Enhanced vitamin K2 production by engineered Bacillus subtilis during leakage fermentation.

Menaquinone-7 (MK-7), a valuable member of the vitamin K2 series, is an essential nutrient for humans. It is used for treating coagulation disorders, and osteoporosis, promoting liver function recovery, and preventing cardiovascular diseases. In this study, to further improve the metabolic synthesis of MK-7 by the mutant strain, the effect of surfactants on the metabolic synthesis of MK-7 by the mutant strain Bacillus subtilis 168 KO-SinR (BS168 KO-SinR) was analyzed. The scanning electron microscopy and flow cytometry results showed that the addition of surfactants changed the permeability of the cell membrane of the mutant strain and the structural components of the biofilm. When 0.7% Tween-80 was added into the medium, the extracellular and intracellular synthesis of MK-7 reached 28.8 mg/L and 59.2 mg/L, respectively, increasing the total synthesis of MK-7 by 80.3%. Quantitative real-time PCR showed that the addition of surfactant significantly increased the expression level of MK-7 synthesis-related genes, and the electron microscopy results showed that the addition of surfactant changed the permeability of the cell membrane. The research results of this paper can serve as a reference for the industrial development of MK-7 prepared by fermentation.

Advances in regulating vitamin K2 production through metabolic engineering strategies.

Vitamin K2 (menaquinone, VK2, MK) is an essential lipid-soluble vitamin that plays critical roles in inhibiting cell ferroptosis, improving blood clotting, and preventing osteoporosis. The increased global demand for VK2 has inspired interest in novel production strategies. In this review, various novel metabolic regulation strategies, including static and dynamic metabolic regulation, are summarized and discussed. Furthermore, the advantages and disadvantages of both strategies are analyzed in-depth to highlight the bottlenecks facing microbial VK2 production on an industrial scale. Finally, advanced metabolic engineering biotechnology for future microbial VK2 production will also be discussed. In summary, this review provides in-depth information and offers an outlook on metabolic engineering strategies for VK2 production.

First report of Rhizopus oryzae causing rhizome rot on ginger in China.

Ginger (Zingiber officinale Rosc.) is a herbal plant, widely grown in China for its medicinal and culinary purposes. In July 2020, a new rhizome rot disease was observed on ginger in Laiwu, Shandong Province, China. The disease symptoms were observed on both above-ground and underground plant parts. The above ground stems and leaves becoming withered and yellow, and water-soaked symptoms were observed on the collar region. The diseased rhizomes were poorly developed with brown lesion and eventually they would rot, without offensive odors. Disease incidence was estimated at approximately 5% across the survey area. To isolate the pathogen, tissues from 30 rhizomes were cut from the border between diseased and healthy tissue, surface sterilized in 75% alcohol for 15 s, soaked in 0.1% mercuric chloride for 1 min, washed with sterile distilled water three times, and plated on potato dextrose agar (PDA) at 25°C for 2-3 days. Twenty nine fungal isolates with similar morphological characteristics were obtained and pure cultures were obtained using single spore isolation. The colony of AQJ-1, a representative isolate, on PDA was cottony, fluffy, white, and beige coloration on the reverse side at first, and subsequently many black sporangia were produced. The sporangia were black, sub-globose, and 45.2-181.7 µm (n = 50) in diameter. The sporangiospores were unequal, globose or sub-globose, about 3.2-8.7 × 4.6-12.3µm (n = 50) in diameter. For the molecular characterization, genomic DNA was extracted by modified CTAB method (Niu et al., 2008). Internal transcribed spacer (ITS) region and translation elongation factor 1-alpha (EF-1α) gene were amplified using the primer pairs ITS1/ITS4 (White et al., 1990) and MEF10/MEF4 (Abe et al., 2007), respectively. The ITS and EF-1α sequences of isolate AQJ-1 were submitted to GenBank (MN606288 and MN735220, respectively). The BLASTn analysis of the sequences showed 99%-100% similarity to the sequences of R. oryzae strain CBS 120.12 (MH854609, AB281529, respectively). Therefore, based on morphological and molecular characteristics, isolate AQJ-1 was identified as R. oryzae. For pathogenicity tests, thirty ginger seedlings (Laiwu Big Ginger) were grown for 30 days in plastic pots and removed from the pots and the rhizomes washed in running tap water. The rhizomes of fifteen ginger seedlings were attached to a 7 mm agar disk from a plate containing 2-day-old mycelium, and the other fifteen seedlings were attached to agar disk without mycelium as control. Then the inoculated and control seedlings were planted in pots and were kept in separate chambers in a greenhouse at 25±2 °C. After 14 days, the same symptoms of rhizome rot were observed in all inoculated plants as previously described, and no symptoms were observed on the control plants. The pathogen was re-isolated from symptomatic tissues, and was identified as R. oryzae, which full-filled the Koch's postulates. To our knowledge, this is the first report of R. oryzae causing rhizome rot on ginger in China. This disease may pose a potential threat to ginger production in China.

Dynamic Changes in Colonic Structure and Protein Expression Suggest Regulatory Mechanisms of Colonic Barrier Function in Torpor-Arousal Cycles of the Daurian Ground Squirrel.

BACKGROUND: Both pathological conditions and hibernation can affect the barrier function of small intestine mucosa. However, the effect of hibernation on the barrier function of colonic mucosa remains unclear. METHODS: We investigated morphological changes in colonic mucosa, the concentrations of specific proteins and molecules, and the enzymatic activity of diamine oxidase (DAO), in serum and colonic tissue; the expression of tight junction proteins and mucin, and the changes in inflammatory, farnesoid X receptor (FXR)-small heterodimer partner (SHP), and apoptosis-related molecules that could play a role in gut permeability changes in Daurian ground squirrels in summer active (SA), late torpor (LT), and interbout arousal (IBA) periods. RESULTS: The results show that hibernation reduced the thickness of the colonic mucosa and the depth of the crypt, decreased the number of goblet cells (GCs), and damaged the structure of some microvilli. The concentrations of proteins and molecules, and the enzymatic activity of DAO, were all increased in the serum and colon, and the localization of tight junction proteins and mucin in the colonic mucosa were altered (compensatory response). Although the ground squirrels ate during the interbout arousal period, the changes remained similar to the response to torpor. Inflammation, apoptosis-anti-apoptosis, and FXR-SHP signaling may be involved in the possible changes in intestinal gut permeability during the torpor-arousal cycle in Daurian ground squirrels. In addition, periodic interbout arousal may play an inflammation-correcting role during the long hibernation season of Daurian ground squirrels.

Differential protein metabolism and regeneration in hypertrophic diaphragm and atrophic gastrocnemius muscles in hibernating Daurian ground squirrels.

NEW FINDINGS: What is the central question of this study? The aim was to investigate whether diaphragm hypertrophy and gastrocnemius atrophy during hibernation of Daurian ground squirrels involve differential regulation of protein metabolism and regeneration. What is the main finding and its importance? We clarified the differences in protein metabolism and muscle regenerative potential in the diaphragm and gastrocnemius of hibernating ground squirrels, reflecting the different adaptability of muscles. ABSTRACT: Are differences in the regulation of protein metabolism and regeneration involved in the different phenotypic adaptation mechanisms of muscle hypertrophy and atrophy in hibernators? Two fast-type muscles (diaphragm and gastrocnemius) in summer active and hibernating Daurian ground squirrels were selected to detect changes in cross-sectional area (CSA) and protein expression indicative of protein synthesis metabolism (protein expression of P-Akt, P-mTORC1, P-S6K1 and P-4E-BP1), protein degradation metabolism (MuRF1, atrogin-1, calpain-1, calpain-2, calpastatin, desmin, troponin T, Beclin1 and LC3-II) and muscle regeneration (MyoD, myogenin and myostatin). In the hibernation group compared with the summer active group, the CSA of the diaphragm muscle increased significantly by 26.1%, whereas the CSA of the gastrocnemius muscle decreased significantly by 20.4%. Our study also indicated that increased protein synthesis, decreased protein degradation and increased muscle regenerative potential contributed to diaphragm muscle hypertrophy, whereas decreased protein synthesis, increased protein degradation and decreased muscle regenerative potential contributed to gastrocnemius muscle atrophy. In conclusion, the differences in muscle regeneration and regulatory pattern of protein metabolism might contribute to the different adaptive changes observed in the diaphragm and gastrocnemius muscles of ground squirrels.

FgPrp4 Kinase Is Important for Spliceosome B-Complex Activation and Splicing Efficiency in Fusarium graminearum.

PRP4 encodes the only kinase among the spliceosome components. Although it is an essential gene in the fission yeast and other eukaryotic organisms, the Fgprp4 mutant was viable in the wheat scab fungus Fusarium graminearum. Deletion of FgPRP4 did not block intron splicing but affected intron splicing efficiency in over 60% of the F. graminearum genes. The Fgprp4 mutant had severe growth defects and produced spontaneous suppressors that were recovered in growth rate. Suppressor mutations were identified in the PRP6, PRP31, BRR2, and PRP8 orthologs in nine suppressor strains by sequencing analysis with candidate tri-snRNP component genes. The Q86K mutation in FgMSL1 was identified by whole genome sequencing in suppressor mutant S3. Whereas two of the suppressor mutations in FgBrr2 and FgPrp8 were similar to those characterized in their orthologs in yeasts, suppressor mutations in Prp6 and Prp31 orthologs or FgMSL1 have not been reported. Interestingly, four and two suppressor mutations identified in FgPrp6 and FgPrp31, respectively, all are near the conserved Prp4-phosphorylation sites, suggesting that these mutations may have similar effects with phosphorylation by Prp4 kinase. In FgPrp31, the non-sense mutation at R464 resulted in the truncation of the C-terminal 130 aa region that contains all the conserved Prp4-phosphorylation sites. Deletion analysis showed that the N-terminal 310-aa rich in SR residues plays a critical role in the localization and functions of FgPrp4. We also conducted phosphoproteomics analysis with FgPrp4 and identified S289 as the phosphorylation site that is essential for its functions. These results indicated that FgPrp4 is critical for splicing efficiency but not essential for intron splicing, and FgPrp4 may regulate pre-mRNA splicing by phosphorylation of other components of the tri-snRNP although itself may be activated by phosphorylation at S289.

Phosphorylation by Prp4 kinase releases the self-inhibition of FgPrp31 in Fusarium graminearum.

Prp31 is one of the key tri-snRNP components essential for pre-mRNA splicing although its exact molecular function is not well studied. In a previous study, suppressor mutations were identified in the PRP31 ortholog in two spontaneous suppressors of Fgprp4 mutant deleted of the only kinase of the spliceosome in Fusarium graminearum. To further characterize the function of FgPrp31 and its relationship with FgPrp4 kinase, in this study we identified additional suppressor mutations in FgPrp31 and determined the suppressive effects of selected mutations. In total, 28 of the 35 suppressors had missense or nonsense mutations in the C terminus 465-594 aa (CT130) region of FgPrp31. The other 7 had missense or deletion mutations in the 7-64 aa region. The nonsense mutation at R464 in FgPRP31 resulted in the truncation of CT130 that contains all the putative Prp4 kinase-phosphorylation sites reported in humans, and partially rescued intron splicing defects of Fgprp4. The CT130 of FgPrp31 displayed self-inhibitory interaction with the N-terminal 1-463 (N463) region, which was reduced or abolished by the L532P, D534G, or G529D mutation in yeast two-hybrid assays. The N463 region, but not full-length FgPrp31, interacted with the N-terminal region of FgBrr2, one main U5 snRNP protein. The L532P mutation in FgPrp31 increased its interaction with FgBrr2. In contrast, suppressor mutations in FgPrp31 reduced its interaction with FgPrp6, another key component of tri-snRNP. Furthermore, we showed that FgPrp31 was phosphorylated by FgPrp4 in vivo. Site-directed mutagenesis analysis showed that phosphorylation at multiple sites in FgPrp31 is necessary to suppress Fgprp4, and S520 and S521 are important FgPrp4-phosphorylation sites. Overall, these results indicated that phosphorylation by FgPrp4 at multiple sites may release the self-inhibitory binding of FgPrp31 and affect its interaction with other components of tri-snRNP during spliceosome activation.

The FgSRP1 SR-protein gene is important for plant infection and pre-mRNA processing in Fusarium graminearum.

The versatile functions of SR (serine/arginine-rich) proteins in pre-mRNA splicing and processing are modulated by reversible phosphorylation. Previous studies showed that FgPrp4, the only protein kinase among spliceosome components, is important for intron splicing and the FgSrp1 SR protein is phosphorylated at five conserved sites in Fusarium graminearum. In this study, we showed that the Fgsrp1 deletion mutant rarely produced conidia and caused only limited symptoms on wheat heads and corn silks. Deletion of FgSRP1 also reduced ascospore ejection and deoxynivalenol (DON) production. Interestingly, FgSRP1 had two transcript isoforms due to alternative splicing and both of them were required for its normal functions in growth and DON biosynthesis. FgSrp1 localized to the nucleus and interacted with FgPrp4 in vivo. Deletion of all four conserved phosphorylation sites but not individual ones affected the FgSRP1 function, suggesting their overlapping functions. RNA-seq analysis showed that the expression of over thousands of genes and splicing efficiency in over 140 introns were affected. Taken together, FgSRP1 is important for conidiation, and pathogenesis and alternative splicing is important for its normal functions. The FgSrp1 SR protein is likely important for pre-mRNA processing or splicing of various genes in different developmental and infection processes.

The phospholipase C (FgPLC1) is involved in regulation of development, pathogenicity, and stress responses in Fusarium graminearum.

Phospholipase C (PLC) is an important phospholipid hydrolase that plays critical roles in various biological processes in eukaryotic cells. To elucidate the functions of PLC in morphogenesis and pathogenesis in Fusarium graminearum, deletion mutants were constructed of all six FgPLC genes identified in this study. Deletion of FgPLC1, but not the other five FgPLC genes, affected hyphal growth and conidiation. The FgPLC1 deletion mutant (Δplc1) also was defective in conidium germination and germ tube growth. It was sterile in selfing crosses and had increased sensitivities to hyperosmotic and cell wall stresses. The Δplc1 mutant showed reduced DON production and virulence during infection in flowering wheat heads. Deletion of FgPLC1 decreased the phosphorylation levels of both Gpmk1 and Mgv1 MAP kinases. qRT-PCR analysis showed that several genes related to defective phenotypes were down-regulated in the Δplc1 mutant. Taken together, these results indicated that FgPLC1 is important for hyphal growth, plant infection, and sexual or asexual reproduction, and it may be functionally related to MAP kinases in F. graminearum.

Functional analysis of the kinome of the wheat scab fungus Fusarium graminearum.

As in other eukaryotes, protein kinases play major regulatory roles in filamentous fungi. Although the genomes of many plant pathogenic fungi have been sequenced, systematic characterization of their kinomes has not been reported. The wheat scab fungus Fusarium graminearum has 116 protein kinases (PK) genes. Although twenty of them appeared to be essential, we generated deletion mutants for the other 96 PK genes, including 12 orthologs of essential genes in yeast. All of the PK mutants were assayed for changes in 17 phenotypes, including growth, conidiation, pathogenesis, stress responses, and sexual reproduction. Overall, deletion of 64 PK genes resulted in at least one of the phenotypes examined, including three mutants blocked in conidiation and five mutants with increased tolerance to hyperosmotic stress. In total, 42 PK mutants were significantly reduced in virulence or non-pathogenic, including mutants deleted of key components of the cAMP signaling and three MAPK pathways. A number of these PK genes, including Fg03146 and Fg04770 that are unique to filamentous fungi, are dispensable for hyphal growth and likely encode novel fungal virulence factors. Ascospores play a critical role in the initiation of wheat scab. Twenty-six PK mutants were blocked in perithecia formation or aborted in ascosporogenesis. Additional 19 mutants were defective in ascospore release or morphology. Interestingly, F. graminearum contains two aurora kinase genes with distinct functions, which has not been reported in fungi. In addition, we used the interlog approach to predict the PK-PK and PK-protein interaction networks of F. graminearum. Several predicted interactions were verified with yeast two-hybrid or co-immunoprecipitation assays. To our knowledge, this is the first functional characterization of the kinome in plant pathogenic fungi. Protein kinase genes important for various aspects of growth, developmental, and infection processes in F. graminearum were identified in this study.

Differential bone remodeling mechanism in hindlimb unloaded and hibernating Daurian ground squirrels: a comparison between artificial and natural disuse within the same species.

Loss of bone mass can occur in mammals after prolonged disuse but the situation for hibernators that are in a state of torpor for many months of the year is not yet fully understood. The present study assesses the bone remodeling mechanisms present in Daurian ground squirrels (Spermophilus dauricus) during hibernation as compared with a model of hindlimb disuse. Differences in microstructure, mechanical properties, bone remodeling-related proteins (Runx2, OCN, ALP, RANKL, CTK and MMP-9) and key proteins of Wnt/ß-catenin signaling pathway (GSK-3ß and phospho-ß-catenin) were evaluated in ground squirrels under 3 conditions: summer active (SA) vs. hibernation (HIB) vs. hindlimb unloaded (HLU). The results indicated that the body weight in HLU ground squirrels was lower than the SA group, and the middle tibia diameter in the HLU group was lower than that in SA and HIB groups. The thickness of cortical and trabecular bone in femurs from HLU ground squirrels was lower than in SA and HIB groups. Most parameters of the tibia in the HLU group were lower than those in SA and HIB groups, which indicated cortical bone loss in ground squirrels. Moreover, our data showed that the changes in microscopic parameters in the femur were more obvious than those in the tibia in HLU and HIB ground squirrels. The levels of Runx2 and ALP were lower in HLU ground squirrels than SA and HIB groups. The protein levels of OCN were unchanged in the three groups, but the protein levels of ALP were lower in the HLU group than in SA and HIB groups. RANKL, CTK and MMP-9 protein levels were significantly decreased in tibia of HLU ground squirrels as compared with SA and HIB groups. In addition, the protein expression levels of RANKL, CTK and MMP-9 showed no statistical difference between SA and HIB ground squirrels. Thus, the mechanisms involved in the balance between bone formation and resorption in hibernating and hindlimb unloading ground squirrels may be different. The present study showed that in femur, the Wnt signaling pathway was inhibited, the protein level of GSK-3ß was increased, and the protein expression of phospho-ß-catenin was decreased in the HIB group as compared with the SA group, which indicates that the Wnt signaling pathway has a great influence on the femur of the HIB group. In conclusion, the natural anti-osteoporosis properties of Daurian ground squirrels are seasonal. The squirrels do not experience bone loss when they are inactive for a long time during hibernation, but the mechanisms of anti-osteoporosis did not work in HLU summer active squirrels.

Biodegradable polymer-curcumin conjugate micelles enhance the loading and delivery of low-potency curcumin.

PURPOSE: To utilize a novel type of polymer-drug conjugate micelle to enhance the delivery of low-potency curcumin. METHODS: Multiple curcumin molecules were conjugated to poly(lactic acid) (PLA) via tris(hydroxymethyl)aminomethane (Tris) linker producing the hydrophobic drug-binding block; methoxy-poly(ethylene glycol) (mPEG) was employed as the hydrophilic block. Micelles were characterized by size, loading capacity, stability, and critical micelle concentration (CMC). Human hepatocellular carcinoma (HepG2) cells were employed to assess cytotoxicity and intracellular targeting ability of micelles. RESULTS: mPEG-PLA-Tris-Cur micelles were within nanorange (<100 nm). CMC of such micelles (2.3 ± 0.4 µg/mL) was 10 times lower than mPEG-PLA micelles (27.4 ± 0.8 µg/mL). Curcumin loading in mPEG-PLA-Tris-Cur micelles reached 18.5 ± 1.3% (w/w), compared to traditional mPEG-PLA micelles at 3.6 ± 0.4% (w/w). IC(50) of mPEG-PLA-Tris-Cur micelles (~22 µg/mL at curcumin-equivalent dose) was similar to unmodified curcumin. Placebo and drug-encapsulated conjugate micelles could be efficiently internalized to cytoplasmic compartment of HepG2 cells. CONCLUSIONS: Micelle-forming polymer-drug conjugates containing multiple drug molecules were an efficient means to increase loading and intracellular delivery of low-potency curcumin.

Differential bone metabolism and protein expression in mice fed a high-fat diet versus Daurian ground squirrels following natural pre-hibernation fattening.

This study compared the effects on bone metabolism and morphology of pathological obesity induced by excessive fat intake in a non-hibernator (mice) versus healthy obesity due to pre-hibernation fattening in a hibernator (ground squirrels). Kunming mice were fed a high-fat diet to provide a model of pathological obesity (OB group). Daurian ground squirrels fattened naturally in their pre-hibernation season (PRE group) were used as a healthy obesity model. Micro-computed tomography (micro-CT) and three-point bending tests were used to determine the microstructure and mechanical properties of bone. Western blots were used to analyze protein expression levels related to bone metabolism (Runt-related transcription factor 2 (RunX2), osteocalcin (OCN), alkaline phosphatase (ALP), osteoprotegerin (OPG), receptor activator of nuclear factor-|κB ligand (RANKL), cathepsin K, matrix metallopeptidase 9 (MMP9), patched protein homolog 1 (Ptch1), phosphorylated ß|-|catenin (P|-|ß|-|catenin), and glycogen synthase kinase-3ß (GSK-3ß)). Compared with controls, there was no obvious bone loss in the OB mice, and the stiffness of the femur was increased significantly. Compared with summer active squirrels, bone formation was enhanced but the mechanical properties did not change in the PRE group squirrels. In OB mice, western blots showed significantly increased expression levels of all proteins except RunX2, OPG, and Ptch1. PRE ground squirrels showed significantly increased expression of most proteins except OCN and Ptch1, which decreased significantly, and P|-|ß|-|catenin and OPG, which did not change. In conclusion, for non-hibernating mice, moderate obesity had a certain protective effect on bones, demonstrating two-way regulation, increasing both bone loss and bone formation. For pre-hibernating ground squirrels, the healthy obesity acquired before hibernation had a positive effect on the microstructure of bones, and also enhanced the expression levels of proteins related to bone formation, bone resorption, and Wnt signaling.

Poly-γ-Glutamic Acid Production by Engineering a DegU Quorum-Sensing Circuit in Bacillus subtilis.

As a natural biological macromolecule, γ-polyglutamic acid (γ-PGA) plays a significant role in medicine, food, and cosmetic industries owing to its unique properties of biocompatibility, biodegradability, water solubility, and viscosity. Although many strategies have been adopted to increase the yield of γ-PGA in Bacillus subtilis, the effectiveness of these common approaches is not high because the strong viscosity affects cell growth. However, dynamic regulation based on quorum sensing (QS) has been extensively applied as a fundamental tool for fine-tuning gene expression in reaction to changes in cell density without adding expensive inducers. A modular PhrQ-RapQ-DegU QS system is developed based on promoter PD4, which is upregulated by phosphorylated DegU (DegU-P). In this study, first, we analyzed the DegU-based gene expression regulation system in B. subtilis 168. We constructed a promoter library of different abilities, selected suitable promoters from the library, and performed mutation screening on the selected promoters and degU region. Furthermore, we constructed a PhrQ-RapQ-DegU QS system to dynamically control the synthesis of γ-PGA in BS168. Cell growth and efficient synthesis of the target product can be dynamically balanced by the QS system. Our dynamic adjustment approach increased the yield of γ-PGA to 6.53-fold of that by static regulation in a 3 L bioreactor, which verified the effectiveness of this strategy. In summary, the PhrQ-RapQ-DegU QS system has been successfully integrated with biocatalytic functions to achieve dynamic metabolic pathway control in BS168, which can be stretched to a large number of microorganisms to fine-tune gene expression and enhance the production of metabolites.

Differential bone remodeling mechanism in hindlimb unloaded rats and hibernating Daurian ground squirrels: a comparison between artificial and natural disuse.

To determine that differential bone remodeling mechanism (especially Wnt signaling) in hindlimb unloaded rats and hibernating Daurian ground squirrels, the bone microstructure, mechanical properties, and expression levels of bone remodeling related proteins and key proteins of Wnt/ß-catenin signaling were analyzed in this study. The thickness of cortical and trabecular bone was decreased in femur of hindlimb unloaded rats, while it was maintained in femur of hibernating ground squirrels. Interestingly, the ultimate bending energy and ultimate normalized displacement were reduced and the bending rigidity was increased in tibia of hibernating ground squirrels. Besides, the protein level of Runx2 was decreased in femur and tibia of unloaded rats, while it was maintained in tibia and even increased in femur of hibernating ground squirrels. The protein levels of RANKL and MMP-9 were increased in femur and tibia in unloaded rats, while they were maintained in both femur and tibia of hibernating ground squirrels. The protein level of GSK-3ß was increased in femur and tibia of unloaded rats, while it was maintained in both femur and tibia of hibernating ground squirrels. The phospho-ß-catenin expression was increased in both femur and tibia of unloaded rats, while it was only decreased in femur, but maintained in tibia of hibernating ground squirrels. In conclusion, the femur and tibia in hindlimb unloaded rats showed obvious bone loss, while they mitigated disuse-induced bone loss in hibernating ground squirrels, involving differential protein expression of key molecules in bone remodeling. In comparison with hindlimb unloaded rats, promoting osteoblast differentiation through activating canonical GSK-3ß/ß-catenin signaling involving Runx2 might be an adaptation to natural disuse in femur of hibernating Daurian ground squirrels. However, there was no statistical change in the protein levels of bone formation related proteins, GSK-3ß and phospho-ß-catenin in tibia of hibernating Daurian ground squirrels.

Differential Protein Metabolism and Regeneration in Gastrocnemius Muscles in High-fat Diet Fed Mice and Pre-hibernation Daurian Ground Squirrels: A Comparison between Pathological and Healthy Obesity.

We focused on pathological obesity induced by excessive fat intake (nutritional obesity) in non-hibernator and healthy obesity due to pre-hibernation (PRE) fat storage in hibernator to study the effects of different types of obesity on skeletal muscle protein metabolism and cell regeneration. Kunming mice were fed with high-fat diet for 3 months to construct a pathological obesity model. Daurian ground squirrels fattened naturally before hibernation were used as a healthy obesity model. Body weight, adipose tissue wet weight, gastrocnemius muscle wet weight, muscle fiber cross-sectional area (CSA) and fiber type distribution were measured. The protein expression levels related to protein degradation (MuRF-1, atrogin-1, calpain1, calpain2, calpastatin, desmin, troponin T, Beclin-1, LC3-II), protein synthesis (P-Akt, P-mTORC1, P-S6K1, P-4E-BP1) and cell regeneration (MyoD, myogenin, myostatin) were detected by Western blot. As a result, the body weight and adipose tissue wet weight were both significantly increased in high fat obese (OB) mice and pre-hibernation fat (PRE) ground squirrels. The muscle wet weight, ratio of muscle wet weight to body weight, and muscle fiber CSA were significantly decreased, while the percentage of MHC I fiber isoform was significantly increased in gastrocnemius muscle of OB mice compared with the control (CON) group. The protein expression levels of P-Akt, P-mTORC1, P-4E-BP1 and myogenin were significantly decreased, while those of calpain1, calpain2, MuRF-1 and myostatin were significantly increased in the OB mice. In the ground squirrels, the muscle wet weight, muscle fiber CSA and percentage of MHC I fiber isoform all showed no change in the gastrocnemius muscle in the PRE group compared with the summer active (SA) group. The protein expression levels of P-Akt, P-mTORC1, P-S6K1 and MyoD were significantly increased, while those of Beclin-1 and LC3-II were significantly decreased in the PRE ground squirrels. This study demonstrated that the decrease in protein expression levels in the Akt/mTOR pathway (P-Akt, P-mTORC1 and P-4E-BP1) and cell regeneration (myogenin) and the increase in protein expression levels of the calpain pathway (calpain1 and calpain2) and ubiquitin-proteasome pathway (MuRF-1) were involved in the mechanism of muscle atrophy in gastrocnemius muscle of the pathologically obese Kunming mice induced by high-fat diet. In contrast, the increased protein expression levels of the Akt/mTOR pathway (P-Akt, P-mTORC1 and P-S6K1) and cell regeneration (MyoD), and the decreased protein expression levels of the autophagy lysosomal pathway (Beclin-1 and LC3-II) were involved in the mechanism of anti-atrophy in gastrocnemius muscle of the healthy obese ground squirrels fattened before hibernation.

Genome-wide characterization, evolutionary analysis of WRKY genes in Cucurbitaceae species and assessment of its roles in resisting to powdery mildew disease.

The WRKY proteins constitute a large family of transcription factors that have been known to play a wide range of regulatory roles in multiple biological processes. Over the past few years, many reports have focused on analysis of evolution and biological function of WRKY genes at the whole genome level in different plant species. However, little information is known about WRKY genes in melon (Cucumis melo L.). In the present study, a total of 56 putative WRKY genes were identified in melon, which were randomly distributed on their respective chromosomes. A multiple sequence alignment and phylogenetic analysis using melon, cucumber and watermelon predicted WRKY domains indicated that melon WRKY proteins could be classified into three main groups (I-III). Our analysis indicated that no recent duplication events of WRKY genes were detected in melon, and strong purifying selection was observed among the 85 orthologous pairs of Cucurbitaceae species. Expression profiles of CmWRKY derived from RNA-seq data and quantitative RT-PCR (qRT-PCR) analyses showed distinct expression patterns in various tissues, and the expression of 16 CmWRKY were altered following powdery mildew infection in melon. Besides, we also found that a total of 24 WRKY genes were co-expressed with 11 VQ family genes in melon. Our comparative genomic analysis provides a foundation for future functional dissection and understanding the evolution of WRKY genes in cucurbitaceae species, and will promote powdery mildew resistance study in melon.