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*

Objective To evaluate the application value of nucleic acid mass spectrometry in detecting ERG11 gene mutations associated with azole resistance in Candida tropicalis(C.tropicalis),thereby providing a scientific basis for the rational clinical use of azole antifungal agents.Methods Clinical isolates of C.tropicalis were obtained from the Affiliated Hospital of Xuzhou Medical University between July 2023 and November 2024.For each isolate,specific ERG11 mutations(A395T and C461T)were analyzed using Sanger sequencing and nucleic acid mass spectrometry.Sanger sequencing was used as the reference standard to evaluate the performance of mass spectrometry in mutation detection.Results The established nucleic acid mass spectrometry method effectively identified four genotypes of ERG11(A395T and C461T),with distinct spectral peaks for each mutation and no cross-interference observed.Among 88 clinical isolates,the sensitivity,specificity,positive predictive value(PPV),and negative predictive value(NPV)for the A395T mutation were 86.7%,100%,100%,and 97.3%,respectively.For the C461T mutation,the corresponding values were 81.3%,100%,100%,and 96.0%,respectively.Kappa statistics demonstrated a high level of agreement between mass spectrometry and sequencing results.Conclusions Nucleic acid mass spectrometry exhibits high sensitivity and specificity in the detection of ERG11 mutations,enabling rapid,accurate,and adaptable high-throughput analysis.This makes it a highly effective method for identifying mutations associated with fungal resistance.

Objective:To explore the predictive value of human cartilage glycoprotein 39 (YKL-40) and growth differentiation factor 15 (GDF-15) for clinical outcomes of patients with Parkinson disease (PD).Methods:A total of 109 patients with PD admitted to Xinxiang Central Hospital from February 2021 to February 2023 were selected and treated with regular anti-PD medications for 4 weeks, with dosage appropriately adjusted according to clinical status and individual response.Clinical outcomes were evaluated after 2 months of treatment, and the predictive value of serum YKL-40 and GDF-15 at admission for clinical outcomes was analyzed.Data were analyzed by independent sample t-test, χ2 test and Logistic regression using SPSS 26.0. Results:PD patients with poor outcomes exhibited higher serum levels of YKL-40((3.18±0.67)mg/L, (2.34±0.41)mg/L) and GDF-15((457.82±142.83)pg/mL, (282.95±105.96)pg/mL) than those with good outcomes, and the differences were statistically significant ( t=8.082, 7.349, both P<0.05).Logistic regression analysis showed that elevated serum levels of YKL-40( B=0.664, OR=1.943, 95% CI=1.237-3.052) and GDF-15( B=0.185, OR=1.787, 95% CI=1.145-2.789) both influenced the clinical outcomes of PD patients(both P<0.05).Serum YKL-40 combined with GDF-15 demonstrated a predictive sensitivity of 87.23%, specificity of 90.32%, and AUC of 0.927(95% CI=0.861-0.968) for clinical outcomes in PD patients.The AUC was significantly higher than that achieved by either indicator alone (YKL-40: AUC (95% CI)=0.722 (0.628-0.804); GDF-15: AUC (95% CI)=0.797 (0.709-0.868)). Conclusion:The elevated levels of YKL-40 and GDF-15 in PD patients are associated with clinical outcomes, which may be the potential markers for predicting clinical outcomes of patients with PD.

Objective:To investigate effect of andrographolide(AG)on neuroinflammation in young epileptic rats by regulating cyclic adenosine monophosphate(cAMP)/protein kinase A(PKA)/cAMP response element binding protein(CREB)signaling pathway.Methods:SPF grade young SD rats were randomly divided into Control group,Model group,low-dose AG group(AG-L,125 mg/kg),high-dose AG group(AG-H,250 mg/kg)and high-dose AG+PKA inhibitor H-89 group(AG-H+H-89,250 mg/kg AG+2 mg/kg H-89),with 12 rats in each group.Epilepsy model of young rats was established by intraperitoneal injection of kainic acid(KA).Morris water maze test was used to detect learning and memory functions of rats.ELISA was used to detect levels of TNF-α,IL-10,malondialdehyde(MDA),superoxide dismutase(SOD)and cAMP in hippocampus of rats in each group.Histomorphology of hippo-campus was detected by Nissl staining.TUNEL test was usd to determine apoptosis rate of neurons in rat hippocampus.PKA and CREB mRNA expressions in hippocampus of rats in each group were detected by RT-qPCR.Western blot was used to detect protein expressions of PKA,Bax,Caspase-3,CREB,p-CREB and brain derived neurotrophic factor(BDNF)in hippocampus of rats in each group.Results:Compared with control group,escape latency,TNF-α and MDA levels in hippocampus,apoptosis rate of nerve cells,Bax and Caspase-3 protein expressions in Model group were obviously increased(P<0.05),target quadrant residence time,SOD,IL-10,cAMP levels,PKA,CREB mRNA and protein expressions,BDNF protein expression in hippocampus were decreased obviously(P<0.05),hippocampal tissue showed pathological damage and a large number of Nissl bodies were lost.Compared with Model group,cor-responding indexes of rats in AG-H group were contrary to the above(P<0.05),loss of Nissl corpuscles was reduced.H-89 alleviated improvement of AG on neuroinflammation in young epileptic rats.Conclusion:AG may reduce neuroinflammation in young epileptic rats by activating cAMP/PKA/CREB signaling pathway.

Objective:To investigate the association between serum thyroid-stimulating hormone(TSH) level and the 10-year risk of atherosclerotic cardiovascular disease(ASCVD) in men over 50 years old with type 2 diabetes mellitus(T2DM).Methods:This study included male T2DM patients aged≥50 years, diagnosed at the First Hospital of Lanzhou University between July 2021 and March 2022. Patients were categorized into three groups based on serum TSH level: elevated TSH group(T3, TSH>5.91 mIU/L) and normal TSH group, which was further divided into T1(0.56 mIU/L≤TSH<3.24 mIU/L) and T2(3.24 mIU/L≤TSH≤5.91 mIU/L) group. The 10-year ASCVD risk index was compared across groups. Spearman correlation and multiple linear regression analyses were used to assess the independent association between TSH level and 10-year ASCVD risk index. Results:A total of 490 male T2DM patients aged≥50 years were included(T1: 310, T2: 131, T3: 49). The 10-year ASCVD risk index was significantly higher in T3 group than that in T1 group(18.40% vs 13.90%, χ2=9.47, P<0.05). Serum TSH level showed a positive correlation with the 10-year ASCVD risk( r=0.144, P<0.05). After adjusting for confounders such as age, hypertension, lipid profile, diabetes duration, aspartate aminotransferase, albumin, lactate dehydrogenase, estimated glomerular filtration rate, creatinine, and phosphorus, multiple linear regression confirmed that TSH level was independently associated with the 10-year ASCVD risk index( β=0.23, 95% CI 0.02-0.45). Conclusions:Higher serum TSH level is independently associated with an increased 10-year ASCVD risk in men over 50 years old with T2DM. Regular TSH monitoring may aid in cardiovascular risk stratification in this population.

Objective:To investigate the neuroprotective effect of vanillin on experimental autoimmune encephalomyelitis(EAE)rats by regulating C-X-C motif chemokine ligand 12(CXCL12)/chemokine(C-X-C motif)receptor(CXCR4)signaling path-way.Methods:A total of 50 rats were injected with 400 μl guinea pig spinal cord and water in oil mixture of complete Freund's adju-vant to establish EAE rat model,and were divided into model group,low-dose vanillin group(50 mg/kg),medium-dose vanillin group(100 mg/kg),high-dose vanillin group(200 mg/kg)and positive drug group(5 mg/kg prednisone acetate),another 10 rats were only injected with the mixture of the same amount of normal saline and complete Freud adjuvant as control group,since the first day of mod-eling,all rats were given corresponding drugs by gavage for 16 consecutive days,and after modeling,rats in each group were scored for neurological function every day;HE and LFB staining were used to observe the pathology and demyelination of spinal cord in rats;levels of serum TNF-α,IL-1β and IL-6 were detected by ELISA;immunohistochemistry was used to detect expressions of CD68 and Iba-1 in spinal cord of rats;Western blot was used to detect expressions of CXCL12 and CXCR4 proteins in spinal cord of rats.Results:Compared with control group,rats in model group showed obvious infiltration of inflammatory cells,a large number of inflammatory cells gathered around the small blood vessels,and the structure of myelin sheath in spinal cord of rats was abnormal,accompanied by a large number of myelin sheath loss,the neurological function score,inflammatory infiltration score and demyelination score of spinal cord,serum TNF-α,IL-1β and IL-6 levels,spinal cord CD68 and Iba-1 proteins,CXCL12 and CXCR4 proteins expressions in-creased obviously(P<0.05);compared with model group,inflammatory cell infiltration and myelin sheath loss of rats in low,medium and high doses groups and positive drug group were obviously alleviated,neurological function score,inflammatory infiltration score and demyelination score of spinal cord,serum TNF-α,IL-1β and IL-6 levels,and expressions of CD68 and Iba-1 proteins in spinal cord decreased obviously(P<0.05),while expressions of CXCL12 and CXCR4 proteins in spinal cord further increased(P<0.05).Conclusion:Vanillin inhibits inflammatory response and alleviates nerve injury in EAE rats,and its mechanism may be related to acti-vation of CXCL12/CXCR4 signaling pathway.

Objective:To explore the predictive value of human cartilage glycoprotein 39 (YKL-40) and growth differentiation factor 15 (GDF-15) for clinical outcomes of patients with Parkinson disease (PD).Methods:A total of 109 patients with PD admitted to Xinxiang Central Hospital from February 2021 to February 2023 were selected and treated with regular anti-PD medications for 4 weeks, with dosage appropriately adjusted according to clinical status and individual response.Clinical outcomes were evaluated after 2 months of treatment, and the predictive value of serum YKL-40 and GDF-15 at admission for clinical outcomes was analyzed.Data were analyzed by independent sample t-test, χ2 test and Logistic regression using SPSS 26.0. Results:PD patients with poor outcomes exhibited higher serum levels of YKL-40((3.18±0.67)mg/L, (2.34±0.41)mg/L) and GDF-15((457.82±142.83)pg/mL, (282.95±105.96)pg/mL) than those with good outcomes, and the differences were statistically significant ( t=8.082, 7.349, both P<0.05).Logistic regression analysis showed that elevated serum levels of YKL-40( B=0.664, OR=1.943, 95% CI=1.237-3.052) and GDF-15( B=0.185, OR=1.787, 95% CI=1.145-2.789) both influenced the clinical outcomes of PD patients(both P<0.05).Serum YKL-40 combined with GDF-15 demonstrated a predictive sensitivity of 87.23%, specificity of 90.32%, and AUC of 0.927(95% CI=0.861-0.968) for clinical outcomes in PD patients.The AUC was significantly higher than that achieved by either indicator alone (YKL-40: AUC (95% CI)=0.722 (0.628-0.804); GDF-15: AUC (95% CI)=0.797 (0.709-0.868)). Conclusion:The elevated levels of YKL-40 and GDF-15 in PD patients are associated with clinical outcomes, which may be the potential markers for predicting clinical outcomes of patients with PD.

Objective:To investigate the neuroprotective effect of vanillin on experimental autoimmune encephalomyelitis(EAE)rats by regulating C-X-C motif chemokine ligand 12(CXCL12)/chemokine(C-X-C motif)receptor(CXCR4)signaling path-way.Methods:A total of 50 rats were injected with 400 μl guinea pig spinal cord and water in oil mixture of complete Freund's adju-vant to establish EAE rat model,and were divided into model group,low-dose vanillin group(50 mg/kg),medium-dose vanillin group(100 mg/kg),high-dose vanillin group(200 mg/kg)and positive drug group(5 mg/kg prednisone acetate),another 10 rats were only injected with the mixture of the same amount of normal saline and complete Freud adjuvant as control group,since the first day of mod-eling,all rats were given corresponding drugs by gavage for 16 consecutive days,and after modeling,rats in each group were scored for neurological function every day;HE and LFB staining were used to observe the pathology and demyelination of spinal cord in rats;levels of serum TNF-α,IL-1β and IL-6 were detected by ELISA;immunohistochemistry was used to detect expressions of CD68 and Iba-1 in spinal cord of rats;Western blot was used to detect expressions of CXCL12 and CXCR4 proteins in spinal cord of rats.Results:Compared with control group,rats in model group showed obvious infiltration of inflammatory cells,a large number of inflammatory cells gathered around the small blood vessels,and the structure of myelin sheath in spinal cord of rats was abnormal,accompanied by a large number of myelin sheath loss,the neurological function score,inflammatory infiltration score and demyelination score of spinal cord,serum TNF-α,IL-1β and IL-6 levels,spinal cord CD68 and Iba-1 proteins,CXCL12 and CXCR4 proteins expressions in-creased obviously(P<0.05);compared with model group,inflammatory cell infiltration and myelin sheath loss of rats in low,medium and high doses groups and positive drug group were obviously alleviated,neurological function score,inflammatory infiltration score and demyelination score of spinal cord,serum TNF-α,IL-1β and IL-6 levels,and expressions of CD68 and Iba-1 proteins in spinal cord decreased obviously(P<0.05),while expressions of CXCL12 and CXCR4 proteins in spinal cord further increased(P<0.05).Conclusion:Vanillin inhibits inflammatory response and alleviates nerve injury in EAE rats,and its mechanism may be related to acti-vation of CXCL12/CXCR4 signaling pathway.

Objective To evaluate the application value of nucleic acid mass spectrometry in detecting ERG11 gene mutations associated with azole resistance in Candida tropicalis(C.tropicalis),thereby providing a scientific basis for the rational clinical use of azole antifungal agents.Methods Clinical isolates of C.tropicalis were obtained from the Affiliated Hospital of Xuzhou Medical University between July 2023 and November 2024.For each isolate,specific ERG11 mutations(A395T and C461T)were analyzed using Sanger sequencing and nucleic acid mass spectrometry.Sanger sequencing was used as the reference standard to evaluate the performance of mass spectrometry in mutation detection.Results The established nucleic acid mass spectrometry method effectively identified four genotypes of ERG11(A395T and C461T),with distinct spectral peaks for each mutation and no cross-interference observed.Among 88 clinical isolates,the sensitivity,specificity,positive predictive value(PPV),and negative predictive value(NPV)for the A395T mutation were 86.7%,100%,100%,and 97.3%,respectively.For the C461T mutation,the corresponding values were 81.3%,100%,100%,and 96.0%,respectively.Kappa statistics demonstrated a high level of agreement between mass spectrometry and sequencing results.Conclusions Nucleic acid mass spectrometry exhibits high sensitivity and specificity in the detection of ERG11 mutations,enabling rapid,accurate,and adaptable high-throughput analysis.This makes it a highly effective method for identifying mutations associated with fungal resistance.

Objective:To investigate the association between serum thyroid-stimulating hormone(TSH) level and the 10-year risk of atherosclerotic cardiovascular disease(ASCVD) in men over 50 years old with type 2 diabetes mellitus(T2DM).Methods:This study included male T2DM patients aged≥50 years, diagnosed at the First Hospital of Lanzhou University between July 2021 and March 2022. Patients were categorized into three groups based on serum TSH level: elevated TSH group(T3, TSH>5.91 mIU/L) and normal TSH group, which was further divided into T1(0.56 mIU/L≤TSH<3.24 mIU/L) and T2(3.24 mIU/L≤TSH≤5.91 mIU/L) group. The 10-year ASCVD risk index was compared across groups. Spearman correlation and multiple linear regression analyses were used to assess the independent association between TSH level and 10-year ASCVD risk index. Results:A total of 490 male T2DM patients aged≥50 years were included(T1: 310, T2: 131, T3: 49). The 10-year ASCVD risk index was significantly higher in T3 group than that in T1 group(18.40% vs 13.90%, χ2=9.47, P<0.05). Serum TSH level showed a positive correlation with the 10-year ASCVD risk( r=0.144, P<0.05). After adjusting for confounders such as age, hypertension, lipid profile, diabetes duration, aspartate aminotransferase, albumin, lactate dehydrogenase, estimated glomerular filtration rate, creatinine, and phosphorus, multiple linear regression confirmed that TSH level was independently associated with the 10-year ASCVD risk index( β=0.23, 95% CI 0.02-0.45). Conclusions:Higher serum TSH level is independently associated with an increased 10-year ASCVD risk in men over 50 years old with T2DM. Regular TSH monitoring may aid in cardiovascular risk stratification in this population.