Transposase-assisted tagmentation: an economical and scalable strategy for single-worm whole-genome sequencing.

AlphaMissense identifies 23 million human missense variants as likely pathogenic, but only 0.1% have been clinically classified. To experimentally validate these predictions, chemical mutagenesis presents a rapid, cost-effective method to produce billions of mutations in model organisms. However, the prohibitive costs and limitations in the throughput of whole-genome sequencing (WGS) technologies, crucial for variant identification, constrain its widespread application. Here, we introduce a Tn5 transposase-assisted tagmentation technique for conducting WGS in Caenorhabditis elegans, Escherichia coli, Saccharomyces cerevisiae, and Chlamydomonas reinhardtii. This method, demands merely 20 min of hands-on time for a single-worm or single-cell clones and incurs a cost below 10 US dollars. It effectively pinpoints causal mutations in mutants defective in cilia or neurotransmitter secretion and in mutants synthetically sterile with a variant analogous to the B-Raf Proto-oncogene, Serine/Threonine Kinase (BRAF) V600E mutation. Integrated with chemical mutagenesis, our approach can generate and identify missense variants economically and efficiently, facilitating experimental investigations of missense variants in diverse species.

Inhibition of METTL3 Alleviates NLRP3 Inflammasome Activation via Increasing Ubiquitination of NEK7.

N6-methyladenosine (m6A) modification, installed by METTL3-METTL14 complex, is abundant and critical in eukaryotic mRNA. However, its role in oral mucosal immunity remains ambiguous. Periodontitis is a special but prevalent infectious disease characterized as hyperinflammation of oral mucosa and bone resorption. Here, it is reported that genetic deletion of Mettl3 alleviates periodontal destruction via suppressing NLRP3 inflammasome activation. Mechanistically, the stability of TNFAIP3 (also known as A20) transcript is significantly attenuated upon m6A modification. When silencing METTL3, accumulated TNFAIP3 functioning as a ubiquitin-editing enzyme facilitates the ubiquitination of NEK7 [NIMA (never in mitosis gene a)-related kinase 7], and subsequently impairs NLRP3 inflammasome assembly. Furtherly, Coptisine chloride, a natural small-molecule, is discovered as a novel METTL3 inhibitor and performs therapeutic effect on periodontitis. The study unveils a previously unknown pathogenic mechanism of METTL3-mediated m6A modifications in periodontitis and indicates METTL3 as a potential therapeutic target.

Neurons dispose of hyperactive kinesin into glial cells for clearance.

Microtubule-based kinesin motor proteins are crucial for intracellular transport, but their hyperactivation can be detrimental for cellular functions. This study investigated the impact of a constitutively active ciliary kinesin mutant, OSM-3CA, on sensory cilia in C. elegans. Surprisingly, we found that OSM-3CA was absent from cilia but underwent disposal through membrane abscission at the tips of aberrant neurites. Neighboring glial cells engulf and eliminate the released OSM-3CA, a process that depends on the engulfment receptor CED-1. Through genetic suppressor screens, we identified intragenic mutations in the OSM-3CA motor domain and mutations inhibiting the ciliary kinase DYF-5, both of which restored normal cilia in OSM-3CA-expressing animals. We showed that conformational changes in OSM-3CA prevent its entry into cilia, and OSM-3CA disposal requires its hyperactivity. Finally, we provide evidence that neurons also dispose of hyperactive kinesin-1 resulting from a clinic variant associated with amyotrophic lateral sclerosis, suggesting a widespread mechanism for regulating hyperactive kinesins.

Bacterial Communities of the Internal Reproductive and Digestive Tracts of Virgin and Mated Tuta absoluta.

Microorganisms can affect host reproduction, defense, and immunity through sexual or opportunistic transmission; however, there are few studies on insect reproductive organs and intestinal bacterial communities and their effects on mating. Tuta absoluta is a worldwide quarantine pest that seriously threatens the production of Solanaceae crops, and the microbial community within tomato leafminers remains unclear. In this study, 16s rRNA sequencing was used to analyze bacterial communities related to the reproductive organs and intestinal tracts of tomato leafminers (the sample accession numbers are from CNS0856533 to CNS0856577). Different bacterial communities were found in the reproductive organs and intestinal tracts of females and males. Community ecological analysis revealed three potential signs of bacterial sexual transmission: (1) Mating increased the similarity between male and female sex organs and intestinal communities. (2) The bacteria carried by mated individuals were found in unmated individuals of the opposite sex but not in unmated individuals of the same sex. (3) The bacteria carried by unmated individuals were lost after mating. In addition, the abundances of bacterial communities carried by eggs were significantly higher than those of adult worms. Our results confirm that mating leads to the transfer of bacterial communities in the reproductive organs and gut of tomato leafminers, and suggest that this community strongly influences the reproductive process.

N6-methyladenosine (m6A) modification of ribosomal RNAs (rRNAs): Critical roles in mRNA translation and diseases.

As key components of the ribosome and the most abundant RNA species, the rRNAs are modified during ribosome formation. N6-methyladenosine (m6A) is a conserved RNA modification occurring on different RNA species including rRNAs. Recently, it has been reported that ZCCHC4 and METTL5 are methyltransferases that mediate m6A modification of human 28S and 18S rRNA, respectively. The newly discovered biological functions of the two methyltransferases include regulation of mRNA translation, cell proliferation, cell differentiation, stress response, and other biological processes. Both of them, especially METTL5, have been proved to be associated with a variety of diseases such as intellectual disability, cancer, congenital dysplasia and have potential clinical application as biomarkers and therapeutic targets.

METTL5 regulates cranial suture fusion via Wnt signaling.

METTL5 is a methyltransferase that mediates eukaryotic 18S ribosomal RNA m6A modification, and its mutations lead to intellectual disability, microcephaly, and facial dysmorphism in patients. However, the role of METTL5 in craniofacial development remains poorly understood. This study demonstrates that Mettl5 knockout mice exhibit poor ossification, widened cranial sutures, and a cleidocranial dysplasia-like phenotype. Deletion of Mettl5 leads to increased proliferation and decreased osteogenic differentiation of suture mesenchymal stem cells. Mechanistically, we find that Wnt signaling is significantly downregulated after Mettl5 knockout. Overall, we reveal an essential role of METTL5 in craniofacial development and osteogenic differentiation of suture mesenchymal stem cells, making METTL5 a potential diagnostic and therapeutic target for craniofacial developmental diseases.

Insights into skeletal stem cells.

The tissue-resident skeletal stem cells (SSCs), which are self-renewal and multipotent, continuously provide cells (including chondrocytes, bone cells, marrow adipocytes, and stromal cells) for the development and homeostasis of the skeletal system. In recent decade, utilizing fluorescence-activated cell sorting, lineage tracing, and single-cell sequencing, studies have identified various types of SSCs, plotted the lineage commitment trajectory, and partially revealed their properties under physiological and pathological conditions. In this review, we retrospect to SSCs identification and functional studies. We discuss the principles and approaches to identify bona fide SSCs, highlighting pioneering findings that plot the lineage atlas of SSCs. The roles of SSCs and progenitors in long bone, craniofacial tissues, and periosteum are systematically discussed. We further focus on disputes and challenges in SSC research.

Hypoxia inducible factor-1 signaling pathway in macrophage involved angiogenesis in materials-instructed osteo-induction.

Although osteo-inductive materials are regarded as promising candidates for critical-sized bone repair, their clinical application is limited by ambiguous mechanisms. The hypoxia-inducible factor (HIF)-1 signaling pathway, which responds to hypoxic conditions, is involved in both angiogenesis and osteogenesis. Strategies harnessing HIF-1 signaling to promote angiogenesis have been applied and have succeeded in repairing segmental bone defects. Meanwhile, macrophages have been shown to have important immunoregulatory effects on material-induced osteo-induction and correlate with HIF-1 activity. Thus, it is reasonable to assume that HIF-activated macrophages may also play important roles in the angiogenesis of material-induced osteo-induction. To verify this assumption, a classical type of osteo-inductive calcium phosphate (TCPs) was utilized. First, using RNA sequencing, we found that hypoxia activated the HIF signaling pathway in macrophages, which contributed to angiogenesis in TCPs. In addition, after treatment with a conditioned medium extracted from the co-culture system of macrophages and TCPs under hypoxic conditions, the migration and tube formation ability of human umbilical vein endothelial cells (HUVECs) significantly increased. In vivo, inhibition of HIF-1 or clearance of macrophages could result in impaired angiogenesis in TCPs. Finally, more blood vessels were formed in the TCPs group than in the control group. In conclusion, this study elucidated the vital role of the HIF signaling pathway in infiltrating macrophages during early vessel growth in material-induced osteo-induction. It is beneficial in advancing the exploration of the related mechanism and providing possible support for optimizing the applicability of osteo-inductive materials in bone repair.

Ciliogenesis requires sphingolipid-dependent membrane and axoneme interaction.

Cilium formation and regeneration requires new protein synthesis, but the underlying cytosolic translational reprogramming remains largely unknown. Using ribosome footprinting, we performed global translatome profiling during cilia regeneration in Chlamydomonas and uncovered that flagellar genes undergo an early transcriptional activation but late translational repression. This pattern guided our identification of sphingolipid metabolism enzymes, including serine palmitoyltransferase (SPT), as essential regulators for ciliogenesis. Cryo-electron tomography showed that ceramide loss abnormally increased the membrane-axoneme distance and generated bulged cilia. We found that ceramides interact with intraflagellar transport (IFT) particle proteins that IFT motors transport along axoneme microtubules (MTs), suggesting that ceramide-IFT particle-IFT motor-MT interactions connect the ciliary membrane with the axoneme to form rod-shaped cilia. SPT-deficient vertebrate cells were defective in ciliogenesis, and SPT mutations from patients with hereditary sensory neuropathy disrupted cilia, which could be restored by sphingolipid supplementation. These results reveal a conserved role of sphingolipid in cilium formation and link compromised sphingolipid production with ciliopathies.

In situ structure of intestinal apical surface reveals nanobristles on microvilli.

Microvilli are actin-bundle-supported membrane protrusions essential for absorption, secretion, and sensation. Microvilli defects cause gastrointestinal disorders; however, mechanisms controlling microvilli formation and organization remain unresolved. Here, we study microvilli by vitrifying the Caenorhabditis elegans larvae and mouse intestinal tissues with high-pressure freezing, thinning them with cryo-focused ion-beam milling, followed by cryo-electron tomography and subtomogram averaging. We find that many radial nanometer bristles referred to as nanobristles project from the lateral surface of nematode and mouse microvilli. The C. elegans nanobristles are 37.5 nm long and 4.5 nm wide. Nanobristle formation requires a protocadherin family protein, CDH-8, in C. elegans. The loss of nanobristles in cdh-8 mutants slows down animal growth and ectopically increases the number of Y-shaped microvilli, the putative intermediate structures if microvilli split from tips. Our results reveal a potential role of nanobristles in separating microvilli and suggest that microvilli division may help generate nascent microvilli with uniformity.

METTL3-mediated m6A RNA methylation regulates dorsal lingual epithelium homeostasis.

The dorsal lingual epithelium, which is composed of taste buds and keratinocytes differentiated from K14+ basal cells, discriminates taste compounds and maintains the epithelial barrier. N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotic cells. How METTL3-mediated m6A modification regulates K14+ basal cell fate during dorsal lingual epithelium formation and regeneration remains unclear. Here we show knockout of Mettl3 in K14+ cells reduced the taste buds and enhanced keratinocytes. Deletion of Mettl3 led to increased basal cell proliferation and decreased cell division in taste buds. Conditional Mettl3 knock-in mice showed little impact on taste buds or keratinization, but displayed increased proliferation of cells around taste buds in a protective manner during post-irradiation recovery. Mechanically, we revealed that the most frequent m6A modifications were enriched in Hippo and Wnt signaling, and specific peaks were observed near the stop codons of Lats1 and FZD7. Our study elucidates that METTL3 is essential for taste bud formation and could promote the quantity recovery of taste bud after radiation.

CVIS: Automated OCT-scan-based software application for the measurements of choroidal vascularity index and choroidal thickness.

PURPOSE: To develop an automated image recognition software for the objective quantification of choroidal vascularity index (CVI) and choroidal thickness (CT) at different choroidal locations on images obtained from enhanced depth imaging optical coherence tomography (EDI-OCT), and to validate its reliability and investigate the difference and correlation between measurements made by manual and software. METHODS: A total of 390 EDI-OCT scans, captured from 130 eligible emmetropic or myopic subjects, were categorized into four grades in terms of their accessibility to identify the choroidal-scleral interface (CSI) and were further assessed for CT and CVI at five locations (subfoveal, nasal, temporal, superior and inferior) by the newly developed Choroidal Vascularity Index Software (CVIS) and three ophthalmologists. Choroidal parameters acquired from CVIS were evaluated for its reliability and correlation with ocular factors, in comparison to manual measurements. Distribution of difference and correlation coefficient between CVIS and manual measurements were also analysed. RESULTS: Choroidal Vascularity Index Software (CVIS) demonstrated excellent intra-session reliability for CT (ICC: 0.992) and CVI (ICC: 0.978) measurements, compared to the relatively lower intra- and inter-observer reliability of manual measurements. Choroidal Vascularity Index Software (CVIS) and manual assessments had the highest correlation at nasal choroid (CT: r = 0.829, p < 0.001; CVI: r = 0.665, p < 0.001). Choroidal parameters identified with CVIS showed stronger correlations with axial length than manual measurements. CONCLUSION: This automated software, CVIS, exhibited excellent reliability compared to manual measurements, which are subject to image quality and clinical experience. With its validated clinical relevance, CVIS holds promise to serve as a flexible and robust tool in future vitreoretinal and chorioretinal studies.

PA28γ, an Accomplice to Malignant Cancer.

PA28γ is a nuclear activator of the 20S proteasome, which is involved in the regulation of several essential cellular processes and angiogenesis. Over the past 20 years, many amino acid sites and motifs have been proven to play important roles in the characteristic functions of PA28γ. The number of binding partners and validated cellular functions of PA28γ have increased, which has facilitated the clarification of its involvement in different biological events. PA28γ is involved in the progression of various diseases, and its aberrant overexpression in cancer is remarkable. Patients with low levels of PA28γ expression have a higher survival rate than those with high levels of PA28γ expression, as has been shown for a wide variety of tumors. The functions of PA28γ in cancer can be divided into five main categories: cell proliferation, cell apoptosis, metastasis and invasion, cell nuclear dynamics that have relevance to angiogenesis, and viral infection. In this review, we focus on the role of PA28γ in cancer, summarizing its aberrant expression, prooncogenic effects and underlying mechanisms in various cancers, and we highlight the possible cancer-related applications of PA28γ, such as its potential use in the diagnosis, targeted treatment and prognostic assessment of cancer.

[Research progress on circular RNA in oral squamous cell carcinoma].

Circular RNA, a non-coding RNA that forms a covalently closed continuous loop, exists widely in eukaryotic cells. The biogenesis and biological function of this type of RNA indicate that it can play a crucial role in diseases such as tumors, neural system diseases, and cardiovascular diseases; moreover, this RNA may have great potential use as a biomarker in these diseases. Oral squamous cell carcinoma (OSCC) is a common malignancy in oral surgery that is difficult to cure, metastasizes easily, and has poor prognosis. In this review, we summarize the loop-forming mechanisms and functions of circular RNA and describe the progress of current research in the development of oral cancer.

Noncoding RNAs in oral premalignant disorders and oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) has the highest mortality rate among all head and neck cancers and a relatively low five-year survival rate. Generally, the development of an oral mucosal malignancy represents a multistep process beginning with normal oral mucosa epithelium and culminating in OSCC after transitioning through intermediary oral premalignant disorders (OPMDs), during which dysplasia is often observed. Noncoding RNAs (ncRNAs) are RNAs that are not translated into proteins, but still can participate in regulating neoplastic cell behavior. Recently, data have emerged on the role of ncRNAs in the progression of oral mucosal malignant diseases, but the exact mechanisms through which ncRNAs are involved remain to be elucidated. CONCLUSIONS: Knowledge on ncRNAs has added an extra layer of complexity to our understanding of the malignant progression of oral mucosal diseases. The identification of ncRNAs in multiple body fluids as biomarkers may provide new diagnostic options that can be used for the diagnosis and prognosis of OPMDs and OSCC, respectively. Despite overall advances that have been made in cancer treatment, the treatment options for OPMDs and OSCC are still limited. Several studies have shown that ncRNA-based treatment regimens may hold promise as alternative methods for treating OPMDs and OSCC. The use of ncRNAs as therapeutic agents, including miR-155, miR-34 and lncRNA HOTAIR, appear promising.

Exo-circRNAs: a new paradigm for anticancer therapy.

CircRNAs, as new members of long noncoding RNAs, have been the focus of recent investigation. CircRNAs feature a closed continuous loop structure without 5'-3' polarity or a poly A tail. Many studies have reported the potential application of circRNAs in the clinic as new biomarkers and therapeutic targets in different diseases, especially for cancer. Additionally, the exosomes are important vehicles in cell-to-cell communication. And exo-circRNAs are circRNAs in exosomes which can be detected to provide additional evidence for conventional diagnostic methods and can be applied to suppress the malignant progress in cancer. In this review, we describe the biogenesis, characteristics, and functions of circRNAs and exosomes. Specifically, we present a comprehensive update of the promising role of exo-circRNAs in anticancer therapy.

The research progress of circRNA in the cardiovascular disease / 中国胸心血管外科临床杂志

@#Circular RNA (circRNA) is a non-coding RNA which exists widely in eukaryotic cells with a structure of covalently closed continuous loop. Its generation, characteristics and functions have received extensive attention, making it one of the hot spots in the field of non-coding RNA research. Many studies have found that circRNA plays an important role in the development of various diseases including cardiovascular disease, nervous system disease and cancer. Cardiovascular disease is a worldwide common disease with high incidence and poor prognosis. Its exact pathogenesis has not been found, which blocks the development of cardiovascular disease treatment. In this review, we summarize the loop-forming mechanisms, the functions and the progress of current researches of circRNA in cardiovascular diseases.

The mechanism and function of circular RNAs in human diseases.

Circular RNAs (circRNAs) are a recently discovered form of RNA. Initially, circRNAs were believed to result from errors during the process of gene transcription. However, after further investigation, scientists suggested that circRNAs are of great biological significance. CircRNAs show stability, conservation, abundance, and tissue and stage specificity. They can also function as miRNA sponges, regulate gene expression, and interact with proteins to affect cell behavior. Emerging evidence has also demonstrated that circRNAs participate or show abnormal expression in diseases, including central nervous system diseases, cardiovascular diseases and cancers, indicating their marked potential in the prediction and prognosis of diseases and clinical treatment.

PA28γ acts as a dual regulator of IL-6 and CCL2 and contributes to tumor angiogenesis in oral squamous cell carcinoma.

PA28γ promotes tumor development and progression and is suggested to play a role in tumor angiogenesis, but the molecular mechanisms have not been investigated. Here, we found that PA28γ enhanced the ability of OSCC cells to promote the migration, invasion, and tube formation of HUVECs and promoted tumor-induced angiogenesis in xenograft mice models in vivo. Then, a mechanism study revealed that the expression and secretion of IL-6 and CCL2 were dependent on PA28γ expression. Furthermore, blocking IL-6 or CCL2 or the transcription factor NF-κB induced the inhibition of tube formation in HUVECs co-cultured with PA28γ-overexpression OSCC cell supernatants. Moreover, we revealed that p-STAT3 and p-AKT, which are downstream of the IL-6 and CCL2 signaling axis, were downregulated in HUVECs co-cultured with the PA28γ-silenced supernatant and were upregulated with the PA28γ-overexpressing supernatant. In addition, IL-6, CCL2 and PA28γ expressions were correlated in a clinical OSCC cohort. Collectively, our study indicates that PA28γ contributes to tumor angiogenesis by regulating IL-6 and CCL2. PA28γ may be a novel therapeutic target as a dual regulator of IL-6 and CCL2 for treating PA28γ-positive OSCC.