Disrupt Protein Synthesis Research Articles

Reduced transcriptome analysis in zebrafish uncovers disruptors of spliceosome and ribosome biosynthesis

Abnormal biosynthesis of spliceosomes and ribosomes can lead to their dysfunction, which in turn disrupts protein synthesis and results in various diseases. While genetic factors have been extensively studied, our understanding of how environmental compounds interfere with spliceosome and ribosome biosynthesis remains limited. In the present study, we employed a Reduced Transcriptome Analysis (RTA) approach, integrating large-scale transcriptome data sets of zebrafish and compiling a specific zebrafish gene panel focusing on the spliceosome and ribosome, to elucidate the potential disruptors targeting their biosynthesis. Transcriptomic data sets for 118 environmental substances and 1400 related gene expression profiles were integrated resulting in 513 exposure signatures. Among these substances, several categories including PCB126, transition metals Lanthanum (La) and praseodymium (Pr), heavy metals Cd2+ and AgNO3 and atrazine were highlighted for inducing the significant transcriptional alterations. Furthermore, we found that the transcriptional patterns were distinct between categories, yet overlapping patterns were generally observed within each group. For instance, over 82 % differentially expressed ribosomal genes were shared between La and Pr within the equivalent concentration range. Additionally, transcriptional complexities were also evident across various organs and developmental stages of zebrafish, with notable differences in the inhibition of the transcription of various spliceosome subunits. Overall, our results provide novel insights into the understanding of the adverse effects of environmental compounds, thereby contributing to their environmental risk assessments.

Drosophila Clueless ribonucleoprotein particles display novel dynamics that rely on the availability of functional protein and polysome equilibrium.

The cytoplasm is populated with many ribonucleoprotein (RNP) particles that post-transcriptionally regulate mRNAs. These membraneless organelles assemble and disassemble in response to stress, performing functions such as sequestering stalled translation pre-initiation complexes or mRNA storage, repression and decay. Drosophila Clueless (Clu) is a conserved multi-domain ribonucleoprotein essential for mitochondrial function that forms dynamic particles within the cytoplasm. Unlike well-known RNP particles, stress granules and Processing bodies, Clu particles completely disassemble under nutritional or oxidative stress. However, it is poorly understood how disrupting protein synthesis affects Clu particle dynamics, especially since Clu binds mRNA and ribosomes. Here, we capitalize on ex vivo and in vivo imaging of Drosophila female germ cells to determine what domains of Clu are necessary for Clu particle assembly, how manipulating translation using translation inhibitors affects particle dynamics, and how Clu particle movement relates to mitochondrial association. Using Clu deletion analysis and live and fixed imaging, we identified three protein domains in Clu, which are essential for particle assembly. In addition, we demonstrated that overexpressing functional Clu disassembled particles, while overexpression of deletion constructs did not. To examine how decreasing translation affects particle dynamics, we inhibited translation in Drosophila germ cells using cycloheximide and puromycin. In contrast to stress granules and Processing bodies, cycloheximide treatment did not disassemble Clu particles yet puromycin treatment did. Surprisingly, cycloheximide stabilized particles in the presence of oxidative and nutritional stress. These findings demonstrate that Clu particles have novel dynamics in response to altered ribosome activity compared to stress granules and Processing bodies and support a model where they function as hubs of translation whose assembly heavily depends on the dynamic availability of polysomes.

Third-Generation Tetracyclines: Current Knowledge and Therapeutic Potential.

Tetracyclines constitute a unique class of antibiotic agents, widely prescribed for both community and hospital infections due to their broad spectrum of activity. Acting by disrupting protein synthesis through tight binding to the 30S ribosomal subunit, their interference is typically reversible, rendering them bacteriostatic in action. Resistance to tetracyclines has primarily been associated with changes in pump efflux or ribosomal protection mechanisms. To address this challenge, tetracycline molecules have been chemically modified, resulting in the development of third-generation tetracyclines. These novel tetracyclines offer significant advantages in treating infections, whether used alone or in combination therapies, especially in hospital settings. Beyond their conventional antimicrobial properties, research has highlighted their potential non-antibiotic properties, including their impact on immunomodulation and malignancy. This review will focus on third-generation tetracyclines, namely tigecycline, eravacycline, and omadacycline. We will delve into their mechanisms of action and resistance, while also evaluating their pros and cons over time. Additionally, we will explore their therapeutic potential, analyzing their primary indications of prescription, potential future uses, and non-antibiotic features. This review aims to provide valuable insights into the clinical applications of third-generation tetracyclines, thereby enhancing understanding and guiding optimal clinical use.

High Concentrations of Se Inhibited the Growth of Rice Seedlings.

Selenium (Se) is crucial for both plants and humans, with plants acting as the main source for human Se intake. In plants, moderate Se enhances growth and increases stress resistance, whereas excessive Se leads to toxicity. The physiological mechanisms by which Se influences rice seedlings' growth are poorly understood and require additional research. In order to study the effects of selenium stress on rice seedlings, plant phenotype analysis, root scanning, metal ion content determination, physiological response index determination, hormone level determination, quantitative PCR (qPCR), and other methods were used. Our findings indicated that sodium selenite had dual effects on rice seedling growth under hydroponic conditions. At low concentrations, Se treatment promotes rice seedling growth by enhancing biomass, root length, and antioxidant capacity. Conversely, high concentrations of sodium selenite impair and damage rice, as evidenced by leaf yellowing, reduced chlorophyll content, decreased biomass, and stunted growth. Elevated Se levels also significantly affect antioxidase activities and the levels of proline, malondialdehyde, metal ions, and various phytohormones and selenium metabolism, ion transport, and antioxidant genes in rice. The adverse effects of high Se concentrations may directly disrupt protein synthesis or indirectly induce oxidative stress by altering the absorption and synthesis of other compounds. This study aims to elucidate the physiological responses of rice to Se toxicity stress and lay the groundwork for the development of Se-enriched rice varieties.

Yeast Small Ubiquitin-Like Modifier (SUMO) Protease Ulp2 is Involved in RNA Splicing.

In eukaryotes, RNA splicing, an essential biological process, is crucial for precise gene expression. Inaccurate RNA splicing can cause aberrant mRNA production, disrupting protein synthesis. To regulate splicing efficiency, some splicing factors are reported to undergo Ubiquitin-like Modifier (SUMO)ylation. Our data indicate that in Saccharomyces cerevisiae, the SUMO protease, Ulp2, is involved in splicing. In the ulp2Δ mutant, some ribosomal protein (RP) transcripts exhibited a significant increase in the levels of intron-containing pre-mRNA because of improper splicing. Moreover, we confirmed Ulp2 protein binding to the intronic regions of RP genes. These findings highlight a critical Ulp2 role in RP transcript splicing.

Mechanical abrasion stimulation: Altered epidermal mucus composition and microbial community in grass carp (Ctenopharyngodon idella)

In modern high-density farming, seasonal change, fishing, transportation, and other conditions easily cause mechanical abrasion of fish skin. This leads to alterations in the material balance of the epidermal mucus composition of fish, preventing normal immune function. Subsequently, the risk of disease increases. In this study, grass carp (Ctenopharyngodon idella) was used as an object to investigate immune-related enzyme activities, structural characteristics, and physiological changes of its epidermal mucous cells caused by mechanical abrasion. Furthermore, we explored the changes in body surface mucus composition and microbial community diversity and structure. Our results showed that mechanical abrasion stimulation, significantly changed the activities of immune-related enzymes, such as lysozyme (LZM), alkaline phosphatase (AKP), and acid phosphatase (ACP) were within 24 h (P < 0.05). Their levels gradually recovered after 48 h. In contrast, no significant change was observed in superoxide dismutase (SOD) levels. The number of mucous cells increased, especially those of type I and type II mucous cells in the dorsal skin, which were significantly increased (P < 0.05). Meanwhile, the stimulation changed the metabolites composition of the fish epidermal mucus in the short term, upregulating 12-OPDA, indole, quinic acid and GSH, which are beneficial to antibacterial and anti-inflammatory, and downregulating L-saccharopine, L-2-aminoadipic acid, spermine, GSSG and ribose 5-phosphate, which disrupt protein synthesis, energy production and DNA synthesis. Additionally, these metabolite changes further affect the composition of microorganism communities, rising the abundance of certain disease-associated microorganisms, such as Acidovorax, Rhizobiaceae, Aurantimicrobium, and Leifsonia, which increases the risk of disease in fish. The findings of the present study provide a deeper understanding of the role of mucus as a component of the immune system in fish and provide a scientific basis for effective grass carp culture management and disease control.

Repurposing an Antioxidant to Kill Mycobacterium tuberculosis by Targeting the 50S Subunit of the Ribosome.

Tuberculosis and drug-resistant TB remain serious threats to global public health. It is urgent to develop novel anti-TB drugs in order to control it. In addition to redesigning and developing new anti-TB drugs, drug repurposing is also an innovative way to develop antibacterial drugs. Based on this method, we discovered SKQ-1 in the FDA-approved drug library and evaluated its anti-TB activity. In vitro, we demonstrated that SKQ-1 engaged in bactericidal activity against drug-sensitive and -resistant Mtb and confirmed the synergistic effects of SKQ1 with RIF and INH. Moreover, SKQ-1 showed a significant Mtb-killing effect in macrophages. In vivo, both the SKQ-1 treatment alone and the treatment in combination with RIF were able to significantly reduce the bacterial load and improve the survival rate of G. mellonella infected with Mtb. We performed whole-genome sequencing on screened SKQ-1-resistant strains and found that the SNP sites were concentrated in the 50S ribosomal subunit of Mtb. Furthermore, we proved that SKQ-1 can inhibit protein translation. In summary, from the perspective of drug repurposing, we discovered and determined the anti-tuberculosis effect of SKQ-1, revealed its synergistic effects with RIF and INH, and demonstrated its mechanism of action through targeting ribosomes and disrupting protein synthesis, thus making it a potential treatment option for DR-TB.

Myostatin as a Prognostic Biomarker in Hepatic Cirrhosis Patients with Sarcopenia

Cirrhosis represents the advanced stage of numerous chronic liver conditions. Sarcopenia is a frequently overlooked complication in cirrhotic patients, resulting from factors such as insufficient protein intake, malabsorption, diminished muscle growth, and increased muscle breakdown. Sarcopenia is a multifaceted, chronic condition associated with elevated risks of morbidity and mortality. In the field of hepatology, sarcopenia is typically described as a phenotypic indication of muscle mass loss, affecting approximately 30-70% of liver cirrhosis patients. Sarcopenia has been scientifically linked to an increased risk of falls, reduced quality of life, the emergence of acute decompensated liver failure, and mortality in cirrhotic patients. Myostatin, classified as a cytokine within the transforming growth factor beta (TGF-β) family, is recognized for its role in disrupting protein synthesis. It functions as a negative regulator of muscle growth, inhibiting myogenesisand is linked to the onset of sarcopenia. Extensive literature supports the prognostic importance of sarcopenia in cirrhosis, and serum myostatin levels have the potential to serve as a valuable biomarker. Elevated myostatin concentrations have been linked to the presence of sarcopenia and decreased survival rates in individuals diagnosed with liver cirrhosis.

Transcriptomic Profiling to Understand Inhibitor Development in Previously Untreated Patients with Severe Hemophilia a

Background: When starting treatment with factor VIII (FVIII) concentrate in individuals with severe hemophilia A, minimizing the risk of FVIII inhibitor development is an important concern for clinicians, patients and their families. Despite increased understanding of some of the genetic and environmental risk factors for inhibitor formation from epidemiological studies, identification of previously untreated patients (PUPs) at risk of developing inhibitors remains a challenge. Prediction of inhibitor development may help optimize care in PUPs with hemophilia A. RNA sequencing (RNA-Seq) with next-generation sequencing (NGS) could be used to investigate the underlying mechanisms of inhibitor development. Aims: Toevaluate peripheral blood transcriptomic profiles prior to and during early FVIII treatment in severe hemophilia A patients who did or did not develop inhibitors to simoctocog alfain the prospective NuProtect study. Methods: The NuProtect study (NCT01712438; EudraCT 2012-002554-23) was a multicenter, multinational, open-label, non-controlled, phase III study that enrolled male PUPs with severe hemophilia A (FVIII:C &amp;lt;1%) of any age and ethnicity. Patients received simoctocog alfa for prophylaxis or on-demand treatment, as well as for surgical prophylaxis. RNA-Seq was performed on blood samples collected from baseline and every 3-4 exposure days (ED) up to 20 EDs or until inhibitor development to assess messenger RNA (mRNA) expression profiles. Bioinformatics analyses were performed for the following timepoints: baseline, early treatment and a later timepoint on treatment. Principal component analysis (PCA) was performed to identify differences in global mRNA expression profiles between inhibitor and non-inhibitor patients. Gene ontology (GO) analyses were performed to detect differences in GO clusters between inhibitor and non-inhibitor patients. Results: Within this first analysis of the full dataset, transcriptomic profiles from 78 of 108 severe hemophilia A PUPs were analyzed (inhibitors n=22, non-inhibitors n=56). Median (range) age at ED1 was 13 (0.5-146) months; 44.9% of patients had the intron 22 inversion F8 gene mutation. Although the PCA indicated no strong global signal for differential expression profiles between non-inhibitor and inhibitor patients, GO analyses demonstrated differences between these patient groups. Inhibitor patients demonstrated increased gene expression of metabolic processes (GO:0008152) and cellular processes (GO:0009987) at baseline. Upregulation of immune system processes (GO:0002376) was seen in inhibitor patients at the early timepoint compared with non-inhibitor patients. Expression of ribosomal proteins, including RPL17 and RPS8 were significantly upregulated at baseline in inhibitor patients (p&amp;lt;0.05). Inhibitor patients further demonstrated upregulation of proteins involved in adaptive immune responses: IGHV1-69-2 at all timepoints (p&amp;lt;0.01) and IGHV2-70D at baseline and the early timepoint (p&amp;lt;0.01) compared with non-inhibitor patients. Upregulation of the B-cell related factors DPP4 at baseline and the early timepoint (p&amp;lt;0.05) and CD20 at the late timepoint (p&amp;lt;0.05) was observed in inhibitor patients compared with non-inhibitor patients. Conclusion: Differential transcriptomic profiles were seen in severe hemophilia A patients who developed inhibitors compared with those who do not, even before commencing FVIII treatment. Inhibitor patients demonstrated alterations in ribosomal protein expression that could disrupt protein synthesis efficiency, protein folding and quality control processes, and trigger cellular stress responses. Inhibitor patients also showed an upregulation of B-cell mediated factors involved in adaptive immune mechanisms. Additional analyses are ongoing to evaluate dynamic changes in gene expression during commencement of FVIII replacement to further identify patients at risk of inhibitor development.

Genetic Determinants of Dependency on Regulated Protein Synthesis in Leukemia Stem Cells

Acute myeloid leukemia (AML) is initiated and sustained by rare self-renewing leukemia stem cells (LSCs). LSCs do not resemble HSCs, but rather more mature hematopoietic progenitor cells that usually have no self-renewal potential. However, during leukemogenesis, LSCs become aberrantly self-renewing, but the mechanisms that allow for this are poorly understood. We recently used models of AML driven by AML-ETO9a and MLL-AF9 to show that LSCs may co-opt from HSCs a dependence on regulation of protein synthesis mediated by CD99 (ASH 2021, #2222), which we identified as a therapeutic target highly expressed in LSCs (PMID: 28123069). However, it remains unclear if LSCs from specific subtypes of AML are particularly dependent on regulated protein synthesis. In an analysis of transcriptome data from the ECOG 1900 cohort, we found that certain mutations ( KIT, PTEN, AML-ETO, IDH1/2 and FLT3-ITD) were associated with the highest levels of CD99, raising the possibility that such AML subtypes are more dependent on CD99. In the TCGA AML cohort, CD99 expression was significantly higher in AML patients with FLT3-ITD mutations but not NRAS mutations ( Fig. 1A). To test if LSCs from AMLs driven by mutations in FLT3 or NRAS differ in their dependence on regulation of protein synthesis by CD99, we crossed Cd99 -/-(CD99 KO) mice with two mouse models of AML driven by these alleles to generate Cd99 -/-; Mx1-Cre; Tet2 fl/fl; Flt3 ITD/+and Cd99 -/-; Mx1-Cre; Tet2 fl/fl; Nras LSL-G12D/+ mice (CD99 KO TET2/FLT3 and CD99 KO TET2/NRAS). TET2 was chosen as a cooperating allele given its neutral association with CD99 in the above human datasets, and it additionally co-occurs frequently with mutations in FLT3 or NRAS. To generate AML in both models, Tet2 was deleted with polyinosine-polycytidylic acid injections, followed by transplant of 4 million bone marrow (BM) cells into irradiated wild-type (WT) mice. At 16 weeks, recipients of CD99 WT TET2/FLT3 BM developed an expansion of aberrant donor-derived Mac-1 + c-kit + cells in the peripheral blood (PB), and BM aspirates revealed an accumulation of LSK CD48 +CD150 - cells that harbor LSC activity in this model ( Fig. 1B). In contrast, recipients of CD99 KO TET2/FLT3 BM exhibited significant depletion of these aberrant populations (P=0.0055 and P=0.019 respectively, n=10 for each group). Recipients of CD99 WT TET2/FLT3 BM also exhibited increased leukocytosis and thrombocytopenia, which were attenuated in recipients of CD99 KO TET2/FLT3 BM (P=0.046 and P=0.042, respectively). In contrast, there was no difference between recipients of CD99 WT TET2/NRAS and CD99 KO TET2/NRAS BM in emergence of the aberrant Mac-1 + c-kit + cells and thrombocytopenia (P=0.3237 and P=0.9752, respectively, n=10 for each group). These data suggest that loss of CD99 may impair leukemogenesis in TET2/FLT3 but not TET2/NRAS AMLs. To determine if CD99 is required for LSC function, we performed secondary transplants of one million BM cells from primary recipients into lethally irradiated WT recipients. Mice transplanted with CD99 KO TET2/FLT3 BM, compared with those transplanted with CD99 WT TET2/FLT3 BM, demonstrated fewer donor-derived cells in the PB. While CD99 WT TET2/FLT3-transplanted mice succumbed to AML at a median of 11 weeks, all CD99 KO TET2/FLT3-transplanted mice survived beyond 16 weeks. PB smears from CD99 KO TET2/FLT3 mice demonstrated leukocytes with condensed chromatin and lobulated nuclei, indicative of restored hematopoietic maturation. Finally, in vivo O-propargyl-puromycin assays showed that loss of CD99 resulted in increased protein synthesis in LSCs (LSK CD48 +CD150 -) (fold-change=1.69±0.18, n=3 for each group). In contrast, mice secondarily transplanted with CD99 KO TET2/NRAS BM exhibited similar engraftment, survival, and morphologic evidence of AML as those transplanted with CD99 WT TET2/NRAS BM. In conclusion, our data demonstrate that LSC function in AML driven by mutated FLT3, but not mutated NRAS, is dependent on maintenance of regulated protein synthesis by CD99. We speculate that this differential dependency may reflect a different cell-of-origin, as recent work has shown that AMLs associated with FLT3-ITD exhibit particularly primitive cellular hierarchies (PMID: 35618837). Finally, our results suggest that certain high-risk AML sub-types associated with elevated CD99 expression may be highly sensitive to strategies disrupting protein synthesis to deplete LSCs.

Dissemination of Methicillin-Resistant Staphylococcus aureus Sequence Type 764 Isolates with Mupirocin Resistance in China.

Mupirocin, a topical antimicrobial agent, is an important component in the eradication of methicillin-resistant Staphylococcus aureus (MRSA) colonization. The molecular characteristics of 46 mupirocin-resistant MRSA (MR-MRSA) clinical isolates were analyzed by multilocus sequence typing (MLST), staphylococcal cassette chromosome mec element (SCCmec) typing, spa typing, and analysis of virulence genes. All 26 MRSA isolates with low-level mupirocin resistance possessed a V588F mutation in ileS. Among 20 MRSA isolates with high-level resistance to mupirocin, all carried mupA; 2 isolates also possessed the V588F mutation in ileS, and 1 possessed the V631F mutation in ileS (isoleucyl-tRNA synthetase). The majority of MR-MRSA isolates were resistant to erythromycin, clindamycin, tetracycline, ciprofloxacin, and gentamicin, but the rates of resistance to rifampin and fusidic acid were 8.7% and 6.5%, respectively. Eight sequence types (STs) were found among the 46 MR-MRSA isolates, of which ST764 was the most prevalent (76.1%). The most frequent spa type identified was t1084 (52.2%). The SCCmec type most frequently found was type II (80.4%). The most common clone among low-level MR-MRSA isolates was ST764-MRSA-SCCmec type II-t1084 (23 isolates), while ST764-MRSA-SCCmec type II-t002 (9 isolates) was the most common clone among high-level MR-MRSA isolates. Additionally, all toxin genes except the seb gene were not identified among ST764 isolates. Among clonal complex 5 (CC5) isolates, immune evasion cluster (IEC)-associated genes (chp, sak, and scn) and seb were present in ST764 but absent in ST5, while sec, sel1, tsst-1, and hlb genes were identified in ST5 but absent in ST764. In conclusion, the spread of CC5 clones, especially a novel ST764-MRSA-SCCmec type II-t1084 clone with high-level resistance to mupirocin, was responsible for the increase in mupirocin resistance. These findings indicated that the emergence of the ST764 MR-MRSA clone involves a therapeutic challenge for treating serious MRSA infections. IMPORTANCE Mupirocin, a topical antibiotic that is commonly used for the nasal decolonization of MRSA and methicillin-sensitive Staphylococcus aureus in hospital settings and nursing homes, was introduced as a highly effective antibiotic against MRSA. Mupirocin acts by competitively binding isoleucyl-tRNA synthetase, thereby disrupting protein synthesis. This drug shows bacteriostatic and bactericidal activity at low and high concentrations, respectively. However, with the increase in mupirocin use, low-level and high-level resistance during nasal mupirocin treatment has been reported. In a previous study, the proportion of MRSA strains with high-level mupirocin resistance in a Canadian hospital increased from 1.6% in the first 5 years of surveillance (1995 to 1999) to 7.0% (2000 to 2004).

A New Combination: Anti Glypican-3 scFv and Diphtheria Toxin with the Best Flexible Linker.

Along with all cancer treatments, including chemotherapy, radiotherapy, and surgery, targeting therapy is a new treatment manner. Immunotoxins are new recombinant structures that kill cancer cells by targeting specific antigens. Immunotoxins are composed of two parts: toxin moiety, which disrupts protein synthesis process, and antigen binding moiety that bind to antigens on the surface of cancer cells. Glypican 3 (GPC3) is an oncofetal antigen on the surface of Hepatocellular carcinoma (HCC) cells. In this study, truncated Diphtheria toxin (DT389) was fused to humanized scFv YP7 by one, two and three repeats of GGGGS linkers (DT389-(GGGGS)1-3YP7). In-silico and experimental investigation were performed to find out how many repeats of linker between toxin and scFv moieties are sufficient. Results of in-silico investigations revealed that the difference in the number of linkers does not have a significant effect on the main structures of the immunotoxin; however, the three-dimensional structure of two repeats of linker had a more appropriate structure compared to others with one and three linker replications. In addition, with enhancing the number of linkers, the probability of protein solubility has increased. Generally, the bioinformatics results of DT389-(GGGGS)2-YP7 structure showed that expression and folding is suitable; and YP7 scFv has appropriate orientation to bind GPC3. The experimental investigations indicated that the fusion protein was expressed as near to 50% soluble. Due to the high binding affinity of YP7 scFv and the proven potency of diphtheria in inhibiting protein synthesis, the proposed DT389-(GGGGS)2-YP7 immunotoxin is expected to function well in inhibiting HCC.

Sex-dependent differential transcript expression in the placenta of growth restricted infants

IntroductionThe pathological decrease of fetal growth during gestation can lead to subsequent poor health outcomes for the fetus. This process is commonly controlled by the placenta, the interface between mother and baby during gestation. Sex-specific gene expression has been implicated in placental function, therefore, there is a need to determine if it is important during reduced fetal growth. We therefore aimed to characterise placental gene expression at term to evaluate sex-specific genetic changes that occur in small for gestational age (SGA) infants. MethodsRNA-sequencing of twelve human placental tissue samples collected from pregnancies yielding either term appropriate for gestational age (AGA) or SGA infants identified at delivery. Candidate genes associated with fetal size and fetal sex were identified using differential gene expression and weighted gene co-expression network analyses. Single-cell sequencing data was used for candidate validation and to estimate candidate transcript expression in specific placental cell populations. ResultsDifferential gene expression and weighted gene co-expression network analyses identified 403 candidate transcripts associated with SGA infants. One hundred and three of these transcripts showed sex-specific expression. . Published placental sequencing datasets were used to validate the key expression results from the twelve placental samples initially studied; the sex-independent transcript expression for genes involved in cell cycle processes in males (7 transcripts) and endoplasmic reticulum stress in females (17 transcripts). DiscussionThis study identified the activation of multiple molecular mechanisms involved in the placental response to an adverse environmental stressor. Mechanisms such as disrupted protein synthesis were shared between infant biological sex when comparing AGA to SGA, whilst other pathways such as cell cycle and endoplasmic reticulum stress appear as independent/specific to either males or females when investigating reduced fetal growth. This data suggests that sexual dimorphism is an important consideration when examining placental dysfunction and poor fetal growth.

Modern methods of detection and identification of microbial toxins that inhibit protein synthesis in cells

Pathogenic microorganisms and products of their metabolism, namely, bacterial protein exotoxins, are considered one of the main sources of biological threat. Microbial toxins are highly active and extremely dangerous to humans. Determining trace amounts of such compounds remains relevant in healthcare and biological protection sector. Timely qualitative- and quantitative-specific indication of biotoxins is a key component in the diagnosis and implementation of therapeutic and preventive measures. Pathogenic microorganisms and products of their metabolism, bacterial protein exotoxins, are considered one of the main sources of biological threat. Microbial toxins are highly active and extremely dangerous to humans. Determining trace amounts of such compounds remains relevant in healthcare and biological protection sector. Timely qualitative- and quantitative-specific indication of biotoxins is a key component in the diagnosis and implementation of therapeutic and preventive measures. The current state and prospects of development in formulating specific indications of microbial toxins that disrupt protein synthesis in cells are analyzed. Modern ideas about the structure and mechanism of action of these toxins are briefly presented. Possibilities were considered, the advantages and disadvantages of classical traditional and modern innovative methods for identifying bacterial toxins that inhibit protein synthesis in cells were compared, and classifications were provided. Examples of the use of various approaches to identify the most significant representatives of this group in both clinical material and in environmental objects, including regulated ones, were given. The review also listed modern domestic and foreign developments in formulating specific indications of microbial toxins inhibiting protein synthesis. The review summarizes the results of studies to determine the current directions in the development of tools and methods for rapid specific indication of microbial toxins. The main trends in the creation of new methods of toxicological screening as part of an effective national system for monitoring biological threats were analyzed. Prospects for the development and introduction to the market of domestic test systems and automatic analysis platforms for the detection of bacterial toxins in environmental objects and biological material were determined.

Conformational sampling of CMT-2D associated GlyRS mutations

During protein synthesis, aminoacyl-tRNA synthetases covalently link amino acids with their cognate tRNAs. Amino acid mutations in glycyl-tRNA synthetase can disrupt protein synthesis and lead to a neurological disorder known as Charcot-Marie-Tooth disease type 2D (CMT-2D). Several studies employing diverse techniques have identified potential disease mechanisms at the molecular level. The majority of CMT-2D mutations in glycyl-tRNA are found within its dimer interface. However, no atomic structures bearing these mutations have been solved. Consequently, the specific disease-causing structural changes that occur in glycyl-tRNA synthetase have not been definitively established. Here we use molecular dynamics simulations to probe conformational changes in glycyl-tRNA synthetase caused by one mutation within the dimer interface: G240R. Our results show that the mutation alters the number of native interactions at the dimer interface and also leads to altered dynamics of two regions of glycyl-tRNA synthetase associated with tRNA binding. Additionally, we use our simulations to make predictions about the effects of other clinically reported CMT-2D mutations. Our results identify a region of the glycyl-tRNA synthetase structure that may be disrupted in a large number of CMT-2D mutations. Structural changes in this region may be a common molecular mechanism in glycyl-tRNA synthetase CMT-2D pathologies. Statement of significanceIn this study, we use molecular dynamics simulations to elucidate structural conformations accessible to glycyl-tRNA synthetase (GlyRS), an enzyme that ligates cytosolic glycine with tRNA-Gly. This protein contains multiple flexible regions with dynamics that elude in vitro structural characterization. Our computational approach provides unparalleled atomistic details of structural changes in GlyRS that contribute to its role in protein synthesis. A number of mutations in GlyRS are associated with a peripheral nerve disorder, Charcot-Marie-Tooth disease type 2D (CMT-2D). Mutation-induced structural and dynamic changes in GlyRS have similarity that elude in vitro structural characterization. Our simulations provide insights into disease mechanisms for one such mutation: G240R. Additionally, we leverage our computational data to identify regions of GlyRS critical to its function and to predict the effects of other disease-associated mutations. These results open up new directions for research into the molecular characterization of GlyRS and into hypothesis-driven studies of CMT-2D disease mechanisms.

Leukemia Stem Cells Depend on Regulated Protein Synthesis

Acute myeloid leukemia (AML) is initiated and sustained by leukemia stem cells (LSCs) which arise from progenitor cells that do not usually self-renew but become aberrantly self-renewing. It is thought that LSCs gain aberrant self-renewal potential by co-opting molecular and cellular programs from hematopoietic stem cells (HSCs) (PMID: 16862118). HSCs have been shown to require tightly regulated protein synthesis rates, where increased or decreased protein synthesis impairs self-renewal (PMID: 24670665), but it is not known if LSCs share this dependence. We have shown that human LSCs reside in the population of AML cells with the highest levels of CD99 (PMID: 28123069). In RNA-sequencing studies, we found that LSCs with high levels of CD99 are depleted for ribosomal protein transcripts. We thus reasoned that similar to HSCs, LSCs may depend on tightly regulated protein synthesis to self-renew.To test if CD99 promotes LSC function by constraining protein synthesis, we transduced c-Kit+ cells from B6-CD99 Gt(pU-21T)44lmeg (CD99 KO) or wild-type (WT) mice to express AML1-ETO9a (AE9a) and transplanted them into WT mice treated with rapamycin or vehicle. There was no difference in leukemogenesis in primary recipients, but CD99 KO-AE9a AMLs exhibited a 72% (p=0.048) increase in protein synthesis compared with WT-AE9a AMLs (Figure 1A), confirming that CD99 negatively regulates protein synthesis in AML. We next performed secondary transplants to assess LSC function, as measured by survival of secondary recipients in the absence of rapamycin treatment (Figure 1B). We furthermore performed these transplants at limiting dilution to quantify LSCs (Figure 1C). CD99 KO-AE9a vehicle treated AMLs demonstrated improved survival and a lower LSC frequency compared with WT-AE9a vehicle treated AMLs, consistent with a self-renewal defect with loss of CD99. Rapamycin treatment completely rescued this defect, leading to decreased survival and increased LSC frequency in CD99 KO-AE9a AMLs compared with vehicle. Conversely, rapamycin treatment depleted LSCs in WT-AE9a AMLs, increasing survival and decreasing LSC frequency compared with vehicle. Thus, similar to HSCs, LSCs are adversely affected by both increases or decreases in protein synthesis.MLL-AF9-induced mouse AMLs initiated in HSCs as compared with granulocyte macrophage progenitors (GMPs) exhibit increased epigenetic imprinting of HSC features resulting in disease features reminiscent of high-risk AML (PMID: 23235717). To test if HSC-derived leukemias exhibit increased dependence on regulated protein synthesis, we transduced HSCs or GMPs from CD99 KO or WT mice to express MLL-AF9 and transplanted them into WT recipients, followed by secondary transplants to assess LSC function. Loss of CD99 led to increased survival indicative of decreased LSC function in HSC-derived but not GMP-derived leukemias (Figure 1D). This suggests that HSC-derived leukemias co-opt from HSCs a more pronounced dependence on tightly regulated protein synthesis. Accordingly, WT HSC-derived leukemias exhibited decreased protein synthesis as compared with their WT GMP-derived counterparts (Figure 1E), as well as increased sensitivity to rapamycin (Figure 1F).To directly study protein synthesis in human LSCs, we transduced primary AML specimens (n=4) to express a destabilized form of GFP (dGFP) from a constitutive promoter followed by xenotransplantation (Figure 1G), allowing us to measure dGFP by flow cytometry as a surrogate for protein synthesis rates in vivo. We validated this assay by measuring protein synthesis using orthogonal O-propargyl-puromycin incorporation assays (Figure 1H). Human AML cells with low levels of dGFP demonstrated increased engraftment in secondary transplants (Figure 1I), demonstrating that human LSCs exhibit low protein synthesis rates.In conclusion, our data demonstrate that LSCs co-opt from HSCs a dependence on tightly regulated protein synthesis. This is the first description of a cellular feature co-opted from HSCs that also represents a therapeutic vulnerability. Furthermore, the types of AML that exhibit the most robust re-activation of HSC programs and increased dependence on regulated protein synthesis are also likely to represent high-risk AMLs most resistant to standard therapies. Our data suggest that such therapy resistant AMLs may be highly sensitive to strategies disrupting protein synthesis to deplete LSCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Chemotherapy agents reduce protein synthesis and ribosomal capacity in myotubes independent of oxidative stress.

Chemotherapeutic agents (CAs) are first-line antineoplastic treatments against a wide variety of cancers. Despite their effectiveness in halting tumor progression, side effects associated with CAs promote muscle loss by incompletely understood mechanisms. To address this problem, we first identified how oxidative stress impairs protein synthesis in C2C12 myotubes. Transient elevations in reactive oxygen species (ROS) resulted in protein synthesis deficits and reduced ribosomal (r)RNA levels. Oxidative stress did not reduce rRNA gene (rDNA) transcription, but it caused an increase in rRNA and protein oxidation. To determine whether CAs affect protein synthesis independent of oxidative stress, we exposed myotubes to Paclitaxel (PTX), Doxorubicin (DXR), or Marizomib (Mzb) at doses that did result in elevated ROS levels (sub-ROS). Exposure to CAs reduced protein synthesis and rRNA levels, but unlike oxidative stress, sub-ROS exposures impaired rDNA transcription. These results indicate that although oxidative stress disrupts protein synthesis by compromising ribosomal quantity and quality, CAs at sub-ROS doses compromise protein synthesis and ribosomal capacity, at least in part, by reducing rDNA transcription. Therefore, CAs negatively impact protein synthesis by causing oxidative stress in addition to directly reducing the ribosomal capacity of myotubes in a ROS-independent manner.

S1960 Acute Pseudomembranous Colitis, Microangiopathic Hemolytic Anemia, and Renal Injury After Pericardiectomy

Introduction: Acute colonic ischemia following surgery is usually a result of colonic hypoperfusion, particularly in watershed regions; however, it is critical to maintain a broad differential diagnosis for alternate etiologies. Case description/methods: A 57-year-old man with lupus on hydroxychloroquine and prednisone underwent elective pericardiectomy for medically refractory constrictive pericarditis. On post-operative day (POD) 3, he developed abdominal pain, nausea, and non-bloody diarrhea. Hematochezia started on POD 4. Computed tomography showed rectosigmoid thickening and perirectal fat stranding (Figure 1A). Sigmoidoscopy revealed edematous, ulcerated, and discolored mucosa from the anal verge to 30 cm proximally that abruptly normalized thereafter (Figure 1B). Sigmoid histology showed lamina propria hemorrhage with crypt withering and focal pseudomembranes (Figure 1C). Clostridioides difficile testing was negative. Colonic ischemia was favored. Given persistent leukocytosis and low fevers, antibiotics were started. From POD 4 to 8, platelets dropped from 142 to 11 × 109/L, and creatinine rose from 1.1 to 2.0 mg/dL (Figure 2). On POD 6, labs showed lactate dehydrogenase 1,107 U/L, indirect bilirubin 2.7 mg/dL, and undetectable haptoglobin, with hemoglobin dropping from 14.0 to 12.9 g/dL from POD 7 to 8. Peripheral smear on POD 8 showed schistocytes (Figure 3). Serum ADAMTS-13, complement levels, and heparin-induced thrombocytopenia panels were normal. Stool PCR detected shiga-toxin producing E. coli and shiga toxin 2, and stool cultures did not grow the 0157:H7 E. coli strain. Antibiotics were stopped due to the diagnosis of shiga-toxin induced hemolytic uremic syndrome (ST-HUS). The hemolysis, thrombocytopenia, and renal injury resolved after one week of supportive measures. Discussion: Infection by shiga-toxin producing E. coli (STEC), especially with shiga toxin 2 (which disrupts protein synthesis, causing apoptosis), presents with hemorrhagic colitis in 80% of cases and ST-HUS in up to 15% of cases. STEC is often acquired from contaminated food/ water. In this case, a hamburger from an external vendor was the suspected source. After a 3-day incubation period, STEC classically presents with abdominal pain and non-bloody diarrhea, followed by bloody diarrhea 1-3 days later. If it occurs, ST-HUS presents 1-2 weeks later from thrombotic microangiopathy. Antibiotics are linked to shiga toxin production by the encoding bacteriophage in the STEC chromosome.Figure 1.: Acute pseudomembranous colitis. A) Rectosigmoid wall thickening (green arrow) and perirectal fat stranding. B) Rectosigmoid colon was edematous, cyanotic, ulcerated, with luminal narrowing by flexible sigmoidoscopy. There was an abrupt transition to normal sigmoid mucosa at 30 cm from anal verge (not shown). C) Pathologic specimens from sigmoid colon showed crypt withering (blue arrow), lamina propria hemorrhage (orange arrow), and pseudomembrane (yellow arrow).Figure 2.: Clinical course of shiga-toxin induced hemolytic uremic syndrome (ST-HUS). Non-bloody diarrhea started on POD 3, followed by bloody diarrhea on POD 4. The patient’s thrombotic microangiopathy, signified by reduction in platelets and renal injury, began 3-4 days later, illustrating the characteristic delay from diarrhea onset and thrombotic microangiopathy in ST-HUS.Figure 3.: Peripheral smear. Peripheral smear was notable for schistocytes (blue arrow), suggestive of hemolysis, and markedly diminished number of platelets (green arrow). The combination of these features was consistent with thrombotic microangiopathy.

Podophyllotoxin Exposure Affects Organelle Distribution and Functions in Mouse Oocyte Meiosis.

Podophyllotoxin (POD) is one of the most characterized lignans that is commonly found in podophyllum, and its preparations and derivatives are widely used in clinical treatment due to strong antitumor and antivirus activities. POD has been reported for its neurotoxicity, liver toxicity, and potential reproductive toxicity. In the present study, we investigated the effects of POD on the organelles of mouse oocytes during meiosis. Our results showed that exposure to POD significantly reduced the developmental competence of mouse oocytes. Further analysis revealed that the endoplasmic reticulum (ER) failed to accumulate to the spindle periphery, suggesting that POD exposure might affect protein synthesis during oocyte meiotic maturation. Similarly, abnormal Golgi apparatus distribution was found after POD exposure, which could be confirmed by the aberrant localization of Rab11a-related vesicles, indicating that POD induced vesicle-based protein transport disorder. We also found the aberrant accumulation of lysosomes in the cytoplasm of POD-exposed oocytes, which implied that POD might lead to aberrant protein degradation. Moreover, the perinuclear distribution of mitochondria was also significantly disturbed, indicating the mitochondrial dysfunction after POD exposure. In all, our study illustrated that exposure to POD might disrupt protein synthesis, transport, degradation, and ATP production by its effects on the distribution and functions of organelles during mouse oocyte meiotic maturation.

Real-time measurements of aminoglycoside effects on protein synthesis in live cells

The spread of antibiotic resistance is turning many of the currently used antibiotics less effective against common infections. To address this public health challenge, it is critical to enhance our understanding of the mechanisms of action of these compounds. Aminoglycoside drugs bind the bacterial ribosome, and decades of results from in vitro biochemical and structural approaches suggest that these drugs disrupt protein synthesis by inhibiting the ribosome's translocation on the messenger RNA, as well as by inducing miscoding errors. So far, however, we have sparse information about the dynamic effects of these compounds on protein synthesis inside the cell. In the present study, we measured the effect of the aminoglycosides apramycin, gentamicin, and paromomycin on ongoing protein synthesis directly in live Escherichia coli cells by tracking the binding of dye-labeled transfer RNAs to ribosomes. Our results suggest that the drugs slow down translation elongation two- to fourfold in general, and the number of elongation cycles per initiation event seems to decrease to the same extent. Hence, our results imply that none of the drugs used in this study cause severe inhibition of translocation.