Microalgae possess high photosynthetic efficiency, robust adaptability, and substantial biomass, serving as excellent biological resources for large-scale cultivation. Malic enzyme (ME), a ubiquitous metabolic enzyme in living organisms, catalyzes the decarboxylation of malate to produce pyruvate, CO₂, and NAD(P)H, playing a role in multiple metabolic pathways including energy metabolism, photosynthesis, respiration, and biosynthesis. In this study, we identified the Scenedesmus quadricauda malic enzyme gene (SqME) and its biological functions, aiming to provide excellent target genes for the genetic improvement of higher plants. Based on the RNA-seq data from S. quadricauda under the biofilm cultivation mode with high CO₂ and light energy transfer efficiency and small water use, a highly expressed gene (SqME) functionally annotated as ME was cloned. The physicochemical properties of the SqME-encoded protein were systematically analyzed by bioinformatics tools. The subcellular localization of SqME was determined via transient transformation in Nicotiana benthamiana leaves. The biological functions of SqME were identified via genetic transformation in Nicotiana tabacum, and the potential of SqME in the genetic improvement of higher plants was evaluated. The ORF of SqME was 1 770 bp, encoding 590 amino acid residues, and the encoded protein was located in chloroplasts. SqME was a NADP-ME, with the typical structural characteristics of ME. The ME activity in the transgenic N. tabacum plant was 1.8 folds of that in the wild-type control. Heterologous expression of SqME increased the content of chlorophyll a, chlorophyll b, and total chlorophyll by 20.9%, 26.9%, and 25.2%, respectively, compared with the control. The transgenic tobacco leaves showed an increase of 54.0% in the fluorescence parameter NPQ and a decrease of 30.1% in Fo compared with the control. Moreover, the biomass, total lipids, and soluble sugars in the transgenic tobacco leaves enhanced by 20.5%, 25.7%, and 9.5%, respectively. On the contrary, the starch and protein content in the transgenic tobacco leaves decreased by 22.4% and 12.2%, respectively. Collectively, the SqME-encoded protein exhibited a strong enzymatic activity. Heterologous expressing of SqME could significantly enhance photosynthetic protection, photosynthesis, and biomass accumulation in the host. Additionally, SqME can facilitate carbon metabolism remodeling in the host, driving more carbon flux towards lipid synthesis. Therefore, SqME can be applied in the genetic improvement of higher plants for enhancing photosynthetic carbon fixation and lipid accumulation. These findings provide scientific references for mining of functional genes from S. quadricauda and application of these genes in the genetic engineering of higher plants.
Nicotiana/genetics* ; Photosynthesis/physiology* ; Malate Dehydrogenase/biosynthesis* ; Plant Leaves/genetics* ; Scenedesmus/enzymology* ; Carbon Cycle/genetics* ; Lipid Metabolism/genetics* ; Plants, Genetically Modified/metabolism*

In order to investigate the molecular mechanism of silk/threonine protein kinase (STK)-mediated blue light response in the algal Chlamydomonas reinhardtii, phenotype identification and transcriptome analysis were conducted for C. reinhardtii STK mutant strain crstk11 (with an AphvIII box reverse insertion in stk11 gene coding region) under blue light stress. Phenotypic examination showed that under normal light (white light), there was a slight difference in growth and pigment contents between the wild-type strain CC5325 and the mutant strain crstk11. Blue light inhibited the growth and chlorophyll synthesis in crstk11 cells, but significantly promoted the accumulation of carotenoids in crstk11. Transcriptome analysis showed that 860 differential expression genes (DEG) (559 up-regulated and 301 down-regulated) were detected in mutant (STK4) vs. wild type (WT4) upon treatment under high intensity blue light for 4 days. After being treated under high intensity blue light for 8 days, a total of 1 088 DEGs (468 upregulated and 620 downregulated) were obtained in STK8 vs. WT8. KEGG enrichment analysis revealed that compared to CC5325, the crstk11 blue light responsive genes were mainly involved in catalytic activity of intracellular photosynthesis, carbon metabolism, and pigment synthesis. Among them, upregulated genes included psaA, psaB, and psaC, psbA, psbB, psbC, psbD, psbH, and L, petA, petB, and petD, as well as genes encoding ATP synthase α, β and c subunits. Downregulated genes included petF and petJ. The present study uncovered that the protein kinase CrSTK11 of C. reinhardtii may participate in the blue light response of algal cells by mediating photosynthesis as well as pigment and carbon metabolism, providing new knowledge for in-depth analysis of the mechanism of light stress resistance in the algae.
Chlamydomonas reinhardtii/genetics* ; Photosynthesis/genetics* ; Plants/metabolism* ; Protein Kinases ; Threonine/metabolism* ; Carbon/metabolism* ; Serine/metabolism*

Purpose: Mixed-lineage leukemia protein 4 (MLL4/KMT2D) is a histone methyltransferase, and its mutation has been reported to be associated with a poor prognosis in many cancers, including lung cancer. We investigated the function of MLL4 in lung carcinogenesis. Materials and Methods: RNA sequencing (RNA-seq) in A549 cells transfected with control siRNA or MLL4 siRNA was performed. Also, we used EdU incorporation assay, colony formation assays, growth curve analysis, transwell invasion assays, immunohistochemical staining, and in vivo bioluminescence assay to investigate the function of MLL4 in lung carcinogenesis. Results: We found that MLL4 expression was downregulated in non–small cell lung cancer (NSCLC) tissues compared to adjacent normal tissues and tended to decrease with disease stage progression. We analyzed the transcriptomes in control and MLL4- deficient cells using high-throughput RNA deep sequencing (RNA-seq) and identified a cohort of target genes, such as SOX2, ATF1, FOXP4, PIK3IP1, SIRT4, TENT5B, and LFNG, some of which are related to proliferation and metastasis. Our results showed that low expression of MLL4 promotes NSCLC cell proliferation and metastasis and is required for the maintenance of NSCLC stem cell properties. Conclusion Our findings identify an important role of MLL4 in lung carcinogenesis through transcriptional regulation of PIK3IP1, affecting the PI3K/AKT/SOX2 axis, and suggest that MLL4 could be a potential prognostic indicator and target for NSCLC therapy.

Objective To explore the effectiveness of the Five Elements Music Therapy(FEMT)in relieving stress in participants with different cultural backgrounds,and to compare the differences between the FEMT and the Western Art Music Therapy(WAMT)in stress relief.Methods This was a comparative pilot randomized pre-post repeated measures study,37 subjects were allocated with 2 dropped out,with an inclusion-ended sample of 35 subjects,23 from Canada and 12 from China.After informed consent,all subjects were randomly assigned to listening to either Five Elements Music or Western Art Music at home for 30 minutes,twice a week for four weeks.Participants were asked to use headphones,measure their pulse rate before and after each session,and fill out five questionnaires,including a background and demographic survey(reporting age,gender,education,cultural background,listening experience).Self-assessment of stress(pre-post after each session),General Hospital Anxiety/Depression Scale(HADS,weekly after 2nd session),Perceived Stress Survey(PSS,weekly after 2nd session),the Music Therapy Intervention Survey(MTIS,pre-post each session).Results ①There was a significant decrease in self-assessed stress scores after the second session in the FEMT compared with the WAMT group(t=-2.057,P=0.046).②In both groups,there was a significant decrease in stress scores pre-post treatment in each group(WAMT t=5.026;FEMT T=7.645,P=0.000).③There was no significant difference between the two groups in post-intervention HADS scores(P>0.05);In the Chinese sample,there was a significant difference in HADS scores after the eighth session in both FEMT and WAMT groups(t=-3.862,P=0.003),and a statistically significant difference in HADS pre-post intervention in the FEMT((t=5.117,P=0.004).There was a significant difference in MTIS pre-post treatment in the WAMT(t=-2.572,P=0.023),but in not the FEMT group(t= 1.331,P=0.207).Conclusion This pilot trial explores a safe and feasible self-administered music therapy approach for stress in two distinct cultural groups,and for the first time provides preliminary comparison and evidence of effectiveness of FEMT and WAMT in alleviating stress and anxiety.Further investigation with bigger randomized samples is needed to elucidate the effects of different kinds of music and cultural groups on stress and anxiety levels.

WRKY is a superfamily of plant-specific transcription factors, playing a critical regulatory role in multiple biological processes such as plant growth and development, metabolism, and responses to biotic and abiotic stresses. Although WRKY genes have been characterized in a variety of higher plants, little is known about them in eukaryotic algae, which are close to higher plants in evolution. To fully characterize algal WRKY family members, we carried out multiple sequence alignment, phylogenetic analysis, and conserved domain prediction to identify the WRKY genes in the genomes of 30 algal species. A total of 24 WRKY members were identified in Chlorophyta, whereas no WRKY member was detected in Rhodophyta, Glaucophyta, or Bacillariophyta. The 24 WRKY members were classified into Ⅰ, Ⅱa, Ⅱb and R groups, with a conserved heptapeptide domain WRKYGQ(E/A/H/N)K and a zinc finger motif C-X4-5-C-X22-23-H-X-H. Haematococcus pluvialis, a high producer of natural astaxanthin, contained two WRKY members (HaeWRKY-1 and HaeWRKY-2). Furthermore, the coding sequences of HaeWRKY-1 and HaeWRKY-2 genes were cloned and then inserted into prokaryotic expression vector. The recombinant vectors were induced to express in Escherichia coli BL21(DE3) cells and the fusion proteins were purified by Ni-NTA affinity chromatography. HaeWRKY-1 had significantly higher expression level than HaeWRKY-2 in H. pluvialis cultured under normal conditions. High light stress significantly up-regulated the expression of HaeWRKY-1 while down-regulated that of HaeWRKY-2. The promoters of HaeWRKY genes contained multiple cis-elements responsive to light, ethylene, ABA, and stresses. Particularly, the promoter of HaeWRKY-2 contained no W-box specific for WRKY binding. However, the W-box was detected in the promoters of HaeWRKY-1 and the key enzyme genes HaeBKT (β-carotene ketolase) and HaePSY (phytoene synthase) responsible for astaxanthin biosynthesis. Considering these findings and the research progress in the related fields, we hypothesized that the low expression of HaeWRKY-2 under high light stress may lead to the up-regulation of HaeWRKY-1 expression. HaeWRKY-1 may then up-regulate the expression of the key genes (HaeBKT, HaePSY, etc.) for astaxanthin biosynthesis, consequently promoting astaxanthin enrichment in algal cells. The findings provide new insights into further analysis of the regulatory mechanism of astaxanthin biosynthesis and high light stress response of H. pluvialis.
Eukaryota ; Gene Expression Regulation, Plant ; Phylogeny ; Plant Proteins/metabolism* ; Plants/metabolism* ; Stress, Physiological/genetics* ; Transcription Factors/metabolism*

Objective:To explore the efficacy of a flat ground exoskeleton robot in improving the walking ability of stroke survivors.Methods:Fifty-eight stroke survivors with mobility difficulties were randomly divided into a robot group ( n=29) and a control group ( n=29). In addition to routine rehabilitation, the control group received conventional walking training, while the robot group underwent exoskeleton robot-assisted gait training. The 30-minute training sessions were held twice a day, 5 days per week for 5 weeks. Before as well as after 2 and 4 weeks of treatment, everyone′s walking ability was tested using the 6-minute walk test (6MWT) and functional ambulation scale (FAC). General lower limb motor function was quantified using the Fugl-Meyer Lower Extremity assessment (FMA-LE). Moreover, gait analysis was conducted before and after 4 weeks of treatment. Results:After 2 and 4 weeks of treatment, the average 6MWT times of both groups were significantly better than before the treatment, with the improvement of the robot group significantly greater than that of the control group after 2 weeks. After 2 and 4 weeks the average FMA-LE and FAC scores of both groups had improved significantly compared with before treatment. After 4 weeks the stride frequency and gait cycle of both groups had improved significantly.Conclusions:Exoskeleton robot-assisted gait training can improve walking ability and lower limb motor function of stroke survivors about as well as conventional walking training.

The unicellular green alga Haematococcus pluvialis is the best source of natural astaxanthin (AST) in the world due to its high content under stress conditions. Although high light (HL) can effectively induce AST biosynthesis, the specific mechanisms of light signal perception and transduction are unclear. In the current study, we used transcriptomic data of normal (N), high white light (W), and high blue light (B) to study the mechanisms of light inducing AST accumulation from the point of photoreceptors. The original data of 4.0 G, 3.8 G, and 3.6 G for N, W, and B were obtained, respectively, by the Illumina Hi-seq 2000 sequencing technology. Totally, 51 954 unigenes (at least 200 bp in length) were generated, of which, 20 537 unigenes were annotated into at least one database (NR, NT, KO, SwissProt, Pfam, GO, or KOG). There were 1 255 DEGs in the W vs N, 1 494 DEGs in the B vs N, and 1 008 DEGs in the both W vs N and B vs N. KEGG enrichment analysis revealed that photosynthesis, oxidative phosphorylation, carotenoid biosynthesis, fatty acids biosynthesis, DNA replication, nitrogen metabolism, and carbon metabolism were the significantly enriched pathways. Moreover, a large number of genes encoding photoreceptors and predicted interacting proteins were predicted in Haematococcus transcriptome data. These genes showed significant differences at transcriptional expression levels. In addition, 15 related DEGs were selected and tested by qRT-PCR and the results were significantly correlated with the transcriptome data. The above results indicate that the signal transduction pathway of "light signal - photoreceptors - interaction proteins - (interaction proteins - transcription factor/transcriptional regulator) - gene expression - AST accumulation" might play important roles in the regulation process, and provide reference for further understanding the transcriptional regulation mechanisms of AST accumulation under HL stress.
Chlorophyta/genetics* ; Gene Expression Profiling ; Signal Transduction/genetics* ; Transcriptome/genetics* ; Xanthophylls

Objective To report our efficacy of one-stage definite treatment of Gustilo type Ⅲ open fractures of long bone by plate osteosynthesis.Methods A retrospective case series study was conducted of the 69 cases who had undergone plate osteosynthesis for one-stage definite treatment of Gustilo type Ⅲ open fractures of long bone from January 2006 to June 2016 at Microsurgery and War Trauma Center of Chengdu Military Command,59 Hospital of Chinese PLA.They were 47 males and 22 females with an average age of 34.2 years (from 2 to 62 years).There were 27 shaft fractures of tibia or fibula (13 cases of type ⅢA,12 cases of type Ⅲ B and 2 cases of type Ⅲ C),4 fractures of distal tibia (2 cases of type Ⅲ A and 2 cases of type ⅢB),14 shaft fractures of ulna or radius (9 cases of type ⅢA,3 cases of type ⅢB and 2 cases of type Ⅲ C),12 factures of humeral shaft (7 cases of type Ⅲ A,3 cases of type Ⅲ B and 3 cases of type Ⅲ C),3 fractures of distal humerus (all type ⅢC),6 fractures of femoral shaft (5 cases of type ⅢA and one type Ⅲ C),and 3 fractures of distal femur (2 cases of type ⅢA and one type ⅢC).The intervals between injury and operation ranged from 4 to 17 hours,averaging 9.6 hours.After thorough debridement,osteosynthesis was performed with locking compression plate,limited contact dynamic compression plate or/and reconstruction locking plate,or 1/3 tubular plate.Direct closure with decreased tension or without tension was used for type Ⅲ A injury;deep open defects were repaired with perforator flaps,neurovascular axis flaps,traditional axis flaps and muscular flaps,or local flaps;limb reconstructions included neurovascular repair in 12 cases,tendon and ligament repair in 5 cases,and muscle reconstruction in 3 cases.Superficial defects were covered by skin grafts simultaneously or secondarily.Results The duration of hospitalization averaged 19 days (from 5 to 37 days).Partial necrosis occurred in one case of sural neurovascular axis flap.Superficial infection with multiple antibiotic-resistant bacteria occurred in 2 cases.Follow-up for the 69 patients ranged from 12 to 27 months (average,19.2 months).No deep bone infection occurred.Implant breaking occurred in 4 cases and implant loosening in one.The implant failures were corrected by change into intramedullary nails or plate refixation (respectively in 2 cases) in addition to bone graft.Bone union was achieved after 5 to 15 months (average,7.7 month)with satisfactory aesthetic and functional outcomes.Conclusion For patients with Gustilo type Ⅲ open fracture of long bone,especially those with metaphyseal,intraarticular or upper limb fracture and pediatric ones,plate osteosynthesis can be a satisfactory one-stage definite treatment besides intramedullar nailing and external fixation,providing that through debridement and satisfactory soft-tissue coverage can be achieved.

Objective To investigate midkine (MK) level in serum of patients with esophageal squamous cell carcinoma (ESCC) before and after operation,detect the expression of MK protein in tissues and analyze its relationship with the malignant biological behavior of ESCC.Methods The MK levels in serum were detected by enzyme-linked immunosorbent assay in 30 healthy cases and 55 patients with ESCC when the day before operation and the tenth-day after operation.Then the expression of MK protein in 55 patients surgically removed ESCC were detected by immunohistochemistry using monoclonal antibodies against human MK.Results The average optical density of MK in serum of ESCC patients before operation was 0.1006±0.0624,0.0455±0.0155 in normal control group,0.0752±0.0267 in postoperative control group (t =6.203,P ＜ 0.001).The MK levels in serum of ESCC patients before operation were significantly higher than those after operation (t =4.357,P ＜ 0.001) and those in healthy controls (P ＜ 0.05).The expression of MK protein was no statistically significant correlation with tumor size (x2 =0.131),TNM stage (x2 =0.315) and lymph node metastasis (x2 =0.282) in ESCC (all P ＞ 0.05).But significant correlation was noted between the expression of MK protein and the vasculature involving of the cutting edge (x2 =4.223),degree of cellular differentiation (x2 =10.326),invasive extent of the carcinoma (x2 =20.556) (all P ＜ 0.05).Conclusion MK is overexpressed in ESCC patient' s serum and tissue.The high level of preoperative serum MK is caused by the tumor.

Objective To study the proliferation inhibition effect by silencing PLCε gene expression with RNA interference in BIU-87 cells. Methods The specific short hairpin RNA recombinant plasmids were constructed by gene clone technology.The expression level of PLCε protein and mRNA were detected by Western blot and RT-PCR respectively after transfected recombinant plasmids into BIU-87 cells.The influence on proliferation was check by MTT.The changes of proliferating cell nuclear antigen(PCNA)were analyzed by immunocytochemical method,and the distribution of cell cycle was analyzed using flow cytometry. Results After transfected with the specific recombinant plasmids,PCNA expression was decreased 33.08%,and the analysis of cell cycle indicated that cells of G0/G1 phase were increased comparision with(40.75±2.30)%and(40.00±1.76)0A,and its G2/M phase cells(8.16±0.51)%were decreased strikingly compared with group control(31.20±1.76)%and group NP(35.94±1.58)%.Cells were blocked at G0/G1 phase,the cell proliferation was inhibited obviously. Conclusion PLCε may play an important role in proliferation of bladder cancer cells,which could be a potential target of biological treatment on bladder cancer in the future.