Establishment of protoplasts transient expression system in Pinellia ternata (Thunb.) Breit.

OBJECTIVE: In this study, we established an efficient and rapid transient expression system in the protoplasts of Pinellia ternata (Thunb.) Breit. (P. ternata). RESULTS: The protoplasts of P. ternata were prepared from plant leaves as the source material by digesting them with the combination of 20 g·l-1 cellulase and 15 g·l-1 macerozyme for 6 h. Based on the screening of PEG concentration, the conditions for PEG-mediated protoplast transformation were improved, and the highest transformation efficiency was found for 40% PEG 4000. Furthermore, we used the subcellular protein localization technique in P. ternata protoplasts to allow further validation of transient expression system. CONCLUSIONS: We present the method that can be applicable for studying both gene verification and expression in P. ternata protoplasts, thus allowing for engineering the improved varieties of P. ternata through molecular plant breeding techniques. This method can also be widely applicable for analyzing protein interactions, detecting promoter activity, for somatic cell fusion in plant breeding, as well as for other related studies.

First report of Fusarium solani (FSSC 6) associated with the root rot of Magnolia denudata in China.

Magnolia denudata (Lilytree or Yulan magnolia) is an important ornamental species of the genus Magnolia. It has considerable economical value because of its beautiful fragrant flowers and excellent tree structure (Wang et al. 2010). In Beijing, nurseries cultivate M. denudata as an ornamental plant and traditional medicine. In May 2020, patches of root rotted plants were observed in a field in Beijing, China, with an estimated incidence of approximately 31%. Early symptoms comprised leaves melanocratic shrunken, and the vascular tissue of roots turned brown. Progressively, the roots rotted and the whole plant died (Fig. 1 a-d). Infected roots tissue was surface disinfested and plated on potato dextrose agar (PDA) medium at 25±2 °C and incubated in the dark for 7 days. Pure cultures were obtained by hyphal tip excision (strain MFR1215.4). Fungal colonies were entire margins, and the aerial mycelium was copious, early white, and gradually developed into cream white. Colonies developed to 45.1 mm in 4 days at 25±2 °C on PDA media. On Spezieller Nährstoffarmer Agar (SNA) medium at 25±2 °C for 10 days. The morphological characteristics including macroconidia, microconidia, and chlamydospores were shown in Fig.1 (i-p). These morphological characteristics of the isolate corresponded to the description given for Fusarium solani sensu lato (Nelson et al. 1983, Summerell, 2003). Molecular identification was confirmed via amplification of translation elongation factor 1α (EF-1α), RNA polymerase I beta subunit gene (RPB1), and RNA polymerase II beta subunit gene (RPB2) regions using EF1/EF2, RPB1-Fa/G2R, RPB2-5f2/7cR, and RPB2-7cF/11aR primers (O'Donnell, 2010). Sequences were registered in GenBank. In the Fusarium-ID database, the EF-1α, RPB1, and RPB2 sequences showed 100% (677/677 bp), 99.8% (1568/1571 bp), and 100% (1457/1457 bp) identity with the F. solani species complex (FSSC). The same species-level identification was also found using Fusarium MLST. A best maximum likelihood tree was constructed using PhyloSuite v1.2.2 (Zhang et al. 2020), and the sequences of the MFR1215.4 isolation showed the same homology with FSSC 6. Pathogenicity tests were conducted on healthy one-year-old M. denudata potted seedlings. 200 ml spore suspension (1×106 spores/ml) was poured over the roots of twenty seedlings, and sterile distilled water was irrigated into twenty seedlings as controls in a greenhouse with 25/15°C day/night temperature and 80% relative humidity. The experiment was repeated three times. All inoculated seedlings showed similar symptoms to those in the field after 65 days, whereas the controls remained symptomless. The reisolating pathogens from symptomatic tissues were identical to the original isolates by morphology and EF-1α sequence identification. Based on morphological, molecular, and pathogenic characterization, the isolated pathogen was identified as FSSC 6. Fusarium species have been recorded in various places of the world and are known to be harmful to numerous plants (Trabelsi et al. 2017). It has been reported that FSSC has infected soybeans (Coleman, 2016, Nelson et al. 1989), oil palm (Hafizi et al. 2013), tobacco (Yang et al. 2020), resulting in sudden death syndrome, crown disease, and root rot. To our knowledge, this is the first report of FSSC-induced root rot in M. denudata in China. This research may contribute to the development of epidemiology and management strategies for root rot caused by FSSC on M. denudata.

First Report of Leaf Spot Caused by Colletotrichum fructicola on Magnolia wufengensis in Hubei, China.

Magnolia wufengensis belongs to the Magnoliaceae family. Its variation-rich flowers (tepal number from 9 to 46, tepal color from pink to bright red) and excellent wood characteristics (strong, straight, texture) have important ornamental and economic value (Duan et al. 2019; Luyi et al. 2006). M. wufengensis is popularly cultivated in parks, courtyards, mountains, and along roadsides. In May 2020, leaf spot symptoms were observed on over 85% of M. wufengensis in Yuyangguan Township, Wufeng County, Hubei Province (110.60°E, 30.21°N). The damaged area was over 18.7 hectares. Early symptoms began as small brown spots with a light-yellow halo. Gradual lesions expanded, and the center was withered, gray, and necrotic with a dark brown border. Eventually, several spots combined with larger irregular lesions, turning the leaves yellow and causing them to fall off. The border of lesions and healthy tissues were cut into small pieces (5×5 mm), and surface sterilized with 1% sodium hypochlorite solution for three minutes, rinsed three times with sterile water, and plated on potato dextrose agar (PDA) medium at 25±2 °C with a 12h photoperiod under fluorescent lighting. Pure isolates (MCS1228.1, MCS1228.4, MCS1228.9) were gray to pale grayish, and their average growth rate was 10.5±1.23 mm/day. Conidiophores were hyaline, aseptate, branched. Conidia were hyaline, aseptate, cylindrical, and 14.00 to 25.17 × 4.74 to 6.56 µm in size (average 17.48 × 5.58 µm) (n=50). Appressoria were brown and showed multivariate shape. The morphological characteristics of the isolates corresponded to the description given for Colletotrichum fructicola (Liu et al. 2015). Molecular identification was accomplished through amplification of the internal transcribed spacer (IST), actin (ACT), calmodulin (CAL), chitin synthase (CHS-1) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin (TUB2) genes (Fu et al. 2018). The ITS (OL800580.1, OL800581.1, OL800582.1), ACT (GenBank accession No. OL873155- OL873157), CAL (GenBank accession No. OL873158- OL873160), CHS-1 (GenBank accession No. OL873161- OL873163), GAPDH (GenBank accession No. OL873164- OL873166) and TUB2 (GenBank accession No. OL873167- OL873169) sequences were deposited in GenBank. A Bayesian inference phylogenetic tree based on multilocus sequences was constructed, and the sequences of the 3 isolations showed the same homology with C. fructicola (Fu et al. 2018). To fulfill Koch's postulates, 30 potted seedlings were inoculated with 1×10^6 conidia/ml suspension of each isolate by spraying the leaves, and 30 potted seedlings were sprayed with sterile distilled water as control. Inoculated and control plants were kept in a greenhouse with 25/15°C (day/night) temperature and 80% relative humidity. In addition, 30 healthy detached leaves free of pests and diseases were washed three times with sterile distilled water, air-dried, and artificially inoculated using a 6 mm (diameter) PDA medium (5 days incubation) with mycelium. 30 leaves were inoculated with sterile PDA medium as control. All leaves were sprayed with sterile water every 24 hours, covered with plastic wrap, and incubated at 25±2 °C, 100% humidity. The experiment was repeated three times. Similar symptoms to those found initially were both observed on all the inoculated potted seedlings and detached leaves after 14 days and 5 days post inoculation (dpi), respectively. Whereas the controls remained symptomless. The reisolated pathogens from symptomatic tissues were identical to the original isolates. In this study, isolated fungi associated with M. wufengensis leaf spot were identified as C. fructicola based on morphological and multiloci phylogenetic analyses, and Koch's postulates. Colletotrichum species are important plant pathogens and cause diseases in a wide variety of woody and herbaceous plants (Cannon et al. 2012). C. fructicola has been identified as a responsible pathogen for apple (Casanova et al. 2016), Fatsia japonica (Shi et al. 2017), and Rubus corchorifolius (Wu et al. 2021) leaf spot. To our knowledge, this is the first report of C. fructicola causing leaf spot in M. wufengensis in China. This research may contribute to the development of management strategies for this disease.

Genomic and physiological analysis reveals versatile metabolic capacity of deep-sea Photobacterium phosphoreum ANT-2200.

Bacteria of the genus Photobacterium thrive worldwide in oceans and show substantial eco-physiological diversity including free-living, symbiotic and piezophilic life styles. Genomic characteristics underlying this variability across species are poorly understood. Here we carried out genomic and physiological analysis of Photobacterium phosphoreum strain ANT-2200, the first deep-sea luminous bacterium of which the genome has been sequenced. Using optical mapping we updated the genomic data and reassembled it into two chromosomes and a large plasmid. Genomic analysis revealed a versatile energy metabolic potential and physiological analysis confirmed its growth capacity by deriving energy from fermentation of glucose or maltose, by respiration with formate as electron donor and trimethlyamine N-oxide (TMAO), nitrate or fumarate as electron acceptors, or by chemo-organo-heterotrophic growth in rich media. Despite that it was isolated at a site with saturated dissolved oxygen, the ANT-2200 strain possesses four gene clusters coding for typical anaerobic enzymes, the TMAO reductases. Elevated hydrostatic pressure enhances the TMAO reductase activity, mainly due to the increase of isoenzyme TorA1. The high copy number of the TMAO reductase isoenzymes and pressure-enhanced activity might imply a strategy developed by bacteria to adapt to deep-sea habitats where the instant TMAO availability may increase with depth.

Comparison of AMG 416 and cinacalcet in rodent models of uremia.

BACKGROUND: AMG 416 is a novel peptide agonist of the calcium-sensing receptor (CaSR). This report describes the activity of AMG 416 in two different rodent models of uremia, compared in each case to cinacalcet, an approved therapeutic for secondary hyperparathyroidism (SHPT) in patients with chronic kidney disease on dialysis. METHODS: AMG 416 was administered as a single intravenous (IV) bolus in a severe, acute model of renal insufficiency (the "1K1C" model) and plasma parathyroid hormone (PTH) and serum calcium levels were monitored for 24 hours. In a chronic, less severe model of renal dysfunction, the 5/6 nephrectomy (5/6 Nx) model, AMG 416 was administered as a once-daily IV bolus for 28 days. Both studies included a control (vehicle) group and a comparison cinacalcet group (po dosing at 30 mg/kg and 10 mg/kg for the 1K1C and 5/6 Nx studies, respectively). RESULTS: Administration of AMG 416 by IV bolus injection into rats with acute renal dysfunction (1K1C model) resulted in a sustained reduction in plasma PTH from the initial elevated values. Following a single IV bolus (0.5 mg/kg), AMG 416 caused a substantial drop in PTH levels which remained approximately 50% below their initial level at 24 hrs. In the same model, oral treatment with cinacalcet (30 mg/kg) resulted in an acute drop in PTH which almost returned to the starting level by 24 hours after dosing. In the 5/6 Nx chronic uremia model, daily IV dosing of AMG 416 over 4 weeks (1 mg/kg) resulted in a sustained reduction in PTH, with approximately 50% of the initial level observed 48 hours post treatment throughout the study. Cinacalcet treatment (10 mg/kg) in the same model resulted in acutely lowered plasma PTH levels which returned to placebo levels by 24 hours post-dose. Consistent with the reductions in plasma PTH, reductions in serum calcium were observed in both AMG 416- and cinacalcet-treated animals. CONCLUSIONS: As a long-acting CaSR agonist suitable for administration by the IV route, AMG 416 is a potential new therapy for the treatment of CKD patients with SHPT receiving hemodialysis.

A meta-analysis of antibiotic residues in the Beibu Gulf.

Antibiotic residue stands as a significant ongoing environmental issue, with aquaculture being a major source of annual antibiotic discharge into the ocean. Nevertheless, there is still an incomplete evaluation of antibiotic residues in the Beibu Gulf, an area encompassed by two prominent aquaculture nations, China and Vietnam. The present systematic review and meta-analysis was conducted to examine the presence antibiotic residues in the Beibu Gulf based on published studies. Data were obtained through eight databases up to December 19th, 2023, and were updated on April 15th, 2024. The pooled concentration of antibiotic residues in seawater was 5.90 (ng/L), ranging from 5.73 to 6.06 (ng/L), and was 8.03 (ng/g), ranging from 7.77 to 8.28 (ng/g) in sediments. Fluoroquinolones, tetracyclines, and macrolides were identified as the main antibiotics found in both seawater and sediment samples. The Beibu Gulf showed higher antibiotic levels in its western and northeastern areas. Additionally, the nearshore mangrove areas displayed the highest prevalence of antibiotic residues. It is strongly advised to conduct regular long-term monitoring of antibiotic residues in the Beibu Gulf. Collaborative surveys covering the entire Beibu Gulf involving China and Vietnam are recommended.

Pharmacology of AMG 416 (Velcalcetide), a novel peptide agonist of the calcium-sensing receptor, for the treatment of secondary hyperparathyroidism in hemodialysis patients.

A novel peptide, AMG 416 (formerly KAI-4169, and with a United States Adopted Name: velcalcetide), has been identified that acts as an agonist of the calcium-sensing receptor (CaSR). This article summarizes the in vitro and in vivo characterization of AMG 416 activity and the potential clinical utility of this novel compound. AMG 416 activates the human CaSR in vitro, acting by a mechanism distinct from that of cinacalcet, the only approved calcimimetic, since it can activate the CaSR both in the presence or the absence of physiologic levels of extracellular calcium. Administration of AMG 416 in vivo into either normal or renally compromised rats results in dose-dependent reductions in parathyroid hormone (PTH) levels and corresponding decreases in serum calcium, regardless of the baseline level of PTH. Treatment of 5/6 nephrectomized rats with AMG 416 resulted in dramatic improvements in their metabolic profile, including lower PTH and serum creatinine levels, reduced amounts of vascular calcification, attenuated parathyroid hyperplasia, and greater expression of the parathyroid gland regulators CaSR, vitamin D receptor, and FGF23 receptor compared with vehicle-treated animals. No drug accumulation was observed under this dosing regimen, and the terminal half-life of AMG 416 was estimated to be 2-4.5 hours. As a long-acting CaSR agonist, AMG 416 is an innovative new therapy for the treatment of hemodialysis patients with secondary hyperparathyroidism.

Complete genome sequence of marine Roseobacter lineage member Ruegeria sp. YS9 with five plasmids isolated from red algae.

Ruegeria sp. YS9, an aerobic and chemoheterotrophic bacterium belonging to marine Roseobacter lineage, was a putative new species isolated from red algae Eucheuma okamurai in the South China Sea (Beihai, Guangxi province). The complete genome sequence in strain YS9 comprised one circular chromosome with 3,244,635 bp and five circular plasmids ranging from 38,085 to 748,160 bp, with a total length of 4.30 Mb and average GC content of 58.39%. In total, 4129 CDSs, 52 tRNA genes and 9 rRNA genes were obtained. Genomic analysis of strain YS9 revealed that 85 CAZymes were organized in 147 PUL-associated CAZymes involved in polysaccharides metabolism, which were the highest among its two closely related Ruegeria strains. Numerous PULs related to degradation on the cell wall of algae, especially agar, indicated its major player role in the remineralization of algal-derived carbon. Further, the existence of multiple plasmids provided strain YS9 with distinct advantages to facilitate its rapid environmental adaptation, including polysaccharide metabolism, denitrification, resistance to heavy metal stresses such as copper and cobalt, type IV secretion systems and type IV toxin-antitoxin systems, which were obviously different from the two Ruegeria strains. This study provides evidence for polysaccharide metabolic capacity and functions of five plasmids in strain YS9, broadening our understanding of the ecological roles of bacteria in the environment around red algae and the function patterns of plasmids in marine Roseobacter lineage members for environmental adaptation.

Species-specificity of the secondary biosynthetic potential in Bacillus.

Introduction: Although Bacillus species have produced a wide variety of structurally diverse and biologically active natural products, the secondary biosynthetic potential of Bacillus species is widely underestimated due to the limited number of biosynthetic gene clusters (BGCs) in this genus. The significant variation in the diversity and novelty of BGCs across different species within the Bacillus genus presents a major obstacle to the efficient discovery of novel natural products from Bacillus. Methods: In this study, the number of each class of BGCs in all 6,378 high-quality Bacillus genomes was predicted using antiSMASH, the species-specificity of BGC distribution in Bacillus was investigated by Principal component analysis. Then the structural diversity and novelty of the predicted secondary metabolites in Bacillus species with specific BGC distributions were analyzed using molecular networking. Results: Our results revealed a certain degree of species-specificity in the distribution of BGCs in Bacillus, which was mainly contributed by siderophore, type III polyketide synthase (T3PKS), and transAT-PKS BGCs. B. wiedmannii, B. thuringiensis, and B. cereus are rich in RiPP-like and siderophore BGCs, but lack T3PKS BGCs, while B. amyloliquefaciens and B. velezensis are abundant in transAT-PKS BGCs. These Bacillus species collectively encode 77,541 BGCs, with NRPS and RiPPs being the two most dominant types, which are further categorized into 4,291 GCFs. Remarkably, approximately 54.5% of GCFs and 93.8% of the predicted metabolite scaffolds are found exclusively in a single Bacillus species. Notably, B. cereus, B. thuringiensis, and B. velezensis exhibit the highest potential for producing species-specific NRPS and PKS bioinformatic natural products. Taking two species-specific NRPS gene clusters as examples, the potential of Bacillus to synthesize novel species-specific natural products is illustrated. Conclusion: This study highlights the species-specificity of the secondary biosynthetic potential in Bacillus and provides valuable insights for the targeted discovery of novel natural products from this genus.

Complete genome of Rossellomorea sp. DA94, an agarolytic and orange-pigmented bacterium isolated from mangrove sediment of the South China Sea.

Rossellomorea sp. DA94, isolated from mangrove sediment in the South China Sea (Beihai, Guangxi province), is an agarolytic and orange-pigmented bacterium. Here, we present the complete genome sequence of strain DA94, which comprises 4.63 Mb sequences with 43.5% GC content. In total, 4589 CDSs, 33 rRNA genes and 110 tRNA genes were obtained. Genomic analysis of strain DA94 revealed that 108 CAZymes were organized in 4578 PULs involved in polysaccharides degradation, transport, and regulation. Further, we performed the diversity of CAZymes and PULs comparison among Rossellomorea strains. Less CAZymes were organized more PULs, indicating highly efficiently polysaccharides utilization in Rossellomorea. Meanwhile, PUL0459, PUL0460 and PUL0316 related to agar degradation, and exolytic beta-agarase GH50, endo-type beta-agarase GH86 and arylsulfatase were identified in the genome of strain DA94. We verified that strain DA94 can degrade agar to form a bright clear zone around the bacterial colonies in the laboratory. Moreover, the carotenoid biosynthetic pathways were proposed, which may be responsible for orange-pigment of Rossellomorea sp. DA94. This study represents a thorough genomic characterization of CAZymes repertoire and carotenoid biosynthetic pathways of Rossellomorea, provides insight into diversity of related enzymes and their potential biotechnological applications.

The eradicating Helicobacter pylori infection in duodenal ulcer patients by three short-term triple therapies in China: a multicenter clinical comparative study.

BACKGROUND/AIMS: This study aimed to compare the 7d triple therapy with 3d and 5d triple therapies, to observe the effect of eradicating Helicobacter pylori (Hp) on treating duodenal ulcers. METHODOLOGY: One hundred and sixteen patients who were confirmed duodenal ulcer active period and Hp positive were enrolled in the study. All the patients were divided into three groups: 3d group (n=39), 5d group (n=37) and 7d control group (n=40). All three groups were provided triple therapy first: rabeprazole, 10mg + furazolidone, 100mg + clarithromycin 250mg, twice a day for three days, five days and seven days, respectively. Then rabeprazole 10mg was provided once a day. Following the treatment, 13C urea breath test was performed to observe the Hp eradication rate. The symptoms of patients such as epigastralgia, burning pain and acidity were evaluated. RESULTS: The Hp eradication rate was: 3d group 76% (28/37), 5d 89% (31/35) and 7d 91% (32/35). There was no significant difference between 5d and 7d group (p>0.05). But the rate of groups 5d and 7d was significantly higher than group 3d (p<0.05). All the three groups showed an improvement in symptoms such as epigastralgia, burning pain and acidity. CONCLUSIONS: All three therapy schemes could alleviate symptoms of duodenal ulcer patients efficiently. But as far as eradicating Hp concerned, 5d and 7d therapies were better than 3d.

Chloroplast genome structure and phylogenetic position of Syringodium isoetifolium (Asch.) Dandy.

Syringodium isoetifolium (noodle seagrass) is a dioecious perennial seagrass. In this study, the complete chloroplast genome of S. isoetifolium was successfully characterized through next-generation sequencing technology. The cp genome was 159,333 bp in length with a GC content of 35.9%, including LSC (89,055 bp), SSC (19,160 bp), and two IRs (25,559 bp). The genome encoded 131 function genes, including 86 protein-coding genes, 37 tRNA genes, and eight rRNA genes. The phylogenetic analysis indicated that S. isoetifolium was clustered with Zostera and Ruppia.

Complete genome sequence and Carbohydrate Active Enzymes (CAZymes) repertoire of Gilvimarinus sp. DA14 isolated from the South China Sea.

Gilvimarinus sp. DA14, a putative new species isolated from mangrove sediment in the South China Sea (Beihai, Guangxi province), is an aerobic and heterotrophic agar degrading bacterium. Here, we present the complete genome sequence of strain DA14, which comprises 3.96 Mb sequences with 53.39% GC content. In total, 3391 CDSs, 6 rRNA genes and 44 tRNA genes were obtained. Genomic analysis of strain DA14 revealed that 218 CAZymes classes were identified and they were organized in 371 CAZymes in PULs involved in polysaccharides degradation, transport and regulation. Further, we performed the genome comparison among Gilvimarinus strains and analysis the diversity of CAZymes and PULs. Meanwhile, ability of agar and alginate degradation in strain DA14 were analyzed. This study represents a thorough genomic characterization of CAZymes repertoire of Gilvimarinus, provides insight into diversity of polysaccharide degrading enzymes existing in Gilvimarinus sp. DA14 and their biotechnological applications.

High Hydrostatic Pressure Inducible Trimethylamine N-Oxide Reductase Improves the Pressure Tolerance of Piezosensitive Bacteria Vibrio fluvialis.

High hydrostatic pressure (HHP) exerts severe effects on cellular processes including impaired cell division, abolished motility and affected enzymatic activities. Transcriptomic and proteomic analyses showed that bacteria switch the expression of genes involved in multiple energy metabolism pathways to cope with HHP. We sought evidence of a changing bacterial metabolism by supplying appropriate substrates that might have beneficial effects on the bacterial lifestyle at elevated pressure. We isolated a piezosensitive marine bacterium Vibrio fluvialis strain QY27 from the South China Sea. When trimethylamine N-oxide (TMAO) was used as an electron acceptor for energy metabolism, QY27 exhibited a piezophilic-like phenotype with an optimal growth at 30 MPa. Raman spectrometry and biochemistry analyses revealed that both the efficiency of the TMAO metabolism and the activity of the TMAO reductase increased under high pressure conditions. Among the two genes coding for TMAO reductase catalytic subunits, the expression level and enzymatic activity of TorA was up-regulated by elevated pressure. Furthermore, a genetic interference assay with the CRISPR-dCas9 system demonstrated that TorA is essential for underpinning the improved pressure tolerance of QY27. We extended the study to Vibrio fluvialis type strain ATCC33809 and observed the same phenotype of TMAO-metabolism improved the pressure tolerance. These results provide compelling evidence for the determinant role of metabolism in the adaption of bacteria to the deep-sea ecosystems with HHP.

[Role of caspase-1 and cytokines activated by caspase-1 in brain injury of the developing rats following recurrent seizures].

OBJECTIVE: The expressions of caspase-1 and cytokines activated by caspase-1 are associated with the pathophysiology of many diseases for its proinflammatory and proapototic peculiarity. However its relationship to brain injury of developing rats following recurrent seizures has not yet been identified. This study aimed to investigate the role of caspase-1 and cytokines activated by caspase-1 in brain injury of developing rats following recurrent seizures. METHODS: A total of 96 postnatal 20 day Sprague-Dawley rats were randomly assigned into Control and Seizure groups. Seizures were induced in the Seizure group by flurothyl inhalation daily for six days. Brain tissues were sampled at 6 hrs, and at 1, 3, and 7 days after last seizure. The expressions of caspase-1, interleukin (IL)-18 and IL-1beta mRNA in the cerebral cortex were detected by RT-PCR. The water content of the brain and the pathological changes of cortex nerve cells were observed. Brain injury was evaluated using a semiquantitative neuropathological scoring system. RESULTS: The levels of caspase-1 and IL-18 mRNA in the cerebral cortex of the Seizure group were obviously higher than those in the Control group at 6 hrs, and at 1, 3, and 7 days after seizure (P < 0.05 or P < 0.01). The expression of IL-1beta mRNA in the Seizure group exhibited a biphasic pattern: increased significantly at 6 hrs, and at 1 and 7 days post-seizure (P < 0.01), but was not significantly different from the Control group at 3 days post-seizure. Edema, degeneration and necrosis of nerve cells in cerebral cortex, accompanying by inflammatory cell infiltration and apoptosis of nerve cells, were observed under a light microscope in the Seizure group after recurrent seizures. The water content of the brain in the Seizure group increased significantly compared with that in the Control group at 6 hrs, and at 1 and 3 days after recurrent seizures (P < 0.01). The Seizure group had significantly higher neuropathological scores than the Control group at each time point (P < 0.01). CONCLUSIONS: Caspase-1 and cytokines activated by caspase-1 play an important role in the developing brain injury after recurrent seizures.

[Effects of neonatal recurrent seizures on gamma-aminobutyric acid B1 receptor expression in the rat brain].

OBJECTIVE: This study investigated the effects of flurothyl-induced neonatal recurrent seizures on gamma-aminobutyric acid B1 receptor (GABAB1R) expression in neonatal and adult rat brain, and explored the possible relationship between the alterations of GABAB1R in mature brain and the changes of spatial memory and seizure susceptibility in adult rats. METHODS: Forty-eight postnatal day (P) 7 Sprague-Dawley rats were randomly assigned into two groups: Control and Seizure group (n=24 each). Seizures were induced by inhalant flurothyl daily for six consecutive days in rat pups from the Seizure group. Twelve rats selected randomly in each group were sacrificed on the 7th day after the last seizure for detecting the expressions of GABAB1R mRNA and protein in cerebral cortex and hippocampus by reverse transcription-polymerase chain reaction (RT-PCR) and immuno-histochemistry method. The spatial memory was tested by using the Morris water maze task during P61 to P64 and the seizure threshold was measured at P75 following intraperitoneal injection of pentylenetetrazol ( PTZ ) in the remaining rats. The rats were then sacrificed for detecting the expressions of GABAB1R mRNA and protein in cerebral cortex and hippocampus. RESULTS: The expressions of GABAB1R mRNA and protein in the cerebral cortex on the 7th day after the last seizure and at P75 decreased significantly in the Seizure group when compared with the Control group (P < 0.05). The GABAB1R protein expression in the dentate gyrus on the 7th day after the last seizure in the Seizure group was significantly lower than that in the Control group (P < 0.05), but the GABAB1R mRNA expression in the hippocampus was not different from that in the Control group. There were no significant differences in the expressions of GABAB1R mRNA and protein in the hippocampus between the two groups at P75. The escape latencies in water maze of the rats in the Seizure group at P64 were significantly longer than those in the Control group (98,533.8 +/- 27,205.4 ms vs 46,723.3 +/- 40,666.5 ms; P <0.05). There were no differences in the seizure threshold between the two groups. CONCLUSIONS: The expressions of GABAB1R mRNA and protein in the cerebral cortex and hippocampus of neonatal rats with recurrent seizures decreased significantly, suggesting the changes of GABAB1R may be related to acute brain injury following neonatal recurrent seizures and the memory deficit in adult rats caused by neonatal recurrent seizures.

[Evaluation method of CT number linearity].

OBJECTIVE: To explore the method for computed tomography (CT) number linearity evaluation. METHODS: The model CTP401 of Catphan500 phantom was adopted to test 43 sets of CT scanners under the same scanning conditions. 1- and 2-order curve fitting of the testing results of each scanner were performed, with the bending of the 2-order fitted curve defined as the degree of CT number non-linearity and the slope of the linear fitting as contrast scale. The 2-norms of the fitting coefficients were calculated for the purpose of fitting quality evaluation. MATLAB 6.1 was employed for programming. RESULTS: The 2-norm of the fitting coefficients of the 2-order fitting curve was approximately 1/10 of that of corresponding linear fitting, indicating that 2-order fitting accorded better to the parent population distribution pattern. The degree of the non-linearity was manifested by the bending of the 2-order fitting curve that increased monotonously with the 2-norm of the corresponding linear fitting. CONCLUSION: It is statistically valid to define CT number non-linearity and contrast scale on the basis of 1 and 2-order fittings of the testing results, which well serves the purpose of quantitative analysis.

Serum tumor markers, hypoxia-inducible factor-1α HIF-1α and vascular endothelial growth factor, in patients with non- small cell lung cancer before and after intervention.

OBJECTIVE: To explore changes in the serum tumor makers, hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) level and their relations in patients with non-small cell lung cancer (NSCLC) before and after intervention. MATERIALS AND METHODS: Forty patients with NSCLC and 40 healthy individuals undergoing physical examination in our hospital provided the observation and control groups. HIF-1α and VEGF levels in serum were detected by enzyme-linked immuno-sorbent assay (ELISA) in the observation group before and after intervention and in control group on the day of physical examination, along with serum carcino-embryonic antigen (CEA), neuron-specific enolase (NSE) and squamous cell carcinoma antigen (SCC) levels in the observation group with a fully automatic biochemical analyzer. Clinical effects and improvement of life quality in the observation group were also evaluated. RESULTS: The total effective rate and improvement of life quality after treatment in observation group were 30.0% and 32.5%, respectively. Serum HIF-1α and VEGF levels in the control group were lower than that in observation group (p<0.01), but remarkably elevated after intervention (p<0.01). In addition, serum CEA, NSE and SCC levels were apparently lowered by treatment (p<0.01). Serum HIF-1α demonstrated a positive relation with VEGF level (p<0.01) and was inversely related with CEA, NSE and SCC levels (p<0.01). CONCLUSIONS: Significant correlations exist between marked increase of serum HIF-1α and VEGF levels and decrease of indexes related to hematological tumor markers in NSCLC patients after intervention.

TNF-α increases bone marrow mesenchymal stem cell migration to ischemic tissues.

The objective of this study was to analyze the influence of TNF-α on rat mesenchymal stem cells (MSCs) and to assess feasibility of MSC transplantation to repair ischemic injury. In this study, adhesion molecules and cell specific surface markers on MSCs were measured after exposure to different concentrations of TNF-α. MSCs stimulated with varying concentrations of TNF-α were cultured with aortic endothelial cells, and the adhesion rate was measured. MSCs were then stimulated with an optimum concentration of TNF-α as determined in vitro, and injected intravenously into rats with ischemic hind limb injury. The number of MSCs in muscle samples from the ischemic area was counted. The results showed that (1) TNF-α induced a concentration-dependent increase in VCAM-1 expression in MSCs, whereas the expression of L-selectin, ICAM-1 and VLA-4 did not change significantly. Expression of MSC-specific antigens was unchanged. (2) MSCs pretreated with 10 ng/ml TNF-α showed significantly increased adhesion to endothelial cells in vitro, and accumulated to a greater extent in the areas of ischemic damage in rat hind limbs. We were able to conclude that TNF-α has no effect on expression of MSC-specific markers, but can increase the expression of VCAM-1 on rat MSCs. Suitable concentrations of TNF-α can promote MSC adhesion to endothelial cells and migration to damaged tissue.