Calmodulin-like protein CML15 interacts with PP2C46/65 to regulate papaya fruit ripening via integrating calcium, ABA and ethylene signals.

It is well known that calcium, ethylene and abscisic acid (ABA) can regulate fruit ripening, however, their interaction in the regulation of fruit ripening has not yet been fully clarified. The present study found that the expression of the papaya calcium sensor CpCML15 was strongly linked to fruit ripening. CpCML15 could bind Ca2+ and served as a true calcium sensor. CpCML15 interacted with CpPP2C46 and CpPP2C65, the candidate components of the ABA signalling pathways. CpPP2C46/65 expression was also related to fruit ripening and regulated by ethylene. CpCML15 was located in the nucleus and CpPP2C46/65 were located in both the nucleus and membrane. The interaction between CpCML15 and CpPP2C46/65 was calcium dependent and further repressed the activity of CpPP2C46/65 in vitro. The transient overexpression of CpCML15 and CpPP2C46/65 in papaya promoted fruit ripening and gene expression related to ripening. The reduced expression of CpCML15 and CpPP2C46/65 by virus-induced gene silencing delayed fruit colouring and softening and repressed the expression of genes related to ethylene signalling and softening. Moreover, ectopic overexpression of CpCML15 in tomato fruit also promoted fruit softening and ripening by increasing ethylene production and enhancing gene expression related to ripening. Additionally, CpPP2C46 interacted with CpABI5, and CpPP2C65 interacted with CpERF003-like, two transcriptional factors in ABA and ethylene signalling pathways that are closely related to fruit ripening. Taken together, our results showed that CpCML15 and CpPP2Cs positively regulated fruit ripening, and their interaction integrated the cross-talk of calcium, ABA and ethylene signals in fruit ripening through the CpCML15-CpPP2Cs-CpABI5/CpERF003-like pathway.

Molecular epidemiological characteristics of Brucella in Guizhou Province, China, from 2009 to 2021.

Introduction: Brucellosis was made statutorily notifiable in 1955, in China, while in Guizhou Province, the pathogen of human brucellosis was isolated for the first time in 2011. However, currently, the brucellosis epidemic is becoming more and more severe in Guizhou Province. The type distribution and genetic characteristics of Brucella in Guizhou Province, as well as its evolutionary relationship with domestic and foreign strains, are still unclear. Methods: MLST, MLVA, and rpoB typing techniques were used for the molecular epidemiological study of the 83 Brucella isolates in Guizhou province. Results: Among the 83 Brucella strains, MLST identified three ST genotypes, of which ST39 is a newly reported type in China. MLVA-16 generated 49 genotypes, and MLVA-11 generated 5 known genotypes and 2 unreported genotypes. Six genotypes were identified by rpoB technology. Discussion: MLVA has a high resolution, but differences at the Bruce 04 and 16 loci cannot exclude associations between epidemics, and combining MLST and rpoB typing methods for epidemiologic tracing can avoid erroneous judgments. Moreover, through the combined analysis of the three typing techniques, the possible origin of the new Brucella can be reasonably inferred, which is also conducive to promoting the subsequent research of the novel Brucella.

Development of a CRISPR/Cas12a-recombinase polymerase amplification assay for visual and highly specific identification of the Congo Basin and West African strains of mpox virus.

Human mpox is a zoonotic disease, similar to smallpox, caused by the mpox virus, which is further subdivided into Congo Basin and West African clades with different pathogenicity. In this study, a novel diagnostic protocol utilizing clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 12a nuclease (CRISPR/Cas12a)-mediated recombinase polymerase amplification (RPA) was developed to identify mpox in the Congo Basin and West Africa (CRISPR-RPA). Specific RPA primers targeting D14L and ATI were designed. CRISPR-RPA assay was performed using various target templates. In the designed CRISPR-RPA reaction system, the exponentially amplified RPA amplification products with a protospacer adjacent motif (PAM) site can locate the Cas12a/crRNA complex to its target regions, which successfully activates the CRISPR/Cas12a effector and achieves ultrafast trans-cleavage of a single-stranded DNA probe. The limit of detection for the CRISPR-RPA assay was 10 copies per reaction for D14L- and ATI-plasmids. No cross-reactivity was observed with non-mpox strains, confirming the high specificity of the CRISPR-RPA assay for distinguishing between the Congo Basin and West African mpox. The CRISPR-RPA assay can be completed within 45 min using real-time fluorescence readout. Moreover, the cleavage results were visualized under UV light or an imaging system, eliminating the need for a specialized apparatus. In summary, the developed CRISPR/RPA assay is a visual, rapid, sensitive, and highly specific detection technique that can be used as an attractive potential identification tool for Congo Basin and West African mpox in resource-limited laboratories.

Development of CRISPR/Cas12b-Based Multiple Cross Displacement Amplification Technique for the Detection of Mycobacterium tuberculosis Complex in Clinical Settings.

Tuberculosis (TB) is a chronic infectious disease with high mortality caused by the Mycobacterium tuberculosis complex (MTC). Its clinical symptoms include a prolonged cough with mucus, pleuritic chest pain, hemoptysis, etc., and predominant complications such as tuberculous meningitis and pleural effusion. Thus, developing rapid, ultrasensitive, and highly specific detection techniques plays an important role in controlling TB. Here, we devised CRISPR/CRISPR-associated 12b nuclease (CRISPR/Cas12b)-based multiple cross displacement amplification technique (CRISPR-MCDA) targeting the IS6110 sequence and used it to detect MTC pathogens. A newly engineered protospacer adjacent motif (PAM) site (TTTC) was modified in the linker region of the CP1 primer. In the CRISPR-MCDA system, the exponentially amplified MCDA amplicons with the PAM sites can guide the Cas12b/gRNA complex to quickly and accurately recognize its target regions, which successfully activates the CRISPR/Cas12b effector and enables ultrafast trans-cleavage of single-stranded DNA reporter molecules. The limit of detection of the CRISPR-MCDA assay was 5 fg/µL of genomic DNA extracted from the MTB reference strain H37Rv. The CRISPR-MCDA assay successfully detected all examined MTC strains and there was no cross-reaction with non-MTC pathogens, confirming that its specificity is 100%. The entire detection process can be completed within 70 min using real-time fluorescence analysis. Moreover, visualization detection (under UV light) was also designed to verify the results, eliminating the use of specialized instruments. In conclusion, the CRISPR-MCDA assay established in this report can be used as a valuable detection technique for MTC infection. IMPORTANCE The Mycobacterium tuberculosis complex pathogen is a crucial infectious agent of tuberculosis. Hence, improving the capability of MTC detection is one of the most urgently required strategies for preventing and controlling TB. In this report, we successfully developed and implemented CRISPR/Cas12b-based multiple cross displacement amplification targeting the IS6110 sequence to detect MTC pathogens. These results demonstrated that the CRISPR-MCDA assay developed in this study was a rapid, ultrasensitive, highly specific, and readily available method which can be used as a valuable diagnostic tool for MTC infection in clinical settings.

Rapid, ultrasensitive, and highly specific identification of Brucella abortus utilizing multiple cross displacement amplification combined with a gold nanoparticles-based lateral flow biosensor.

Brucella abortus (B. abortus) as an important infectious agent of bovine brucellosis cannot be ignored, especially in countries/regions dominated by animal husbandry. Thus, the development of an ultrasensitive and highly specific identification technique is an ideal strategy to control the transmission of bovine brucellosis. In this report, a novel detection protocol, which utilizes multiple cross displacement amplification (MCDA) combined with a gold nanoparticles-based lateral flow biosensor (AuNPs-LFB) targeting the BruAb2_0168 gene was successfully devised and established for the identification of B. abortus (termed B. abortus-MCDA-LFB). Ten specific primers containing engineered C1-FAM (carboxyfluorescein) and D1-biotin primers were designed according to the MCDA reaction mechanism. These genomic DNA extracted from various bacterial strains and whole blood samples were used to optimize and evaluate the B. abortus-MCDA-LFB assay. As a result, the optimal reaction conditions for the B. abortus-MCDA-LFB assay were 66°C for 40 min. The limit of detection of the B. abortus-MCDA-LFB was 10 fg/µl (~3 copies/µl) for genomic DNA extracted from pure cultures of B. abortus isolate. Meanwhile, the B. abortus-MCDA-LFB assay accurately identified all tested B. abortus strains, and there was no cross-reaction with non-B. abortus pathogens. Moreover, the detection workflow of the B. abortus-MCDA-LFB assay for whole blood samples can be completed within 70 min, and the cost of a single test is approximately 5.0 USD. Taken together, the B. abortus-MCDA-LFB assay is a visual, fast, ultrasensitive, low-cost, easy-to-operate, and highly specific detection method, which can be used as a rapid identification tool for B. abortus infections.

Effects of Resistant Starch on Patients with Chronic Kidney Disease: A Systematic Review and Meta-Analysis.

Background: Chronic kidney disease (CKD) is a main health problem associated with increased risk of cardiovascular disease, morbidity, and mortality. Recent studies shown that the progression of CKD may be related to the change of intestinal flora. Resistant starch (RS) is a type of dietary fiber that can act as a substrate for microbial fermentation. Some studies have found that the supplementation of RS can improve the intestinal flora disorder in CKD patients. However, the specific effect of RS on CKD patients remains controversial. Objective: We designed this meta-analysis to identify and assess the effects of RS on patients with CKD. Methods: A comprehensive search of MEDLINE, Embase, Web of Science, and Cochrane systematic review databases was conducted in January 2020, and all new trials were updated in August 2021. Randomized trials were collected to assess the effects of RS on patients with CKD. The weighted average effect size of the net change was calculated by using the random-effects model. Results: The meta-analysis included 8 studies involving 301 participants. RS intake significantly reduced serum indolephenol sulfate (IS), blood phosphorus, IL-6, and uric acid levels in dialysis patients. The mean difference (MD) of serum IS (P = 0.0002) in the dialysis subgroup was -12.57 µmol/L (95% CI: -19.28, -5.86 µmol/L). The MD of blood phosphorus (P = 0.03) was -0.39 mg/dl (95% CI: -0.78, -0.01 mg/dl). The MD of serum uric acid (P = 0.004) between the dialysis subgroup and the nondialysis subgroup was -31.58 mmol/L (95% CI: -52.99, -10.17 mmol/L). The mean difference (MD) of IL-6 (P = 0.02) in the dialysis subgroup was -1.16 µmol/L (95% CI: -2.16, -0.16 µmol/L). However, there was no significant change of RS on hs-CRP, serum creatinine, blood urea nitrogen (BUN), blood paracresol sulfate, and blood lipid. Conclusions: The intake of RS reduced the serum IS, serum phosphorus, IL-6, and uric acid levels significantly in dialysis patients, while hs-CRP, serum creatinine, BUN, serum paracresol sulfate, and blood lipid showed no significant changes.

Melatonin Treatment Improves Postharvest Preservation and Resistance of Guava Fruit (Psidium guajava L.).

Guava fruit has a short postharvest shelf life at room temperature. Melatonin is widely used for preservation of various postharvest fruit and vegetables. In this study, an optimal melatonin treatment (600 µmol·L-1, 2 h) was identified, which effectively delayed fruit softening and reduced the incidence of anthracnose on guava fruit. Melatonin effectively enhanced the antioxidant capacity and reduced the oxidative damage to the fruit by reducing the contents of superoxide anions, hydrogen peroxide and malondialdehyde; improving the overall antioxidant capacity and enhancing the enzymatic antioxidants and non-enzymatic antioxidants. Melatonin significantly enhanced the activities of catalase, superoxide dismutase, ascorbate peroxidase and glutathione reductase. The contents of total flavonoids and ascorbic acid were maintained by melatonin. This treatment also enhanced the defense-related enzymatic activities of chitinase and phenylpropanoid pathway enzymes, including phenylalanine ammonia lyase and 4-coumaric acid-CoA-ligase. The activities of lipase, lipoxygenase and phospholipase D related to lipid metabolism were repressed by melatonin. These results showed that exogenous melatonin can maintain the quality of guava fruit and enhance its resistance to disease by improving the antioxidant and defense systems of the fruit.

A Label-Based Polymer Nanoparticles Biosensor Combined with Loop-Mediated Isothermal Amplification for Rapid, Sensitive, and Highly Specific Identification of Brucella abortus.

Brucella abortus (B. abortus), an important zoonotic pathogen in Brucella spp., is the major causative agent of abortion in cattle (namely, bovine brucellosis). Currently, although the isolation and identification of the Brucella abortus were commonly accepted as the gold standard method, it cannot meet the requirements for early diagnostic strategies. Conventional PCR techniques and immunological tests can realize rapid detection of B. abortus, but the demands for PCR thermal cyclers and/or specific antibodies hinder their application in basic laboratories. Thus, rapid, sensitive, and specific diagnostic strategies are essential to prevent and control the spread of the bovine brucellosis. In this work, a novel detection method for the rapid identification of B. abortus, which uses loop-mediated isothermal amplification (LAMP) combined with a label-based polymer nanoparticles lateral flow immunoassay biosensor (LFIA), was established. One set of specific B. abortus-LAMP primers targeting the BruAb2_0168 gene was designed by the online LAMP primer design tool. The B. abortus-LAMP-LFIA assay was optimized and evaluated using various pathogens and whole blood samples. The optimal amplification temperature and time for B. abortus-LAMP-LFIA were determined to be 65°C and 50 min, respectively. The B. abortus-LAMP-LFIA method limit of detection (LoD) was 100 fg per reaction for pure genomic DNA of B. abortus. Meanwhile, the detection specificity was 100%, and there was no cross-reactivity for other Brucella members and non-Brucella strains. Furthermore, the entire procedure, including the DNA preparation for whole blood samples (30 min), isothermal incubation (50 min), and LFIA detection (2-5 min), can be completed in approximately 85 min. Thus, the B. abortus-LAMP-LFIA assay developed was a simple, rapid, sensitive, and reliable detection technique, which can be used as a screening and/or diagnostic tool for B. abortus in the field and basic laboratories.

Interactions between calcium and ABA signaling pathways in the regulation of fruit ripening.

Fruit ripening and senescence are finely controlled by plant hormones such as ethylene and abscisic acid (ABA) but also by calcium ions and by calcium-dependent signaling pathways. Although there are extensive data supporting an interaction between ethylene and calcium in fruit ripening, the regulatory mechanisms resulting from the interaction between ABA and calcium have not yet been fully clarified. In this article, we have reviewed the full roles of calcium and its sensors (CaM, CMLs, CDPKs, CBLs) as well as ABA and the interactions between the two signaling pathways in the regulation of stress responses and in fruit ripening. To illustrate the possible interaction between calcium sensors and ABA signaling components in the control of fruit ripening, we propose an interaction model between the calcium and ABA signaling pathways.

Transcriptomic analysis reveals key factors in fruit ripening and rubbery texture caused by 1-MCP in papaya.

BACKGROUND: Ethylene promotes fruit ripening whereas 1-methylcyclopropene (1-MCP), a non-toxic antagonist of ethylene, delays fruit ripening via the inhibition of ethylene receptor. However, unsuitable 1-MCP treatment can cause fruit ripening disorders. RESULTS: In this study, we show that short-term 1-MCP treatment (400 nL•L- 1, 2 h) significantly delays papaya fruit ripening with normal ripening characteristics. However, long-term 1-MCP treatment (400 nL•L- 1, 16 h) causes a "rubbery" texture of fruit. The comparative transcriptome analysis showed that a total of 5529 genes were differently expressed during fruit ripening compared to freshly harvested fruits. Comprehensive functional enrichment analysis showed that the metabolic pathways of carbon metabolism, plant hormone signal transduction, biosynthesis of amino acids, and starch and sucrose metabolism are involved in fruit ripening. 1-MCP treatment significantly affected fruit transcript levels. A total of 3595 and 5998 differently expressed genes (DEGs) were identified between short-term 1-MCP, long-term 1-MCP treatment and the control, respectively. DEGs are mostly enriched in the similar pathway involved in fruit ripening. A large number of DEGs were also identified between long-term and short-term 1-MCP treatment, with most of the DEGs being enriched in carbon metabolism, starch and sucrose metabolism, plant hormone signal transduction, and biosynthesis of amino acids. The 1-MCP treatments accelerated the lignin accumulation and delayed cellulose degradation during fruit ripening. Considering the rubbery phenotype, we inferred that the cell wall metabolism and hormone signal pathways are closely related to papaya fruit ripening disorder. The RNA-Seq output was confirmed using RT-qPCR by 28 selected genes that were involved in cell wall metabolism and hormone signal pathways. CONCLUSIONS: These results showed that long-term 1-MCP treatment severely inhibited ethylene signaling and the cell wall metabolism pathways, which may result in the failure of cell wall degradation and fruit softening. Our results reveal multiple ripening-associated events during papaya fruit ripening and provide a foundation for understanding the molecular mechanisms underlying 1-MCP treatment on fruit ripening and the regulatory networks.

DFT calculations of the defect structures, electronic structures, and EPR parameters for three Rh2+ centers in AgCl.

The local structures for various Rh2+ centers in AgCl are theoretically studied using density functional theory (DFT) with periodic CP2K program. Through geometry optimizing, the stable ground states with minimal energies and electronic structures are obtained for the tetragonally elongated (TE ), orthorhombically elongated (OE ), and tetragonally compressed (TC ) centers, and the corresponding g and hyperfine coupling tensors are calculated in ORCA level. The calculations reveal obvious Jahn-Teller elongation distortions of about 0.109 and 0.110 Å along [001] axis for TE and OE centers without and with 1 next nearest neighbor (nnn) cation vacancy VAg in [100] axis, respectively. Whereas TC center with 1 nnn VAg along [001] axis exhibits moderate axial compression of about 0.066 Å due to the Jahn-Teller effect. For OE and TC centers with 1 nnn VAg , the ligand intervening in the central Rh2+ and the VAg is found to displace away from the VAg by about 0.028 and 0.024 Å, respectively. The present results are discussed and compared with those of the previous calculations based on the perturbation formulas by using the improved ligand field theory.