an exploratory sequential design, beginning with the quantitative data collection (up to 18 closed questions) immediately followed by a semi-structured interview. HCPs were recruited from 11 PCUs in the Sao Paulo region and included managers, physicians, pharmacists, nurses and community health agents. Descriptive statistics were used to present findings from the quantitative questionnaire and inductive analysis was used to identify themes from the qualitative data. RESULTS: One hundred seven HCPs were interviewed between September 2019 and May 2020. Three main themes were identified that encapsulated barriers and facilitators to AF care: access to care (appointments, equipment/tests and medication), HCP and patient roles (HCP/patient relationship and patient adherence) and the role of the organisation/system (infrastructure, training and protocols/guidelines). Findings from the qualitative analysis reinforced the quantitative findings, including a lack of AF-specific training for HCPs, protocols/guidelines on AF management, INR tests in the PCUs, patient knowledge of AF management and novel oral anticoagulants (NOACs) as key barriers to optimal AF care. CONCLUSIONS: Development and implementation of AF-specific training for PCU HCPs are needed in Brazil, along with evidence-based protocols and guidelines, educational programmes for patients, better access to INR tests for patients taking warfarin and availability of NOACs.

Transient ascending ST-segment depression and widening of the S wave in 3-channel Holter monitoring-A sign of dromotropic disturbance in the right ventricular outflow tract in the Brugada syndrome: A report of five cases.

BACKGROUND: Brugada syndrome (BrS) is somewhat a challenging diagnosis, due to its dynamic pattern. One of the aspects of this disease is a significant conduction disorder located in the right ventricular outflow tract (RVOT), which can be explained as a consequence of low expression of Connexin-43. This decreased conduction speed is responsible for the typical electrocardiographic pattern. Opposite leads located preferably in inferior leads of the electrocardiogram may show a deep and widened S wave associated with ascending ST segment depression. Holter monitoring electrocardiographic (ECG) aspects is still a new frontier of knowledge in BrS, especially in intermittent clinical presentations. METHODS: We describe, as an exploratory analysis, five case series of intermittent type 1 BrS to demonstrate the appearance of ascending ST segment depression and widening of the S wave, during 3-channel 24h-Holter monitoring (C1, C2 and C3) with bipolar leads. RESULTS: In the five cases described, the ST segment depression was observed mainly in C2, but in some cases also in C1 and C3. Only case 1 presented concomitant intermittent elevation of the ST segment in C1. All cases were intermittent. CONCLUSION: The recognition of an ECG pattern with ascending ST-segment depression and widening of the S wave in 3-channel Holter described in this case series should raise a suspicion of the BrS and suggests the counterpart of a dromotropic disturbance registered in the RVOT and/or reciprocal changes.

Stochastic Petri net model describing the relationship between reported maternal and congenital syphilis cases in Brazil.

INTRODUCTION: Syphilis is a sexually transmitted disease (STD) caused by Treponema pallidum subspecies pallidum. In 2016, it was declared an epidemic in Brazil due to its high morbidity and mortality rates, mainly in cases of maternal syphilis (MS) and congenital syphilis (CS) with unfavorable outcomes. This paper aimed to mathematically describe the relationship between MS and CS cases reported in Brazil over the interval from 2010 to 2020, considering the likelihood of diagnosis and effective and timely maternal treatment during prenatal care, thus supporting the decision-making and coordination of syphilis response efforts. METHODS: The model used in this paper was based on stochastic Petri net (SPN) theory. Three different regressions, including linear, polynomial, and logistic regression, were used to obtain the weights of an SPN model. To validate the model, we ran 100 independent simulations for each probability of an untreated MS case leading to CS case (PUMLC) and performed a statistical t-test to reinforce the results reported herein. RESULTS: According to our analysis, the model for predicting congenital syphilis cases consistently achieved an average accuracy of 93% or more for all tested probabilities of an untreated MS case leading to CS case. CONCLUSIONS: The SPN approach proved to be suitable for explaining the Notifiable Diseases Information System (SINAN) dataset using the range of 75-95% for the probability of an untreated MS case leading to a CS case (PUMLC). In addition, the model's predictive power can help plan actions to fight against the disease.

Effects of flaxseed cake fortification on bread shelf life, and its possible use as feed for Tenebrio molitor larvae in a circular economy: preliminary results.

BACKGROUND: Bread represents a significant share of food waste worldwide. The extension of the bread shelf life together with innovative systems of food waste treatment might decrease waste biomass decay, the need for transportation, and the need for storage. In recent years, insects have been selected as a valuable tool for food waste treatment owing to their capability to transform low-value food waste into biomass with high nutritional value. Bakery wastes can be used profitably for this purpose. This work had two objectives: (i) to measure the impact of flaxseed cake fortification on bread shelf life depending on the leavening agent (baker's yeast vs sourdough); (ii) to evaluate the possible reuse of the stale bread fortified with flaxseed cake for Tenebrio molitor rearing. RESULTS: Our results showed that fortification seemed to slow the hardening rate of bread, particularly if baker's yeast was used. The time necessary for mold to appear in sourdough bread doubled (from 2 to 4 days). The addition of flaxseed cake to the recipe determined an increase of its scrap consumption by T. molitor larvae. We also observed a significant increase in the body mass of the T. molitor larvae fed with bread obtained with the brewer's yeast with respect to larvae fed with the sourdough. CONCLUSION: Taken together, these preliminary data can indicate that sourdough bread fortified with 5% of flaxseed cake can represent a promising tool to reduce food waste and to recycle bread scraps by a novel zero-waste approach. © 2021 Society of Chemical Industry.

Determination of the internal dose due to the intake of 226Ra and 210Pb in drinking water from deep wells in the Metropolitan Region of Recife-Brazil.

The Metropolitan Region of Recife, the capital of the state of Pernambuco in northeastern Brazil, has a high demographic density and developed under a region of marine phosphorus with high concentrations of phosphate that naturally contains uranium ore, producing ionizing radiation from descendants of the radioisotope 238U where 226Ra and 210Pb are of great importance in verifying the probable harmful effects on human health due to environmental radioactivity. The supply of drinking water is the responsibility of the state-owned company COMPESA which uses wells of great depth to complete the supply of drinking water for the entire population. COMPESA and the RAE Group of the Federal University of Pernambuco developed a joint project to assess the concentrations of 226Ra and 210Pb and estimate the equivalent and effective doses caused by ingesting these radiation sources. According to the above, this research aimed to evaluate concentrations of 226Ra and 210Pb in drinking water samples from 110 deep wells in Recife. The activities of 226Ra and 210Pb ranged from 1.4 ± 0.3 to 119.3 ± 12.9 and from 25.6 ± 3.3 to 563.2 ± 45.6 mBq.L-1, with arithmetic means of 48.1 ± 3.8 and 231.1 ± 20 mBq.L-1, respectively. The equivalent doses average in bone tissue due to 226Ra and 210Pb were 0.45 ± 0.04 and 3.9 ± 0.37 mSv.y-1, and the annual average effective doses were 0.01 ± 0.00 and 0.13 ± 0.01 mSv.y-1, respectively.

Whole-exome sequencing of non-BRCA1/BRCA2 mutation carrier cases at high-risk for hereditary breast/ovarian cancer.

The current study aimed to identify new breast and/or ovarian cancer predisposition genes. For that, whole-exome sequencing (WES) was performed in the germline DNA of 52 non-BRCA1/BRCA2/TP53 mutation carrier women at high-risk for hereditary breast and ovarian cancer (HBOC). All variants were classified using information from population and disease specific databases, in silico prediction tools and the American College of Medical Genetics and Genomics (ACMG) criteria. Loss of heterozygosity (LOH) of tumor samples and segregation analyses were performed whenever possible. The variants identified were investigated in a second, independent cohort of 17 BC cases. Pathogenic/Likely Pathogenic variants were identified in known cancer genes such as CHEK2, MUTYH, PMS2, and RAD51C. Rare and potentially pathogenic variants were identified in DNA repair genes (FAN1, POLQ, and RAD54L) and other cancer-related genes such as DROSHA and SLC34A2. Interestingly, the variant c.149T>G in the FAN1 gene was identified in two unrelated families, and exhibited LOH in the tumor tissue of one of them. In conclusion, this is the largest Brazilian WES study involving families at high-risk for HBOC which has brought novel insights into the role of potentially new genetic risk factors for hereditary breast and ovarian cancer.

A Systems-level Characterization of the Differentiation of Human Embryonic Stem Cells into Mesenchymal Stem Cells.

Mesenchymal stem/stromal cells (MSCs) are self-renewing multipotent cells with regenerative, secretory and immunomodulatory capabilities that are beneficial for the treatment of various diseases. To avoid the issues that come with using tissue-derived MSCs in therapy, MSCs may be generated by the differentiation of human embryonic stems cells (hESCs) in culture. However, the changes that occur during the differentiation process have not been comprehensively characterized. Here, we combined transcriptome, proteome and phosphoproteome profiling to perform an in-depth, multi-omics study of the hESCs-to-MSCs differentiation process. Based on RNA-to-protein correlation, we determined a set of high confidence genes that are important to differentiation. Among the earliest and strongest induced proteins with extensive differential phosphorylation was AHNAK, which we hypothesized to be a defining factor in MSC biology. We observed two distinct expression waves of developmental HOX genes and an AGO2-to-AGO3 switch in gene silencing. Exploring the kinetic of noncoding ORFs during differentiation, we mapped new functions to well annotated long noncoding RNAs (CARMN, MALAT, NEAT1, LINC00152) as well as new candidates which we identified to be important to the differentiation process. Phosphoproteome analysis revealed ESC and MSC-specific phosphorylation motifs with PAK2 and RAF1 as top predicted upstream kinases in MSCs. Our data represent a rich systems-level resource on ESC-to-MSC differentiation that will be useful for the study of stem cell biology.

Influence of nascent polypeptide positive charges on translation dynamics.

Protein segments with a high concentration of positively charged amino acid residues are often used in reporter constructs designed to activate ribosomal mRNA/protein decay pathways, such as those involving nonstop mRNA decay (NSD), no-go mRNA decay (NGD) and the ribosome quality control (RQC) complex. It has been proposed that the electrostatic interaction of the positively charged nascent peptide with the negatively charged ribosomal exit tunnel leads to translation arrest. When stalled long enough, the translation process is terminated with the degradation of the transcript and an incomplete protein. Although early experiments made a strong argument for this mechanism, other features associated with positively charged reporters, such as codon bias and mRNA and protein structure, have emerged as potent inducers of ribosome stalling. We carefully reviewed the published data on the protein and mRNA expression of artificial constructs with diverse compositions as assessed in different organisms. We concluded that, although polybasic sequences generally lead to lower translation efficiency, it appears that an aggravating factor, such as a nonoptimal codon composition, is necessary to cause translation termination events.

Codon stabilization coefficient as a metric to gain insights into mRNA stability and codon bias and their relationships with translation.

The codon stabilization coefficient (CSC) is derived from the correlation between each codon frequency in transcripts and mRNA half-life experimental data. In this work, we used this metric as a reference to compare previously published Saccharomyces cerevisiae mRNA half-life datasets and investigate how codon composition related to protein levels. We generated CSCs derived from nine studies. Four datasets produced similar CSCs, which also correlated with other independent parameters that reflected codon optimality, such as the tRNA abundance and ribosome residence time. By calculating the average CSC for each gene, we found that most mRNAs tended to have more non-optimal codons. Conversely, a high proportion of optimal codons was found for genes coding highly abundant proteins, including proteins that were only transiently overexpressed in response to stress conditions. We also used CSCs to identify and locate mRNA regions enriched in non-optimal codons. We found that these stretches were usually located close to the initiation codon and were sufficient to slow ribosome movement. However, in contrast to observations from reporter systems, we found no position-dependent effect on the mRNA half-life. These analyses underscore the value of CSCs in studies of mRNA stability and codon bias and their relationships with protein expression.