Synthesis Of Specific Proteins Research Articles

MicroRNAs (miRNAs) role in hypertension: pathogenesis and promising therapeutics.

MicroRNAs (miRNAs) are small, non-coding RNA molecules that play a crucial role in regulating various cellular processes, including cell proliferation, differentiation, apoptosis, and disease development. Recent studies have highlighted the importance of miRNAs in the development and progression of essential hypertension, a common form of high blood pressure that affects millions of individuals worldwide. The molecular mechanisms by which miRNAs regulate hypertension are complex and multifaceted. MiRNAs target the 3' untranslated regions of mRNA molecules, thereby regulating the synthesis of specific proteins involved in cardiovascular function. For instance, miRNAs are known to regulate the expression of genes involved in blood vessel tone, cardiac function, and inflammation. The growing body of research on miRNAs in hypertension has highlighted their potential as therapeutic targets for managing this condition. Studies have shown that miRNA-based therapies can modulate the expression of key genes involved in hypertension, leading to improvements in blood pressure and cardiovascular function. However, more research is needed to fully understand the mechanisms of miRNA-mediated hypertension and to develop effective therapeutic strategies. In summary, this review highlights the current understanding of the role of miRNAs in essential hypertension, including their molecular mechanisms and potential therapeutic applications. Further research is needed to fully understand the impact of miRNAs on hypertension and to develop new treatments for this common and debilitating condition.

Impacts of Water Logging on Soil Microbial Intentions and Mung Bean (Vigna radiata L.) Physiology

The mung bean (Vigna radiata L.) is an important pulse crop known for its high protein content and adaptability to tropical and subtropical regions, particularly in Asia. Despite of their importance as a source of protein, most noticeably that, waterlogging negatively affects their growth and yield. Waterlogging, resulting from factors like compacted soil and excessive irrigation, leads to hypoxic conditions and negatively affects various physiological and morphological aspects of mung bean plants. The impact of waterlogging on mung beans at different growth stages emphasises its detrimental effects on root growth, leaf area, chlorophyll content, photosynthesis, and biomass. Waterlogging disrupts nutrient uptake, causing deficiencies and hindering overall plant adaptation. The molecular responses and metabolic changes in mung beans under waterlogging stress can upset the shift to anaerobic metabolism and the synthesis of specific proteins. Microbial interactions in the rhizosphere soil and their influence on mung bean growth during waterlogging also play an important role in mung bean growth and yield. Tolerance mechanisms, including morphological adaptations and physiological changes, are explored, offering insights into selecting tolerant genotypes. Agronomic strategies, such as the use of cytokinin and gibberellin acid used for mitigating waterlogging effects and enhancing mung bean resilience.

STN7 is not essential for developmental acclimation of Arabidopsis to light intensity.

Plants respond to changing light intensity in the short term through regulation of light harvesting, electron transfer, and metabolism to mitigate redox stress. A sustained shift in light intensity leads to a long-term acclimation response (LTR). This involves adjustment in the stoichiometry of photosynthetic complexes through de novo synthesis and degradation of specific proteins associated with the thylakoid membrane. The light-harvesting complex II (LHCII) serine/threonine kinase STN7 plays a key role in short-term light harvesting regulation and was also suggested to be crucial to the LTR. Arabidopsis plants lacking STN7 (stn7) shifted to low light experience higher photosystem II (PSII) redox pressure than the wild type or those lacking the cognate phosphatase TAP38 (tap38), while the reverse is true at high light, where tap38 suffers more. In principle, the LTR should allow optimisation of the stoichiometry of photosynthetic complexes to mitigate these effects. We used quantitative label-free proteomics to assess how the relative abundance of photosynthetic proteins varied with growth light intensity in wild-type, stn7, and tap38 plants. All plants were able to adjust photosystem I, LHCII, cytochrome b6 f, and ATP synthase abundance with changing white light intensity, demonstrating neither STN7 nor TAP38 is crucial to the LTR per se. However, stn7 plants grown for several weeks at low light (LL) or moderate light (ML) still showed high PSII redox pressure and correspondingly lower PSII efficiency, CO2 assimilation, and leaf area compared to wild-type and tap38 plants, hence the LTR is unable to fully ameliorate these symptoms. In contrast, under high light growth conditions the mutants and wild type behaved similarly. These data are consistent with the paramount role of STN7-dependent LHCII phosphorylation in tuning PSII redox state for optimal growth in LL and ML conditions.

Deciphering the antibacterial mechanism of monocaprin against methicillin-resistant Staphylococcus aureus by integrated transcriptomic and metabolomic analyses and its application in pork preservation

Methicillin-resistant Staphylococcus aureus (MRSA) is a drug-resistant pathogen generally derived from pork and other animal foods. This study investigated the antibacterial mechanism of monocaprin against MRSA and its preservation effect on pork. The results showed that monocaprin had a strong anti-MRSA activity with visible cell surface damage and a minimum inhibitory concentration of 0.16 mg/mL. The transcriptomic and metabolomic analyses showed that 255 genes, 45 metabolites in positive ion mode, and 50 in negative ion mode were significantly changed after monocaprin treatment (0.08 mg/mL). The integrative analysis of omics suggested that monocaprin led to osmotic imbalance, amino acid metabolism inhibition, energy metabolism disorder and aerobic respiration obstruction of MRSA. Sublethally damaged MRSA required increased specific protein synthesis and pyrimidine metabolism, enhanced glycolysis, activation of anaerobic respiration, and promotion of biofilm formation for survival under monocaprin stress. Moreover, monocaprin obviously restrained the growth of MRSA in pork, delayed the spoilage of pork during storage at 4 °C for 8 days, and had no adverse effects on its sensory properties. Overall, these results revealed the anti-MRSA mechanism of monocaprin and suggested that monocaprin can be used to control MRSA in pork.

Do COVID-19 RNA-Injections Affect Male Fertility? Latest Facts and Perspective

Background: Based on available data on male fertility adverse effects after COVID-19 injections, we draw the reader´s attention to open questions and undeniable risks of the new RNA-based vaccine technology. Methods: Review and reanalysis of published data on pre- and post-injection semen analyses. Evaluation of UK Yellow Card and US VAERS databases for male fertility adverse effects and of the German deStatis database for monthly live birth rates after start of the governmental vaccination program. Results: The deStatis database demonstrates a time shift of exactly nine months between the start of the governmental vaccination campaign (April 2021) and an abrupt decline in live births (January 2022). Frequency of male fertility adverse effects is approx. 100 times lower than that of female fertility. Remarkably, report numbers per one million doses are similar between AstraZeneca and BionTech/Pfizer, but significantly increased for Moderna despite overall numbers of administered doses are smaller than that of the other two manufacturers. Both increase in and correlation between erectile dysfunction and heart failure could be demonstrated. Review and reanalysis of published data on pre- and post-injection semen analyses identified a number of limitations of the currently available studies. Conclusion: There remain still far more questions than answers. Due to the principle “primum non nocere,” any new medical therapy must be banned until harmlessness beyond doubt has been proven. Most importantly, it must be realized that the active ingredient of RNA-based vaccines is not simply mRNA promoting the synthesis of a nota bene viral specific protein, but modRNA specifically designed for longevity and encapsulated in LNPs to bypass biological barriers and get access to all cells, possibly also germ cells. As mRNA is involved in regulation of gene expression, cells have mechanisms at hand to silence mRNA species not required, however, theses protective mechanisms will not work with modRNA.

Escherichia coli methionine-tRNAi/methionyl tRNA synthetase pairs induced protein initiation of interest (PII) expression

The precise regulatory role in protein synthesis by facilitating interactions with mRNA codons for various tRNA modifications is unclear. We previously reported that enhanced green fluorescent protein (GFP) reduced enhanced GFP mRNA expression in human methionine-conjugated initiator tRNA (tRNAi)/tRNA synthetase pairs under methionine-deficient conditions. Here, we investigated the effect of non-formylated methionine-conjugated Escherichia coli tRNAi on the synthesis of the protein initiation of interest (PII) in HeLa cells under intracellular L-methionine levels. We found that E. coli methionine-tRNAi counteracts human methionine-tRNAi, indicating that E. coli methionyl tRNA synthetase can induce enhanced GFP expression due to increased stability of enhanced GFP mRNA. Both complexes could support translation initiation without being employed to introduce methionine residues in the subsequent elongation steps. The results indicated that E. coli methionine-tRNAi could offset human methionine-tRNAi, and E. coli methionine-tRNAi/methionyl tRNA synthetase pairs can drive enhanced GFP mRNA expression. Unlike the human methionine-tRNAi/methionyl tRNA synthetase pairs that were used as a positive control, the non-formylated E. coli methionine-tRNAi/methionyl tRNA synthetase pairs reduced the expression of enhanced GFP mRNA, resulting in reduced HeLa cell survival. Using tRNAs functions causes of heterologous origin, such as from prokaryotes, and modified, to enhance or suppress the synthesis of specific proteins in eukaryotic organisms into the potential may possess a more prominent advantage of E. coli methionine-tRNAi as approaches that can control PII. This study provides new insights on the E. coli methionine- tRNAi/methionyl tRNA synthetase pair induced PII synthesis and the relative viability of cells could pave the way to regulate ecological/biological systems.

Investigating Post-transcriptional Mechanism of Neuropathic Pain

Neuropathic pain is a chronic condition which can arise following damage to the somatosensory system and often involves both hyperalgesia and allodynia. The molecular mechanisms of neuropathic pain remain incompletely understood but require enduring alterations in specific gene programs and protein synthesis affecting neuronal signaling and excitability. We investigate non-coding RNA and RNA-binding protein regulatory pathways in impacting hyperalgesia and neuropathic pain using the mouse spared nerve injury model. Nerve injury alters the expression of many miRNAs, including the highly conserved let-7 family miRNAs, which repress pro-growth mRNAs and are implicated in axon growth, neuronal plasticity, and brain circuit development. The Lin28 RNA binding protein can prevent maturation of let-7 precursor RNAs; consequently, increased Lin28 signaling promotes pro-growth gene expression. The regulation and potential roles of Lin28/let-7 pathway in neuropathic pain remain largely unexplored. In preliminary data, we find that Lin28a loss of function in some, but not other, sensory neuron populations can result in a deficit in mechanical hypersensitivity post-injury. In the SNI mouse model, we evaluate molecular mechanisms underlying pain using single molecule detection. A specific RNA imaging assay, RNAscope in situ hybridization (ISH), is used to amplify single RNA target signals in fixed tissues to allow mapping of the spatiotemporal patterns and cell type specificity of changes. We find that Lin28 mRNAs are elevated in classes of injured neurons relative to uninjured neurons in dorsal route ganglia (DRG) which are ipsilateral at early, 3 day, timepoints post-SNI surgery. In contrast, Lin28 mRNAs are highly elevated following a more protracted time courses post-surgery in the compartment of injured neuronal processes. The spatiotemporal differences in Lin28 mRNA levels across compartments of the nervous system following nerve injury could indicate potential trafficking or local stabilization of Lin28 mRNAs and are the subject of ongoing investigations. Grant support from NIH NS103974.

Functional nuclear retention of pre-mRNA involving Cajal bodies during meiotic prophase in European larch (Larix decidua).

Gene regulation ensures that the appropriate genes are expressed at the proper time. Nuclear retention of incompletely spliced or mature mRNAs is emerging as a novel, previously underappreciated layer of posttranscriptional regulation. Studies on this phenomenon indicated that it exerts a significant influence on the regulation of gene expression by regulating export and translation delay, which allows the synthesis of specific proteins in response to a stimulus or at strictly controlled time points, for example, during cell differentiation or development. Here, we show that transcription in microsporocytes of European larch (Larix decidua) occurs in a pulsatile manner during prophase of the first meiotic division. Transcriptional activity was then silenced after each pulse. However, the transcripts synthesized were not exported immediately to the cytoplasm but were retained in the nucleoplasm and Cajal bodies (CBs). In contrast to the nucleoplasm, we did not detect mature transcripts in CBs, which only stored nonfully spliced transcripts with retained introns. Notably, the retained introns were spliced at precisely defined times, and fully mature mRNAs were released into the cytoplasm for translation. As similar processes have been observed during spermatogenesis in animals, our results illustrate an evolutionarily conserved mechanism of gene expression regulation during generative cells development in Eukaryota.

Enrichment of Newly Synthesized Proteins following treatment of C2C12 Myotubes with Endotoxin and Interferon-γ.

Inflammation in muscle induces the synthesis of mediators that can impair protein synthesis and enhance proteolysis, and when sustained lead to muscle atrophy. Furthermore, muscle-derived mediators that are secreted may participate in disrupting the function of other peripheral organs. Selective identification of newly synthesized proteins can provide insight on biological processes that depend on the continued synthesis of specific proteins to maintain homeostasis as well as those proteins that are up- or down-regulated in response to inflammation. We used puromycin-associated nascent chain proteomics (PUNCH-P) to characterize new protein synthesis in C2C12 myotubes and changes resulting from their exposure to the inflammatory mediators lipopolysaccharide (LPS) and interferon (IFN)-γ for either a short (4h) or prolonged (16h) time period. We identified sequences of nascent polypeptide chains belonging to a total of 1523 proteins and report their detection from three independent samples of each condition at each time point. The identified nascent proteins correspond to approximately 15% of presently known proteins in C2C12 myotubes and are enriched in specific cellular components and pathways. A subset of these proteins was identified only in treated samples and has functional characteristics consistent with the synthesis of specific new proteins in response to LPS/IFNγ. Thus, the identification of proteins from their nascent polypeptide chains provides a resource to analyze the role of new synthesis of proteins in both protein homeostasis and in proteome responses to stimuli in C2C12 myotubes. Our results reveal a profile of actively translating proteins for specific cellular components and biological processes in normal C2C12 myotubes and a different enrichment of proteins in response to LPS/IFNγ. Collectively, our data disclose a highly interconnected network that integrates the regulation of cellular proteostasis and reveal a diverse immune response to inflammation in muscle which may underlie the concomitantly observed atrophy and be important in inter-organ communication.

Relational model of the standard genetic code

The genetic code is a set of rules that establishes mapping between triplets in messenger RNA and amino acids in proteins. The most common way to display these rules is the Standard Genetic Code (SGC) table. This paper takes an alternative approach, based on the relational data model by Edgar F. Codd (Commun. ACM, 13:377–387, 1970). The relational model (RM) proposes a distributed storage of data into a collection of tables (called relations), that can be connected by shared communality. Basic elements of the table are rows (called records or tuples), and columns (called fields or attributes). The SGC table, according to the relational data model, represents the so called unnormalized form of a table. Using normalization rules it is possible to subdivide the SGC table into four tables. The rows and columns of single tables are defined by the first and second base and individual tables by the third codon base. The result of this model is an approach to managing genetic code data, represented in terms of tuples and grouped into relations, with table structure and language consistent with first-order (predicate) logic. The RM explains that the final step in the development of the SGC was the adoption of coding function by the third base, which makes an informational/functional unit with the first base, despite the different physical location in a triplet. This enabled the synthesis of specific proteins without ambiguity, in accordance with the concept of ambiguity reduction and five phases of the general model on the origin of biological codes by Marcello Barbieri (BioSystems 181:11–19, 2019).

Influence of crude oil pollution on the content and electrophoretic spectrum of proteins in Carex hirta plants at the initial stages of vegetative development

The role of proteins in the general adaptive response of Carex hirta plants to soil pollution by crude oil has been studied. It was established that a possible element of the process of adaptation of C. hirta plants to combined stress – conditions of soil polluted by crude oil – may be the synthesis of stress proteins – high molecular weight of more than 60 kD and low molecular weight, not exceeding 22–45 kD. The synthesis of all 5 HSP families was detected in the leaves and rhizomes, and only sHSP (starting from Mr 32 kD), Hsp 60 and Hsp 100 proteins were synthesized in the roots under the influence of crude oil pollution. The development of C. hirta adaptation syndrome under the influence of crude oil pollution of the soil was promoted by enhanced synthesis of proteins with Mr 85, 77, 64, 60 and 27 kD in the leaves, 118 and 41 kD in the rhizomes and proteins with Mr 105, 53, 50 and 43 kD in the roots of the plants. The decrease in the amount of proteins with Mr 91, 45, 28 kD in the leaves, proteins with Mr 85, 76 and 23 kD in rhizomes and proteins with Mr 64 and 39 in the roots of C. hirta plants under conditions of crude oil polluted soil could be a consequence of inhibition of synthesis or degradation of protein molecules providing the required level of low molecular weight protective compounds in cells. The root system and rhizomes of C. hirta plants undergo a greater crude oil load, which leads to increased protein synthesis in these organs and decreased in the leaves, correspondingly. However, a decrease in protein content in the leaves may indicate their outflow in the roots and rhizomes. Сrude oil contaminated soil as a polycomponent stressor accelerated the aging of leaves of C. hirta plants, which could be caused by increased synthesis of ABA. ABA in its turn induced the synthesis of leaf-specific protein with Mr 27 kD. These proteins bind significant amounts of water with their hydrate shells maintaining the high water holding capacity of the cytoplasm under drought conditions. ABA inhibits the mRNA synthesis and their corresponding proteins, which are characteristic under normal conditions, and induces the expression of genes and, consequently, the synthesis of specific proteins including 27 kD protein. By stimulating the expression of individual genes and the synthesis of new polypeptides, ABA promotes the formation of protective reactions and increases the resistance of plants to crude oil pollution.

Exportin-1 Connects Dynamic Transcription and Translation of Genotoxic Stress Genes in Diffuse Large B-Cell Lymphoma Patients

Exportin-1 (XPO1), that exports a subset of proteins from the nucleus to the cytoplasm, is amplified in 30% of DLBCL. Regardless of XPO1 gene status, DLBCLs require XPO1 to proliferate and survive as demonstrated in pre-clinical (Marullo et al. AACR 2015) and clinical (Kuruvilla at al. Blood 2017) studies with the covalent XPO1 inhibitor selinexor. However, the mechanism underlying such dependency remains unknown.We analyzed the expression of XPO1 protein in a cohort of 57 DLBCL patients by IHC and correlated with XPO1 gene status and clinical outcome. XPO1 increased copy number is associated with elevated protein expression (p = 0.03) and, importantly, patients with elevated XPO1 levels (XPO1 high) are less likely to respond to RCHOP as refractory/early relapse diseases (p = 0.005). This suggests that XPO1 high DLBCL could be biologically fitter to tolerate genotoxic stress. To explore this hypothesis, we compared gene expression profiles (RNA-sequencing) of XPO1 high (n = 15) vs. XPO1 low (n = 16) DLBCLs by DESeq2. XPO1 high DLBCLs were enriched (q<0.005) for genes regulating “cell proliferation”, “DNA damage response and repair”, “mRNA processing” and “RNA export”; whereas depleted (q<0.05) for genes regulating “immune response”. Gene Set Enrichment Analysis also revealed that XPO1 high were enriched for transcripts up-regulated in highly aneuploid tumors (FDR = 0, NES = 2.19). All these findings were validated in an independent cohort of 72 DLBCL patients.To gain insight into the mechanism by which XPO1 enhances genotoxic stress tolerance, we exposed a panel of 5 DLBCL cell lines to DNA damaging agents (etoposide, doxorubicin, mechloretamine) with or without XPO1 inhibition (selinexor). Comet assay experiments revealed that selinexor did not affect the amount of DNA damage induced by chemotherapy, but delayed cell's ability to repair the DNA damage (P>0.005). This translated into a significant increase of sensitivity toward otherwise sub-lethal effect of chemotherapy, as all the combinations tested showed synergistic effect on growth inhibition. To determine the existence of a therapeutic window to capitalize on this effect, we evaluated the combination of CHOP +/- selinexor in an XPO1 high DLBCL patient-derived xenograft mice model (n = 40). Combination treatment achieved higher anti-lymphoma effect compared to either selinexor or CHOP alone (p>0.005 and p= 0.0015, respectively). Importantly, no increase in toxicity was observed by biochemistry or pathological examination. Thus, proliferating DLBCL cells but not normal cells rely on XPO1 to survive exogenous genotoxic stress.To characterize the molecular consequences of XPO1 inhibition, we quantified the expression of proteins involved in DNA damage response and repair (immunoblotting). Exposure to selinexor decreased the expression of a subset of these proteins including CHEK1, BRCA1, BCL6 and RAD51. This reduction was neither due to decreased gene transcription (qRT-PCR) nor to increased protein degradation (cycloheximide chase). Instead, analysis of nascent proteins by L-azido-homo-alanine labeling revealed that XPO1 inhibition decreases the synthesis of specific DNA damage repair proteins whose increased turnover is required to survive genotoxic stress. Since XPO1 inhibition affected only a subset of proteins, the mechanism may involve selective decrease in nuclear export of mRNA templates. Thus, we quantified the nuclear/cytoplasmic ratio of mRNAs encoding for DNA damage repair proteins in DLBCL cells treated with selinexor. XPO1 inhibition resulted in nuclear retention of BRCA1, RAD51, BCL6 and CHEK1 mRNAs but not of β-actin. Since XPO1 is unable to directly bind mRNAs, we reasoned that its inhibition may impair the export of proteins carrying mRNA (ribonucleoproteins). Indeed, we identified eIF4E as one of these cargoes. Immunofluorescence experiments revealed that eIF4E is retained in the nucleus of DLBCL cells upon XPO1 inhibition and its nuclear retention is enhanced in etoposide-treated cells. Moreover, RIP-qPCR experiments revealed that eIF4E binds several DNA damage repair mRNAs such as CHEK1, BCL6 and BRCA1 in the nucleus of DLBCL cells and this binding increases significantly in response to DNA damage.Overall, our data demonstrate that XPO1, by regulating the selective export of mRNA-binding proteins, promotes genotoxic stress tolerance thus conferring a survival advantage to XPO1high DLBCLs. DisclosuresYang:Regeneron Pharmaceuticals: Employment. Cerchietti:Leukemia and Lymphoma Society: Research Funding; Lymphoma Research Foundation: Research Funding; Celgene: Research Funding; Weill Cornell Medicine - New York Presbyterian Hospital: Employment.

Investigating post-transcriptional mechanisms of neuropathic pain

Neuropathic pain is a chronic condition which can arise following damage to the somatosensory system and often involves both hyperalgesia and allodynia. The molecular mechanisms of neuropathic pain remain incompletely understood but require enduring alterations in specific gene program expression and protein synthesis affecting neuronal signaling and excitability. We investigate the roles of non-coding RNA regulatory pathways in impacting hyperalgesia and determining the mRNA complement recruited during the protein synthesis response in neuropathic pain. Nerve injury alters the expression of many miRNAs, including the highly conserved let-7 family miRNAs, which repress pro-growth mRNAs and are implicated in axon growth, neuronal plasticity, and brain circuit development. The Lin28 RNA binding protein can prevent maturation of let-7 precursor RNAs; consequently, increased Lin28 signaling promotes pro-growth gene expression. The regulation and potential roles role of Lin28/let-7 pathway in neuropathic pain remain largely unexplored. Using spared nerve injury (SNI) mouse models of neuropathic pain, we find in preliminary data that Lin28a loss of function in sensory neuron populations can result in a deficit in mechanical hypersensitivity post-surgery. In SNI and sciatic nerve transection (SNT) mouse models, we evaluate molecular mechanisms underlying pain using single molecule detection and genetic manipulation. A sensitive RNA imaging assays, RNAScope in situ hybridization (ISH), is used to amplify single RNA target signals in fixed tissues to allow mapping of the spatiotemporal patterns and cell type specificity of changes in non-coding RNA regulatory pathways. Digital PCR is used to provide sensitive and quantitative validation. We find lin28 mRNA level are elevated in injured dorsal root ganglion cells, injured sciatic nerves and their surrounding Schwann cells at 3 days post SNI and SNT surgery accordingly. Neuropathic pain is a chronic condition which can arise following damage to the somatosensory system and often involves both hyperalgesia and allodynia. The molecular mechanisms of neuropathic pain remain incompletely understood but require enduring alterations in specific gene program expression and protein synthesis affecting neuronal signaling and excitability. We investigate the roles of non-coding RNA regulatory pathways in impacting hyperalgesia and determining the mRNA complement recruited during the protein synthesis response in neuropathic pain. Nerve injury alters the expression of many miRNAs, including the highly conserved let-7 family miRNAs, which repress pro-growth mRNAs and are implicated in axon growth, neuronal plasticity, and brain circuit development. The Lin28 RNA binding protein can prevent maturation of let-7 precursor RNAs; consequently, increased Lin28 signaling promotes pro-growth gene expression. The regulation and potential roles role of Lin28/let-7 pathway in neuropathic pain remain largely unexplored. Using spared nerve injury (SNI) mouse models of neuropathic pain, we find in preliminary data that Lin28a loss of function in sensory neuron populations can result in a deficit in mechanical hypersensitivity post-surgery. In SNI and sciatic nerve transection (SNT) mouse models, we evaluate molecular mechanisms underlying pain using single molecule detection and genetic manipulation. A sensitive RNA imaging assays, RNAScope in situ hybridization (ISH), is used to amplify single RNA target signals in fixed tissues to allow mapping of the spatiotemporal patterns and cell type specificity of changes in non-coding RNA regulatory pathways. Digital PCR is used to provide sensitive and quantitative validation. We find lin28 mRNA level are elevated in injured dorsal root ganglion cells, injured sciatic nerves and their surrounding Schwann cells at 3 days post SNI and SNT surgery accordingly.

Optically transparent electrodes to study living cells: A mini review.

The use of electrochemical methods to study living systems, including cells, has been of interest to researchers for a long time. Thus, controlling the polarization of the electrode contacting living cells, one can influence, for example, their proliferation or the synthesis of specific proteins. Moreover, the electrochemical approach formed the basis of the biocompatibility improvement of the materials contacting with body tissues that use in carbon hemosorbents and implants development. It became possible to reach a fundamentally new level in the study of cell activity with the introduction of optically transparent electrodes in this area. The advantage of the using of optically transparent electrodes is the possibility of simultaneous analysis of living cells by electrochemical and microscopic methods. The use of such materials allowed approaching to the study of the influence of the electrode potential on adhesion activity and morphology of the different cell types (HeLa cells, endothelial cell, etc.) more detailed. There are a negligible number of publications in this area despite the advantages of the usage of optically transparent electrodes to study living cells. This mini-review is devoted to some aspects of the interaction of living cells with conductive materials and current advances in the use of optically transparent electrodes for the study of living cells, as well as the prospects for their use in cellular technologies.

An Esrrb and Nanog Cell Fate Regulatory Module Controlled by Feed Forward Loop Interactions

Cell fate decisions during development are governed by multi-factorial regulatory mechanisms including chromatin remodeling, DNA methylation, binding of transcription factors to specific loci, RNA transcription and protein synthesis. However, the mechanisms by which such regulatory “dimensions” coordinate cell fate decisions are currently poorly understood. Here we quantified the multi-dimensional molecular changes that occur in mouse embryonic stem cells (mESCs) upon depletion of Estrogen related receptor beta (Esrrb), a key pluripotency regulator. Comparative analyses of expression changes subsequent to depletion of Esrrb or Nanog, indicated that a system of interlocked feed-forward loops involving both factors, plays a central part in regulating the timing of mESC fate decisions. Taken together, our meta-analyses support a hierarchical model in which pluripotency is maintained by an Oct4-Sox2 regulatory module, while the timing of differentiation is regulated by a Nanog-Esrrb module.

Fish cartilage: A promising source of biomaterial for biological scaffold fabrication in cartilage tissue engineering.

Here, engineered cartilage-like scaffold using an extracellular matrix (ECM) from sturgeon fish cartilage provided a chondroinductive environment to stimulate cartilaginous matrix synthesis in human adipose stem cells (hASCs). Three dimensional porous and degradable fish cartilage ECM-derived scaffold (FCS) was produced using a protocol containing chemical decellularization, enzymatic solubilization, freeze-drying and EDC-crosslinking treatments and the effect of different ECM concentrations (10, 20, 30, and 40 mg/ml) on prepared scaffolds was investigated through physical, mechanical and biological analysis. The histological and scanning electron microscopy analysis revealed the elimination of the cell fragments and a 3-D interconnected porous structure, respectively. Cell viability assay displayed no cytotoxic effects. The prepared porous constructs of fish cartilage ECM were seeded with hASCs for 21 days and compared to collagen (Col) and collagen-10% hyaluronic acid (Col-HA) scaffolds. Cell culture results evidenced that the fabricated scaffolds could provide a proper 3-D structure to support the adhesion, proliferation and chondrogenic differentiation of hASCs considering the synthesis of specific proteins of cartilage, collagen type II (Col II) and aggrecan (ACAN). Based on the results of the present study, it can be concluded that the porous scaffold derived from fish cartilage ECM possesses an excellent potential for cartilage tissue engineering.

Translational Regulation of Clock Genes BMAL1 and REV-ERBα by Polyamines.

Polyamines stimulate the synthesis of specific proteins at the level of translation, and the genes encoding these proteins are termed as the “polyamine modulon”. The circadian clock generates daily rhythms in mammalian physiology and behavior. We investigated the role of polyamines in the circadian rhythm using control and polyamine-reduced NIH3T3 cells. The intracellular polyamines exhibited a rhythm with a period of about 24 h. In the polyamine-reduced NIH3T3 cells, the circadian period of circadian clock genes was lengthened and the synthesis of BMAL1 and REV-ERBα was significantly reduced at the translation level. Thus, the mechanism of polyamine stimulation of these protein syntheses was analyzed using NIH3T3 cells transiently transfected with genes encoding enhanced green fluorescent protein (EGFP) fusion mRNA with normal or mutated 5′-untranslated region (5′-UTR) of Bmal1 or Rev-erbα mRNA. It was found that polyamines stimulated BMAL1 and REV-ERBα synthesis through the enhancement of ribosomal shunting during the ribosome shunting within the 5′-UTR of mRNAs. Accordingly, the genes encoding Bmal1 and Rev-erbα were identified as the members of “polyamine modulon”, and these two proteins are significantly involved in the circadian rhythm control.

Stress Tolerance Effect of Three Different Sub-aerial Cyanobacteria sp. Isolated from Exteriors of Buildings under Adverse Environmental Conditions

The current interest of scientific study aims at survival mechanisms of the cyanobacteria on the extreme habitats (i.e. building facades and monuments) growing under adverse conditions. The present investigation points towards finding out indigenous which are tolerant of conflicting environmental conditions, such as pH, temperature and calcium carbonate. Three isolates of cyanobacteria Scytonema coactile, Scytonema geitleri and Lyngbya aerugineo–coerulea from a cave, building façade, and temple, Orissa respectively were examined. Tolerance to stress at different pH and temperature were evaluated by quantifying cyanobacteria growth at different time intervals. Tolerance to CaCO3 was studied by subjecting the isolates to the desired concentration 0.0001 – 1% w/v. Each organism was grown for 15 days at 25°C ± 1°C under continuous light intensity (7.5 W/m2) and then harvested, succeeded by SDS gel-electrophoresis protein analysis. Results revealed that three isolated cyanobacteria species from different sub-aerial habitats responded in a specific manner to different stress conditions and to various concentration of CaCO3 concerning protein synthesis. A 30 and 38 kDa protein was overproduced by all isolates under pH and temperature stress, whereas for CaCO3 stress, the protein of 16 and 22 kDa was overproduced by Lyngbya aerugineo–coerulea respectively which concluded that the survival of the isolates under stress conditions depends on specific protein synthesis. Generally, isolates tolerant to different stress may be due to specific protein synthesis for their survival to extreme habitats.

System-wide molecular dynamics of endothelial dysfunction in Gram-negative sepsis

BackgroundInflammation affecting whole organism vascular networks plays a central role in the progression and establishment of several human diseases, including Gram-negative sepsis. Although the molecular mechanisms that control inflammation of specific vascular beds have been partially defined, knowledge lacks on the impact of these on the molecular dynamics of whole organism vascular beds. In this study, we have generated an in vivo model by coupling administration of lipopolysaccharide with stable isotope labeling in mammals to mimic vascular beds inflammation in Gram-negative sepsis and to evaluate its effects on the proteome molecular dynamics. Proteome molecular dynamics of individual vascular layers (glycocalyx (GC), endothelial cells (EC), and smooth muscle cells (SMC)) were then evaluated by coupling differential systemic decellularization in vivo with unbiased systems biology proteomics.ResultsOur data confirmed the presence of sepsis-induced disruption of the glycocalyx, and we show for the first time the downregulation of essential molecular maintenance processes in endothelial cells affecting this apical vascular coating. Similarly, a novel catabolic phenotype was identified in the newly synthesized EC proteomes that involved the impairment of protein synthesis, which affected multiple cellular mechanisms, including oxidative stress, the immune system, and exacerbated EC-specific protein turnover. In addition, several endogenous molecular protective mechanisms involving the synthesis of novel antithrombotic and anti-inflammatory proteins were also identified as active in EC. The molecular dynamics of smooth muscle cells in whole organism vascular beds revealed similar patterns of impairment as those identified in EC, although this was observed to a lesser extent. Furthermore, the dynamics of protein posttranslational modifications showed disease-specific phosphorylation sites in the EC proteomes.ConclusionsTogether, the novel findings reported here provide a broader picture of the molecular dynamics that take place in whole organism vascular beds in Gram-negative sepsis inflammation. Similarly, the obtained data can pave the way for future therapeutic strategies aimed at intervening in specific protein synthesis mechanisms of the vascular unit during acute inflammatory processes.

Selective inhibition of human translation termination by a drug-like compound

Methods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation. These molecules also inhibit translation termination by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 inhibits translation termination by arresting the nascent chain (NC) in the ribosome exit tunnel. The arrested NC adopts a compact α-helical conformation that induces 28 S rRNA nucleotide rearrangements that suppress the peptidyl transferase center (PTC) catalytic activity stimulated by eukaryotic release factor 1 (eRF1). These data support a mechanism of action for a small molecule targeting translation that suppresses peptidyl-tRNA hydrolysis promoted by eRF1, revealing principles of eukaryotic translation termination and laying the foundation for new therapeutic strategies.