Search

Introduction: Solute carrier (SLC) transport proteins play a crucial role in maintaining cellular nutrient and metabolite homeostasis and are implicated in various human diseases, making them potential targets for therapeutic interventions. However, the study of SLCs has been limited due to the lack of suitable tools, particularly cell-based substrate uptake assays, necessary for understanding their biological functions and for drug discovery purposes.Methods: In this study, a cell-based uptake assay was developed using a stable isotope-labeled compound as the substrate for SLCs, with detection facilitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay aimed to address the limitations of existing assays, such as reliance on hazardous radiolabeled substrates and limited availability of fluorescent biosensors.Results: The developed assay was successfully applied to detect substrate uptakes by two specific SLCs: L-type amino acid transporter 1 (LAT1) and sodium taurocholate co-transporting polypeptide (NTCP). Importantly, the assay demonstrated comparable results to the radioactive method, indicating its reliability and accuracy. Furthermore, the assay was utilized to screen for novel inhibitors of NTCP, leading to the identification of a potential NTCP inhibitor compound.Discussion: The findings highlight the utility of the developed cell-based uptake assay as a rapid, simple, and environmentally friendly tool for investigating SLCs’ biological roles and for drug discovery purposes. This assay offers a safer alternative to traditional methods and has the potential to contribute significantly to advancing our understanding of SLC function and identifying therapeutic agents targeting SLC-mediated pathways.

Abstract Microbial production of monoterpenoid indole alkaloids (MIAs) provides a sustainable and eco‐friendly means to obtain compounds with high pharmaceutical values. However, efficient biosynthesis of MIAs in heterologous microorganisms is hindered due to low supply of key precursors such as geraniol and its derivative 8‐hydroxygeraniol catalyzed by geraniol 8‐hydroxylase (G8H). In this study, we developed a facile evolution platform to screen strains with improved yield of geraniol by using the SCRaMbLE system embedded in the Sc2.0 synthetic yeast and confirmed the causal role of relevant genomic targets. Through genome mining, we identified several G8H enzymes that perform much better than the commonly used CrG8H for 8‐hydroxygeraniol production in vivo. We further showed that the N‐terminus of these G8H enzymes plays an important role in cellular activity by swapping experiments. Finally, the combination of the engineered chassis, optimized biosynthesis pathway, and utilization of G8H led to the final strain with more than 30‐fold improvement in producing 8‐hydroxygeraniol compared with the starting strain. Overall, this study will provide insights into the construction and optimization of yeast cells for efficient biosynthesis of 8‐hydroxygeraniol and its derivatives.

Abstract Synthetic auxotrophy in which cell viability depends on the presence of an unnatural amino acid (unAA) provides a powerful strategy to restrict unwanted propagation of genetically modified organisms (GMOs) in open environments and potentially prevent industrial espionage. Here, we describe a generic approach for robust biocontainment of budding yeast dependent on unAA. By understanding escape mechanisms, we specifically optimize our strategies by introducing designed “immunity” to the generation of amber-suppressor tRNAs and developing the transcriptional- and translational-based biocontainment switch. We further develop a fitness-oriented screening method to easily obtain multiplex safeguard strains that exhibit robust growth and undetectable escape frequency (

Abstract Induction of hypothermia during hibernation/torpor enables certain mammals to survive under extreme environmental conditions. However, pharmacological induction of hypothermia in most mammals remains a huge challenge. Here we show that a natural product P57 promptly induces hypothermia and decreases energy expenditure in mice. Mechanistically, P57 inhibits the kinase activity of pyridoxal kinase (PDXK), a key metabolic enzyme of vitamin B6 catalyzing phosphorylation of pyridoxal (PL), resulting in the accumulation of PL in hypothalamus to cause hypothermia. The hypothermia induced by P57 is significantly blunted in the mice with knockout of PDXK in the preoptic area (POA) of hypothalamus. We further found that P57 and PL have consistent effects on gene expression regulation in hypothalamus, and they may activate medial preoptic area (MPA) neurons in POA to induce hypothermia. Taken together, our findings demonstrate that P57 has a potential application in therapeutic hypothermia through regulation of vitamin B6 metabolism and PDXK serves as a previously unknown target of P57 in thermoregulation. In addition, P57 may serve as a chemical probe for exploring the neuron circuitry related to hypothermia state in mice.

Objective: This study aimed to investigate the role and mechanism of circular RNA PVT1 (circPVT1) in patients with acute myeloid leukemia (AML). Materials and Methods: The expression of circPVT1 in 23 patients with de novo AML (not acute promyelocytic leukemia, not APL) and cell lines were detected by RT-qPCR. Loss of function assays were carried out to explore the influence of silenced circPVT1 on the proliferation, migration, and apoptosis in the THP-1 cell line. CCK-8 assays, transwell assays, and annexin V/PI staining assays were performed to assess proliferation, migration, and apoptosis, respectively. Results: CircPVT1 was highly expressed in AML patients and myeloid cell lines compared to healthy controls. Higher expression of circPVT1 was related to shorter overall survival (OS) and relapse-free survival (RFS) in AML patients. Cell viability and migration were inhibited and apoptosis was increased when circPVT1 was knocked down in THP-1 cells. Knockdown of circPVT1 resulted in marked suppression of c-Myc protein with no significant change in mRNA levels. We also found that circPVT1 knockdown markedly increased the phosphorylation of c-Myc Thr-58, which was responsible for c-Myc degradation. Silencing of c-Myc caused a significant decrease in CXCR4 mRNA and protein expression, whereas the overexpression of c-Myc caused the opposite result, suggesting that CXCR4 is a target molecule of c-Myc. Finally, we found that overexpression of c-Myc could partially reverse circPVT1 knockdown-induced anti-tumor effects on THP-1 cells in vitro. Conclusion: Our findings showed that circPVT1 was highly expressed in AML patients and was related to shorter OS and RFS. CircPVT1 may exert an oncogenic effect in THP-1 cells by stabilizing c-Myc protein expression and downstream target CXCR4 expression. These data indicate that circPVT1 may be a promising therapeutic target for AML.

This paper addresses the issue of artificial visual inspection being overly reliant on subjective experience and the difficulty for the human eye to accurately identify dense and non-significant defects. To solve this problem, we have implemented an automatic object detection algorithm based on an improved version of YOLOv5.First, we use the K-means++ clustering algorithm to automatically calculate the Anchor of the model to reduce the effect of the close location of the initial clustering centers on the clustering of the sample data.Second, we add the Coordinate Attention (CA) attention mechanism to the model to allow the model to better capture and understand important features in the images. Then, we add a new detection layer with a downsampling multiplier of 4 to the Neck network to improve the precision of the model. Finally, we use the lightweight network MobileNetV3 instead of YOLOv5's backbone network to reduce the model detection time overhead.Our model achieves 85.87% mAP, which is 6.44% better than the YOLOv5 network, and the detection time for a single image is only 54ms, which is 50% faster than the YOLOv5 network. After testing, we have proven that our proposed algorithm can quickly and accurately detect the condition of bearing appearance defects, improving detection efficiency and reducing costs.

Applications of the SCRaMbLE process are hindered due to the lack of facile and tight regulation and limited understanding of key factors that may affect the rearrangement outcomes. Here the authors present an approach to precisely regulate SCRaMbLE recombination in a dose-dependent manner using genetic code expansion technology with low basal activity.

PurposeTo investigate cerebrovascular hemodynamics, including critical closing pressure (CrCP) and pulsatility index (PI), and their independent relationship with cerebral small vessel disease (CSVD) burden in patients with small-vessel occlusion (SVO).MethodsWe recruited consecutive patients with SVO of acute cerebral infarction who underwent brain magnetic resonance imaging (MRI), transcranial Doppler (TCD) and CrCP during admission. Cerebrovascular hemodynamics were assessed using TCD. We used the CSVD score to rate the total MRI burden of CSVD. Multiple regression analysis was used to determine parameters related to CSVD burden or CrCP.ResultsNinety-seven of 120 patients (mean age, 64.51 ± 9.99 years; 76% male) completed the full evaluations in this study. We observed that CrCP was an independent determinant of CSVD burden in four models [odds ratio, 1.41; 95% confidence interval (CI), 1.17–1.71; P < 0.001] and correlated with CSVD burden [β (95% CI): 0.05 (0.04–0.06); P < 0.001]. In ROC analysis, CrCP was considered as a predictor of CSVD burden, and AUC was 86.2% (95% CI, 78.6–93.9%; P < 0.001). Multiple linear regression analysis showed that CrCP was significantly correlated with age [β (95% CI): 0.27 (0.06 to 0.47); P = 0.012], BMI [β (95% CI): 0.61 (0.00–1.22)] and systolic BP [β (95% CI): 0.16 (0.09–0.23); P < 0.001].ConclusionsCrCP representing cerebrovascular tension is an independent determinant and predictor of CSVD burden. It was significantly correlated with age, BMI and systolic blood pressure. These results provide new insights in the mechanism of CSVD development.

Dyslipidemia due to renal insufficiency is a common complication in patients with chronic kidney diseases (CKD), and a major risk factor for the development of cardiovascular events. Atorvastatin (AT) is mainly used in the treatment of dyslipidemia in patients with CKD. However, response to the atorvastatin varies inter-individually in clinical applications. We examined the association between polymorphisms in genes involved in drug metabolism and transport, and plasma concentrations of atorvastatin and its metabolites (2-hydroxy atorvastatin (2-AT), 2-hydroxy atorvastatin lactone (2-ATL), 4-hydroxy atorvastatin (4-AT), 4-hydroxy atorvastatin lactone (4-ATL), atorvastatin lactone (ATL)) in kidney diseases patients. Genotypes were determined using TaqMan real time PCR in 212 CKD patients, treated with 20 mg of atorvastatin daily for 6 weeks. The steady state plasma concentrations of atorvastatin and its metabolites were quantified using ultraperformance liquid chromatography in combination with triple quadrupole mass spectrometry (UPLC−MS/MS). Univariate and multivariate analyses showed the variant in ABCC4 (rs3742106) was associated with decreased concentrations of AT and its metabolites (2-AT+2-ATL: β = -0.162, p = 0.028 in the dominant model; AT+2-AT+4-AT: β = -0.212, p = 0.028 in the genotype model), while patients carrying the variant allele ABCC4-rs868853 (β = 0.177, p = 0.011) or NR1I2-rs6785049 (β = 0.123, p = 0.044) had higher concentrations of 2-AT+2-ATL in plasma compared with homozygous wildtype carriers. Luciferase activity was enhanced in HepG2 cells harboring a construct expressing the rs3742106-T allele or the rs868853-G allele (p < 0.05 for each) compared with a construct expressing the rs3742106G or the rs868853-A allele. These findings suggest that two functional polymorphisms in the ABCC4 gene may affect transcriptional activity, thereby directly or indirectly affecting release of AT and its metabolites from hepatocytes into the circulation.

Oxylipins are important biological regulators that have received extensive research attention. Due to the extremely low concentrations, large concentration variations, and high structural similarity of many oxylipins, the quantitative analysis of oxylipins in biological samples is always a great challenge. Here, we developed a liquid chromatography-tandem mass spectrometry-based method with high sensitivity, wide linearity, and acceptable resolution for quantitative profiling of oxylipins in multiple biological samples. A total of 104 oxylipins, some with a high risk of detection crosstalk, were well separated on a 150 mm column over 20 min. The method showed high sensitivity with lower limits of quantitation for 87 oxylipins, reaching 0.05–0.5 pg. Unexpectedly, we found that the linear range for 16, 18, and 17 oxylipins reached 10,000, 20,000, and 40,000 folds, respectively. Due to the high sensitivity, while reducing sample consumption to below half the volume of previous methods, 74, 78, and 59 low-abundance oxylipins, among which some were difficult to detect like lipoxins and resolvins, were well quantified in the tested mouse plasma, mouse liver, and human plasma samples, respectively. Additionally, we determined that analytes with multifarious concentrations of over a 1,000-fold difference could be well quantified simultaneously due to the wide linearity. In conclusion, most likely due to the instrumental advancement, this method effectively improves the quantitative sensitivity and linear range over existing methods, which will facilitate and advance the study of the physiological and pathophysiological functions of oxylipins.

Lung cancer has the highest tumor incidence in China. Lung squamous cell carcinoma (LUSC) is the most common type, accounting for 40–51% of primary lung cancers. LUSC is slow in growth and late in metastasis. Immune-related genes (IRGs) and immune infiltrating cells play a vital role in the clinical outcomes of LUSC. It is important to systematically study its immune gene map to help the prognosis of cancer patients. In this study, we combined the prognostic landscape and expression status of IRGs downloaded from the TCGA and InnatedDB databases and systematically analyzed the prognostic information of LUSC patients to obtain IRGs. After systematically exploring the survival analysis, prognosis-related genes were found, and the PPI network revealed that a total of 11 genes were hub genes. A two-gene prognosis risk model was established by multivariate Cox analysis. Two IRGs were closely correlated with the prognosis of LUSC. Based on these two genes, a new independent prognostic risk model was established, and this model was further verified in the GEO database. Moreover, the risk score of the model was correlated with sex, survival status, and lymphatic metastasis in LUSC patients, and the predictive risk of the prognostic risk model was significantly positively correlated with five kinds of immune cells (CD4 T cells, CD8 T cells, neutrophils, macrophages, and dendritic cells). This study comprehensively analyzed immunogenomics and presented immune-related prognostic biomarkers for LUSC.

To boost energy efficiency and assure more wise resource allocation, China has developed a manufacturing power strategy, meaningfully implemented green manufacturing and intelligent manufacturing projects, and promoted the green, intelligent, and high-end manufacturing sector. China’s illogical energy structure, significant pollution problems, low energy consumption efficiency, and other crucial challenges are now seriously impeding the integration of China’s industrialization and informationization processes. Investigating whether intelligence may have a direct impact on energy efficiency is beneficial in order to attain intelligent energy and smart energy in the era of intelligent development. Using panel data from 30 provinces from 2011 to 2019, this study constructs a fixed-effects model to evaluate the aggregate and mediating impacts of energy efficiency. The findings demonstrate that intelligence may greatly increase energy efficiency, but the effect is defined by geographical variability, as opposed to improving energy efficiency through fostering technical advancement.

The site-specific incorporation of the noncanonical amino acid (ncAA) into proteins via genetic code expansion (GCE) has enabled the development of new and powerful ways to learn, regulate, and evolve biological functions in vivo. However, cellular biosynthesis of ncAA-containing proteins with high efficiency and fidelity is a formidable challenge. In this review, we summarize up-to-date progress towards improving the efficiency and orthogonality of GCE and enhancing intracellular compatibility of introduced translation machinery in the living cells by creation and optimization of orthogonal translation components, constructing genomically recoded organism (GRO), utilization of unnatural base pairs (UBP) and quadruplet codons (four-base codons), and spatial separation of orthogonal translation.

Universally present aminoacyl-tRNA synthetases (aaRSs) stringently recognize their cognate tRNAs and acylate them with one of the proteinogenic amino acids. However, some organisms possess aaRSs that deviate from the accurate translation of the genetic code and exhibit relaxed specificity toward their tRNA and/or amino acid substrates. Typically, these aaRSs are part of an indirect pathway in which multiple enzymes participate in the formation of the correct aminoacyl-tRNA product. The indirect cysteine (Cys)-tRNA pathway, originally thought to be restricted to methanogenic archaea, uses the unique O-phosphoseryl-tRNA synthetase (SepRS), which acylates the non-proteinogenic amino acid O-phosphoserine (Sep) onto tRNACys. Together with Sep-tRNA:Cys-tRNA synthase (SepCysS) and the adapter protein SepCysE, SepRS forms a transsulfursome complex responsible for shuttling Sep-tRNACys to SepCysS for conversion of the tRNA-bound Sep to Cys. Here, we report a comprehensive bioinformatic analysis of the diversity of indirect Cys encoding systems. These systems are present in more diverse groups of bacteria and archaea than previously known. Given the occurrence and distribution of some genes consistently flanking SepRS, it is likely that this gene was part of an ancient operon that suffered a gradual loss of its original components. Newly identified bacterial SepRS sequences strengthen the suggestion that this lineage of enzymes may not rely on the m1G37 identity determinant in tRNA. Some bacterial SepRSs possess an N-terminal fusion resembling a threonyl-tRNA synthetase editing domain, which interestingly is frequently observed in the vicinity of archaeal SepCysS genes. We also found several highly degenerate SepRS genes that likely have altered amino acid specificity. Cross-analysis of selenocysteine (Sec)-utilizing traits confirmed the co-occurrence of SepCysE and the Sec-utilizing machinery in archaea, but also identified an unusual O-phosphoseryl-tRNASec kinase fusion with an archaeal Sec elongation factor in some lineages, where it may serve in place of SepCysE to prevent crosstalk between the two minor aminoacylation systems. These results shed new light on the variations in SepRS and SepCysS enzymes that may reflect adaptation to lifestyle and habitat, and provide new information on the evolution of the genetic code.

Abstract To explore the mechanism of microRNA‐155 (miR‐155) deficiency, protecting against experimental autoimmune prostatitis (EAP) in a toll‐like receptor 4 (TLR4)‐dependent manner. After wild‐type (WT) and miR‐155−/− mice were injected with complete Freund's adjuvant and prostate antigen to establish EAP model, half were randomly selected for injection with lipopolysaccharide (LPS, a TLR4 ligand). The following experiments were then performed: von Frey filaments, hematoxylin‐eosin (HE) staining, real time quantitative polymerase chain reaction (qRT‐PCR), Western blotting, and enzyme‐linked immunosorbent assay (ELISA). And the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH‐Px) and the level of Malondialdehyde (MDA) were detected by corresponding kits.miR‐155−/− mice with prostatitis exhibited the attenuated pelvic tactile allodynia/hyperalgesia and the suppressed TLR4/nuclear factor‐kappa B (NF‐κB) pathway as compared with the WT mice with prostatitis. In addition, LPS enhanced the upregulation of miR‐155 and the activation of the TLR4/NF‐κB pathway in the prostatic tissues of WT mice with EAP. Furthermore, prostatitis mice had aggravated inflammation scores accompanying the increased interleukin (IL)‐1β, tumor necrosis factor‐α, IL‐6, interferon‐γ, IL‐12, and MDA in prostatic tissues with the decreased IL‐10, SOD and GSH‐Px, and the unaltered IL‐4. Compared with the mice from the WT + EAP group and the miR‐155−/− + EAP + LPS group, mice from the miR‐155−/− + EAP group had decreased inflammation and oxidative stress. miR‐155 deficiency ameliorated pelvic tactile allodynia/hyperalgesia in EAP mice and improved inflammation and oxidative stress in prostatic tissues in a TLR4‐dependent manner involving NF‐κB activation, thereby exerting a therapeutic effect in chronic prostatitis treatment.

BackgroundBladder cancer (Bca) is the most common malignant tumor of the urinary system. Circular RNAs (circRNAs) have been recognized as key regulators in tumorigenesis. However, the molecular mechanisms underlying circRNAs involved in the progression of BCa remain largely unknown.MethodsWe detected the expression level of circular RNA TAF4B (circTAF4B) by qRT-PCR assay. Cell proliferation was evaluated by CCK-8 and colony formation assays. Wound healing and Transwell assays were performed to measure cell migration and invasion capability. Moreover, we performed qRT-PCR and western blotting assays to determine the expression levels of epithelial-mesenchymal transition (EMT) markers. A nuclear/cytoplasmic fractionation assay was used to measure the subcellular location of circTAF4B. RNA pull-down and dual-luciferase reporter assays were used to detect the target microRNA of circTAF4B. A mouse xenograft model was produced to analyze the effect of circTAF4B on the tumorigenesis of BCa.ResultsIn this study, we identified a novel circular RNA, circTAF4B, that is significantly upregulated in BCa and correlated with poor prognosis. Downregulated circTAF4B abolished the growth, metastasis and EMT process in BCa cells. Mechanistically, we found that circTAF4B facilitated the expression of transforming growth factor A (TGFA) by sponging miR-1298-5p. Finally, circTAF4B enhanced the proliferation and EMT process of BCa cells in vivo.ConclusionIn summary, our study demonstrated that circTAF4B played a carcinogenic role in the growth, metastasis, and EMT process of BCa by regulating the miR-1298-5p/TGFA axis. Thus, circTAF4B may become a diagnostic and therapeutic target for BCa.

Researches involving arterial pressure measurements in mice have primarily relied on carotid arterial catheterization. However, in some circumstances, measuring arterial pressure through the carotid arterial impairs accuracy. This study was aimed to evaluate whether femoral artery could displace carotid artery for the blood pressure (BP) measurements in mice. Fifty-six Swiss mice (n = 14 in each group) were randomized into four groups: control, left femoral artery, right femoral artery, and union group, in which BP was measured through left carotid, left femoral, right femoral artery, and simultaneously from right femoral artery and left carotid artery, respectively. Arterial pressure was recorded for 5 min after catheterization. There was no significant difference of the success rate and mortality rate among four groups (P > 0.05), and no obvious difference (P > 0.05) of catheterization time among the first three groups. For intergroup comparison of arterial pressure, there was no significant difference (P > 0.05) of the systolic blood pressure (SBP), diastolic BP, mean arterial pressure, and pulse pressure among the first three groups. For intragroup comparison, SBP, diastolic blood pressure (DBP), mean arterial pressure (MAP) monitored from right femoral artery were similar (P > 0.05) with those from left carotid artery, with significantly positive correlation. The mean values of difference of SBP, DBP, and MAP were −1.3, 1.2, and 0.5 mmHg, respectively. Our results indicated that femoral artery catheterization could be a safe, feasible, reliable, and accuracy alternative for the direct measurement of arterial pressure in anesthesia mice.

Curcumin exerts a neuroprotective effect on Alzheimer’s disease; however, it is not known whether microRNAs are involved in this protective effect. This study was conducted using swAPP695-HEK293 cells as an Alzheimer’s disease cell model. swAPP695-HEK293 cells were treated with 0, 0.5, 1, 2, 5, and 10 μM curcumin for 24 hours. The changes in miR-15b-5p, miR-19a-3p, miR-195-5p, miR-101-3p, miR-216b-5p, miR-16-5p and miR-185-5p expression were assessed by real-time quantitative polymerase chain reaction. The mRNA and protein levels of amyloid precursor protein, amyloid-β40 and amyloid-β42 were evaluated by quantitative real-time polymerase chain reaction, western blot assays and enzyme-linked immunosorbent assays. swAPP695-HEK293 cells were transfected with miR-15b-5p mimic, or treated with 1 μM curcumin 24 hours before miR-15b-5p inhibitor transfection. The effects of curcumin on amyloid precursor protein, amyloid-β40 and amyloid-β42 levels were evaluated by western blot assays and enzyme-linked immunosorbent assay. Luciferase assays were used to analyze the interaction between miR-15b-5p and the 3′-untranslated region of amyloid precursor protein. The results show that amyloid precursor protein and amyloid-β expression were enhanced in swAPP695-HEK293 cells compared with HEK293 parental cells. Curcumin suppressed the expression of amyloid precursor protein and amyloid-β and up-regulated the expression of miR-15b-5p in swAPP695-HEK293 cells. In addition, we found a negative association of miR-15b-5p expression with amyloid precursor protein and amyloid-β levels in the curcumin-treated cells. Luciferase assays revealed that miR-15b-5p impaired the luciferase activity of the plasmid harboring the 3′-untranslated region of amyloid precursor protein. These findings indicate that curcumin down-regulates the expression of amyloid precursor protein and amyloid-β in swAPP695-HEK293 cells, which was partially mediated by miR-15b-5p via targeting of the 3′-untranslated region of amyloid precursor protein.

Filaggrin, encoded by the FLG gene, plays a crucial role in the barrier function of epidermis, but the association between FLG loss-of-function mutations and infectious skin diseases has not been systematically studied. FLG coding sequences from 945 patients with leprosy and 916 healthy controls were captured and enriched using an array-based high-throughput system, and subjected to next-generation sequencing. The loss-of-function mutations found were further validated by Sanger sequencing. A total of 21 loss-of-function mutations were found in 945 patients with leprosy, with a carrier rate of 17.53%, while the prevalence of these mutations in 916 healthy controls was 14.77%, which was significantly lower than in patients. Two individual FLG loss-of-function mutations (K4022X and Q1790X) were found to be significantly associated with leprosy. These results suggest a possible role for filaggrin in defending against leprosy pathogens.

Thyroid storm is a rare but life-threatening condition mainly triggered by infection and abrupt discontinuation of antithyroid drug therapy for Graves’ disease. Pancytopenia is a rare adverse reaction to antithyroid drugs. We present a 13-year-old girl with thyroid storm and pancytopenia with symptoms similar to those of methimazole-induced pancytopenia. Although in this context the use of methimazole is still under debate, due to multiple normal complete blood counts (CBC) monitored during fever, sepsis-induced pancytopenia with thyroid storm was considered, and methimazole treatment combined with methylprednisolone and meropenem was able to resolve both pancytopenia and thyroid storm. During the period of infection and antithyroid drug therapy, close monitoring of CBC may help differentiate the aetiology of pancytopenia. This is the first paediatric case report that outlines the use of methimazole in the management of thyroid storm with pancytopenia.

The ubiquitin-like (Ubl) protein is widely distributed in Archaea and involved in many cellular pathways. A well-established method to reconstitute archaeal Ubl protein conjugation in vitro is important to better understand the process of archaeal Ubl protein modification. This protocol describes the in vitro reconstitution of Ubl protein modification and following analysis of this modification in Haloferax volcanii, a halophilic archaeon serving as the model organism.

ABSTRACT Methionine sulfoxide reductase A (MsrA) is an antioxidant enzyme found in all domains of life that catalyzes the reduction of methionine-S-sulfoxide (MSO) to methionine in proteins and free amino acids. We demonstrate that archaeal MsrA has a ubiquitin-like (Ubl) protein modification activity that is distinct from its stereospecific reduction of MSO residues. MsrA catalyzes this Ubl modification activity, with the Ubl-activating E1 UbaA, in the presence of the mild oxidant dimethyl sulfoxide (DMSO) and in the absence of reductant. In contrast, the MSO reductase activity of MsrA is inhibited by DMSO and requires reductant. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis reveals that MsrA-dependent Ubl conjugates are associated with DNA replication, protein remodeling, and oxidative stress and include the Ubl-modified MsrA, Orc3 (Orc1/Cdc6), and Cdc48d (Cdc48/p97 AAA+ ATPase). Overall, we found archaeal MsrA to have opposing MSO reductase and Ubl modifying activities that are associated with oxidative stress responses and controlled by exposure to mild oxidant. IMPORTANCE Proteins that are damaged by oxidative stress are often targeted for proteolysis by the ubiquitin-proteasome system (UPS). The mechanisms that control this response are poorly understood, especially under conditions of mild oxidative stress when protein damage is modest. Here, we discovered a novel function of archaeal MsrA in guiding the Ubl modification of target proteins in the presence of mild oxidant. This newly reported activity of MsrA is distinct from its stereospecific reduction of methionine-S-sulfoxide to methionine residues. Our results are significant steps forward, first, in elucidating a protein factor that guides Ubl modification in archaea, and second, in providing an insight into oxidative stress responses that can trigger Ubl modification in a cell.

Highly regulated and targeted protein degradation plays a fundamental role in almost all cellular processes. Determination of the protein half-life by the chase assay serves as a powerful and popular strategy to compare the protein stability and study proteolysis pathways in cells. Here, we describe a chase assay in Haloferax volcanii, a halophilic archaeon as the model organism.

Selenoproteins that contain selenocysteine (Sec) are found in all kingdoms of life. Although they constitute a small proportion of the proteome, selenoproteins play essential roles in many organisms. In photosynthetic eukaryotes, selenoproteins have been found in algae but are missing in land plants (embryophytes). In this study, we explored the evolutionary dynamics of Sec incorporation by conveying a genomic search for the Sec machinery and selenoproteins across Archaeplastida. We identified a complete Sec machinery and variable sizes of selenoproteomes in the main algal lineages. However, the entire Sec machinery was missing in the Bangiophyceae-Florideophyceae clade (BV) of Rhodoplantae (red algae) and only partial machinery was found in three species of Archaeplastida, indicating parallel loss of Sec incorporation in different groups of algae. Further analysis of genome and transcriptome data suggests that all major lineages of streptophyte algae display a complete Sec machinery, although the number of selenoproteins is low in this group, especially in subaerial taxa. We conclude that selenoproteins tend to be lost in Archaeplastida upon adaptation to a subaerial or acidic environment. The high number of redox-active selenoproteins found in some bloom-forming marine microalgae may be related to defense against viral infections. Some of the selenoproteins in these organisms may have been gained by horizontal gene transfer from bacteria.

Micro–nano-plastic (MNP) particles (p) in the environment can enter the human body and pose a potential threat to human health. However, it is unknown whether these substances are present in polypropylene (PP) plastic-bottled injections, which are used as high-frequency intravenous infusions to treat diseases. Therefore, the objective of this study was to identify and quantify insoluble MNP particles in 16 batches of injectable formulations within the validity period. Primarily, ethylene–propylene copolymer or P(E–P) micro-plastic (MP) particles (2–10 μm, 216 p/mL) were identified by micro-Raman spectroscopy, and nano-particles (

ABSTRACT The molecular mechanisms of targeted proteolysis in archaea are poorly understood, yet they may have deep evolutionary roots shared with the ubiquitin-proteasome system of eukaryotic cells. Here, we demonstrate in archaea that TBP2, a TATA-binding protein (TBP) modified by ubiquitin-like isopeptide bonds, is phosphorylated and targeted for degradation by proteasomes. Rapid turnover of TBP2 required the functions of UbaA (the E1/MoeB/ThiF homolog of archaea), AAA ATPases (Cdc48/p97 and Rpt types), a type 2 JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) homolog (JAMM2), and 20S proteasomes. The ubiquitin-like protein modifier small archaeal modifier protein 2 (SAMP2) stimulated the degradation of TBP2, but SAMP2 itself was not degraded. Analysis of the TBP2 fractions that were not modified by ubiquitin-like linkages revealed that TBP2 had multiple N termini, including Met1-Ser2, Ser2, and Met1-Ser2(p) [where (p) represents phosphorylation]. The evidence suggested that the Met1-Ser2(p) form accumulated in cells that were unable to degrade TBP2. We propose a model in archaea in which the attachment of ubiquitin-like tags can target proteins for degradation by proteasomes and be controlled by N-terminal degrons. In support of a proteolytic mechanism that is energy dependent and recycles the ubiquitin-like protein tags, we find that a network of AAA ATPases and a JAMM/MPN+ metalloprotease are required, in addition to 20S proteasomes, for controlled intracellular proteolysis. IMPORTANCE This study advances the fundamental knowledge of signal-guided proteolysis in archaea and sheds light on components that are related to the ubiquitin-proteasome system of eukaryotes. In archaea, the ubiquitin-like proteasome system is found to require function of an E1/MoeB/ThiF homolog, a type 2 JAMM/MPN+ metalloprotease, and a network of AAA ATPases for the targeted destruction of proteins. We provide evidence that the attachment of the ubiquitin-like protein is controlled by an N-terminal degron and stimulates proteasome-mediated proteolysis.

While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNA(Lys)UUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.