Processing and decay of 6S-1 and 6S-2 RNAs in Bacillus subtilis.

Noncoding 6S RNAs regulate transcription by binding to the active site of bacterial RNA polymerase holoenzymes. Processing and decay of 6S-1 and 6S-2 RNA were investigated in Bacillus subtilis by northern blot and RNA-seq analyses using different RNase knockout strains, as well as by in vitro processing assays. For both 6S RNA paralogs, we identified a key-but mechanistically different-role of RNase J1. RNase J1 catalyzes 5'-end maturation of 6S-1 RNA, yet relatively inefficient and possibly via the enzyme's "sliding endonuclease" activity. 5'-end maturation has no detectable effect on 6S-1 RNA function, but rather regulates its decay: The generated 5'-monophosphate on matured 6S-1 RNA propels endonucleolytic cleavage in its apical loop region. The major 6S-2 RNA degradation pathway is initiated by endonucleolytic cleavage in the 5'-central bubble to trigger 5'-to-3'-exoribonucleolytic degradation of the downstream fragment by RNase J1. The four 3'-exonucleases of B. subtilis-RNase R, PNPase, YhaM, and particularly RNase PH-are involved in 3'-end trimming of both 6S RNAs, degradation of 6S-1 RNA fragments, and decay of abortive transcripts (so-called product RNAs, ∼14 nt in length) synthesized on 6S-1 RNA during outgrowth from stationary phase. In the case of the growth-retarded RNase Y deletion strain, we were unable to infer a specific role of RNase Y in 6S RNA decay. Yet, a participation of RNase Y in 6S RNA decay still remains possible, as evidence for such a function may have been obscured by overlapping substrate specificities of RNase Y, RNase J1, and RNase J2.

The torpedo effect in Bacillus subtilis: RNase J1 resolves stalled transcription complexes.

RNase J1 is the major 5'-to-3' bacterial exoribonuclease. We demonstrate that in its absence, RNA polymerases (RNAPs) are redistributed on DNA, with increased RNAP occupancy on some genes without a parallel increase in transcriptional output. This suggests that some of these RNAPs represent stalled, non-transcribing complexes. We show that RNase J1 is able to resolve these stalled RNAP complexes by a "torpedo" mechanism, whereby RNase J1 degrades the nascent RNA and causes the transcription complex to disassemble upon collision with RNAP. A heterologous enzyme, yeast Xrn1 (5'-to-3' exonuclease), is less efficient than RNase J1 in resolving stalled Bacillus subtilis RNAP, suggesting that the effect is RNase-specific. Our results thus reveal a novel general principle, whereby an RNase can participate in genome-wide surveillance of stalled RNAP complexes, preventing potentially deleterious transcription-replication collisions.

New meroterpenoids and polyketides from the endophytic fungus Paraphaeosphaeria sp. C-XB-J-1 and their anti-inflammatory and SARS-CoV-2 Mpro inhibitory activities.

Seven new meroterpenoids, paraphaeones A-G (1-7), and two new polyketides, paraphaeones H-I (8-9), along with eight known compounds (10-17), were isolated from the endophytic fungus Paraphaeosphaeria sp. C-XB-J-1. The structures of 1-9 were identified through the analysis of 1H, 13C, and 2D NMR spectra, assisted by HR-ESI-MS data. Compounds 1 and 7 exhibited a dose-dependent decrease in lactate dehydrogenase levels, with IC50 values of 1.78 µM and 1.54 µM, respectively. Moreover, they inhibited the secretion of IL-1ß and CASP-1, resulting in a reduction in the activity levels of NLRP3 inflammasomes. Fluorescence microscopy results indicated that compound 7 concentration-dependently attenuated cell pyroptosis. Additionally, compounds 4 and 7 showed potential inhibitory effects on the severe acute respiratory syndrome coronavirus-2 main protease (SARS-CoV-2 Mpro), with IC50 values of 10.8 ± 0.9 µM and 12.9 ± 0.7 µM, respectively.

Characterization of NR1J1 Paralog Responses of Marine Mussels: Insights from Toxins and Natural Activators.

The pregnane X receptor (PXR) is a nuclear hormone receptor that plays a pivotal role in regulating gene expression in response to various ligands, particularly xenobiotics. In this context, the aim of this study was to shed light on the ligand affinity and functions of four NR1J1 paralogs identified in the marine mussel Mytilus galloprovincialis, employing a dual-luciferase reporter assay. To achieve this, the activation patterns of these paralogs in response to various toxins, including freshwater cyanotoxins (Anatoxin-a, Cylindrospermopsin, and Microcystin-LR, -RR, and -YR) and marine algal toxins (Nodularin, Saxitoxin, and Tetrodotoxin), alongside natural compounds (Saint John's Wort, Ursolic Acid, and 8-Methoxypsoralene) and microalgal extracts (Tetraselmis, Isochrysis, LEGE 95046, and LEGE 91351 extracts), were studied. The investigation revealed nuanced differences in paralog response patterns, highlighting the remarkable sensitivity of MgaNR1J1γ and MgaNR1J1δ paralogs to several toxins. In conclusion, this study sheds light on the intricate mechanisms of xenobiotic metabolism and detoxification, particularly focusing on the role of marine mussel NR1J1 in responding to a diverse array of compounds. Furthermore, comparative analysis with human PXR revealed potential species-specific adaptations in detoxification mechanisms, suggesting evolutionary implications. These findings deepen our understanding of PXR-mediated metabolism mechanisms, offering insights into environmental monitoring and evolutionary biology research.

The JMJD3 histone demethylase inhibitor GSK-J1 ameliorates lipopolysaccharide-induced inflammation in a mastitis model.

Jumonji domain-containing 3 (JMJD3/KDM6B) is a histone demethylase that plays an important role in regulating development, differentiation, immunity, and tumorigenesis. However, the mechanisms responsible for the epigenetic regulation of inflammation during mastitis remain incompletely understood. Here, we aimed to investigate the role of JMJD3 in the lipopolysaccharide (LPS)-induced mastitis model. GSK-J1, a small molecule inhibitor of JMJD3, was applied to treat LPS-induced mastitis in mice and in mouse mammary epithelial cells in vivo and in vitro. Breast tissues were then collected for histopathology and protein/gene expression examination, and mouse mammary epithelial cells were used to investigate the mechanism of regulation of the inflammatory response. We found that the JMJD3 gene and protein expression were upregulated in injured mammary glands during mastitis. Unexpectedly, we also found JMJD3 inhibition by GSK-J1 significantly alleviated the severity of inflammation in LPS-induced mastitis. These results are in agreement with the finding that GSK-J1 treatment led to the recruitment of histone 3 lysine 27 trimethylation (H3K27me3), an inhibitory chromatin mark, in vitro. Furthermore, mechanistic investigation suggested that GSK-J1 treatment directly interfered with the transcription of inflammatory-related genes by H3K27me3 modification of their promoters. Meanwhile, we also demonstrated that JMJD3 depletion or inhibition by GSK-J1 decreased the expression of toll-like receptor 4 and negated downstream NF-κB proinflammatory signaling and subsequently reduced LPS-stimulated upregulation of Tnfa, Il1b, and Il6. Together, we propose that targeting JMJD3 has therapeutic potential for the treatment of inflammatory diseases.

The soothing effect of phlorotannins on cedar pollinosis in Cry j 1-stimulated mice.

The antiallergic properties of phlorotannins, algal polyphenols, have been widely reported. This study examined the soothing effect of phlorotannin concentrate (PTC) from Eisenia nipponica on cedar pollinosis in Cry j 1-stimulated mice. PTC reduced the mice's sneezing and nasal rubbing, which was attributed to decreased levels of immunoglobulin E and Th2-type cytokines [interleukin (IL)-4, IL-5, and IL-13].

Chromatin interactions in differentiating keratinocytes reveal novel atopic dermatitis- and psoriasis-associated genes.

BACKGROUND: Hundreds of variants associated with atopic dermatitis (AD) and psoriasis, 2 common inflammatory skin disorders, have previously been discovered through genome-wide association studies (GWASs). The majority of these variants are in noncoding regions, and their target genes remain largely unclear. OBJECTIVE: We sought to understand the effects of these noncoding variants on the development of AD and psoriasis by linking them to the genes that they regulate. METHODS: We constructed genomic 3-dimensional maps of human keratinocytes during differentiation by using targeted chromosome conformation capture (Capture Hi-C) targeting more than 20,000 promoters and 214 GWAS variants and combined these data with transcriptome and epigenomic data sets. We validated our results with reporter assays, clustered regularly interspaced short palindromic repeats activation, and examination of patient gene expression from previous studies. RESULTS: We identified 118 target genes of 82 AD and psoriasis GWAS variants. Differential expression of 58 of the 118 target genes (49%) occurred in either AD or psoriatic lesions, many of which were not previously linked to any skin disease. We highlighted the genes AFG1L, CLINT1, ADO, LINC00302, and RP1-140J1.1 and provided further evidence for their potential roles in AD and psoriasis. CONCLUSIONS: Our work focused on skin barrier pathology through investigation of the interaction profile of GWAS variants during keratinocyte differentiation. We have provided a catalogue of candidate genes that could modulate the risk of AD and psoriasis. Given that only 35% of the target genes are the gene nearest to the known GWAS variants, we expect that our work will contribute to the discovery of novel pathways involved in AD and psoriasis.

New dot-blot method for evaluating the effect of inactivators on mite and Japanese cedar pollen allergens.

As a method of evaluating the effect of inactivators on allergens while suppressing the effect of inactivator on the assay, we developed new dot-blot method that combines immunostaining and protein detection methods. This method is useful for evaluating whether the inactivator can inactivate allergens rather than removing them from the assay.

Formononetin, J1 and J2 have different effects on endothelial cells via EWSAT1-TRAF6 and its downstream pathway.

Formononetin is a natural isoflavone compound found mainly in Chinese herbal medicines such as astragalus and red clover. It is considered to be a typical phytooestrogen. In our previous experiments, it was found that formononetin has a two-way regulatory effect on endothelial cells (ECs): low concentrations promote the proliferation of ECs and high concentrations have an inhibitory effect. To find a specific mechanism of action and provide a better clinical effect, we performed a structural transformation of formononetin and selected better medicinal properties for formononetin modifier J1 and J2 from a variety of modified constructs. The MTT assay measured the effects of drugs on human umbilical vein endothelial cell (HUVEC) activity. Scratch and transwell experiments validated the effects of the drugs on HUVEC migration and invasion. An in vivo assessment effect of the drugs on ovariectomized rats. Long-chain non-coding RNA for EWSAT1, which is abnormally highly expressed in HUVEC, was screened by gene chip, and the effect of the drug on its expression was detected by PCR after the drug was applied. The downstream factors and their pathways were analysed, and the changes in the protein levels after drug treatment were evaluated by Western blot. In conclusion, the mechanism of action of formononetin, J1 and J2 on ECs may be through EWSAT1-TRAF6 and its downstream pathways.

The anti-parasitic drug suramin potently inhibits formation of seminal amyloid fibrils and their interaction with HIV-1.

Seminal amyloid fibrils are made up of naturally occurring peptide fragments and are key targets for the development of combination microbicides or antiviral drugs. Previously, we reported that the polysulfonic compound ADS-J1 is a potential candidate microbicide that not only inhibits HIV-1 entry, but also seminal fibrils. However, the carcinogenic azo moieties in ADS-J1 preclude its clinical application. Here, we screened several ADS-J1-like analogs and found that the antiparasitic drug suramin most potently inhibited seminal amyloid fibrils. Using various biochemical methods, including Congo red staining, CD analysis, transmission EM, viral infection assays, surface plasmon resonance imaging, and molecular dynamics simulations, we investigated suramin's inhibitory effects and its putative mechanism of action. We found that by forming a multivalent interaction, suramin binds to proteolytic peptides and mature fibrils, thereby inhibiting seminal fibril formation and blocking fibril-mediated enhancement of viral infection. Of note, suramin exhibited potent anti-HIV activities, and combining suramin with several antiretroviral drugs produced synergistic effects against HIV-1 in semen. Suramin also displayed a good safety profile for vaginal application. Moreover, suramin inhibited the semen-derived enhancer of viral infection (SEVI)/semen-mediated enhancement of HIV-1 transcytosis through genital epithelial cells and the subsequent infection of target cells. Collectively, suramin has great potential for further development as a combination microbicide to reduce the spread of the AIDS pandemic by targeting both viral and host factors involved in HIV-1 sexual transmission.

Immunological effects of sublingual immunotherapy with Japanese cedar pollen extract in patients with combined Japanese cedar and Japanese cypress pollinosis.

Sublingual immunotherapy (SLIT) with Japanese cedar (JCe) pollinosis was expected to be effective for Japanese cypress (JCy) pollinosis. However, only a half of JCy pollinosis patients clinically improved. Therefore, we examined the immunological effect of SLIT for JCy pollinosis. Peripheral blood mononuclear cells (PBMCs) from patients with JCe and JCy pollinosis who did and did not receive SLIT were incubated with Cry j 1, Cha o 1 and Cha o 3 antigens. Basophil activation test (BAT) were performed. Production of IL-5 and IL-17 induced by antigens was inhibited in the SLIT group. Cry j 1-specific production of IL-10 was increased, and serum Cry j 1-specific IgE and -IgG4 were elevated. However, Cha o 1- or Cha o 3-specific production of IL-10 and specific IgG4 was not increased. Antigens-specific BAT did not decrease after SLIT. New SLIT with JCe and JCy is needed for patients with combined JCe and JCy pollinosis.

Research update and opportunity of non-hormonal male contraception: Histone demethylase KDM5B-based targeting.

With the continued increase in global human population, diverse contraception approaches have become increasingly essential, including non-hormonal male contraception. Non-hormonal approaches to contraception are very convenient; however, such options are limited because data regarding the identification and characterization of tissue/cell-specific targets and appropriate small molecule candidate contraceptives are lacking. Based on in-silico studies of genomics, transcriptomics, and proteomics, performed by mining datasets in PubMed, we first reviewed testis-, epididymis-, and germline cell-specific genes/proteins, with the aim of presenting evidence that many of these could become 'druggable' targets for the development of non-hormonal male contraceptives in the future. Although many hurdles remain before the successful therapeutic use of non-hormonal contraceptive, to facilitate this approach, we describe here the changing perspectives on several potential non-hormonal contraceptives (e.g. small molecules, plant extracts, etc.) that are under development; continued effort may yield marketable products. Further, we highlight specific enzymes within the histone lysine demethylase subfamily that play a central role in germ line regulation. In particular, we focused on several prospective candidate small-molecules suggested to interact with the catalytic domain of histone lysine demethylase KDM5B, which is ubiquitously expressed in the testis/spermatozoa of both mice and human.

Parietaria major allergens vs pollen in the air we breathe.

BACKGROUND: Parietaria and Urtica are the genera from the Urticaceae family more frequent in Mediterranean and Atlantic areas. Moreover, both genera share pollination periods, and their pollen (of the main species) is so similar that there is no aerobiological evidence of the proportion of each of them in the airborne pollen identification, except in the case of U. membranacea. However, Parietaria is one of the most important causes of pollinosis and Urtica is not. Our aim is determine if airborne Urticaceae pollen concentrations show the aerodynamics of the two major allergens of Parietaria (Par j 1 and Par j 2) as well as the allergen distribution in the different-sized particles. METHODS: The air was sampled during the pollination period of Urticaceae using Hirst Volumetric Sampler and Andersen Cascade Impactor in two cities of Southern Spain (Córdoba and Granada). The samples were analysed by the methodology proposed by the Spanish Aerobiology Network (REA) and the minimum requirements of the European Aeroallergen Society (EAS) for pollen, and by ELISA immunoassay for allergens. RESULTS: The patterns of airborne pollen and Par j 1-Par j 2 were present in the air during the studied period, although with irregular oscillations. Urticaceae pollen and Par j 1-Par j 2 allergens located in PM2.5 showed positive and significant correlation during the period with maximum concentrations (March to April). CONCLUSION: Parietaria aeroallergens show similar pattern of Urticaceae airborne pollen. Urticaceae pollen calendar is as a good tool for allergy prevention. On the other hand, important concentrations of Par j 1 and Par j 2 were located in the breathable fraction (PM2.5), which could explain the asthmatic symptoms in the allergic population to Parietaria.

A facile solvent-free three-component domino synthesis of novel 2,4-diaryl-5,6-dihydrobenzo[j][1,7]phenanthrolines.

A simple, efficient and green procedure for the synthesis of novel 2,4-diaryl-5,6-dihydrobenzo[j][1,7]phenanthrolines has been developed via a Krohnke-type one-pot three-component reaction of 2-[arylmethylidene]-3,4-dihydro-1(2H)-acridinones and (2-aryl-2-oxoethyl)pyridinium bromides in the presence of excess ammonium acetate in good yields under solvent-free conditions. Good functional group tolerance, high substrate scope and no column purification are the practical advantages of this methodology.

Genome mining in Trichoderma viride J1-030: discovery and identification of novel sesquiterpene synthase and its products.

Sesquiterpene synthases in Trichoderma viride have been seldom studied, despite the efficiency of filamentous fungi for terpenoid production. Using the farnesyl diphosphate-overexpressing Saccharomyces cerevisiae platform to produce diverse terpenoids, we herein identified an unknown sesquiterpene synthase from T. viride by genome mining and determined the structure of its corresponding products. One new 5/6 bicyclic sesquiterpene and its esterified derivative were characterised by GC-MS and 1D and 2D NMR spectroscopy. To the best of our knowledge, this is the first well-identified sesquiterpene synthase from T. viride to date.

Downregulation and Aberrant Localization of Forkhead Box J1 in Allergic Nasal Mucosa.

BACKGROUND: Forkhead box J1 (FOXJ1) plays pivotal roles in motile cilia formation. However, it remains unclear whether abnormal expression or localization of FOXJ1 in nasal mucosa tissues is associated with allergic rhinitis (AR), in which impaired mucociliary clearance is implicated. OBJECTIVE: We sought to investigate the expression and localization of FOXJ1 in inferior turbinate from patients with AR and controls. METHODS: We assayed mRNA levels of FOXJ1, DNAI1, DNALI1, and DNAH9 by using whole-genome expression array and quantitative real-time polymerase chain reaction. We elucidated the localization of FOXJ1 by using immunofluorescence assays in paraffin sections and primary single cells. Four patterns of FOXJ1 localization (normal, N; intermediate, I; mislocalization, M; absence, A) were defined. We developed a semiquantitative scoring system to elucidate their localization in 5 areas per paraffin section, with individual sections being assigned a score between 0 and 2. RESULTS: The mRNA levels of FOXJ1, DNAI1, DNALI1, and DNAH9 were significantly reduced in patients with AR compared with controls (all p < 0.05). The median (1st and 3rd quartile) of the FOXJ1 score was 0.4 (0.0 and 0.85) in patients with AR, and 0.2 (0.0 and 0.4) in controls (p < 0.05). For primary cytospin samples, the mean percentages of FOXJ1 localization patterns N, I, M, and A were 46.7, 10.0, 30.0, and 26.7% in patients with AR, and 82.5, 5.0, 5.0, and 7.5% in controls, respectively (p < 0.05). CONCLUSION: Downregulation and aberrant localization of FOXJ1 may be crucial characteristics of the allergic nasal mucosa.

Ubiquitin-conjugating enzyme UBE2J1 negatively modulates interferon pathway and promotes RNA virus infection.

BACKGROUND: Viral infection activates innate immune pathways and interferons (IFNs) play a pivotal role in the outcome of a viral infection. Ubiquitin modifications of host and viral proteins significantly influence the progress of virus infection. Ubiquitin-conjugating enzyme E2s (UBE2) have the capacity to determine ubiquitin chain topology and emerge as key mediators of chain assembly. METHODS: In this study, we screened the functions of 34 E2 genes using an RNAi library during Dengue virus (DENV) infection. RNAi and gene overexpression approaches were used to study the gene function in viral infection and interferon signaling. RESULTS: We found that silencing UBE2J1 significantly impaired DENV infection, while overexpression of UBE2J1 enhanced DENV infection. Further studies suggested that type I IFN expression was significantly increased in UBE2J1 silenced cells and decreased in UBE2J1 overexpressed cells. Reporter assay suggested that overexpression of UBE2J1 dramatically suppressed RIG-I directed IFNß promoter activation. Finally, we have confirmed that UBE2J1 can facilitate the ubiquitination and degradation of transcription factor IFN regulatory factor 3 (IRF3). CONCLUSION: These results suggest that UBE2 family member UBE2J1 can negatively regulate type I IFN expression, thereby promote RNA virus infection.

The Eastern side of the Westernmost Europeans: Insights from subclades within Y-chromosome haplogroup J-M304.

OBJECTIVES: We examined internal lineages and haplotype diversity in Portuguese samples belonging to J-M304 to improve the spatial and temporal understanding of the introduction of this haplogroup in Iberia, using the available knowledge about the phylogeography of its main branches, J1-M267 and J2-M172. METHODS: A total of 110 males of Portuguese descent were analyzed for 17 Y-chromosome bi-allelic markers and seven Y-chromosome short tandem repeats (Y-STR) loci. RESULTS: Among J1-M267 individuals (n = 36), five different sub-haplogroups were identified, with the most common being J1a2b2-L147.1 (∼72%), which encompassed the majority of representatives of the J1a2b-P58 subclade. One sample belonged to the rare J1a1-M365.1 lineage and presented a core Y-STR haplotype consistent with the Iberian settlement during the fifth century by the Alans, a people of Iranian heritage. The analysis of J2-M172 Portuguese males (n = 74) enabled the detection of the two main subclades at very dissimilar frequencies, J2a-M410 (∼80%) and J2b-M12 (∼20%), among which the most common branches were J2a1(xJ2a1b,h)-L26 (22.9%), J2a1b(xJ2a1b1)-M67 (20.3%), J2a1h-L24 (27%), and J2b2-M241 (20.3%). CONCLUSIONS: While previous inferences based on modern haplogroup J Y-chromosomes implicated a main Neolithic dissemination, here we propose a later arrival of J lineages into Iberia using a combination of novel Portuguese Y-chromosomal data and recent evidence from ancient DNA. Our analysis suggests that a substantial tranche of J1-M267 lineages was likely carried into the Iberian Peninsula as a consequence of the trans-Mediterranean contacts during the first millennium BC, while most of the J2-M172 lineages may be associated with post-Neolithic population movements within Europe.

Both exo- and endo-nucleolytic activities of RNase J1 from Staphylococcus aureus are manganese dependent and active on triphosphorylated 5'-ends.

RNA decay and RNA maturation are important steps in the regulation of bacterial gene expression. RNase J, which is present in about half of bacterial species, has been shown to possess both endo- and 5' to 3' exo-ribonuclease activities. The exonucleolytic activity is clearly involved in the degradation of mRNA and in the maturation of at least the 5' end of 16S rRNA in the 2 Firmicutes Staphylococcus aureus and Bacillus subtilis. The endoribonuclease activity of RNase J from several species has been shown to be weak in vitro and 3-D structural data of different RNase J orthologs have not provided a clear explanation for the molecular basis of this activity. Here, we show that S. aureus RNase J1 is a manganese dependent homodimeric enzyme with strong 5' to 3' exo-ribonuclease as well as endo-ribonuclease activity. In addition, we demonstrated that SauJ1 can efficiently degrade 5' triphosphorylated RNA. Our results highlight RNase J1 as an important player in RNA turnover in S. aureus.