Direct RNA sequencing in plants: practical applications and future perspectives.

The transcriptome serves as a bridge that links genomic variation and phenotype diversity. A vast number of studies using next-generation RNA sequencing (RNA-seq) in the last two decades emphasize the essential roles of plant transcriptome in response to developmental and environmental conditions, leading to numerous insights into the dynamic change, evolutionary trace and elaborate regulation of plant transcriptome. With substantial improvement in accuracy and throughput, direct RNA sequencing (DRS) has emerged as a new and powerful sequencing platform for the precise detection of native and full-length transcripts, which overcomes many limitations such as read length and PCR bias that are inherent to short-read RNA-seq. Here, we reviewed recent advances in dissecting the complexity and diversity of plant transcriptome utilizing DRS as a main technological mean from many aspects of RNA metabolism, including novel isoforms, poly(A) tail and RNA modification, and proposed a comprehensive workflow for the data process of plants DRS. Many challenges concerning the application of DRS in plants, such as machine learning tools tailored to plant transcriptome, remain to be solved, and together we prospect the future biological questions that can be potentially answered by DRS such as allele-specific RNA modification. This technology provides convenient support on which the connection of distinct RNA features is tightly built, sustainably refining our understanding of the biological functions of plant transcriptome.

Circ_BBS9 as an early diagnostic biomarker for lung adenocarcinoma: direct interaction with IFIT3 in the modulation of tumor immune microenvironment.

Background: Introduction: Circular RNAs (circRNAs) have been identified as significant contributors to the development and advancement of cancer. The objective of this study was to examine the expression and clinical implications of circRNA circ_BBS9 in lung adenocarcinoma (LUAD), as well as its potential modes of action. Methods: The expression of Circ_BBS9 was examined in tissues and cell lines of LUAD through the utilization of microarray profiling, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis. In this study, we assessed the impact of circ_BBS9 on the proliferation of LUAD cells, as well as its influence on ferroptosis and tumor formation. To analyze these effects, we employed CCK-8 assays and ferroptosis assays. The identification of proteins that interact with Circ_BBS9 was achieved through the utilization of RNA pull-down and mass spectrometry techniques. A putative regulatory network comprising circ_BBS9, miR-7150, and IFIT3 was established using bioinformatics study. The investigation also encompassed the examination of the correlation between the expression of IFIT3 and the invasion of immune cells. Results: Circ_BBS9 was significantly downregulated in LUAD tissues and cell lines. Low circ_BBS9 expression correlated with poor prognosis. Functional experiments showed that circ_BBS9 overexpression inhibited LUAD cell proliferation and promoted ferroptosis in vitro and suppressed tumor growth in vivo. Mechanistically, circ_BBS9 was found to directly interact with IFIT3 and regulate its expression by acting as a sponge for miR-7150. Additionally, IFIT3 expression correlated positively with immune infiltration in LUAD. Conclusion: Circ_BBS9 has been identified as a tumor suppressor in lung adenocarcinoma (LUAD) and holds promise as a diagnostic biomarker. The potential mechanism of action involves the modulation of ferroptosis and the immunological microenvironment through direct interaction with IFIT3 and competitive binding to miR-7150. The aforementioned findings offer new perspectives on the pathophysiology of LUAD and highlight circ_BBS9 as a potentially valuable target for therapeutic interventions.

Genome sequencing and CAZymes repertoire analysis of Diaporthe eres P3-1W causing postharvest fruit rot of 'Hongyang' kiwifruit in China.

Postharvest rot caused by various fungal pathogens is a damaging disease affecting kiwifruit production and quality, resulting in significant annual economic losses. This study focused on isolating the strain P3-1W, identified as Diaporthe eres, as the causal agent of 'Hongyang' postharvest rot disease in China. The investigation highlighted cell wall degrading enzymes (CWDEs) as crucial pathogenic factors. Specially, the enzymatic activities of cellulase, ß-galactosidase, polygalacturonase, and pectin methylesterases peaked significantly on the second day after infection of D. eres P3-1W. To gain a comprehensive understanding of these CWDEs, the genome of this strain was sequenced using PacBio and Illumina sequencing technologies. The analysis revealed that the genome of D. eres P3-1W spans 58,489,835 bp, with an N50 of 5,939,879 bp and a GC content of 50.7%. A total of 15,407 total protein-coding genes (PCGs) were predicted and functionally annotated. Notably, 857 carbohydrate-active enzymes (CAZymes) were identified in D. eres P3-1W, with 521 CWDEs consisting of 374 glycoside hydrolases (GHs), 108 carbohydrate esterase (CEs) and 91 polysaccharide lyases (PLs). Additionally, 221 auxiliary activities (AAs), 91 glycosyltransferases (GTs), and 108 carbohydrate binding modules (CBMs) were detected. These findings offer valuable insights into the CAZymes of D. eres P3-1W.

A CRISPR/RfxCas13d-mediated strategy for efficient RNA knockdown in mouse embryonic development.

The growing variety of RNA classes, such as mRNAs, lncRNAs, and circRNAs, plays pivotal roles in both developmental processes and various pathophysiological conditions. Nonetheless, our comprehension of RNA functions in live organisms remains limited due to the absence of durable and effective strategies for directly influencing RNA levels. In this study, we combined the CRISPR-RfxCas13d system with sperm-like stem cell-mediated semi-cloning techniques, which enabled the suppressed expression of different RNA species. This approach was employed to interfere with the expression of three types of RNA molecules: Sfmbt2 mRNA, Fendrr lncRNA, and circMan1a2(2,3,4,5,6). The results confirmed the critical roles of these RNAs in embryonic development, as their loss led to observable phenotypes, including embryonic lethality, delayed embryonic development, and embryo resorption. In summary, our methodology offers a potent toolkit for silencing specific RNA targets in living organisms without introducing genetic alterations.

Treatment of Refractory Segmental Vitiligo and Alopecia Areata in a Child with Upadacitinib and NB-UVB: A Case Report.

Vitiligo and alopecia areata are both autoimmune skin diseases, and the chances of co-occurrence are very low. Conventional treatments often include glucocorticoids, which have many adverse reactions with long-term use and are difficult to achieve satisfactory results. Upadacitinib has been found to be effective in both vitiligo and alopecia areata due to partial overlap in pathogenic pathways. We report the successful treatment of vitiligo combined with alopecia areata in a nine-year-old child with upadacitinib in combination with UVB. The area of vitiligo and alopecia areata decreased significantly, and satisfactory results were obtained. It provides a new idea for the treatment of vitiligo complicated with alopecia areata in children.

α-1 antitrypsin is promising for the identification of glaucoma severity and is associated with glaucomatous neural damage.

Aim: To investigate the association between plasma AAT level and glaucoma. Methods: 163 glaucoma patients and 111 healthy controls were recruited. The plasma AAT levels were measured by ELISA. Results: Plasma AAT level was significantly higher in glaucoma patients than those in healthy controls (p < 0.001). Patients with higher plasma AAT level exhibited severer disease stage (early vs. severe: p < 0.05; H-P-A; early vs. severe: p < 0.05; early vs. end-stage: p < 0.01; AGIS). ROC curves yielded that AAT can distinguish patients with early glaucoma from those with advanced glaucoma (early vs. severe: AUC: 0.616; H-P-A; early vs. severe: AUC: 0.763; early vs. end-stage: AUC: 0.660; AGIS). Conclusion: Plasma AAT is a useful biomarker for the identification of glaucoma severity.

[Box: see text].

Opening Pandora's box - key facilitators of practice change in detecting and responding to childhood adversity - a practitioner perspective.

BACKGROUND: Childhood adversities worsen physical and mental health across the lifespan. Health and social care practitioners play a key role in identifying and responding to childhood adversity, however, may be reluctant to do so due to a perceived lack of services to refer to, time pressures and a deficit of training and confidence. We aimed to (1) quantify changes in practitioner comfort and confidence to identify and respond to childhood adversity following a multimodal intervention within an integrated child and family health and social care hub and (2) to understand barriers and facilitators of practice change. METHODS: Hub practitioners were surveyed about their competence and comfort to directly ask about and confidence to respond to adversity at baseline and then at six and twelve months post training. Interviews were undertaken to explore practitioner barriers and enablers of practice change. Interviews were recorded, transcribed verbatim, and analysed using reflexive thematic analysis. The theoretical domains framework was used to identify the key drivers of practice change. RESULTS: Fifteen of 18 practitioners completed all three surveys and 70% reported increased competence and comfort to directly ask, and confidence to respond across a range of adversities over the 12-month intervention. Twenty-one practitioners completed interviews. Six themes were identified as either facilitators or barriers to practice change. Facilitator themes included (1) connection matters, (2) knowledge provides assurance, (3) confidence in ability and (4) choosing change. Barrier themes were (1) never enough time and (2) opening Pandora's box. Following analysis, key drivers of practice change were 'social influence', 'belief in capability', 'knowledge' and 'behaviour regulation' while barriers to practice change were 'environmental context and resources' and 'emotion'. CONCLUSIONS: Practitioners reported improved confidence in identifying and responding to adversity through a multimodal intervention delivered in an integrated Child and Family Hub. Changing practice requires more than just education and training. Opportunities for social connection and coaching to improve self-confidence and perceived competence are needed to overcome the fear of opening Pandora's box.

Experimental Study on the Application of Bioactive Xenogeneic Porcine Cancellous Bone for Cervical Intervertebral Fusion in Goats.

STUDY DESIGN: An experimental study in a cervical intervertebral fusion goat model. OBJECTIVE: To investigate the effect of bioactive xenogeneic porcine cancellous bone applied to the intervertebral fusion of goat cervical vertebrae. SUMMARY OF BACKGROUND DATA: Although autogenous bone achieves satisfied outcome in cervical intervertebral fusion, it is limited and cause several complications. The application of xenogeneic bone has potential to solve these problems. METHODS: Thirty local goats were randomly divided into 3 groups: group A (12 goats): autogenous tricortical iliac bone group; group B (6 goats): polyetheretherketone (PEEK) cage with autologous bone; and group C (12 goats): PEEK cage with bioactive xenogeneic porcine cancellous bone. C3-C4 discectomy was performed in each group and the above bone graft and bone graft substitutes were implanted. Lateral cervical spine x-rays were taken at preoperative; immediately postoperative; and 4, 8, 12, and 24 weeks postoperatively every goat. Disc space heights (DSHs) were measured on lateral x-rays. CT examination was performed at 12 and 24 weeks after surgery for the fusion score. After 4 and 8 weeks after surgery, 3 goats were euthanized in both groups A and C to evaluate the immune rejection response through histology. At 12 and 24 weeks after surgery, 3 goats were euthanized in each group. The cervical implants fusion outcome was evaluated through specimen histology observation. RESULT: As time extended, the immune rejection of bioactive xenogeneic porcine cancellous bone gradually subsided. Radiology, specimen observation, and histology manifested that the C3-4 vertebral bodies of goats in each group gradually fused. All the goats in each group achieved bony fusion at 24 weeks after surgery. In terms of preventing intervertebral space collapse, the PEEK cage could achieve better results. There was no significant difference in the remaining experimental data (P>0.05). CONCLUSIONS: Bioactive xenogeneic porcine cancellous bone can obtain satisfied fusion outcomes in cervical intervertebral fusion and is an ideal intervertebral fusion material in goats.

ASFV infection induces macrophage necroptosis and releases proinflammatory cytokine by ZBP1-RIPK3-MLKL necrosome activation.

African swine fever (ASF) is an infectious disease characterized by hemorrhagic fever, which is highly pathogenic and causes severe mortality in domestic pigs. It is caused by the African swine fever virus (ASFV). ASFV is a large DNA virus and primarily infects porcine monocyte macrophages. The interaction between ASFV and host macrophages is the major reason for gross pathological lesions caused by ASFV. Necroptosis is an inflammatory programmed cell death and plays an important immune role during virus infection. However, whether and how ASFV induces macrophage necroptosis and the effect of necroptosis signaling on host immunity and ASFV infection remains unknown. This study uncovered that ASFV infection activates the necroptosis signaling in vivo and macrophage necroptosis in vitro. Further evidence showed that ASFV infection upregulates the expression of ZBP1 and RIPK3 to consist of the ZBP1-RIPK3-MLKL necrosome and further activates macrophage necroptosis. Subsequently, multiple Z-DNA sequences were predicted to be present in the ASFV genome. The Z-DNA signals were further confirmed to be present and colocalized with ZBP1 in the cytoplasm and nucleus of ASFV-infected cells. Moreover, ZBP1-mediated macrophage necroptosis provoked the extracellular release of proinflammatory cytokines, including TNF-α and IL-1ß induced by ASFV infection. Finally, we demonstrated that ZBP1-mediated necroptosis signaling inhibits ASFV replication in host macrophages. Our findings uncovered a novel mechanism by which ASFV induces macrophage necroptosis by facilitating Z-DNA accumulation and ZBP1 necrosome assembly, providing significant insights into the pathogenesis of ASFV infection.

CRISPR-array-mediated imaging of non-repetitive and multiplex genomic loci in living cells.

Dynamic imaging of genomic loci is key for understanding gene regulation, but methods for imaging genomes, in particular non-repetitive DNAs, are limited. We developed CRISPRdelight, a DNA-labeling system based on endonuclease-deficient CRISPR-Cas12a (dCas12a), with an engineered CRISPR array to track DNA location and motion. CRISPRdelight enables robust imaging of all examined 12 non-repetitive genomic loci in different cell lines. We revealed the confined movement of the CCAT1 locus (chr8q24) at the nuclear periphery for repressed expression and active motion in the interior nucleus for transcription. We uncovered the selective repositioning of HSP gene loci to nuclear speckles, including a remarkable relocation of HSPH1 (chr13q12) for elevated transcription during stresses. Combining CRISPR-dCas12a and RNA aptamers allowed multiplex imaging of four types of satellite DNA loci with a single array, revealing their spatial proximity to the nucleolus-associated domain. CRISPRdelight is a user-friendly and robust system for imaging and tracking genomic dynamics and regulation.

Rational design of ROS scavenging and fluorescent gold nanoparticles to deliver siRNA to improve plant resistance to Pseudomonas syringae.

Bacterial diseases are one of the most common issues that result in crop loss worldwide, and the increasing usage of chemical pesticides has caused the occurrence of resistance in pathogenic bacteria and environmental pollution problems. Nanomaterial mediated gene silencing is starting to display powerful efficiency and environmental friendliness for improving plant disease resistance. However, the internalization of nanomaterials and the physiological mechanisms behind nano-improved plant disease resistance are still rarely understood. We engineered the polyethyleneimine (PEI) functionalized gold nanoparticles (PEI-AuNPs) with fluorescent properties and ROS scavenging activity to act as siRNA delivery platforms. Besides the loading, protection, and delivery of nucleic acid molecules in plant mature leaf cells by PEI-AuNPs, its fluorescent property further enables the traceability of the distribution of the loaded nucleic acid molecules in cells. Additionally, the PEI-AuNPs-based RNAi delivery system successfully mediated the silencing of defense-regulated gene AtWRKY1. Compared to control plants, the silenced plants performed better resistance to Pseudomonas syringae, showing a reduced bacterial number, decreased ROS content, increased antioxidant enzyme activities, and improved chlorophyll fluorescence performance. Our results showed the advantages of AuNP-based RNAi technology in improving plant disease resistance, as well as the potential of plant nanobiotechnology to protect agricultural production.

Association of plasma lactoferrin levels with disease severity in glaucoma patients.

Objective: To explore the relationship between plasma lactoferrin (Lf) and glaucoma, assessing the clinical utility of Lf in glaucoma. Methods: A cross-sectional study involved 161 glaucoma patients and 115 healthy controls, with a follow-up of 14 subjects after approximately 2 years. Plasma Lf markers were quantified using ELISA, comparing levels between glaucoma patients and healthy controls, and analyzing plasma Lf across different glaucoma severity grades. Results: Glaucoma patients had significantly elevated plasma Lf levels compared to healthy controls (p < 0.001). Higher plasma Lf levels correlated with more severe disease stages (HPA grades showed ρ = 0.435, p < 0.001; AGIS grades showed ρ = 0.436, p < 0.001) and reduced retinal nerve fiber layer (RNFL) thickness (RNFL thickness showed ρ = -0.204, p = 0.024). ROC curve analysis demonstrated the efficacy of glaucoma markers in differentiating early-stage from advanced glaucoma. Conclusion: Plasma Lf levels are significantly associated with glaucoma severity and may be involved in the pathogenic progression of the disease.

ZBTB7A interferes with the RPL5-P53 feedback loop and reduces endoplasmic reticulum stress-induced apoptosis of pancreatic cancer cells.

Endoplasmic reticulum (ER) stress is a primary mechanism leading to cell apoptosis, making it of great research interests in cancer management. This study delves into the function of ribosomal protein L5 (RPL5) in ER stress within pancreatic cancer (PCa) cells and investigates its regulatory mechanisms. Bioinformatics predictions pinpointed RPL5 as an ER stress-related gene exhibiting diminished expression in PCa. Indeed, RPL5 was found to be poorly expressed in PCa tissues and cells, with this reduced expression correlating with an unfavorable prognosis. Moreover, RPL5 overexpression led to heightened levels of p-PERK, p-eIF2α, and CHOP, bolstering the proapoptotic effect of Tunicamycin, an ER stress activator, on PCa cells. Additionally, the RPL5 overexpression curbed cell proliferation, migration, and invasion. Tunicamycin enhanced the binding between RPL5 and murine double minute 2 (MDM2), thus suppressing MDM2-mediated ubiquitination and degradation of P53. Consequently, P53 augmentation intensified ER stress, which further enhanced the binding between RPL5 and MDM2 through PERK-dependent eIF2α phosphorylation, thereby establishing a positive feedback loop. Zinc finger and BTB domain containing 7A (ZBTB7A), conspicuously overexpressed in PCa samples, repressed RPL5 transcription, thereby reducing P53 expression. Silencing of ZBTB7A heightened ER stress and subdued the malignant attributes of PCa cells, effects counteracted upon RPL5 silencing. Analogous outcomes were recapitulated in vivo employing a xenograft tumor mouse model, where ZBTB7A silencing dampened the tumorigenic potential of PCa cells, an effect reversed by additional RPL5 silencing. In conclusion, this study suggests that ZBTB7A represses RPL5 transcription, thus impeding the RPL5-P53 feedback loop and mitigating ER-induced apoptosis in PCa cells.

Development of Broad-Spectrum Nanobodies for the Therapy and Diagnosis of SARS-CoV-2 and Its Multiple Variants.

The continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evaded the efficacy of previously developed antibodies and vaccines, thus remaining a significant global public health threat. Therefore, it is imperative to develop additional antibodies that are capable of neutralizing emerging variants. Nanobodies, as the smallest functional single-domain antibodies, exhibit enhanced stability and penetration ability, enabling them to recognize numerous concealed epitopes that are inaccessible to conventional antibodies. Herein, we constructed an immune library based on the immunization of alpaca with the S1 subunit of the SARS-CoV-2 spike protein, from which two nanobodies, Nb1 and Nb2, were selected using phage display technology for further characterization. Both nanobodies, with the binding residues residing within the receptor-binding domain (RBD) region of the spike, exhibited high affinity toward the S1 subunit. Moreover, they displayed cross-neutralizing activity against both wild-type SARS-CoV-2 and 10 ο variants, including BA.1, BA.2, BA.3, BA.5, BA.2.75, BF.7, BQ.1, EG.5.1, XBB.1.5, and JN.1. Molecular modeling and dynamics simulations predicted that both nanobodies interacted with the viral RBD through their complementarity determining region 1 (CDR1) and CDR2. These two nanobodies are novel tools for the development of therapeutic and diagnostic countermeasures targeting SARS-CoV-2 variants and potentially emerging coronaviruses.

Altered nucleocytoplasmic export of adenosine-rich circRNAs by PABPC1 contributes to neuronal function.

Circular RNAs (circRNAs) are upregulated during neurogenesis. Where and how circRNAs are localized and what roles they play during this process have remained elusive. Comparing the nuclear and cytoplasmic circRNAs between H9 cells and H9-derived forebrain (FB) neurons, we identify that a subset of adenosine (A)-rich circRNAs are restricted in H9 nuclei but exported to cytosols upon differentiation. Such a subcellular relocation of circRNAs is modulated by the poly(A)-binding protein PABPC1. In the H9 nucleus, newly produced (A)-rich circRNAs are bound by PABPC1 and trapped by the nuclear basket protein TPR to prevent their export. Modulating (A)-rich motifs in circRNAs alters their subcellular localization, and introducing (A)-rich circRNAs in H9 cytosols results in mRNA translation suppression. Moreover, decreased nuclear PABPC1 upon neuronal differentiation enables the export of (A)-rich circRNAs, including circRTN4(2,3), which is required for neurite outgrowth. These findings uncover subcellular localization features of circRNAs, linking their processing and function during neurogenesis.

Enhanced Interface Charge Carrier Transport of SnO2/CeO2 via Oxygen Vacancy Synergized Heterojunction for Triethylamine Sensing Property.

Efficient charge carrier transport characteristics are critical to achieving the excellent performance of metal-oxide semiconductor gas sensors. Herein, SnO2/CeO2 heterojunction layered nanosheets with abundant oxygen vacancies were successfully synthesized through a simple solvothermal assisted high-temperature calcination method. The synergistic effect of oxygen vacancies and heterojunctions promoting the charge carrier transport properties at the SnO2/CeO2 interface for the enhanced sensing properties of triethylamine (TEA) was highlighted. As a result, the optimized SnO2/CeO2 exhibits improved gas sensing performance at 173 °C to 50 ppm of TEA. These include high response (205), excellent selectivity, low detection limit, and good long-term stability. This enhanced gas sensing property of SnO2/CeO2 is mainly attributed to the fact that the heterojunction and oxygen vacancies act as dual active sites synergistically inducing electron transfer, thereby effectively modulating the transport properties of the interfacial charge carriers, and thus facilitate the surface reactions efficiently. In this work, the dual-engineering strategy of synergistic interaction of heterojunction and oxygen vacancies can provide new perspectives for the design of advanced gas sensing materials.

Long non-coding RNAs in the nucleolus: Biogenesis, regulation, and function.

The nucleolus functions as a multi-layered regulatory hub for ribosomal RNA (rRNA) biogenesis and ribosome assembly. Long noncoding RNAs (lncRNAs) in the nucleolus, originated from transcription by different RNA polymerases, have emerged as critical players in not only fine-tuning rRNA transcription and processing, but also shaping the organization of the multi-phase nucleolar condensate. Here, we review the diverse molecular mechanisms by which functional lncRNAs operate in the nucleolus, as well as their profound implications in a variety of biological processes. We also highlight the development of emerging molecular tools for characterizing and manipulating RNA function in living cells, and how application of such tools in the nucleolus might enable the discovery of additional insights and potential therapeutic strategies.

Preparation of bacterial fertilizer from biogas residue after anaerobic co-digestion of kitchen waste and residual sludge.

Azotobacter chroococcum and Bacillus subtilis were selected as fermentation strains, and biogas residue after anaerobic digestion of kitchen waste and residual sludge was used as fermentation substrate. A single factor optimization test was used to optimize the solid-state fermentation parameters of biogas residue with the number of viable bacteria and the number of spores as indexes. The results showed that the optimum inoculation conditions involved the following: 55% initial moisture content, 15% initial inoculation amount, 30 ℃, and 1:1 initial inoculation ratio for 13 days. Pot experiment showed that the prepared three kinds of bacterial fertilizers could not only effectively promote the growth of white clover, improve the composition of soil nutrients, but also change the structure of soil bacterial community, which is of great significance to the health of soil ecosystem in white clover.