Application of CRISPR/Cas12i.3 for targeted mutagenesis in broomcorn millet (Panicum miliaceum L.).

In the present study, we have successfully established a gene editing platform in broomcorn millet, one of the oldest crops originating from China, by using our CRISPR/Cas12i.3, and we also created new elite germplasm for this crop.

Genomic variation in weedy and cultivated broomcorn millet accessions uncovers the genetic architecture of agronomic traits.

Large-scale genomic variations are fundamental resources for crop genetics and breeding. Here we sequenced 1,904 genomes of broomcorn millet to an average of 40× sequencing depth and constructed a comprehensive variation map of weedy and cultivated accessions. Being one of the oldest cultivated crops, broomcorn millet has extremely low nucleotide diversity and remarkably rapid decay of linkage disequilibrium. Genome-wide association studies identified 186 loci for 12 agronomic traits. Many causative candidate genes, such as PmGW8 for grain size and PmLG1 for panicle shape, showed strong selection signatures during domestication. Weedy accessions contained many beneficial variations for the grain traits that are largely lost in cultivated accessions. Weedy and cultivated broomcorn millet have adopted different loci controlling flowering time for regional adaptation in parallel. Our study uncovers the unique population genomic features of broomcorn millet and provides an agronomically important resource for cereal crops.

Modeling Mathematical Relationship with Weight Loss and Texture on Table Grapes of 'Red Globe' and 'Wink' during Cold and Ambient Temperature Storage.

Weight loss associated with fruit texture during storage has received numerous reports; however, no research has been conducted on the mathematical relationships between weight loss and textural traits of table grapes stored at cold and ambient temperatures. In this study, it was found that the weight loss of 'Red Globe' was in the range of 0 to 0.0487, 0 to 0.0284 and 0 to 0.0199 compared to 0 to 0.0661, 0 to 0.0301 and 0 to 0.028 of 'Wink' at 13 °C, 3 °C, and 0 °C of storage for 13 days. Stored for 13 days at 13 °C, 3 °C, and 0 °C, the range of the textural traits of failure force, strain and penetration work in 'Red Globe' were 6.274 to 3.765, 6.441 to 3.867, 6.321 to 4.014; 51.931 to 11.114, 51.876 to 13.002, 51.576 to 20.892; 21.524 to 13.225, 21.432 to 14.234, 21.321 to 15.198 in contrast to in 'Wink' of 4.4202 to 2.2292, 4.4197 to 2.653, 4.4371 to 2.8199 and 15.674 to 2.7881, 15.776 to 4.1431, 15.704 to 5.702 and 12.922 to 7.754, 12.909 to 8.021, 12.915 to 8.407. Meanwhile, the weight loss and textural traits of two table grapes were examined using time-dependent and weight loss-dependent modeling at 13 °C, 3 °C, and 0 °C of storage. The Logistic, ExpDec1, and ExpDec2 models, as well as the Boltzmann model, were identified as the best fit for the obtained data. The equations proved to be more effective in characterizing the change in weight loss and texture of 'Red Globe' and 'Wink,' with the best equations suited to the weight loss and textural parameters having an average mean standard error of 2.89%. The viability of the established models was evaluated, and parametric confidence intervals of the equations were proposed to fit different grape cultivars. According to the findings, the weight loss and texture of the two grape cultivars could be accurately predicted by the established models; additionally, the results showed that cold storage is better for the quality of table grapes and that weight loss can predict the textural quality of table grapes. This study provides a theoretical framework for optimum storage temperature together with a significantly convenient and quick approach to measure the texture of grapes for fruit dealers and enterprises.

Structural characterization and induction of tumor cell apoptosis of polysaccharide from purple sweet potato (Ipomoea batatas (L.) Lam).

The carbohydrate is the main ingredient of purple sweet potato. A polysaccharide, named PSP, was separated and purified from purple sweet potato by extraction with hot water, precipitation with ethanol, deproteinization with Sevag reagent and column chromatography with Sephadex G-100. The purity and structure were studied with HPLC, UV-Vis, GC-MS and NMR. The PSP is a neutral polysaccharide with Mw of 470 kDa. The monosaccharide composition of PSP contained D-xylose, d-glucose, D-galactose with ratio of 1.0: 8.3: 1.3. The backbone of PSP was composed of the residues of →6)-D-Glcp-(1 â†’ and →2, 6)-D-Glcp-(1→. The branches of PSP contained the residues of →3)-D-Galp-(1→, and D-Xylp-(1→. The antitumor activity in vitro of PSP was analyzed with HT-29 cells. And the SEM, AO staining, MDC staining and hoechst 33342 staining were performed to study the effect on apoptosis of HT-29 cells by PSP. The results revealed that the PSP can significantly inhibit the proliferation of HT-29 cells from induction apoptosis. The manuscript provided valuable knowledges on structural characteristics of the polysaccharides from purple sweet potato.

A polysaccharide from Pleurotus citrinopileatus mycelia enhances the immune response in cyclophosphamide-induced immunosuppressed mice via p62/Keap1/Nrf2 signal transduction pathway.

The manuscript aimed to study the immunoregulatory activity and the mechanism of the polysaccharide (CMP) from Pleurotus citrinopileatus mycelia. The mice were divided into normal group, model group, different dosage of CMP (50, 100 and 200 mg/kg, respectively) groups and levamisole hydrochloride treated group. The results showed that, compared with the model group, CMP could significantly improve the auricle swelling rate, half hemolysis value and phagocytic index in mice. The indices of immune organs were raised, and tissue damage of spleen was relieved. Splenic Th1 cells were decreased, while Th2 cells were increased, furthermore the proliferation of splenic lymphocytes and the cytotoxicity of NK cells were increased. The levels of interleukin-12 (IL-12), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in spleen were decreased, while interleukin-4 (IL-4) and interleukin-10 (IL-10) were increased. In serum and spleen, the levels of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities were increased, while the level of malondialdehyde (MDA) was decreased. And the levels of Immunoglobulin were also increased. Western blot showed that CMP had immunoregulatory activity by activating Nrf2, Keap1, p62, HO-1, and NQO1 in the p62/Keap1/Nrf2 signaling pathway. The study proved that CMP could be used as a biological Immune regulating agent.

A TaqMan Probe-Based Multiplex Real-Time PCR for Simultaneous Detection of Porcine Epidemic Diarrhea Virus Subtypes G1 and G2, and Porcine Rotavirus Groups A and C.

Porcine viral diarrhea diseases affect the swine industry, resulting in significant economic losses. Porcine epidemic diarrhea virus (PEDV) genotypes G1 and G2, and groups A and C of the porcine rotavirus, are major etiological agents of severe gastroenteritis and profuse diarrhea, particularly among piglets, with mortality rates of up to 100%. Based on the high prevalence rate and frequent co-infection of PEDV, RVA, and RVC, close monitoring is necessary to avoid greater economic losses. We have developed a multiplex TaqMan probe-based real-time PCR for the rapid simultaneous detection and differentiation of PEDV subtypes G1 and G2, RVA, and RVC. This test is highly sensitive, as the detection limits were 20 and 100 copies/µL for the G1 and G2 subtypes of PEDV, respectively, and 50 copies/µL for RVA and RVC, respectively. Eighty-eight swine clinical samples were used to evaluate this new test. The results were 100% in concordance with the standard methods. Since reassortment between porcine and human rotaviruses has been reported, this multiplex test not only provides a basis for the management of swine diarrheal viruses, but also has the potential to impact public health as well.

Identification and characterization of long non-coding RNAs in juvenile and adult skeletal muscle of largemouth bass (Micropterus salmoides).

Long non-coding RNAs (lncRNAs) are a class of transcriptional RNA molecules, which play critical roles in diverse biological processes. However, little is known about the overall expression pattern and roles of lncRNAs in skeletal muscle of largemouth bass (LMB). Here, we constructed two skeletal muscle RNA libraries to find lncRNAs that may involve in the regulation of skeletal muscle development between juvenile and adult LMB. A total of 16,147 lncRNAs and 4611 differentially expressed lncRNAs were identified. Among these identified lncRNAs, 10 lncRNAs were randomly selected to confirm their expression by real-time qPCR both in libraries, which were consistent with the RNA sequencing results. The target mRNAs of lncRNAs were predicted for GO enrichment analysis. Results showed that these targets associated with growth and development of muscle, such as skeletal muscle fiber development, myoblast proliferation and differentiation. Importantly, correlation analysis of lncRNA-miRNA-mRNA regulatory network revealed that several lncRNAs targeted miRNAs which are closely involved in the regulation of muscle development. It is the first time to identify a number of lncRNA that correlate with skeletal muscle development in LMB. Our results not only provide a comprehensive expression profile of muscle lncRNAs in this species, but also provide a theoretical basis for further elaborating genetic regulation mechanism of muscle growth and development, and pave the way for the future molecular assisted breeding in carnivorous fishes.

Identification and functional analysis of circRNAs in the skeletal muscle of juvenile and adult largemouth bass (Micropterus salmoides).

Circular RNA (circRNA) is a novel emerging type of endogenous regulatory non-coding RNA molecules with a covalent closed-loop configuration, which exerts important functions in multiple biological processes. CircRNAs are known to regulate gene expression as functional regulators interacting with miRNAs by sponge, which have been reported to regulate skeletal muscle development. Nevertheless, the information of circRNAs involved in regulating muscle growth and development in fish is largely unknown. Here, we first identified 312 and 511 circRNAs in skeletal muscle of juvenile and adult largemouth bass (LMB) using RNA sequencing, respectively. The differentially expressed circRNAs (DE-circRNAs) analysis showed that there are 44 DE-circRNAs at two different skeletal muscle growth stages. Six circRNAs were chosen randomly and their relative expression levels in juvenile and adult LMB were confirmed by real-time PCR, indicating that these circRNAs were existed authenticity. In addition, Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis showed that these hose genes (their linear mRNAs) of DE-circRNAs were mainly enriched in the regulation of actin cytoskeleton signaling pathways. The circRNA-miRNA interaction regulatory networks indicated that one circRNA can regulate one or more miRNA. For instance, more than 30 miRNAs were regulated by two circRNAs (circRNA389 and circRNA399). Of them, the muscle-related miRNAs including the let-7 family, miR-133 and miR-26 and so on were found acting as miRNAs sponge regulated by circRNAs, indicating the roles of circRNAs in regulating muscle growth-related genes expression. Overall, these findings will not only broaden our understanding of circRNAs regulation mechanisms underlying muscle growth and development in LMB but also provides a novel clue for further functional research in carnivorous fish genetic breeding.

hWJECM-Derived Oriented Scaffolds with Autologous Chondrocytes for Rabbit Cartilage Defect Repairing.

Previously, we synthesized an articular cartilage extracellular matrix (ECM)-derived oriented scaffold for cartilage tissue engineering, which was biomimetic in terms of structure and biochemical composition. However, the limit resource of the cartilage-derived ECM is a hindrance for its application. In this study, we developed a new material for cartilage tissue engineering-human umbilical cord Wharton's jelly-derived ECM (hWJECM). The hWJECM has an abundant resource and similar biochemistry with cartilage ECM, and the use of it is not associated with ethical controversy. We adopted the method previously used in cartilage ECM-derived oriented scaffold preparation to generate the oriented hWJECM-derived scaffold, and the scaffold properties were tested in vitro and in vivo. The three-dimensional scaffold has a porous and well-oriented structure, with a mean pore diameter of ∼104 µm. Scanning electron microscopy and cell viability staining results demonstrated that the oriented scaffold has good biocompatibility and cell alignment. In addition, we used functional autologous chondrocytes to seed the hWJECM-derived oriented scaffold and tested the efficacy of the cell-scaffold constructs to repair the full-thickness articular cartilage defect in a rabbit model. Defects of 4 mm diameter were generated in the patellar grooves of the femurs of both knees and were implanted with chondrocyte-scaffold constructs (group A) or scaffolds alone (group B); rabbits with untreated defects were used as a control (group C). Six months after surgery, all defects in group A were filled completely with repaired tissue, and most of which were hyaline cartilage. In contrast, the defects in group B were filled partially with repaired tissue, and approximately half of these repaired tissues were hyaline cartilage. The defects in group C were only filled with fibrotic tissue. Histological grading score of group A was lower than those of groups B and C. Quantification of glycosaminoglycan indicated that newly formed cartilage in group A rabbits was comparable with normal cartilage. In conclusion, hWJECM-derived oriented scaffolds loaded with autologous chondrocytes induced cartilage repair in rabbit knees, which was comparable with native cartilage in terms of macroscopic view, microstructure, and biochemical composition.

[Effects of residual paralysis after a single intubating dose of rocuronium on postoperative pulmonary function of patients undergoing laparoscopic gynecological surgeries].

OBJECTIVE: To evaluate the residual paralysis after a single intubating dose of rocuronium and its effect of residual paralysis after a single dose of rocuronium on the postoperative pulmonary function of patients undergoing laparoscopic gynecological surgeries. METHODS: Sixty American Society of Anesthesiologists (ASA) I - II patients undergoing laparoscopic gynecological surgeries were randomly divided into rocuronium (R) group (n = 30) and rocuronium + neostigmine (R + N) group (n = 30).All patients received midazolam (0.02 mg/kg), fentanyl (1 microg/kg), propofol(1.5-2 mg/kg), and rocuronium (0.6 mg/kg) to facilitate tracheal intubation and no more relaxant thereafter. Anesthesia was maintained with isoflurane and nitrous oxide in oxygen (N(2)O:O(2) = 1:1). At the end of the procedure, neuromuscular blockade was not reversed in R group, while antagonism was accomplished with neostigmine (0.04 mg/kg) and atropine (0.02 mg/kg) in R + N group. Immediately after tracheal extubation and on arrival in the PACU, the train-of-four (TOF) ratio at the adductor pollicis of all patients were measured using acceleromyography. Forced vital capacity (FVC), forced expiratory volume in one second (FEV(1)), and peak expiratory flow rate (PEFR) of all patients were measured using spirometry before surgery, after administration of midazolam and fentanyl, immediately after tracheal extubation, on arrival in the PACU, and after the TOF ratio recovered to 1.0. The TOF ratio and pulmonary function between two groups were compared. RESULTS: Immediately after tracheal extubation and on arrival in the PACU, the mean TOF ratio in R group was significantly lower than that in R + N group (P < 0.05). The mean time to achieve TOF ratio of 0.9 and 1.0 in R group was significantly longer than in R + N group (P < 0.05). Immediately after tracheal extubation and on arrival in the PACU, FVC, FEV(1), and PEFR were significantly lower in R group than in R + N group (P < 0.05). FVC, FEV(1), and PEFR after administration of midazolam and fentanyl and after TOF ratio recovered to 1.0 were significantly lower than the baseline values in all patients (P < 0.01). CONCLUSIONS: After a single intubating dose of rocuronium, residual paralysis exists in the majority of patients undergoing laparoscopic gynecological surgeries. The pulmonary function is impaired after the surgery, even after recovery of TOF ratio to 1.0.