Integrated metabolomic and transcriptomic analysis reveals the regulatory mechanisms of flavonoid and alkaloid biosynthesis in the new and old leaves of Murraya tetramera Huang.

BACKGROUND: Murraya tetramera Huang is a traditional Chinese woody medicine. Its leaves contain flavonoids, alkaloids, and other active compounds, which have anti-inflammatory and analgesic effects, as well as hypoglycemic and lipid-lowering effects, and anti-tumor effects. There are significant differences in the content of flavonoids and alkaloids in leaves during different growth cycles, but the synthesis mechanism is still unclear. RESULTS: In April 2021, new leaves (one month old) and old leaves (one and a half years old) of M. tetramera were used as experimental materials to systematically analyze the changes in differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) with transcriptomics and metabolomics technology. This was done to identify the signaling pathways of flavonoid and alkaloid synthesis. The results showed that the contents of total alkaloids and flavonoids in old leaves were significantly higher than those in new leaves. Thirteen flavonoid compounds, three isoflavone compounds, and nineteen alkaloid compounds were identified, and 125 and 48 DEGs related to flavonoid and alkaloid synthesis were found, respectively. By constructing the KEGG (Kyoto Encyclopedia of Genes and Genomes) network of DEGs and DAMs, it was shown that the molecular mechanism of flavonoid biosynthesis in M. tetramera mainly focuses on the "flavonoid biosynthetic pathway" and the "flavonoid and flavonol biosynthetic pathway". Among them, p-Coumaryl alcohol, Sinapyl alcohol, Phloretin, and Isoquercitrin were significantly accumulated in old leaves, the up-regulated expression of CCR (cinnamoyl-CoA reductase) might promote the accumulation of p-Coumaryl alcohol, upregulation of F5H (ferulate-5-hydroxylase) might promote Sinapyl alcohol accumulation. Alkaloids, including indole alkaloids, pyridine alkaloids, imidazole alkaloids, and quinoline alkaloids, were significantly accumulated in old leaves, and a total of 29 genes were associated with these substances. CONCLUSIONS: These data are helpful to better understand the biosynthesis of flavonoids and alkaloids in M. tetramera and provide a scientific basis for the development of medicinal components in M. tetramera.

Preoperative and intraoperative tirofiban during endovascular thrombectomy in large vessel occlusion stroke due to large artery atherosclerosis.

BACKGROUND AND PURPOSE: The aim of this study is to investigate the efficacy and safety of preoperative versus intraoperative tirofiban in patients with large vessel occlusion (LVO) due to large artery atherosclerosis (LAA). METHODS: This is a retrospective multicenter cohort study based on the RESCUE-RE (Registration Study for Critical Care of Acute Ischemic Stroke After Recanalization) trial enrolling patients with anterior circulation LVO classified as LAA within 24 h of onset. Patients were divided into three groups: preoperative tirofiban (PT), intraoperative tirofiban (IT), and no tirofiban (NT). Propensity score matching (PSM) was used to balance baseline characteristics. The efficacy outcomes included 90-day functional independence (modified Rankin Scale score = 0-2) and early partial recanalization (EPR; defined as a modified Thrombolysis in Cerebral Infarction score = 1-2a). The safety outcomes included symptomatic intracranial hemorrhage (sICH). RESULTS: A total of 104 matched triplets were obtained through PSM. Compared with NT, PT increased 90-day functional independence (60.8% vs. 42.3%, p = 0.008) and EPR (42.7% vs. 18.3%, p < 0.001) rate, with a tendency to increase the asymptomatic intracranial hemorrhage (aICH) proportion (28.8% vs. 18.3%, p = 0.072). Compared with IT, PT had a higher 90-day functional independence (60.8% vs. 45.2%, p = 0.025) and EPR (42.7% vs. 20.2%, p = 0.001) rate, with no significant difference in sICH (14.4% vs. 7.7%, p = 0.122) and aICH (28.8% vs. 21.2%, p = 0.200). Compared with NT, IT had a lower 90-day mortality rate (9.6% vs. 24.0%, p = 0.005). CONCLUSIONS: Tirofiban shows good adjuvant therapy potential in acute ischemic stroke-LVO due to LAA patients. PT is associated with higher rates of EPR and better therapeutic efficacy. In addition, EPR may be a potential way to improve prognosis.

[Dongbai Tonglin Mixture for the treatment of chronic prostatitis].

OBJECTIVE: To evaluate the efficacy of Dongbai Tonglin Mixture (DTM) in the treatment of chronic prostatitis (CP) with the damp-heat downward diffusion syndrome. METHODS: We randomly selected 76 cases of CP with the damp-heat downward diffusion syndrome, equally divided them into a DTM and a control group, and treated them by oral administration of DTM and Qianlie Tai Tablets, respectively, both for 8 weeks. We obtained the NIH-CPSI and TCM Syndrome Scores of the patients, recorded the counts of white blood cells (WBC) and small particles of lecithin (SPL) in the prostate fluid, and compared them between the two groups before and after treatment. RESULTS: Compared with the baseline, the total NIH-CPSI scores were significantly reduced in both groups after treatment (P<0.05), particularly the scores on urination symptoms, pain / discomfort and quality of life (P<0.05), even more significantly in the DTM than in the control group (P<0.05), and so were the TCM Syndrome Scores (P<0.05), especially the scores on urinary incontinence, abdominal pain, perineal pain, and scrotal dampness (P<0.05), even more significantly in the former than in the latter group (P<0.05). The count of WBC in the prostate fluid was remarkably decreased (P<0.05), while that of SPL markedly increased in both groups after treatment (P<0.05), with an even more significant improvement in the DTM than in the control group (P<0.05), and the overall effectiveness rate of treatment was significantly higher in the former group than in the latter (88.89% vs 70.27%, P<0.05). CONCLUSION: Dongbai Tonglin Mixture is effective for the treatment of CP with the damp-heat downward diffusion syndrome.

Transition metal-free [3 + 3] annulation of cyclopropanols with ß-enamine esters to assemble nicotinate derivatives.

A metal-free and efficient approach for the synthesis of structurally important nicotinates through 4-HO-TEMPO-mediated [3 + 3] annulation of cyclopropanols with ß-enamine esters is presented. This protocol features high atom efficiency, green waste, simple operation and broad substrate scope. Moreover, the experiments of gram-scale synthesis and recovery of oxidants make this strategy more sustainable and practical.

The Importance of Tumor Necrosis Factor-α-Induced Protein-8 Like-2 in the Pathogenesis of Cervical Cancer and Preeclampsia via Regulation of Cell Invasion.

Tumor necrosis factor-α-induced protein-8 like-2 (TIPE2) as a novel negative immune regulator plays an important role in several human diseases. However, its influences in cervical cancer and preeclampsia (PE) remain unclear. This study aims to explore the important role of TIPE2 in cervical cancer and PE via regulating cell invasion. TIPE2 expression in the cervical cancer tissues or the placenta of PE patients was detected. Human cervical cancer cell lines and trophoblasts were transfected with adenovirus expressing human TIPE2 and green fluorescent protein (GFP) (Ad-TIPE2), or the control adenovirus expressing GFP (Ad-GFP). Xenograft models were also constructed on nude mice, aiming to clarify how TIPE2 affects in vivo growth of cervical cancer cells. TIPE2 was down-regulated in the tumor tissues or placenta of patients with cervical cancer or PE. As a result, CaSKi and Hela cells in the Ad-TIPE2 group had decreased migration and invasion, with significant up-regulations of TIPE2 and E-cadherin, but down-regulations of ß-catenin and N-cadherin. Ad-TIPE2 decreased the volume and weight of xenograft tumors in the nude mice, with the down-regulation of Ki67. The quantity of cells (HTR8/SVneo and JEG3 cells) transfected with Ad-TIPE2 had increased, with up-regulations of TIPE2, matrix metalloproteinase (MMP)-2 and MMP-9. TIPE2 overexpression could reduce the invasion and migration of cervical cancer cells via inhibiting the epithelial-mesenchymal transition (EMT) process, and promote trophocyte invasion via upregulating the expression of MMPs, and it may be used as a potential therapeutic target for cervical cancer and PE.

[Role of innate immunity in the pathogenesis of chronic prostatitis/chronic pelvic pain syndrome: Advances in studies].

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common disease in men, which has a high incidence and seriously affects the quality of life of the patients. The possible pathogenic factors of the disease include urine reflux, hidden infection, central sensitization, oxidative stress, and abnormal immune response, among which abnormal immune response plays a significant role in its development and progression. Immune response involves innate immunity and adaptive immunity, and most previous studies focused on adaptive immunity. In recent years, more and more attention has been paid to the role of the innate immune system in the pathogenesis of CP/CPPS. Studies show that mast cells, macrophages, Toll-like receptors and related cytokines in the innate immune system are all involved in the development and progression of CP/CPPS. As the innate immune system is the first barrier of the immune response of the body, studies on innate immunity will provide some new ideas for the diagnosis and treatment of CP/CPPS.

Comparison of Biochemical Characteristics, Action Models, and Enzymatic Mechanisms of a Novel Exolytic and Two Endolytic Lyases with Mannuronate Preference.

Recent explorations of tool-like alginate lyases have been focused on their oligosaccharide-yielding properties and corresponding mechanisms, whereas most were reported as endo-type with α-L-guluronate (G) preference. Less is known about the ß-D-mannuronate (M) preference, whose commercial production and enzyme application is limited. In this study, we elucidated Aly6 of Flammeovirga sp. strain MY04 as a novel M-preferred exolytic bifunctional lyase and compared it with AlgLs of Pseudomonas aeruginosa (Pae-AlgL) and Azotobacter vinelandii (Avi-AlgL), two typical M-specific endolytic lyases. This study demonstrated that the AlgL and heparinase_II_III modules play indispensable roles in determining the characteristics of the recombinant exo-type enzyme rAly6, which is preferred to degrade M-enriched substrates by continuously cleaving various monosaccharide units from the nonreducing end, thus yielding various size-defined ΔG-terminated oligosaccharides as intermediate products. By contrast, the endolytic enzymes Pae-rAlgL and Avi-rAlgL varied their action modes specifically against M-enriched substrates and finally degraded associated substrate chains into various size-defined oligosaccharides with a succession rule, changing from ΔM to ΔG-terminus when the product size increased. Furthermore, site-directed mutations and further protein structure tests indicated that H195NHSTW is an active, half-conserved, and essential enzyme motif. This study provided new insights into M-preferring lyases for novel resource discoveries, oligosaccharide preparations, and sequence determinations.

Predictive value of the surgical pleth index for the hemodynamic responses to trachea intubation and skin incision.

Surgical pleth index (SPI) has been widely investigated in assessing the nociceptive level. The aim of this study was to investigate the relationship between SPI level and patient responses to trachea intubation and skin incision. A total of 40 patients undergoing open abdominal general surgery were recruited for analyses. The patients were monitored with electrocardiogram, non-invasive blood pressure, SpO2, invasive blood pressure and SPI before anesthesia induction. Anesthesia was induced with midazolam, propofol, sufentanil and rocuronium and maintained with sufentanil and sevoflurane. Blood pressure, heart rate and SPI were recorded for analyses during the peri-intubation and peri-incision periods. A receiver operating characteristic (ROC) curve analysis was performed to analyze the predictive value of blood pressure, heart rate (HR) and SPI for hemodynamic responses for trachea intubation and skin incision. SPI had a similar changing trend to systolic blood pressure (SBP) and diastolic blood pressure (DBP). The SPI level was linearly correlated with SBP, DBP and HR. SPI increased significantly after intubation and incision in patients with positive but not negative responses to intubation and incision. The ROC analysis showed that only SBP level is predictive of intubation responses. These data suggested that SPI elevated under the noxious stimulation by intubation and incision, but it was not predictive of the hemodynamic responses to intubation and incision.

A chondroitin sulfate and hyaluronic acid lyase with poor activity to glucuronyl 4,6-O-disulfated N-acetylgalactosamine (E-type)-containing structures.

GlcUAß1-3GalNAc(4S,6S) (E unit)-rich domains have been shown to play key roles in various biological functions of chondroitin sulfate (CS). However, an enzyme that can specifically isolate such domains through the selective digestion of other domains in polysaccharides has not yet been reported. Here, we identified a glycosaminoglycan lyase from a marine bacterium Vibrio sp. FC509. This enzyme efficiently degraded hyaluronic acid (HA) and CS variants, but not E unit-rich CS-E, into unsaturated disaccharides; therefore, we designated this enzyme a CS-E-resisted HA/CS lyase (HCLase Er). We isolated a series of resistant oligosaccharides from the final product of a low-sulfated CS-E exhaustively digested by HCLase Er and found that the E units were dramatically accumulate in these resistant oligosaccharides. By determining the structures of several resistant tetrasaccharides, we observed that all of them possessed a Δ4,5HexUAα1-3GalNAc(4S,6S) at their non-reducing ends, indicating that the disulfation of GalNAc abrogates HCLase Er activity on the ß1-4 linkage between the E unit and the following disaccharide. Δ4,5HexUAα1-3GalNAc(4S,6S)ß1-4GlcUAß1-3GalNAc(4S,6S) was most strongly resistant to HCLase Er. To our knowledge, this study is the first reporting a glycosaminoglycan lyase specifically inhibited by both 4-O- and 6-O-sulfation of GalNAc. Site-directed and truncation mutagenesis experiments indicated that HCLase Er may use a general acid-base catalysis mechanism and that an extra domain (Gly739-Gln796) is critical for its activity. This enzyme will be a useful tool for structural analyses and for preparing bioactive oligosaccharides of HA and CS variants, particularly from E unit-rich CS chains.

Sequencing of chondroitin sulfate oligosaccharides using a novel exolyase from a marine bacterium that degrades hyaluronan and chondroitin sulfate/dermatan sulfate.

Glycosaminoglycans (GAGs) are a family of chemically heterogeneous polysaccharides that play important roles in physiological and pathological processes. Owing to the structural complexity of GAGs, their sophisticated chemical structures and biological functions have not been extensively studied. Lyases that cleave GAGs are important tools for structural analysis. Although various GAG lyases have been identified, exolytic lyases with unique enzymatic property are urgently needed for GAG sequencing. In the present study, a putative exolytic GAG lyase from a marine bacterium was recombinantly expressed and characterized in detail. Since it showed exolytic lyase activity toward hyaluronan (HA), chondroitin sulfate (CS), and dermatan sulfate (DS), it was designated as HCDLase. This novel exolyase exhibited the highest activity in Tris-HCl buffer (pH 7.0) at 30°C. Especially, it showed a specific activity that released 2-aminobenzamide (2-AB)-labeled disaccharides from the reducing end of 2-AB-labeled CS oligosaccharides, which suggest that HCDLase is not only a novel exolytic lyase that can split disaccharide residues from the reducing termini of sugar chains but also a useful tool for the sequencing of CS chains. Notably, HCDLase could not digest 2-AB-labeled oligosaccharides from HA, DS, or unsulfated chondroitin, which indicated that sulfates and bond types affect the catalytic activity of HCDLase. Finally, this enzyme combined with CSase ABC was successfully applied for the sequencing of several CS hexa- and octasaccharides with complex structures. The identification of HCDLase provides a useful tool for CS-related research and applications.

A Model Design for Risk Assessment of Line Tripping Caused by Wildfires.

A power line is particularly vulnerable to wildfires in its vicinity, and various damage including line tripping can be caused by wildfires. Using remote sensing techniques, a novel model developed to assess the risk of line tripping caused by the wildfire occurrence in high-voltage power line corridors is presented. This model mainly contains the wildfire risk assessment for power line corridors and the estimation of the probability of line tripping when a wildfire occurs in power line corridors. For the wildfire risk assessment, high-resolution satellite data, Moderate Resolution Imaging Spectroradiometer (MODIS) data, meteorological data, and digital elevation model (DEM) data were employed to infer the natural factors. Human factors were also included to achieve good reliability. In the estimation of the probability of line tripping, vegetation characteristics, meteorological status, topographic conditions, and transmission line parameters were chosen as influencing factors. According to the above input variables and observed historical datasets, the risk levels for wildfire occurrence and line tripping were obtained with a logic regression approach. The experimental results demonstrate that the developed model can provide good results in predicting wildfire occurrence and line tripping for high-voltage power line corridors.

Biochemical Characteristics and Variable Alginate-Degrading Modes of a Novel Bifunctional Endolytic Alginate Lyase.

Bifunctional alginate lyases can efficiently degrade alginate comprised of mannuronate (M) and guluronate (G), but their substrate-degrading modes have not been thoroughly elucidated to date. In this study, we present Aly1 as a novel bifunctional endolytic alginate lyase of the genus Flammeovirga The recombinant enzyme showed optimal activity at 50°C and pH 6.0. The enzyme produced unsaturated disaccharide (UDP2) and trisaccharide fractions as the final main alginate digests. Primary substrate preference tests and further structure identification of various size-defined final oligosaccharide products demonstrated that Aly1 is a bifunctional alginate lyase and prefers G to M. Tetrasaccharide-size fractions are the smallest substrates, and M, G, and UDP2 fractions are the minimal product types. Remarkably, Aly1 can vary its substrate-degrading modes in accordance with the terminus types, molecular sizes, and M/G contents of alginate substrates, producing a series of small size-defined saturated oligosaccharide products from the nonreducing ends of single or different saturated sugar chains and yielding unsaturated products in distinct but restricted patterns. The action mode changes can be partially inhibited by fluorescent labeling at the reducing ends of oligosaccharide substrates. Deletion of the noncatalytic region (NCR) of Aly1 caused weak changes of biochemical characteristics but increased the degradation proportions of small size-defined saturated M-enriched oligosaccharide substrates and unsaturated tetrasaccharide fractions without any size changes of degradable oligosaccharides, thereby enhancing the M preference and enzyme activity. Therefore, our results provided insight into the variable action mode of a novel bifunctional endolytic alginate lyase to inform accurate enzyme use.IMPORTANCE The elucidated endolytic alginate lyases usually degrade substrates into various size-defined unsaturated oligosaccharide products (≥UDP2), and exolytic enzymes yield primarily unsaturated monosaccharide products. However, it is poorly understood whether endolytic enzymes can produce monosaccharide product types when degrading alginate. In this study, we demonstrated that Aly1, a bifunctional alginate lyase of Flammeovirga sp. strain MY04, is endolytic and monosaccharide producing. Using various sugar chains as testing substrates, we also proved that key factors causing Aly1's action mode changes are the terminus types, molecular sizes, and M/G contents of substrates. Furthermore, the NCR fragment's effects on Aly1's biochemical characteristics and alginate-degrading modes and corresponding mechanisms were discovered by gene truncation and enzyme comparison. In summary, this study provides a novel bifunctional endolytic tool and a variable action mode for accurate use in alginate degradation.

Cloning and characterization of a novel chondroitin sulfate/dermatan sulfate 4-O-endosulfatase from a marine bacterium.

Sulfatases are potentially useful tools for structure-function studies of glycosaminoglycans (GAGs). To date, various GAG exosulfatases have been identified in eukaryotes and prokaryotes. However, endosulfatases that act on GAGs have rarely been reported. Recently, a novel HA and CS lyase (HCLase) was identified for the first time from a marine bacterium (Han, W., Wang, W., Zhao, M., Sugahara, K., and Li, F. (2014) J. Biol. Chem. 289, 27886-27898). In this study, a putative sulfatase gene, closely linked to the hclase gene in the genome, was recombinantly expressed and characterized in detail. The recombinant protein showed a specific N-acetylgalactosamine-4-O-sulfatase activity that removes 4-O-sulfate from both disaccharides and polysaccharides of chondroitin sulfate (CS)/dermatan sulfate (DS), suggesting that this sulfatase represents a novel endosulfatase. The novel endosulfatase exhibited maximal reaction rate in a phosphate buffer (pH 8.0) at 30 °C and effectively removed 17-65% of 4-O-sulfates from various CS and DS and thus significantly inhibited the interactions of CS and DS with a positively supercharged fluorescent protein. Moreover, this endosulfatase significantly promoted the digestion of CS by HCLase, suggesting that it enhances the digestion of CS/DS by the bacterium. Therefore, this endosulfatase is a potential tool for use in CS/DS-related studies and applications.

Novel Alginate Lyase (Aly5) from a Polysaccharide-Degrading Marine Bacterium, Flammeovirga sp. Strain MY04: Effects of Module Truncation on Biochemical Characteristics, Alginate Degradation Patterns, and Oligosaccharide-Yielding Properties.

Alginate lyases are important tools for oligosaccharide preparation, medical treatment, and energy bioconversion. Numerous alginate lyases have been elucidated. However, relatively little is known about their substrate degradation patterns and product-yielding properties, which is a limit to wider enzymatic applications and further enzyme improvements. Herein, we report the characterization and module truncation of Aly5, the first alginate lyase obtained from the polysaccharide-degrading bacterium Flammeovirga. Aly5 is a 566-amino-acid protein and belongs to a novel branch of the polysaccharide lyase 7 (PL7) superfamily. The protein rAly5 is an endolytic enzyme of alginate and associated oligosaccharides. It prefers guluronate (G) to mannuronate (M). Its smallest substrate is an unsaturated pentasaccharide, and its minimum product is an unsaturated disaccharide. The final alginate digests contain unsaturated oligosaccharides that generally range from disaccharides to heptasaccharides, with the tetrasaccharide fraction constituting the highest mass concentration. The disaccharide products are identified as ΔG units. While interestingly, the tri- and tetrasaccharide fractions each contain higher proportions of ΔG to ΔM ends, the larger final products contain only ΔM ends, which constitute a novel oligosaccharide-yielding property of guluronate lyases. The deletion of the noncatalytic region of Aly5 does not alter its M/G preference but significantly decreases the enzymatic activity and enzyme stability. Notably, the truncated protein accumulates large final oligosaccharide products but yields fewer small final products than Aly5, which are codetermined by its M/G preference to and size enlargement of degradable oligosaccharides. This study provides novel enzymatic properties and catalytic mechanisms of a guluronate lyase for potential uses and improvements.

Biochemical Characteristics and Substrate Degradation Pattern of a Novel Exo-Type ß-Agarase from the Polysaccharide-Degrading Marine Bacterium Flammeovirga sp. Strain MY04.

UNLABELLED: Exo-type agarases release disaccharide units (3,6-anhydro-l-galactopyranose-α-1,3-d-galactose) from the agarose chain and, in combination with endo-type agarases, play important roles in the processive degradation of agarose. Several exo-agarases have been identified. However, their substrate-degrading patterns and corresponding mechanisms are still unclear because of a lack of proper technologies for sugar chain analysis. Herein, we report the novel properties of AgaO, a disaccharide-producing agarase identified from the genus Flammeovirga AgaO is a 705-amino-acid protein that is unique to strain MY04. It shares sequence identities of less than 40% with reported GH50 ß-agarases. Recombinant AgaO (rAgaO) yields disaccharides as the sole final product when degrading agarose and associated oligosaccharides. Its smallest substrate is a neoagarotetraose, and its disaccharide/agarose conversion ratio is 0.5. Using fluorescence labeling and two-stage mass spectrometry analysis, we demonstrate that the disaccharide products are neoagarobiose products instead of agarobiose products, as verified by (13)C nuclear magnetic resonance spectrum analysis. Therefore, we provide a useful oligosaccharide sequencing method to determine the patterns of enzyme cleavage of glycosidic bonds. Moreover, AgaO produces neoagarobiose products by gradually cleaving the units from the nonreducing end of fluorescently labeled sugar chains, and so our method represents a novel biochemical visualization of the exolytic pattern of an agarase. Various truncated AgaO proteins lost their disaccharide-producing capabilities, indicating a strict structure-function relationship for the whole enzyme. This study provides insights into the novel catalytic mechanism and enzymatic properties of an exo-type ß-agarase for the benefit of potential future applications. IMPORTANCE: Exo-type agarases can degrade agarose to yield disaccharides almost exclusively, and therefore, they are important tools for disaccharide preparation. However, their enzymatic mechanisms and agarose degradation patterns are still unclear due to the lack of proper technologies for sugar chain analysis. In this study, AgaO was identified as an exo-type agarase of agarose-degrading Flammeovirga bacteria, representing a novel branch of glycoside hydrolase family 50. Using fluorescence labeling, high-performance liquid chromatography, and mass spectrum analysis technologies, we provide a useful oligosaccharide sequencing method to determine the patterns of enzyme cleavage of glycosidic bonds. We also demonstrate that AgaO produces neoagarobiose by gradually cleaving disaccharides from the nonreducing end of fluorescently labeled sugars. This study will benefit future enzyme applications and oligosaccharide studies.

[Efficacy and safety of Tonglin Powder in the treatment of benign prostatic hyperplasia].

OBJECTIVE: To investigate the effect and safety of oral Tonglin Powder in the treatment of benign prostatic hyperplasia (BPH). METHODS: We conducted a randomized controlled study on 100 BPH patients, aged 40－85 years, treated with Tonglin Powder (treatment group, n=50) or terazosin (control group, n=50), all for 3 months. Then we obtained the International Prostate Symptom Score (IPSS), quality of life score (QoL), prostate volume, postvoid residual urine volume (PVR), urine routine indexes, and liver and kidney function indexes from the patients and compared them between the two groups before and after treatment. RESULTS: The baseline data of the patients in the treatment and control groups were as follows, IPSS (22.24±7.33) vs (21.40±8.24), QoL 4 (2－6) vs 4 (2－6), prostate length 45 (30－65) vs 45 (39－65) mm, prostate width 35 (21－54) vs 36 (26－57) mm, and PVR 10 (5－100) vs 10 (10－100) ml, none with statistically significant difference between the two groups (P>0.05). After treatment, the patients of the treatment group, in comparison with those of the control, showed remarkable decreases in IPSS (11.60±6.49 vs 15.38±7.34, P=0.008) and QoL (2 ï¼»0－5ï¼½ vs 3 ï¼»1－6ï¼½, P=0.01). No statistically significant differences were observed between the treatment and control groups in prostate length (47 ï¼»38－67ï¼½ vs 47.5 ï¼»38－67ï¼½ mm), prostate width (36 ï¼»26－57ï¼½ vs 36.5 ï¼»31－57ï¼½ mm), and PVR (10 ï¼»8－100ï¼½ vs 10 ï¼»8－70ï¼½ ml) (P>0.05). The Nimodipine method of evaluation showed that the excellence rate of therapeutic effectiveness was significantly higher in the treatment than in the control group (40% vs 8%, P<0.001), and so was the total effectiveness rate (82% vs 64%, P=0.043). CONCLUSIONS: Tonglin Powder can effectively improve the symptoms of BPH, such as difficult urination, and hence the patient's quality of life.

A novel eliminase from a marine bacterium that degrades hyaluronan and chondroitin sulfate.

Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even though marine animals are rich in GAGs with unique structures and functions. Herein we isolated a novel GAG lyase for the first time from the marine bacterium Vibrio sp. FC509 and then recombinantly expressed and characterized it. It showed strong lyase activity toward hyaluronan (HA) and chondroitin sulfate (CS) and was designated as HA and CS lyase (HCLase). It exhibited the highest activities to both substrates at pH 8.0 and 0.5 m NaCl at 30 °C. Its activity toward HA was less sensitive to pH than its CS lyase activity. As with most other marine enzymes, HCLase is a halophilic enzyme and very stable at temperatures from 0 to 40 °C for up to 24 h, but its activity is independent of divalent metal ions. The specific activity of HCLase against HA and CS reached a markedly high level of hundreds of thousands units/mg of protein under optimum conditions. The HCLase-resistant tetrasaccharide Δ(4,5)HexUAα1-3GalNAc(6-O-sulfate)ß1-4GlcUA(2-O-sulfate)ß1-3GalNAc(6-O-sulfate) was isolated from CS-D, the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated d-glucuronic acid (GlcUA) in CS chains. Site-directed mutagenesis indicated that HCLase may work via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid. Thus, the identification of HCLase provides a useful tool for HA- and CS-related research and applications.

Supercharged fluorescent protein as a versatile probe for the detection of glycosaminoglycans in vitro and in vivo.

Glycosaminoglycans (GAGs) are linear acidic heteropolysaccharides that are ubiquitously expressed in animal tissues and participate in various life processes. To date, the detection and visualization of GAGs in complex biological samples and living organisms remain a challenge because of the lack of powerful biocompatible probes. In this study, a superpositively charged green fluorescent protein (ScGFP) was shown great potential in GAG detection for the first time. First, on the basis of the phenomenon of GAGs dose-dependently inhibiting the fluorescence quenching of ScGFP by graphene oxide, a simple and highly sensitive signal-on homogeneous platform was established for detecting and quantifying GAGs, even in complex samples such as heparin in citrated plasma and oversulfated chondroitin sulfate in heparin. Furthermore, ScGFP with excellent stability and biocompatibility could be easily used as a highly sensitive and selective probe to visualize different types of GAGs in vitro and in vivo through combination with specific GAG-degrading enzymes. This study introduces a versatile probe for GAG detection, which is easy to prepare and which shows a high practical value in basic research and medical applications.

An extra peptide within the catalytic module of a ß-agarase affects the agarose degradation pattern.

Agarase hydrolyzes agarose into a series of oligosaccharides with repeating disaccharide units. The glycoside hydrolase (GH) module of agarase is known to be responsible for its catalytic activity. However, variations in the composition of the GH module and its effects on enzymatic functions have been minimally elucidated. The agaG4 gene, cloned from the genome of the agarolytic Flammeovirga strain MY04, encodes a 503-amino acid protein, AgaG4. Compared with elucidated agarases, AgaG4 contains an extra peptide (Asn(246)-Gly(302)) within its GH module. Heterologously expressed AgaG4 (recombinant AgaG4; rAgaG4) was determined to be an endo-type ß-agarase. The protein degraded agarose into neoagarotetraose and neoagarohexaose at a final molar ratio of 1.5:1. Neoagarooctaose was the smallest substrate for rAgaG4, whereas neoagarotetraose was the minimal degradation product. Removing the extra fragment from the GH module led to the inability of the mutant (rAgaG4-T57) to degrade neoagarooctaose, and the final degradation products of agarose by the truncated protein were neoagarotetraose, neoagarohexaose, and neoagarooctaose at a final molar ratio of 2.7:2.8:1. The optimal temperature for agarose degradation also decreased to 40 °C for this mutant. Bioinformatic analysis suggested that tyrosine 276 within the extra fragment was a candidate active site residue for the enzymatic activity. Site-swapping experiments of Tyr(276) to 19 various other amino acids demonstrated that the characteristics of this residue were crucial for the AgaG4 degradation of agarose and the cleavage pattern of substrate.

[Study on monosaccharide composition of intracellular polysacchride and contents of cordycepin and cordyceps polysacchride produced by Cordyceps militaris induced by blue light].

OBJECTIVE: To study effects of blue light irradiation on monosaccharide composition of intracellular polysacchride and contents of cordycepin and cordyceps polysacchride of mycelium and sporocarp in Cordyceps militaris. METHODS: The monosaccharide composition of intracellular polysacchride of mycelium and sporocarp in Cordyceps militaris as materials were determined by gas chromatography after 144 h blue light irradiation. The contents of cordycepin and cordyceps polysacchride of mycelium and sporocarp in Cordyceps militaris were detected at different blue light irradiation periods. At the same time, the growth of mycelium and sporocarp in Cordyceps militaris were observed during blue light irradiation. RESULTS: Mycelium polysaccharide in Cordyceps militaris was a kind of heteropolysaccharide containing four kinds of monosaccharide and fruiting body polysaccharide was a kind of heteropolysaccharide containing five kinds of monosaccharide. Whether blue light irradiation or dark culture, the content changes of cordyceps polysacchride in two groups showed similar patterns in the test of mycelium polysaccharides. The content changes of cordyceps polysacchride in two groups were basically the same in the detection of sporocarp polysacchride. Cordycepin content in the two set of experiments of blue light irradiation all showed a clear upward trend in the detection of mycelium and sporocarp in Cordyceps militaris. CONCLUSION: The blue light irradiation has certain effect on the species and quantity of monosaccharide in intracellular polysaccharide. The content increase of cordycepin and cordyceps polysacchride in Cordyceps militaris are promoted by blue light irradiation. Blue light can help the morphogenesis and promote the differentiation and growth of sporocarp in Cordyceps militaris. This study is the first report about the effect of blue light on the type and quantity of the monosaccharide composition in polysaccharide of Cordyceps militaris, which will lay the foundation for further study on the metabolism of active substance in Cordyceps militaris by blue light irradiation.