Alternate routes to mnm5s2U synthesis in Gram-positive bacteria.

The wobble bases of tRNAs that decode split codons are often heavily modified. In bacteria, tRNAGlu, Gln, Asp contains a variety of xnm5s2U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negative Escherichia coli K12 model. Despite the ubiquitous presence of mnm5s2U modification, genomic analysis shows the absence of mnmC orthologous genes, suggesting the occurrence of alternate biosynthetic schemes for the conversion of cmnm5s2U to mnm5s2U. Using a combination of comparative genomics and genetic studies, a member of the YtqA subgroup of the radical Sam superfamily was found to be involved in the synthesis of mnm5s2U in both Bacillus subtilis and Streptococcus mutans. This protein, renamed MnmL, is encoded in an operon with the recently discovered MnmM methylase involved in the methylation of the pathway intermediate nm5s2U into mnm5s2U in B. subtilis. Analysis of tRNA modifications of both S. mutans and Streptococcus pneumoniae shows that growth conditions and genetic backgrounds influence the ratios of pathway intermediates owing to regulatory loops that are not yet understood. The MnmLM pathway is widespread along the bacterial tree, with some phyla, such as Bacilli, relying exclusively on these two enzymes. Although mechanistic details of these newly discovered components are not fully resolved, the occurrence of fusion proteins, alternate arrangements of biosynthetic components, and loss of biosynthetic branches provide examples of biosynthetic diversity to retain a conserved tRNA modification in Nature.IMPORTANCEThe xnm5s2U modifications found in several tRNAs at the wobble base position are widespread in bacteria where they have an important role in decoding efficiency and accuracy. This work identifies a novel enzyme (MnmL) that is a member of a subgroup of the very versatile radical SAM superfamily and is involved in the synthesis of mnm5s2U in several Gram-positive bacteria, including human pathogens. This is another novel example of a non-orthologous displacement in the field of tRNA modification synthesis, showing how different solutions evolve to retain U34 tRNA modifications.

Factors associated with burnout among frontline nurses in the post-COVID-19 epidemic era: a multicenter cross-sectional study.

BACKGROUND: The COVID-19 pandemic has significantly increased the risk of burnout among frontline nurses. However, the prevalence of burnout and its associated factors in the post-pandemic era remain unclear. This research aims to investigate burnout prevalence among frontline nurses in the post-pandemic period and pinpoint associated determinants in China. METHODS: From April to July 2023, a cross-sectional study was carried out across multiple centers, focusing on frontline nurses who had been actively involved in the COVID-19 pandemic. The data collection was done via an online platform. The Maslach Burnout Inventory-Human Services Survey was utilized to evaluate symptoms of burnout. A multivariable logistic regression analysis was used to pinpoint factors associated with burnout. RESULTS: Of the 2210 frontline nurses who participated, 75.38% scored over the cut-off for burnout. Multivariable logistic regression revealed that factors like being female [odds ratio (OR) = 0.41, 95%CI = 0.29-0.58] and exercising 1-2 times weekly[OR = 0.53, 95%CI = 0.42-0.67] were protective factors against burnout. Conversely, having 10 or more night shifts per month[OR = 1.99, 95%CI = 1.39-2.84], holding a master's degree or higher[OR = 2.86, 95% CI = 1.59-5.15], poor health status[OR = 2.43, 95% CI = 1.93-3.08] and [OR = 2.82, 95%CI = 1.80-4.43], under virus infection[OR = 7.12, 95%CI = 2.10-24.17], and elevated work-related stress[OR = 1.53, 95% CI = 1.17-2.00] were all associated with an elevated risk of burnout. CONCLUSION: Our findings indicate that post-pandemic burnout among frontline nurses is influenced by several factors, including gender, monthly night shift frequency, academic qualifications, weekly exercise frequency, health condition, and viral infection history. These insights can inform interventions aimed at safeguarding the mental well-being of frontline nurses in the post-pandemic period.

Apocarotenoids are allosteric effectors of a dimeric plant glycosyltransferase involved in defense and lignin formation.

Glycosyltransferases are nature's versatile tools to tailor the functionalities of proteins, carbohydrates, lipids, and small molecules by transferring sugars. Prominent substrates are hydroxycoumarins such as scopoletin, which serve as natural plant protection agents. Similarly, C13-apocarotenoids, which are oxidative degradation products of carotenoids/xanthophylls, protect plants by repelling pests and attracting pest predators. We show that C13-apocarotenoids interact with the plant glycosyltransferase NbUGT72AY1 and induce conformational changes in the enzyme catalytic center ultimately reducing its inherent UDP-α-d-glucose glucohydrolase activity and increasing its catalytic activity for productive hydroxycoumarin substrates. By contrast, C13-apocarotenoids show no effect on the catalytic activity toward monolignol lignin precursors, which are competitive substrates. In vivo studies in tobacco plants (Nicotiana benthamiana) confirmed increased glycosylation activity upon apocarotenoid supplementation. Thus, hydroxycoumarins and apocarotenoids represent specialized damage-associated molecular patterns, as they each provide precise information about the plant compartments damaged by pathogen attack. The molecular basis for the C13-apocarotenoid-mediated interplay of two plant protective mechanisms and their function as allosteric enhancers opens up potential applications of the natural products in agriculture and pharmaceutical industry.

A new and improved method of library preparation for non-invasive prenatal testing: plasma to library express technology.

OBJECTIVES: This study aims to develop a novel library preparation method, plasma to library express technology (PLET), to construct next-generation sequencing (NGS) libraries directly from plasma without cell-free DNA (cfDNA) isolation. METHODS: Peripheral blood samples (600) were obtained from a retrospective cohort of 300 pregnant women prior to invasive diagnostic testing. The samples were subsequently distributed between library preparation methodologies, with 300 samples prepared by PLET and 300 by conventional methods for non-invasive prenatal testing (NIPT) to screen for common trisomies using low-pass whole genome next generation sequencing. RESULTS: NIPT conducted on PLET libraries demonstrated comparable metrics to libraries prepared using conventional methods, including 100% sensitivity and specificity. CONCLUSIONS: Our study demonstrates the potential utility of PLET in the clinical setting and highlights its significant advantages, including dramatically reduced process complexity and markedly decreased turnaround time.

Acceptors and Effectors Alter Substrate Inhibition Kinetics of a Plant Glucosyltransferase NbUGT72AY1 and Its Mutants.

One of the main obstacles in biocatalysis is the substrate inhibition (SI) of enzymes that play important roles in biosynthesis and metabolic regulation in organisms. The promiscuous glycosyltransferase UGT72AY1 from Nicotiana benthamiana is strongly substrate-inhibited by hydroxycoumarins (inhibitory constant Ki < 20 µM), but only weakly inhibited when monolignols are glucosylated (Ki > 1000 µM). Apocarotenoid effectors reduce the inherent UDP-glucose glucohydrolase activity of the enzyme and attenuate the SI by scopoletin derivatives, which could also be achieved by mutations. Here, we studied the kinetic profiles of different phenols and used the substrate analog vanillin, which has shown atypical Michaelis-Menten kinetics in previous studies, to examine the effects of different ligands and mutations on the SI of NbUGT72AY1. Coumarins had no effect on enzymatic activity, whereas apocarotenoids and fatty acids strongly affected SI kinetics by increasing the inhibition constant Ki. Only the F87I mutant and a chimeric version of the enzyme showed weak SI with the substrate vanillin, but all mutants exhibited mild SI when sinapaldehyde was used as an acceptor. In contrast, stearic acid reduced the transferase activity of the mutants to varying degrees. The results not only confirm the multi-substrate functionality of NbUGT72AY1, but also reveal that the enzymatic activity of this protein can be fine-tuned by external metabolites such as apocarotenoids and fatty acids that affect SI. Since these signals are generated during plant cell destruction, NbUGT72AY1 likely plays an important role in plant defense by participating in the production of lignin in the cell wall and providing direct protection through the formation of toxic phytoalexins.

Cardiovascular Disease Risk Stratification in Wrist Wearable Devices and e-Health App Users: A Large-Scale Retrospective Study.

Introduction: Wearable devices, including smart wristbands and watches, are often used with e-health applications (apps). The users' characteristics of wrist wearable devices currently lack description, and the cardiovascular disease (CVD) high-risk rate of users remains unknown. Purpose: This study aimed to (1) describe the basic characteristics and habits of users of the "Amazfit Health" app and Huami wrist wearable devices and (2) analyze the proportion and define the population characteristics of users with a high risk of developing CVD. Subjects and Methods: This study included users >18 years of age, residing in mainland China, using the "Amazfit Health" app and Huami wearable devices. Devices data and users' self-reported information were collected in the app. The risk stratification was based on WHO/ISH cardiovascular risk prediction charts for the Western Pacific Region. Subjects with CVD history, total cholesterol ≥8 mmol/L, or ≥10% predicted CVD risk and those with <10% predicted CVD risk were considered to be at high and low risk of developing CVD, respectively. Results: Data were obtained from 80,098 (total users) and 10,866 users (subjects) for risk stratification. The age of the total users and subjects were 45.6 ± 15.4 and 50.7 ± 14.0 years, respectively. The number of male and female users was 50,024, and 30,074 in total users, and 7,284, and 3,582 in subjects, respectively. The body mass index of total users and subjects was 24.0 ± 4.6 kg/m2 and 24.6 ± 3.8 kg/m2, respectively. By classifying users' residences into first-tier cities, municipalities and provincial capitals, and other areas, the numbers of total users were 20,179, 28,213, and 31,137, and subjects were 2,587, 3,966, and 4,269, respectively. The number of subjects with high CVD risk was 1,161, accounting for 10.7% of all subjects. Conclusions: Users with high CVD risk only accounted for a small proportion of the population of wearable devices users.

Six Uridine-Diphosphate Glycosyltransferases Catalyze the Glycosylation of Bioactive C13-Apocarotenols.

C13-apocarotenoids (norisoprenoids) are carotenoid-derived oxidation products that perform important physiological functions in plants. Although their biosynthetic pathways have been extensively studied, their metabolism including glycosylation remains poorly understood. Candidate uridine-diphosphate glycosyltransferase genes (UGTs) were selected based on their high transcript abundance in comparison with other UGTs in vegetative tissues of Nicotiana benthamiana and peppermint (Mentha × piperita), as these tissues are rich sources of apocarotenoid glucosides. Hydroxylated C13-apocarotenol substrates were produced by P450-catalyzed biotransformation and microbial/plant enzyme systems were established for the synthesis of glycosides. Natural substrates were identified by physiological aglycone libraries prepared from isolated plant glycosides. In total, we identified six UGTs that catalyze the glucosylation of C13-apocarotenols, where Glc is bound either to the cyclohexene ring or the butane side chain. MpUGT86C10 is a superior novel enzyme that catalyzes the glucosylation of allelopathic 3-hydroxy-α-damascone, 3-oxo-α-ionol, 3-oxo-7,8-dihydro-α-ionol (Blumenol C), and 3-hydroxy-7,8-dihydro-ß-ionol, whereas a germination test demonstrated the higher phytotoxic potential of a norisoprenoid glucoside in comparison to its aglycone. Glycosylation of C13-apocarotenoids has several functions in plants, including increased allelopathic activity of the aglycone, facilitating exudation by roots and allowing symbiosis with arbuscular mycorrhizal fungi. The results enable in-depth analysis of the roles of glycosylated norisoprenoid allelochemicals, the physiological functions of apocarotenoids during arbuscular mycorrhizal colonization, and the associated maintenance of carotenoid homeostasis.

Glucosylation of the phytoalexin N-feruloyl tyramine modulates the levels of pathogen-responsive metabolites in Nicotiana benthamiana.

Enzyme promiscuity, a common property of many uridine diphosphate sugar-dependent glycosyltransferases (UGTs) that convert small molecules, significantly hinders the identification of natural substrates and therefore the characterization of the physiological role of enzymes. In this paper we present a simple but effective strategy to identify endogenous substrates of plant UGTs using LC-MS-guided targeted glycoside analysis of transgenic plants. We successfully identified natural substrates of two promiscuous Nicotiana benthamiana UGTs (NbUGT73A24 and NbUGT73A25), orthologues of pathogen-induced tobacco UGT (TOGT) from Nicotiana tabacum, which is involved in the hypersensitive reaction. While in N. tabacum, TOGT glucosylated scopoletin after treatment with salicylate, fungal elicitors and the tobacco mosaic virus, NbUGT73A24 and NbUGT73A25 produced glucosides of phytoalexin N-feruloyl tyramine, which may strengthen cell walls to prevent the intrusion of pathogens, and flavonols after agroinfiltration of the corresponding genes in N. benthamiana. Enzymatic glucosylation of fractions of a physiological aglycone library confirmed the biological substrates of UGTs. In addition, overexpression of both genes in N. benthamiana produced clear lesions on the leaves and led to a significantly reduced content of pathogen-induced plant metabolites such as phenylalanine and tryptophan. Our results revealed some additional biological functions of TOGT enzymes and indicated a multifunctional role of UGTs in plant resistance.

MicroRNA-325-3p inhibits cell proliferation and induces apoptosis in hepatitis B virus-related hepatocellular carcinoma by down-regulation of aquaporin 5.

BACKGROUND: Hepatitis B virus (HBV) infection is acknowledged as the main cause of hepatocellular carcinoma (HCC). Moreover, previous studies have revealed that microRNAs (miRNAs) widely participate in regulation of various cellular processes, such as viral replication. Hence, the purpose of this study was to investigate the roles of aquaporin 5 (AQP5) and miR-325-3p in the proliferation and apoptosis of HBV-related HCC cells. METHODS: AQP5 and miR-325-3p expression in both normal and HBV-HCC tissues or cells (both Huh7-1.3 and HepG2.2.15) was detected using qRT-PCR. AQP5 expression was knocked down in HBV-related Huh7-1.3 and HepG2.2.15 cells using small interfering RNA (siRNA) technology. Down-regulation was confirmed using real-time PCR and Western blot analysis. Effects of AQP5 down-regulation on the proliferation and apoptosis were assessed. Dual luciferase reporter gene assay, Western blot and qRT-PCR were employed to evaluate the effect of miR-325-3p on the luciferase activity and expression of AQP5. Moreover, miR-325-3p mimic-induced changes in cellular proliferation and apoptosis were detected through CCK-8 assay, BrdU assay, flow cytometry analysis and ELISA. RESULTS: In this study, the expression of AQP5 was up-regulated in human HBV-HCC tissue, Huh7-1.3 and HepG2.2.15 cells. Knockdown of AQP5 significantly inhibited the proliferation and promoted apoptosis of HBV-HCC cells. Next, miR-325-3p was obviously down-regulated in HBV-HCC. In concordance with this, MiR-325-3p directly targeted AQP5, and reduced both mRNA and protein levels of AQP5, which promoted cell proliferation and suppressed cell apoptosis in HCC cells. Overexpression of miR-325-3p dramatically inhibited cell proliferation and induced cell apoptosis. CONCLUSIONS: Our findings clearly demonstrated that introduction of miR-325-3p inhibited proliferation and induced apoptosis of Huh7-1.3 and HepG2.2.15 cells by directly decreasing AQP5 expression, and that silencing AQP5 expression was essential for the pro-apoptotic effect of miR-325-3p overexpression on Huh7-1.3 and HepG2.2.15 cells. It is beneficial to gain insight into the mechanism of HBV infection and pathophysiology of HBV-related HCC.

Structural and Functional Analysis of UGT92G6 Suggests an Evolutionary Link Between Mono- and Disaccharide Glycoside-Forming Transferases.

Glycosylation mediated by UDP-dependent glycosyltransferase (UGT) is one of the most common reactions for the biosynthesis of small molecule glycosides. As glycosides have various biological roles, we characterized UGT genes from grapevine (Vitis vinifera). In silico analysis of VvUGT genes that were highly expressed in leaves identified UGT92G6 which showed sequence similarity to both monosaccharide and disaccharide glucoside-forming transferases. The recombinant UGT92G6 glucosylated phenolics, among them caffeic acid, carvacrol, eugenol and raspberry ketone, and also accepted geranyl glucoside and citronellyl glucoside. Thus, UGT92G6 formed mono- and diglucosides in vitro from distinct compounds. The enzyme specificity constant Vmax/Km ratios indicated that UGT92G6 exhibited the highest specificity towards caffeic acid, producing almost equal amounts of the 3- and 4-O-glucoside. Transient overexpression of UGT92G6 in Nicotiana benthamiana leaves confirmed the production of caffeoyl glucoside; however, the level of geranyl diglucoside was not elevated upon overexpression of UGT92G6, even after co-expression of genes encoding geraniol synthase and geraniol UGT to provide sufficient precursor. Comparative sequence and 3-D structure analysis identified a sequence motif characteristic for monoglucoside-forming UGTs in UGT92G6, suggesting an evolutionary link between mono- and disaccharide glycoside UGTs. Thus, UGT92G6 functions as a mono- and diglucosyltransferase in vitro, but acts as a caffeoyl glucoside UGT in N. benthamiana.

[Mining and characterization of miRNAs closely associated with the pathogenicity in tomato].

MicroRNAs (miRNAs) are a class of small endogenously RNA with an approximate length of 22nt which are known to be ubiquitous in eukaryotes. More importantly, miRNAs are key regulators of gene expression in eukaryotic cells through the degradation of target mRNA. The investigation on miRNAs in tomato that is an important model plant has made a great progress in recent years. Herein, by collecting the reported literature and miRBase, we found that 34 miRNAs in tomato are closely associated with pathogenicity. Subsequently, we predicted their target genes through bioinformatics approaches and built a disease-related regulatory network of miRNA and its target genes using Cytoscope program. This has led us to identify 13 miRNAs that are closely associated with pathogenicity in tomato from which we selected miR169, miR482, miR5300, miR6024, miR6026 and miR6027 for further analysis based on the association between miRNAs and the number of target genes. Lastly, we performed the analysis of target gene, promoter and real time quantitative PCR verification for these 6 miRNAs. Our study may pave the way for future in-depth analysis of biological action of miRNAs.

A ROS-responsive microsphere capsule encapsulated with NADPH oxidase 4 inhibitor ameliorates macrophage inflammation and ferroptosis.

Inflammatory macrophages within the synovium play a pivotal role in the progression of arthritis inflammation. Effective drug therapy targeting inflammatory macrophages has long been a goal for clinicians and researchers. The standard approach for treating osteoarthritis (OA) involves systemic treatment and local injection. However, the high incidence of side effects associated with long-term drug administration increases the risk of complications in patients. Additionally, the rapid clearance of the joint cavity poses a biological barrier to the therapeutic effect. NADPH oxidase 4 (NOX4) is an enzyme protein regulating the cellular redox state by generating reactive oxygen species (ROS) within the cell. In this study, we designed and fabricated a hydrogel microsphere consisting of methyl methacrylate (MMA) and polyvinyl acetate (PVA) as the outer layer structure. We then loaded GLX351322 (GLX), a novel selective NOX4 inhibitor, into hydrogel microspheres through self-assembly with the compound polyethylene glycol ketone mercaptan (mPEG-TK) containing a disulfide bond, forming nanoparticles (mPEG-TK-GLX), thus creating a two-layer drug-loaded microspheres capsule with ROS-responsive and slow-releasing capabilities. Our results demonstrate that mPEG-TK-GLX@PVA-MMA effectively suppressed TBHP-induced inflammation, ROS production, and ferroptosis, indicating a promising curative strategy for OA and other inflammatory diseases in the future.

Mapping the tRNA Modification Landscape of Bartonella henselae Houston I and Bartonella quintana Toulouse.

Transfer RNA (tRNA) modifications play a crucial role in maintaining translational fidelity and efficiency, and they may function as regulatory elements in stress response and virulence. Despite their pivotal roles, a comprehensive mapping of tRNA modifications and their associated synthesis genes is still limited, with a predominant focus on free-living bacteria. In this study, we employed a multidisciplinary approach, incorporating comparative genomics, mass spectrometry, and next-generation sequencing, to predict the set of tRNA modification genes responsible for tRNA maturation in two intracellular pathogens- Bartonella henselae Houston I and Bartonella quintana Toulouse, which are causative agents of cat-scratch disease and trench fever, respectively. This analysis presented challenges, particularly because of host RNA contamination, which served as a potential source of error. However, our approach predicted 26 genes responsible for synthesizing 23 distinct tRNA modifications in B. henselae and 22 genes associated with 23 modifications in B. quintana . Notably, akin to other intracellular and symbiotic bacteria, both Bartonella species have undergone substantial reductions in tRNA modification genes, mostly by simplifying the hypermodifications present at positions 34 and 37. B. quintana exhibited the additional loss of four modifications and these were linked to examples of gene decay, providing snapshots of reductive evolution.

Mapping the tRNA modification landscape of Bartonella henselae Houston I and Bartonella quintana Toulouse.

Transfer RNA (tRNA) modifications play a crucial role in maintaining translational fidelity and efficiency, and they may function as regulatory elements in stress response and virulence. Despite their pivotal roles, a comprehensive mapping of tRNA modifications and their associated synthesis genes is still limited, with a predominant focus on free-living bacteria. In this study, we employed a multidisciplinary approach, incorporating comparative genomics, mass spectrometry, and next-generation sequencing, to predict the set of tRNA modification genes responsible for tRNA maturation in two intracellular pathogens-Bartonella henselae Houston I and Bartonella quintana Toulouse, which are causative agents of cat-scratch disease and trench fever, respectively. This analysis presented challenges, particularly because of host RNA contamination, which served as a potential source of error. However, our approach predicted 26 genes responsible for synthesizing 23 distinct tRNA modifications in B. henselae and 22 genes associated with 23 modifications in B. quintana. Notably, akin to other intracellular and symbiotic bacteria, both Bartonella species have undergone substantial reductions in tRNA modification genes, mostly by simplifying the hypermodifications present at positions 34 and 37. Bartonella quintana exhibited the additional loss of four modifications and these were linked to examples of gene decay, providing snapshots of reductive evolution.

Multifunctional Prosthesis Surface: Modification of Titanium with Cinnamaldehyde-Loaded Hierarchical Titanium Dioxide Nanotubes.

Orthopedic prostheses are the ultimate therapeutic solution for various end-stage orthopedic conditions. However, aseptic loosening and pyogenic infections remain as primary complications associated with these devices. In this study, a hierarchical titanium dioxide (TiO2) nanotube drug delivery system loaded with cinnamaldehyde for the surface modification of titanium implants, is constructed. These specially designed dual-layer TiO2 nanotubes enhance material reactivity and provide an extensive drug-loading platform within a short time. The introduction of cinnamaldehyde enhances the bone integration performance of the scaffold (simultaneously promoting bone formation and inhibiting bone resorption), anti-inflammatory capacity, and antibacterial properties. In vitro experiments have demonstrated that this system promoted osteogenesis by upregulating both Wnt/ß-catenin and MAPK signaling pathways. Furthermore, it inhibits osteoclast formation, suppresses macrophage-mediated inflammatory responses, and impedes the proliferation of Staphylococcus aureus and Escherichia coli. In vivo experiments shows that this material enhances bone integration in a rat model of femoral defects. In addition, it effectively enhances the antibacterial and anti-inflammatory properties in a subcutaneous implant in a rat model. This study provides a straightforward and highly effective surface modification strategy for orthopedic Ti implants.

tRNA modification reprogramming contributes to artemisinin resistance in Plasmodium falciparum.

Plasmodium falciparum artemisinin (ART) resistance is driven by mutations in kelch-like protein 13 (PfK13). Quiescence, a key aspect of resistance, may also be regulated by a yet unidentified epigenetic pathway. Transfer RNA modification reprogramming and codon bias translation is a conserved epitranscriptomic translational control mechanism that allows cells to rapidly respond to stress. We report a role for this mechanism in ART-resistant parasites by combining tRNA modification, proteomic and codon usage analyses in ring-stage ART-sensitive and ART-resistant parasites in response to drug. Post-drug, ART-resistant parasites differentially hypomodify mcm5s2U on tRNA and possess a subset of proteins, including PfK13, that are regulated by Lys codon-biased translation. Conditional knockdown of the terminal s2U thiouridylase, PfMnmA, in an ART-sensitive parasite background led to increased ART survival, suggesting that hypomodification can alter the parasite ART response. This study describes an epitranscriptomic pathway via tRNA s2U reprogramming that ART-resistant parasites may employ to survive ART-induced stress.

Queuosine Salvage in Bartonella henselae Houston 1: A Unique Evolutionary Path.

Queuosine (Q) stands out as the sole tRNA modification that can be synthesized via salvage pathways. Comparative genomic analyses identified specific bacteria that showed a discrepancy between the projected Q salvage route and the predicted substrate specificities of the two identified salvage proteins: 1) the distinctive enzyme tRNA guanine-34 transglycosylase (bacterial TGT, or bTGT), responsible for inserting precursor bases into target tRNAs; and 2) Queuosine Precursor Transporter (QPTR), a transporter protein that imports Q precursors. Organisms like the facultative intracellular pathogen Bartonella henselae, which possess only bTGT and QPTR but lack predicted enzymes for converting preQ1 to Q, would be expected to salvage the queuine (q) base, mirroring the scenario for the obligate intracellular pathogen Chlamydia trachomatis. However, sequence analyses indicate that the substrate-specificity residues of their bTGTs resemble those of enzymes inserting preQ1 rather than q. Intriguingly, mass spectrometry analyses of tRNA modification profiles in B. henselae reveal trace amounts of preQ1, previously not observed in a natural context. Complementation analysis demonstrates that B. henselae bTGT and QPTR not only utilize preQ1, akin to their Escherichia coli counterparts, but can also process q when provided at elevated concentrations. The experimental and phylogenomic analyses suggest that the Q pathway in B. henselae could represent an evolutionary transition among intracellular pathogens-from ancestors that synthesized Q de novo to a state prioritizing the salvage of q. Another possibility that will require further investigations is that the insertion of preQ1 has fitness advantages when B. henselae is growing outside a mammalian host.

Translational response to mitochondrial stresses is orchestrated by tRNA modifications.

Mitochondrial stress and dysfunction play important roles in many pathologies. However, how cells respond to mitochondrial stress is not fully understood. Here, we examined the translational response to electron transport chain (ETC) inhibition and arsenite induced mitochondrial stresses. Our analysis revealed that during mitochondrial stress, tRNA modifications (namely f5C, hm5C, queuosine and its derivatives, and mcm5U) dynamically change to fine tune codon decoding, usage, and optimality. These changes in codon optimality drive the translation of many pathways and gene sets, such as the ATF4 pathway and selenoproteins, involved in the cellular response to mitochondrial stress. We further examined several of these modifications using targeted approaches. ALKBH1 knockout (KO) abrogated f5C and hm5C levels and led to mitochondrial dysfunction, reduced proliferation, and impacted mRNA translation rates. Our analysis revealed that tRNA queuosine (tRNA-Q) is a master regulator of the mitochondrial stress response. KO of QTRT1 or QTRT2, the enzymes responsible for tRNA-Q synthesis, led to mitochondrial dysfunction, translational dysregulation, and metabolic alterations in mitochondria-related pathways, without altering cellular proliferation. In addition, our analysis revealed that tRNA-Q loss led to a domino effect on various tRNA modifications. Some of these changes could be explained by metabolic profiling. Our analysis also revealed that utilizing serum deprivation or alteration with Queuine supplementation to study tRNA-Q or stress response can introduce various confounding factors by altering many other tRNA modifications. In summary, our data show that tRNA modifications are master regulators of the mitochondrial stress response by driving changes in codon decoding.

ß-amyloid accumulation enhances microtubule associated protein tau pathology in an APPNL-G-F/MAPTP301S mouse model of Alzheimer's disease.

Introduction: The study of the pathophysiology study of Alzheimer's disease (AD) has been hampered by lack animal models that recapitulate the major AD pathologies, including extracellular -amyloid (A) deposition, intracellular aggregation of microtubule associated protein tau (MAPT), inflammation and neurodegeneration. Methods: The humanized APPNL-G-F knock-in mouse line was crossed to the PS19 MAPTP301S, over-expression mouse line to create the dual APPNL-G-F/PS19 MAPTP301S line. The resulting pathologies were characterized by immunochemical methods and PCR. Results: We now report on a double transgenic APPNL-G-F/PS19 MAPTP301S mouse that at 6 months of age exhibits robust A plaque accumulation, intense MAPT pathology, strong inflammation and extensive neurodegeneration. The presence of A pathology potentiated the other major pathologies, including MAPT pathology, inflammation and neurodegeneration. MAPT pathology neither changed levels of amyloid precursor protein nor potentiated A accumulation. Interestingly, study of immunofluorescence in cleared brains indicates that microglial inflammation was generally stronger in the hippocampus, dentate gyrus and entorhinal cortex, which are regions with predominant MAPT pathology. The APPNL-G-F/MAPTP301S mouse model also showed strong accumulation of N6-methyladenosine (m6A), which was recently shown to be elevated in the AD brain. m6A primarily accumulated in neuronal soma, but also co-localized with a subset of astrocytes and microglia. The accumulation of m6A corresponded with increases in METTL3 and decreases in ALKBH5, which are enzymes that add or remove m6A from mRNA, respectively. Discussion: Our understanding of the pathophysiology of Alzheimer's disease (AD) has been hampered by lack animal models that recapitulate the major AD pathologies, including extracellular -amyloid (A) deposition, intracellular aggregation of microtubule associated protein tau (MAPT), inflammation and neurodegeneration. The APPNL-G-F/MAPTP301S mouse recapitulates many features of AD pathology beginning at 6 months of aging, and thus represents a useful new mouse model for the field.