β-hydroxy-β-methylbutyric acid (HMB) is widely applied in sports nutrition, disease prevention and other fields. However, chemical synthesis methods, limited by toxic reagents and violent reactions, can hardly meet the demands of green production. The biosynthesis method mainly utilizes enzymatic catalysis or metabolic engineering techniques for synthesis, and has the advantages of high efficiency, low cost, and sustainability. Therefore, the production of HMB by the biosynthesis method has a good application prospect. In this research, a biosynthesis-based production strategy for HMB was developed. By using L-leucine as the substrate and constructing a dual-enzyme co-expression system, we established an efficient catalytic process. At first, the enzymatic properties of L-amino acid deaminase (PvL-AAD) from Proteus vulgaris and 4-hydroxyphenylpyruvate dioxygenase (Rn4-HPPD) from Rattus norvegicus were characterized. Rn4-HPPD had low relative activity and required an acidic environment for catalysis. Based on the surface charge modification strategy of the enzyme protein, site-directed mutagenesis and combinatorial mutagenesis were conducted on 10 sites of Rn4-HPPD. A double mutant Rn4-HPPD^H18R/N302R was thus obtained, with the enzyme activities being 2.00 times and 2.39 times that of the wild type at pH 5.5 and pH 6.5, respectively. Subsequently, the expression of the two enzymes in Escherichia coli was optimized. After the optimal expression ratio of the two enzymes was determined as 1:3 and under the conditions of OD₆₀₀ of 70, pH 6.0, 35 ℃, Fe²⁺ concentration of 1.5 mmol/L, and feeding of the substrate in batches in a 5 L fermenter, the maximum yield of HMB reached 8.60 g/L. This study not only enhances the optimal pH and activity of Rn4-HPPD but also provides new approaches for the efficient microbial synthesis of HMB.
Escherichia coli/metabolism* ; Valerates/metabolism* ; Recombinant Proteins/biosynthesis* ; Animals ; Metabolic Engineering/methods* ; Rats ; Catalysis

Objective To design a tabletop exercise teaching toolkit for emergency rescue training for mass wounded at sea.Methods Guided by the competency-based education(CBE)theory,a preliminary tabletop exercise teaching toolkit was constructed by combining field research and literature analysis.The toolkit was tested and modified through expert meetings.Results A set of tabletop exercise teaching toolkit including tabletop exercise scheme,casualty pool,teaching aids and teaching evaluation tools was formed.Conclusion The tabletop exercise teaching toolkit developed in this study can be used for the training of medical personnels on large surface warships,and it provides new ideas for our military medical training.

Objective:To explore the clinical efficacy and safety of endoscopic rubber band ligation (ERBL) and endoscopic injection sclerotherapy (EIS) in the treatment of grade I-III internal hemorrhoids, to provide a basis for the individualized treatment of patients with internal hemorrhoids in clinical practice.Methods:A retrospective cohort study was conducted. A total of 613 patients with grade I to III internal hemorrhoids who underwent ERBL or EIS at The Sixth Affiliated Hospital of Sun Yat-sen University from December 2019 to November 2023 were retrospectively collected. Inclusion criteria: (1) Patients diagnosed with symptomatic grade I-III internal hemorrhoids who failed conservative treatments such as diet adjustment and medication; (2) Patients who were unable or unwilling to receive surgical treatment due to multiple underlying systemic diseases. Exclusion criteria: (1) Patients with grade I-III internal hemorrhoids complicated with incarceration, thrombosis or other complications; (2) Patients with a history of hemorrhoid surgery; (3) Patients complicated with perianal abscess, anal fistula, active proctitis, rectal tumor, polyp, radiation proctitis or inflammatory bowel disease; (4) Patients with incomplete clinical data or lost to follow-up. This study was divided into the ERBL group and the EIS group based on different treatment. Baseline characteristics, postoperative effective rate, recurrence rate, pain score, anal distension, anal edema, complication rate, and treatment satisfaction were compared between the two groups.Results:After balancing with propensity score matching (PSM), a total of 313 patients were included, including 200 in the ERBL group and 113 in the EIS group. There were no statistically significant differences in baseline characteristics, such as gender, age, body mass index, Goligher classification, and laboratory test indicators, between the two groups (all P > 0.05), indicating that the two groups were comparable. The cure rates of the ERBL group and the EIS group were 64.0% (128/200) and 62.8% (71/113), respectively. The marked effective rates were 31.5% (63/200) and 34.5% (39/113), and the ineffective rates were 4.5% (9/200) and 2.7% (3/113), respectively. Statistical analysis revealed no statistically significant difference in efficacy between the two groups within 3 months after the operation (χ2=0.858, P=0.651). The recurrence rate of the ERBL group was lower than that of the EIS group [13.1% (25/191) vs. 18.2% (20/110)], but the difference was not statistically significant (χ2=1.424, P=0.233). Subgroup analysis showed that the recurrence rates of ERBL and EIS were similar in grade I-II internal hemorrhoids [10.3% (15/146) vs. 10.5% (9/86), χ2=0.002, P=0.963]; in grade III internal hemorrhoids, the recurrence rate of the ERBL group was lower than that of the EIS group [22.2% (10/45) vs. 45.8% (11/24), χ2=4.121, P=0.042]. Still, the difference was not statistically significant after Bonferroni correction (χ2=4.121, corrected P>0.025). Compared with the ERBL group, the EIS group had a lower pain score on the first day after operation [0 (0, 0) vs. 1 (0, 3), Z=-8.211, P<0.001] and a lower incidence of anal distension [25.7% (29/113) vs. 61.5% (123/200), χ2=37.122, P<0.001], with statistically significant differences. The total incidence of complications in the ERBL group was significantly higher than that in the EIS group [29.5% (59/200) vs. 4.4% (5/113), χ2=27.910, P<0.001], mainly reflected in postoperative bleeding [18.0% (36/200) vs. 3.5% (4/113), χ2=13.544, P<0.001] and urinary retention [8.5% (17/200) vs. 0, χ2=10.157, P=0.001], with statistically significant differences. There were no statistically significant differences in postoperative satisfaction, health status score, and the proportion of returning to everyday life within 1 month between the two groups (all P>0.05). Conclusions:Both ERBL and EIS are effective minimally invasive therapies for grade I-III internal hemorrhoids. EIS is preferred for grade I and II internal hemorrhoids to reduce complications, while ERBL shows a trend in reducing the recurrence rate of grade III internal hemorrhoids. Clinical decisions should be made comprehensively based on the classification, patient tolerance, and prognosis.

Objective:To explore the clinical efficacy and safety of endoscopic rubber band ligation (ERBL) and endoscopic injection sclerotherapy (EIS) in the treatment of grade I-III internal hemorrhoids, to provide a basis for the individualized treatment of patients with internal hemorrhoids in clinical practice.Methods:A retrospective cohort study was conducted. A total of 613 patients with grade I to III internal hemorrhoids who underwent ERBL or EIS at The Sixth Affiliated Hospital of Sun Yat-sen University from December 2019 to November 2023 were retrospectively collected. Inclusion criteria: (1) Patients diagnosed with symptomatic grade I-III internal hemorrhoids who failed conservative treatments such as diet adjustment and medication; (2) Patients who were unable or unwilling to receive surgical treatment due to multiple underlying systemic diseases. Exclusion criteria: (1) Patients with grade I-III internal hemorrhoids complicated with incarceration, thrombosis or other complications; (2) Patients with a history of hemorrhoid surgery; (3) Patients complicated with perianal abscess, anal fistula, active proctitis, rectal tumor, polyp, radiation proctitis or inflammatory bowel disease; (4) Patients with incomplete clinical data or lost to follow-up. This study was divided into the ERBL group and the EIS group based on different treatment. Baseline characteristics, postoperative effective rate, recurrence rate, pain score, anal distension, anal edema, complication rate, and treatment satisfaction were compared between the two groups.Results:After balancing with propensity score matching (PSM), a total of 313 patients were included, including 200 in the ERBL group and 113 in the EIS group. There were no statistically significant differences in baseline characteristics, such as gender, age, body mass index, Goligher classification, and laboratory test indicators, between the two groups (all P > 0.05), indicating that the two groups were comparable. The cure rates of the ERBL group and the EIS group were 64.0% (128/200) and 62.8% (71/113), respectively. The marked effective rates were 31.5% (63/200) and 34.5% (39/113), and the ineffective rates were 4.5% (9/200) and 2.7% (3/113), respectively. Statistical analysis revealed no statistically significant difference in efficacy between the two groups within 3 months after the operation (χ2=0.858, P=0.651). The recurrence rate of the ERBL group was lower than that of the EIS group [13.1% (25/191) vs. 18.2% (20/110)], but the difference was not statistically significant (χ2=1.424, P=0.233). Subgroup analysis showed that the recurrence rates of ERBL and EIS were similar in grade I-II internal hemorrhoids [10.3% (15/146) vs. 10.5% (9/86), χ2=0.002, P=0.963]; in grade III internal hemorrhoids, the recurrence rate of the ERBL group was lower than that of the EIS group [22.2% (10/45) vs. 45.8% (11/24), χ2=4.121, P=0.042]. Still, the difference was not statistically significant after Bonferroni correction (χ2=4.121, corrected P>0.025). Compared with the ERBL group, the EIS group had a lower pain score on the first day after operation [0 (0, 0) vs. 1 (0, 3), Z=-8.211, P<0.001] and a lower incidence of anal distension [25.7% (29/113) vs. 61.5% (123/200), χ2=37.122, P<0.001], with statistically significant differences. The total incidence of complications in the ERBL group was significantly higher than that in the EIS group [29.5% (59/200) vs. 4.4% (5/113), χ2=27.910, P<0.001], mainly reflected in postoperative bleeding [18.0% (36/200) vs. 3.5% (4/113), χ2=13.544, P<0.001] and urinary retention [8.5% (17/200) vs. 0, χ2=10.157, P=0.001], with statistically significant differences. There were no statistically significant differences in postoperative satisfaction, health status score, and the proportion of returning to everyday life within 1 month between the two groups (all P>0.05). Conclusions:Both ERBL and EIS are effective minimally invasive therapies for grade I-III internal hemorrhoids. EIS is preferred for grade I and II internal hemorrhoids to reduce complications, while ERBL shows a trend in reducing the recurrence rate of grade III internal hemorrhoids. Clinical decisions should be made comprehensively based on the classification, patient tolerance, and prognosis.

Oxalate is an organic dicarboxylic acid that is a common component of plant foods.The kidneys are essential organs for oxalate excretion,but excessive oxalates may induce kidney stones.Jupiter micro-tubule associated homolog 2(JPT2)is a critical molecule in Ca2+mobilization,and its intrinsic mecha-nism in oxalate exposure and kidney stones remains unclear.This study aimed to reveal the mechanism of JPT2 in oxalate exposure and kidney stones.Genetic approaches were used to control JPT2 expression in cells and mice,and theJPT2 mechanism of action was analyzed using transcriptomics and untargeted metabolomics.The results showed that oxalate exposure triggered the upregulation of JPT2,which is involved in nicotinic acid adenine dinucleotide phosphate(NAADP)-mediated Ca2+mobilization.Tran-scriptomic analysis revealed that cell adhesion and macrophage inflammatory polarization were inhibited by JPT2 knockdown,and these were dominated by phosphatidylinositol 3-kinase(PI3K)/AKT signaling,respectively.Untargeted metabolomics indicated that JPT2 knockdown inhibited the produc-tion of succinic acid semialdehyde(SSA)in macrophages.Furthermore,JPT2 deficiency in mice inhibited kidney stones mineralization.In conclusion,this study demonstrates that oxalate exposure facilitates kidney stones by promoting crystal-cell adhesion,and modulating macrophage metabolism and in-flammatory polarization via JPT2/PI3K/AKT signaling.

Candida albicans is the most abundant fungal species in oral cavity. As a smart opportunistic pathogen, it increases the virulence by switching its forms from yeasts to hyphae and becomes the major pathogenic agent for oral candidiasis. However, the overuse of current clinical antifungals and lack of new types of drugs highlight the challenges in the antifungal treatments because of the drug resistance and side effects. Anti-virulence strategy is proved as a practical way to develop new types of anti-infective drugs. Here, seven artemisinins, including artemisinin, dihydroartemisinin, artemisinic acid, dihydroartemisinic acid, artesunate, artemether and arteether, were employed to target at the hyphal development, the most important virulence factor of C. albicans. Artemisinins failed to affect the growth, but significantly inhibited the hyphal development of C. albicans, including the clinical azole resistant isolates, and reduced their damage to oral epithelial cells, while arteether showed the strongest activities. The transcriptome suggested that arteether could affect the energy metabolism of C. albicans. Seven artemisinins were then proved to significantly inhibit the productions of ATP and cAMP, while reduced the hyphal inhibition on RAS1 overexpression strain indicating that artemisinins regulated the Ras1-cAMP-Efg1 pathway to inhibit the hyphal development. Importantly, arteether significantly inhibited the fungal burden and infections with no systemic toxicity in the murine oropharyngeal candidiasis models in vivo caused by both fluconazole sensitive and resistant strains. Our results for the first time indicated that artemisinins can be potential antifungal compounds against C. albicans infections by targeting at its hyphal development.
Animals ; Mice ; Candida albicans ; Candidiasis, Oral/drug therapy* ; Antifungal Agents/pharmacology* ; Hyphae ; Artemisinins/pharmacology*

Objective To investigate the influences of herpes simplex virus 1 and 2 ( HSV1 and HSV2) infection on the expression of signaling molecules associated with innate immune response in respira-tory and vaginal epithelial cells for bettering understanding of HSV infection and pathological characteristics in the primary infection site, namely mucosal epithelial tissues. Methods KMB17 and VK2 cells were in-fected with HSV. Changes in cell morphology and inner structure after HSV infection were observed under optical microscope and scanning electron microscope, respectively. Viral proliferation in KMB17 and VK2 cells was detected by plaque assay, microcytopathic assay and real-time quantitative PCR. Expression of sig-naling molecules associated with innate immune response in virus-infected KMB17 and VK2 cells were ana-lyzed by real-time quantitative PCR. Results Both HSV1 and HSV2 could infect KMB17 and VK2 cells, and cause damage to cell morphology and inner structure after 12 hours. Both of the two viruses formed simi-lar plaque on the single layer of KMB17 and VK2 cells, although HSV2 proliferated slower than HSV1. There were differences in the expression of signaling molecules associated with innate immune response in-duced by the two viruses in KMB17 and VK2 cells. Conclusion Both HSV1 and HSV2 could infect and proliferate in epithelial cells ( KMB17 and VK2 cells) . Although there were slight differences in viral prolif-eration between them, significant differences in the expression of signaling molecules associated with innate immune response induced by the two viruses were observed.

Objective To investigate the potential dosimetric advantages of half jaw volumetric modulated arc therapy ( H-VMAT) applied to the Oropharyngeal Cancer, comparing with full jaw VMAT (F-VMAT) and intensity modulated radiotherapy ( IMRT ). Methods Planning CT images of 10 oropharyngeal cancer patients were retrospectively chosen and transferred to Eclipse treatment planning system v. 11. 0 (Varian Medical Systems, Pala Alto, USA), based on which H-VMAT, W-VMAT, and IMRT plans were created. Two full arcs (360°) were adopted for VMAT planning, and the 7 beams were equally distributed for IMRT planning. The optimization constraints remained the same for the three kinds of plans. The dosimetric parameters such as D2 , D98 , D50 , HI, and CI were evaluated for PGTV, PCTV1, PCTV2, PGTVln, and PCTVln. In addition, the maximum dose (Dmax) and D1 cc(minimum dose received by 1cc) of the brainstem and spinal cord were analyzed respectively. The mean dose ( Dmean ) to the parotids, oral cave, larynx, and cervical normal tissues were also reviewed. The monitor units ( MU) for all treatment plans were recorded. Results Comparisons of the three planning techniques showed that H-VAMT improved the HI and CI of the targets (except PCTV2) significantly (HI: F =3. 959, 6. 764, 10. 581, 6. 770, 13. 040, P<0. 05;CI:F=6. 594, 4. 138, 0. 842, 4. 031, 5. 388, P<0. 05);reduced Dmax(F=4. 509, 20. 331, P<0. 05) and D1 cc for brainstem and spinal cord (F=27. 432, 26. 314, P<0. 05) significantly;reduced Dmean(F=4. 279, 29. 498, 19. 295, P<0. 05) to the normal tissues of the mouth, throat and neck significantly. The V50 of the mouth and throat were slightly lower in IMRT plans (F=8. 140, P<0. 05). IMRT was slightly better than W-VMAT in sparing oral cavity and larynx, but the dose distribution was the worst. The H-VMAT plans showed the best dose distribution in the cervical normal tissues, especially for the lower and posterior parts, where IMRT plans displayed high dose curves. Conclusions H-VMAT is dosimetrically superior than W-VMAT and IMRT for oropharyngeal cancer, which could be considered for clinical applications.

Objective To explore the characters of lower urinary tract symptoms (LUTS) in patients with Parkinson's Disease (PD).Methods From Oct 2013 to Jun 2016,after evaluating of movement disorder by modified Hoehn-Yahr(H-Y) scale and LUTS by international prostate symptom score (IPSS),urodynamic study was performed in PD patients with LUTS.The incidence of every symptom of LUTS,the relationships between the IPSS categories and urodynamic study were analyzed.Results 64 patients (containing 26 male and 38 female) with 40-80 (62.7 ± 10.2) years old were included.2,4,30,19,12 and 6 patients were belonged to modified H-Y scale 1-4,respectively.Frequency (50 patients,78.1％) was the most common LUTS,while frequency all day (20 patients,31.3％) was the most common symptom for the most severe LUTS.IPSS was 1 7.5 ± 7.8 (4-35) and quality of life was 5.1 ± 0.6 (4-6) for the patients.There was no significant correlation between modified H-Y scale and IPSS (P ＞ 0.05).According to the criteria of IPSS,28 patients (43.8％) only had irritative symptoms,3 patients (4.7％) only had obstructive symptoms,while 26 patients (40.6％) had mixed symptoms and 7 patients (10.9％) belonged to no one.Urodynamic study showed 11 patients (39.3％) with only irritative symptoms had detrusor overactivity(DO),6 patients (23.1％) with mixed symptoms had DO + bladder outlet obstruction (BOO) or DO + detrusor underactivity,however,there was no one with BOO in the three patients with only obstructive symptoms.Conclusion Frequency was the most common LUTS,while frequency all day was the most common symptom for the most severe LUTS in PD patients.Irritative and mixed symptoms were common in PD patients with LUTS,but the urodynamic results were not consistent with the category of LUTS in most of the patients.LUTS severity was not correlated with movement disorders in PD patients.

OBJECTIVETo study the promoter methylation status of SFRP genes and the effect of 5- aza- 2'- deoxycytidine (5- Aza- CdR)induced apoptosis via Wnt/β- catenin pathway by demethylation in Jurkat cells.

METHODSJurkat cells were treated with different concentrations of 5- Aza- CdR. The cell proliferation level of Jurkat cells was detected by MTT assay. Apoptosis was evaluated by flow cytometry. Methylation- spcific PCR (MSP) was used to determine the methylation status of SFRP genes. The expressions of SFRP genes were detected by real time fluorescence quantitative PCR. The mRNA expression levels of survivin, c- myc and cyclin- D1 were analyzed by RT- PCR. Western blot was used to detect the levels of β-catenin protein.

RESULTSCompared with control group, the different concentrations of 5-Aza-CdR could significantly inhibit the proliferation of Jurkat cells in a time-dose dependent manner (P<0.05). After being treated by 5- Aza- CdR for 48 hours, the cell early apoptosis rate in experiment group was significantly higher than that in control group (P<0.05). The promoters of SFRP1, SFRP2, SFRP4, SFRP5 genes were hypermethylation state in the control group, after being treated by 5-Aza-CdR for 72 hours, the brightness of SFRP1, SFRP2, SFRP4, SFRP5 genes' methylation strips weakened in a dose- dependent manner. SFRP mRNA expression increased (P<0.05) when 5- Aza- CdR concentration increased, and the level of β- catenin protein was dampened in a dose- dependent manner (P<0.05). As compared to the control group, the mRNA expressions of associated apoptosis genes survivin, c-myc and cyclin- D1, respectively were obviously down- regulated in a dose- dependent manner (P<0.05).

CONCLUSIONThe effect of demethylation could up- regulate SFRP genes expressions by reversing its hypermethylation and induced apoptosis by down-regulation of β-catenin and associated apoptosis genes.

Apoptosis ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Proliferation ; DNA Methylation ; Down-Regulation ; Gene Expression ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; Jurkat Cells ; Membrane Proteins ; genetics ; Promoter Regions, Genetic ; Wnt Signaling Pathway ; beta Catenin ; metabolism