Genome-wide identification and expression analysis of calmodulin and calmodulin-like genes in passion fruit (Passiflora edulis) and their involvement in flower and fruit development.

BACKGROUND: The calmodulin (CaM) and calmodulin-like (CML) proteins play regulatory roles in plant growth and development, responses to biotic and abiotic stresses, and other biological processes. As a popular fruit and ornamental crop, it is important to explore the regulatory mechanism of flower and fruit development of passion fruit. RESULTS: In this study, 32 PeCaM/PeCML genes were identified from passion fruit genome and were divided into 9 groups based on phylogenetic analysis. The structural analysis, including conserved motifs, gene structure and homologous modeling, illustrates that the PeCaM/PeCML in the same subgroup have relative conserved structural features. Collinearity analysis suggested that the expansion of the CaM/CML gene family likely took place mainly by segmental duplication, and the whole genome replication events were closely related with the rapid expansion of the gene group. PeCaM/PeCMLs were potentially required for different floral tissues development. Significantly, PeCML26 had extremely high expression levels during ovule and fruit development compared with other PeCML genes, suggesting that PeCML26 had potential functions involved in the development of passion fruit flowers and fruits. The co-presence of various cis-elements associated with growth and development, hormone responsiveness, and stress responsiveness in the promoter regions of these PeCaM/PeCMLs might contribute to their diverse regulatory roles. Furthermore, PeCaM/PeCMLs were also induced by various abiotic stresses. This work provides a comprehensive understanding of the CaM/CML gene family and valuable clues for future studies on the function and evolution of CaM/CML genes in passion fruit. CONCLUSION: A total of 32 PeCaM/PeCML genes were divided into 9 groups. The PeCaM/PeCML genes showed differential expression patterns in floral tissues at different development stages. It is worth noting that PeCML26, which is highly homologous to AtCaM2, not only interacts with multiple BBR-BPC TFs, but also has high expression levels during ovule and fruit development, suggesting that PeCML26 had potential functions involved in the development of passion fruit flowers and fruits. This research lays the foundation for future investigations and validation of the potential function of PeCaM/PeCML genes in the growth and development of passion fruit.

In-locus gene silencing in plants using genome editing.

Gene silencing is crucial in crop breeding for desired trait development. RNA interference (RNAi) has been used widely but is limited by ectopic expression of transgenes and genetic instability. Introducing an upstream start codon (uATG) into the 5'untranslated region (5'UTR) of a target gene may 'silence' the target gene by inhibiting protein translation from the primary start codon (pATG). Here, we report an efficient gene silencing method by introducing a tailor-designed uATG-containing element (ATGE) into the 5'UTR of genes in plants, occupying the original start site to act as a new pATG. Using base editing to introduce new uATGs failed to silence two of the tested three rice genes, indicating complex regulatory mechanisms. Precisely inserting an ATGE adjacent to pATG achieved significant target protein downregulation. Through extensive optimization, we demonstrated this strategy substantially and consistently downregulated target protein expression. By designing a bidirectional multifunctional ATGE4, we enabled tunable knockdown from 19% to 89% and observed expected phenotypes. Introducing ATGE into Waxy, which regulates starch synthesis, generated grains with lower amylose, revealing the value for crop breeding. Together, we have developed a programmable and robust method to knock down gene expression in plants, with potential for biological mechanism exploration and crop enhancement.

A novel exoskeletal-derived C-type lectin facilitates phagocytosis of hemocytes in the oriental river prawn Macrobrachium nipponense.

C-type lectins (CTLs) execute critical functions in multiple immune responses of crustaceans as a member of pattern recognition receptors (PRRs) family. In this study, a novel CTL was identified from the exoskeleton of the oriental river prawn Macrobrachium nipponense (MnLec3). The full-length cDNA of MnLec3 was 1150 bp with an open reading frame of 723 bp, encoding 240 amino acids. MnLec3 protein contained a signal peptide and one single carbohydrate-recognition domain (CRD). MnLec3 transcripts were widely distributed at the exoskeleton all over the body. Significant up-regulation of MnLec3 in exoskeleton after Aeromonas hydrophila challenged suggested the involvement of MnLec3 as well as the possible function of the exoskeleton in immune response. In vitro tests with recombinant MnLec3 protein (rMnLec3) manifested that it had polysaccharide binding activity, a wide spectrum of bacterial binding activity and agglutination activity only for tested Gram-negative bacteria (Escherichia coli, Vibrio anguillarum and A. hydrophila). Moreover, rMnLec3 significantly promoted phagocytic ability of hemocytes against A. hydrophila in vivo. What's more, MnLec3 interference remarkably impaired the survivability of the prawns when infected with A. hydrophila. Collectively, these results ascertained that MnLec3 derived from exoskeleton took an essential part in immune defense of the prawns against invading bacteria as a PRR.

The Effects of Near-Infrared Phototherapy Preirradiation on Lower-Limb Muscle Strength and Injury After Exercise: A Systematic Review and Meta-analysis.

OBJECTIVE: To assess near-infrared preirradiation effects on postexercise lower-limb muscle damage and function and determine optimal dosage. DATA SOURCES: PubMed, Embase, Cochrane Library, EBSCO, Web of Science, China National Knowledge Infrastructure, and Wanfang Data were systematically searched (2009-2023). STUDY SELECTION: Randomized controlled trials of near-infrared preirradiation on lower-limb muscles after fatigue exercise were incorporated into the meta-analysis. Out of 4550 articles screened, 21 met inclusion criteria. DATA EXTRACTION: The included studies' characteristics were independently extracted by 2 authors, with discrepancies resolved through discussion or by a third author. Quality assessment was performed using the Cochrane risk of bias tool and the Grading of Recommendations, Assessment, Development, and Evaluation System. DATA SYNTHESIS: In 21 studies, near-infrared preirradiation on lower-limb muscles inhibited the decline in peak torque (standardized mean difference [SMD], 1.33; 95% confidence interval [CI], 1.08-1.59; p<.001; increasing 27.97±4.87N·m), reduced blood lactate (SMD, -0.2; 95% CI, -0.37 to -0.03; p=.272; decreasing 0.54±0.42mmol/L), decreased creatine kinase (SMD, -2.11; 95% CI, -2.57 to -1.65; p<.001; decreasing 160.07±27.96U/L), and reduced delayed-onset muscle soreness (SMD, -0.53; 95% CI, -0.81 to 0.24; p<.001). Using a 24-hour cutoff revealed 2 trends: treatment effectiveness depended on power and energy density, with optimal effects at 24.16 J/cm2 and 275 J/cm2 for energy, and 36.81 mW/cm2 and 5495 mW/cm2 for power. Noting that out of 21 studies, 19 are from Brazil, 1 from the United States, and 1 from Australia, and the results exhibit high heterogeneity. CONCLUSIONS: Although we would have preferred a more geographic dispersion of laboratories, our findings indicate that near-infrared preirradiation mitigates peak torque decline in lower-limb muscles. Influenced by energy and power density with a 24-hour threshold, optimal energy and power densities are observed at 24.16 J/cm2, 275 J/cm2, 36.81 mW/cm2, and 5495 mW/cm2, respectively. Laser preirradiation also reduces blood lactate, creatine kinase, and delayed-onset muscle soreness.

High-Throughput CRISPR/Cas9 Mutagenesis Streamlines Trait Gene Identification in Maize.

Maize (Zea mays) is one of the most important crops in the world. However, few agronomically important maize genes have been cloned and used for trait improvement, due to its complex genome and genetic architecture. Here, we integrated multiplexed CRISPR/Cas9-based high-throughput targeted mutagenesis with genetic mapping and genomic approaches to successfully target 743 candidate genes corresponding to traits relevant for agronomy and nutrition. After low-cost barcode-based deep sequencing, 412 edited sequences covering 118 genes were precisely identified from individuals showing clear phenotypic changes. The profiles of the associated gene-editing events were similar to those identified in human cell lines and consequently are predictable using an existing algorithm originally designed for human studies. We observed unexpected but frequent homology-directed repair through endogenous templates that was likely caused by spatial contact between distinct chromosomes. Based on the characterization and interpretation of gene function from several examples, we demonstrate that the integration of forward and reverse genetics via a targeted mutagenesis library promises rapid validation of important agronomic genes for crops with complex genomes. Beyond specific findings, this study also guides further optimization of high-throughput CRISPR experiments in plants.

Characteristics, prognostic determinants of monocytes, macrophages and T cells in acute coronary syndrome: protocol for a multicenter, prospective cohort study.

BACKGROUND: Acute coronary syndrome(ACS) is the leading cause of mortality and disability worldwide. Immune response has been confirmed to play a vital role in the occurrence and development of ACS. The objective of this prospective, multicenter, observational study is to define immune response and their relationship to the occurrence and progressive of ACS. METHODS: This is a multicenter, prospective, observational longitudinal cohort study. The primary outcome is the incidence of major adverse cardiovascular events (MACE) including in-stent restenosis, severe ventricular arrhythmia, heart failure, recurrent angina pectoris, and sudden cardiac death, and stroke one year later after ACS. Demographic characteristics, clinical data, treatments, and outcomes are collected by local investigators. Furthermore, freshly processed samples will be stained and assessed by flow cytometry. The expression of S100A4, CD47, SIRPα and Tim-3 on monocytes, macrophages and T cells in ACS patients were collected. FOLLOW-UP: during hospitalization, 3, 6 and 12 months after discharge. DISCUSSION: It is expected that this study will reveal the possible targets to improve the prognosis or prevent from occurrence of MACE in ACS patients. Since it's a multicenter study, the enrollment rate of participants will be accelerated and it can ensure that the collected data are more symbolic and improve the richness and credibility of the test basis. ETHICS AND DISSEMINATION: This study has been registered in Chinese Clinical Trial Registry Center. Ethical approval was obtained from the Affiliated Hospital of Guizhou Medical University. The dissemination will occur through the publication of articles in international peer-reviewed journals. TRIAL REGISTRATION: Chinese Clinical Trial Registry: ChiCTR2200066382.

High-throughput genome editing in rice with a virus-based surrogate system.

With the widespread use of clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) technologies in plants, large-scale genome editing is increasingly needed. Here, we developed a geminivirus-mediated surrogate system, called Wheat Dwarf Virus-Gate (WDV-surrogate), to facilitate high-throughput genome editing. WDV-Gate has two parts: one is the recipient callus from a transgenic rice line expressing Cas9 and a mutated hygromycin-resistant gene (HygM) for surrogate selection; the other is a WDV-based construct expressing two single guide RNAs (sgRNAs) targeting HygM and a gene of interest, respectively. We evaluated WDV-Gate on six rice loci by producing a total of 874 T0 plants. Compared with the conventional method, the WDV-Gate system, which was characterized by a transient and high level of sgRNA expression, significantly increased editing frequency (66.8% vs. 90.1%), plantlet regeneration efficiency (2.31-fold increase), and numbers of homozygous-edited plants (36.3% vs. 70.7%). Large-scale editing using pooled sgRNAs targeting the SLR1 gene resulted in a high editing frequency of 94.4%, further demonstrating its feasibility. We also tested WDV-Gate on sequence knock-in for protein tagging. By co-delivering a chemically modified donor DNA with the WDV-Gate plasmid, 3xFLAG peptides were successfully fused to three loci with an efficiency of up to 13%. Thus, by combining transiently expressed sgRNAs and a surrogate selection system, WDV-Gate could be useful for high-throughput gene knock-out and sequence knock-in.

Genome editing for plant research and crop improvement.

The advent of clustered regularly interspaced short palindromic repeat (CRISPR) has had a profound impact on plant biology, and crop improvement. In this review, we summarize the state-of-the-art development of CRISPR technologies and their applications in plants, from the initial introduction of random small indel (insertion or deletion) mutations at target genomic loci to precision editing such as base editing, prime editing and gene targeting. We describe advances in the use of class 2, types II, V, and VI systems for gene disruption as well as for precise sequence alterations, gene transcription, and epigenome control.

Method for monitoring environmental flows with high spatial and temporal resolution satellite data.

Monitoring environmental flows is crucial to maintaining the function and stability of river and lake ecosystems. However, current methods for monitoring environmental flows are expensive and ground based, and the accuracy of the results needs to be verified to evaluate the environmental flows. This evaluation is hampered by the problem of data shortages, such as hydrological and ecological data. In this study, a method for monitoring environmental flows is proposed using multisource high spatial and temporal resolution satellite data. A case study in the Yongding River Basin demonstrates that the method is feasible for monitoring the environmental flows of rivers in semiarid and arid areas. The results show that the environmental flows and months with large water discharges and shortages in the three control sections of the Yongding River Basin were different. Moreover, the downstream river width rarely met the environmental water demand, achieving this only for one period from 2017 to 2019 according to the three typical types of years (an average water year, a dry year, and an extremely dry year). This method and the results have applications in planning environmental flows and could improve the comprehensive management of the ecological environment in river basins.

Multiplex gene editing in rice with simplified CRISPR-Cpf1 and CRISPR-Cas9 systems.

We developed simplified single transcriptional unit (SSTU) CRISPR systems for multiplex gene editing in rice using FnCpf1, LbCpf1 or Cas9, in which the nuclease and its crRNA array are co-expressed from a single Pol II promoter, without any additional processing machinery. Our SSTU systems are easy to construct and effective in mediating multiplex genome editing.

Editing of endogenous tubulins reveals varying effects of tubulin posttranslational modifications on axonal growth and regeneration.

Tubulin posttranslational modifications (PTMs) modulate the dynamic properties of microtubules and their interactions with other proteins. However, the effects of tubulin PTMs were often revealed indirectly through the deletion of modifying enzymes or the overexpression of tubulin mutants. In this study, we directly edited the endogenous tubulin loci to install PTM-mimicking or -disabling mutations and studied their effects on microtubule stability, neurite outgrowth, axonal regeneration, cargo transport, and sensory functions in the touch receptor neurons of Caenorhabditis elegans. We found that the status of ß-tubulin S172 phosphorylation and K252 acetylation strongly affected microtubule dynamics, neurite growth, and regeneration, whereas α-tubulin K40 acetylation had little influence. Polyglutamylation and detyrosination in the tubulin C-terminal tail had more subtle effects on microtubule stability likely by modulating the interaction with kinesin-13. Overall, our study systematically assessed and compared several tubulin PTMs for their impacts on neuronal differentiation and regeneration and established an in vivo platform to test the function of tubulin PTMs in neurons.

Effect of Low-Intensity Bloodflow Restriction Training on Nontraumatic Knee Joint Conditions: A Systematic Review and Meta-analysis.

CONTEXT: Nontraumatic knee conditions are common in clinical practice. Existing pharmaceutical and immobilization approaches provide limited pain relief and functional enhancement. Low-intensity bloodflow restriction training (LI-BFRT) is being investigated as a nonpharmacological alternative; however, its efficacy is uncertain. OBJECTIVE: To assess the effectiveness of LI-BFRT for nontraumatic knee conditions and compare it with high-intensity resistance training (HI-RT) and low-intensity resistance training (LI-RT). DATA SOURCES: PubMed, EBSCO, Science Direct, Cochrane Library, China Knowledge Infrastructure, Wanfang Data, and VIP databases were searched until May 30, 2023. STUDY SELECTION: Original randomized controlled trials involving nontraumatic knee joint conditions with interventions consisting mainly of LI-BFRT, HI-RT, or LI-RT. The results assessed mainly pain and muscle performance. STUDY DESIGN: Systematic review and meta-analysis. LEVEL OF EVIDENCE: Level 1. DATA EXTRACTION: Sample characteristics, study design, country, disease, groups, evaluation time, duration, and outcomes were extracted. RESULTS: A total of 13 randomized controlled trials were included in the systematic review. Compared with pretreatment, LI-BFRT significantly alleviated pain (weighted standardized mean difference [SMD], -1.33; 95% CI, -1.62 to -1.05), with better additional effects on hip muscle training (SMD, -3.14; 95% CI, -4.07 to -2.75). Compared with LI-RT, LI-BFRT significantly relieved pain in male patients (SMD, -1.47; 95% CI, -1.92 to -1.01). LI-BFRT significantly increased quadriceps cross-sectional area (SMD, 0.53; 95% CI, 0.27-0.78), knee extension strength (SMD, 0.84; 95% CI, 0.48-1.2), and leg press strength (SMD, 0.64; 95% CI, 0.34-0.94) compared with pretreatment. Its effects were superior to those of LI-RT and similar to those of HI-RT. However, sex differences in muscle strength improvement were observed. CONCLUSION: In patients with nontraumatic knee joint conditions, LI-BFRT effectively alleviated pain, increased muscle cross-sectional area, and enhanced muscle strength. LI-BFRT showed pain relief comparable with that of LI-RT while surpassing LI-RT in muscle growth and strength improvement.

Chemical composition of soil carbon is governed by microbial diversity during understory fern removal in subtropical pine forests.

Understory vegetation has an important impact on soil organic carbon (SOC) accumulation. However, little is known about how understory vegetation alters soil microbial community composition and how microbial diversity contributes to SOC chemical composition and persistence during subtropical forest restoration. In this study, removal treatments of an understory fern (Dicranopteris dichotoma) were carried out within pine (Pinus massoniana) plantations restored in different years in subtropical China. Soil microbial community composition and microbial diversity were measured using phospholipid fatty acids (PLFAs) biomarkers and high-throughput sequencing, respectively. The chemical composition of SOC was also measured via solid-state 13C nuclear magnetic resonance (13C NMR). Our results showed that fern removal decreased alkyl C by 4.2 % but increased O-alkyl C by 15.6 % on average, leading to a decline of alkyl C/O-alkyl C ratio, suggesting altered chemical composition of SOC and lowered SOC recalcitrance without fern. Fern removal significantly lowered the fungi-to-bacteria ratio, and it also reduced fungal and bacterial diversity. Partial correlation analysis revealed that soil nitrogen availability was a key factor influencing microbial diversity. Bacterial diversity showed a close relationship with the Alkyl C/O-alkyl C ratio following fern removal. Furthermore, the microbial community structure and bacterial diversity were responsible for 18 % and 55 % of the explained variance in the chemical composition of SOC, respectively. Taken together, these analyses jointly suggest that bacterial diversity exerts a greater role than microbial community structure in supporting SOC persistence during understory fern removal. Our study emphasizes the significance of understory ferns in supporting microbial abundance and diversity as a means of altering SOC persistence during subtropical forest restoration.

Rapid and dynamic detection of endogenous proteins through in-locus tagging in rice.

Understanding the behavior of endogenous proteins is crucial for functional genomics, yet their dynamic characterization in plants presents substantial challenges. While mammalian studies have leveraged in-locus tagging with the luminescent HiBiT peptide and genome editing for rapid quantification of native proteins, this approach remained unexplored in plants. Here, we introduce the in-locus HiBiT tagging of rice proteins and demonstrate its feasibility in plants. We found that although traditional HiBiT blotting works in rice, it failed to detect two of the three tagged proteins, which is attributed to the low luminescence activity in plants. To overcome this limitation, we engaged in extensive optimization, culminating in a new luciferin substrate coupled with a refined reaction protocol that enhanced luminescence by up to 6.9-fold. This innovation led to the development of TagBIT (tagging with HiBiT), a robust method for high-sensitivity protein characterization in plants. Our application of TagBIT to seven rice genes illustrates its versatility on endogenous proteins, enabling antibody-free protein blotting, real-time protein quantification via luminescence, in-situ visualization using a cross-breeding strategy, and effective immunoprecipitation for protein interaction analysis. The heritable nature of this system, confirmed across T1 to T3 generations, positions TagBIT as a powerful tool for protein study in plant biology.

The inhibitory effect of ABA on floral transition is mediated by ABI5 in Arabidopsis.

Seed germination and flowering initiation are both transitions responding to similar seasonal cues. This study shows that ABSCISIC ACID-INSENSITIVE MUTANT 5 (ABI5), a bZIP transcription factor, which plays an important role in the abscisic acid (ABA)-arrested seed germination, is robustly associated with the floral transition in Arabidopsis. Under long-day conditions, overexpression of ABI5 could delay floral transition through upregulating FLOWERING LOCUS C (FLC) expression. In contrast, ectopically overexpressing FLC in an abi5 mutant reversed the earlier flowering phenotype. Further analysis indicated that transactivation of FLC could be promoted by ABI5 and/or other abscisic acid-responsive element (ABRE)-binding factors (ABFs). The expression of FLC that was promoted by ABI5 and/or other ABFs could be blocked in a triple SNF1-related protein kinase (SnRK) mutant, snrk2.2/2.3/2.6, despite the presence of ABA. In sharp contrast, when SnRK2.6 was coexpressed, the reduction of transactivity of FLC was reverted in mesophyll protoplasts of snrk2.2/2.3/2.6. Additional results from analysing transgenic plants carrying mutations of phosphoamino acids (ABI5 ( S42AS145AT201A )), which are conserved in ABI5, suggested that SnRK2-mediated ABI5 and/or ABF phosphorylation may be crucial for promoting FLC expression. The transgenic plants ABI5 ( S42AS145AT201A ) were insensitive to ABA in seed germination, in addition to having an earlier flowering phenotype. Direct binding of ABI5 to the ABRE/G-box promoter elements existing in FLC was demonstrated by chromatin immunoprecipitation. Mutations at the ABRE/G-box regions in FLC promoter sequences abolished the ABI5-promoted transactivation of FLC. In summary, these results may decipher the inhibitory effect of ABA on floral transition in Arabidopsis.