The T120P or M172V mutation on rv2172c confers high level para-aminosalicylic acid resistance in Mycobacterium tuberculosis.

AbstractAlthough para-aminosalicylic acid (PAS) has been used to treat tuberculosis for decades, mechanisms of resistance to this drug in Mycobacterium tuberculosis (M. tuberculosis) clinical isolates have not been thoroughly investigated. Previously, we found that decreased methylenetetrahydrofolate reductase (MTHFR) activity of Rv2172c led to increased sensitivity to antifolates in M. tuberculosis. In this study, we collected the genome-sequencing data of 173 PAS-resistant and 803 PAS-sensitive clinical isolates and analyzed rv2172c mutations in those 976 isolates. The results showed that two mutations (T120P and M172 V) on rv2172c could be identified in a certain proportion (6.36%) of PAS-resistant isolates. The results of AlphaFold2 prediction indicated that the T120P or M172 V mutation might affect the enzymatic activity of Rv2172c by influencing nicotinamide adenine dinucleotide (NADH) binding, and this was verified by subsequent biochemical analysis, demonstrating the role of residues Thr120 and Met172 on NADH binding and enzymatic activity of Rv2172c. In addition, the effect of rv2172c T120P or M172 V mutation on methionine production and PAS resistance was determined in M. tuberculosis. The results showed that both T120P and M172 V mutations caused increased intracellular methionine concentrations and high level PAS resistance. In summary, we discovered new molecular markers and also a novel mechanism of PAS resistance in M. tuberculosis clinical isolates and broadened the understanding of the NADH-dependent MTHFR catalytic mechanism of Rv2172c in M. tuberculosis, which will facilitate the molecular diagnosis of PAS resistance and also the development of new drugs targeting Rv2172c.

Gadd45b mediates environmental enrichment-induced neurogenesis in the SVZ of rats following ischemia stroke via BDNF.

Ischemic stroke is a major cause of mortality and disability. Subventricular zone (SVZ) neurogenesis following an ischemic stroke may be beneficial for improving the outcomes. Environmental enrichment (EE) has been reported to increase neurogenesis following stroke. Growth arrest and DNA-damage-inducible protein 45 ß (Gadd45b) is a crucial gene for activity-correlated neurogenesis in the adult hippocampus of mice. This study examined whether Gadd45b inhibition affects adult SVZ neurogenesis after an ischemic injury and explored the role of Gadd45b in EE-induced SVZ neurogenesis in adult male Sprague Dawley rats following middle cerebral artery occlusion (MCAO). Gadd45b expression was silenced by a lentivirus with RNA interference (RNAi). The 5-ethynyl-2-deoxyuridine (EdU) staining test was performed to detect cell proliferation. Gadd45b-RNAi after MCAO decreased SVZ proliferation and differentiation in the infarction boundary following ischemic injury, accompanied by the depressed expression of the brain-derived neurotrophic factor (BDNF). Treatment with EE following ischemic stroke upregulated Gadd45b and BDNF expressions and increased neurogenesis in the SVZ. Inhibition of Gadd45b markedly ameliorated the increased neurogenesis induced by EE. These data indicated that Gadd45b is related to SVZ neurogenesis following ischemic stroke, and Gadd45b mediates EE-induced neurogenesis via BDNF in the SVZ of rats following an ischemia stroke. These results implicate that Gadd45b can be a potential therapeutic target to enhance adult neurogenesis following cerebral ischemia.

Genome-wide characterization and expression profiling of the NAC genes under abiotic stresses in Cucumis sativus.

The NAC (standing for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF] and cup-shaped cotyledon [CUC]) proteins pertain to one of the plant-specific transcription factor families that play important roles in plant development, abiotic stress resistance and signalling transduction. In the present study, the genomic features of the NAC genes in cucumber were analysed in depth using in silico tools. To reveal a tissue-specific, abiotic stress and hormone-responsive expression profile of CsNAC genes, RT-qPCR was performed under different treatments. Phylogenetic analyses and genome-wide annotation indicated that 82 high-confidence CsNAC genes were clustered into 13 sub-groups with uneven distribution in the cucumber genome. Furthermore, the CsNAC genes exhibited different tissue-specific expression patterns in 10 tissues under normal growth conditions, while 13 (16%) and 28 (34%) genes displayed preferential expression in roots and flowers, respectively. Moreover, CsNAC genes were more sensitive to salinity than other stresses; however, their responses were relatively rapid and transient to nutrition deprivation. Several CsNAC genes, including CsNAC35, which is an orthologue of the known stress-responsive Arabidopsis RD26, were identified as highly responsive to abiotic stresses and hormones. Overall, our findings revealed the genomic landscape and expression profiling of the CsNAC genes in response to multiple stresses and hormones, offering clues for further function analyses and molecular breeding.

Leaf Volatile Compounds and Associated Gene Expression during Short-Term Nitrogen Deficient Treatments in Cucumis Seedlings.

Nitrogen (N) is an important macronutrient for plant growth and development, but the regulatory mechanism of volatile compounds in response to N deficiency is not well understood, especially in cucumber, which consumes excessive N during growth. In this study, the major volatile compounds from cucumber leaves subjected to N deficiency were analyzed by GC-MS. A total of 24 volatile components were identified including 15 aldehydes, two ketones, two alkenes, and five other volatile compounds in 9930 leaves. Principal component analysis using volatile compounds from cucumber leaves provided good separation between N-sufficient and N-deficient treatments. The main volatiles in cucumber leaves were found to be C6 and C9 aldehydes, especially (E)-2-hexanal and (E,Z)-2,6-nonadienal. (E)-2-hexanal belonged to the C6 aldehyde and was the most abundant compound, whereas (E,Z)-2,6-nonadienal was the chief component of C9 aldehydes. During N-deficient treatment, short-chain volatile content was significantly improved at 5 day, other volatiles displayed significant reduction or no significantly changes in all sampling points. Improvement of short-chain volatiles was confirmed in the six other inbred lines at 5 day after N-deficient treatments. The expression analysis of 12 cucumber LOX genes and two HPL genes revealed that CsLOX19, CsLOX20, and CsLOX22 had common up-regulated expression patterns in response to N-deficient stress in most inbred lines; meanwhile, most sample points of CsHPL1 also had significant up-regulated expression patterns. This research focused on the relationship between volatiles in cucumber and different nitrogen environments to provide valuable insight into the effect of cultivation and management of the quality of cucumber and contributes to further research on volatile metabolism in cucumber.

Effect of nitrogen deficiency on ascorbic acid biosynthesis and recycling pathway in cucumber seedlings.

L-Ascorbic acid (AsA, ascorbate) is one of the most abundant natural antioxidants, and it is an important factor in the nutritional quality of cucumber. In this work, key enzymes involved in the ascorbic acid biosynthesis and recycling pathway in cucumber seedlings under nitrogen deficiency were investigated at the levels of transcription and enzyme activity. The activities of myo-inositol oxygenase (MIOX) and transcript levels of MIOXs increased dramatically, while the activities of ascorbate oxidase (AO) and glutathione reductase (GR) and transcript levels of AOs and GR2 decreased significantly in N-limited leaves, as did the ascorbate concentration, in nitrogen-deficient cucumber seedlings. The activities of other enzymes and transcript levels of other genes involved in the ascorbate recycling pathway and ascorbate synthesis pathways decreased or remained unchanged under nitrogen deficiency. These results indicate that nitrogen deficiency induced genes involved in the ascorbate-glutathione recycling and myo-inositol pathway in cucumber leaves. Thus, the AO, GR and MIOX involved in the pathways might play roles in AsA accumulation.

[Overview of organic agriculture development.] / 有机农业发展概述.

This paper introduced the concepts of organic agriculture as defined by different international organizations, origin and theoretical development of organic agriculture, as well as its developing trajectory in China (i.e. a late start followed by rapid growth compared to developed countries). The differences between domestic and international organic agriculture were illustrated by scale, crop types, production standards, inputs and planting techniques. Constraints limiting improvements to organic agriculture in aspects of standards, technology, marketing, certification, environmental pollution, enterprise reputation, and national policies were discussed. Future directions and strategies for developing healthy organic agriculture in China were provided.

Effects of human ß-defensin-3 on biofilm formation­regulating genes dltB and icaA in Staphylococcus aureus.

An understanding of the regulatory mechanisms that drive Staphylococcus aureus biofilm formation may lead to the development of an effective strategy to control the increasing number of refractory clinical infections it causes. The present study examined the effects of the antimicrobial agent human ß­defensin 3 (hBD­3) and the antibiotics vancomycin and clindamycin on the expression of the S. aureus biofilm formation­regulating genes, icaA and dltB, during bacterial adhesion and biofilm formation. Transcription (mRNA) levels of dlt and ica genes were measured using quantitative polymerase chain reaction, and slimes of S. aureus biofilm were examined with confocal scanning laser microscopy during S. aureus adhesion and biofilm formation. Although hBD­3, vancomycin and clindamycin led to significantly attenuated biofilm formation, their treatment­associated effects on the mRNA expression of dlt and ica were not identical. Vancomycin and clindamycin induced sustained expression of the dlt and ica genes, which may be harnessed to induce biofilm formation. However, hBD­3 did not have a significant affect on the transcription level of dltB during either bacterial adhesion or biofilm formation. Therefore, the mechanism of hBD­3 that regulated the suppression of biofilm formation appears to differ from the mechanisms of vancomycin and clindamycin.

Human beta-defensin-3 for the diagnosis of periprosthetic joint infection and loosening.

In this study, the difference in expression of human beta-defensin-3 in periprosthetic tissue and cancellous bone was observed in the periprosthetic tissue and cancellous bone of patients in the periprosthetic joint infection group, the aseptic loosening group, and the spacer treatment group as well as the synovial membrane and ilium of the normal control group. Hematoxylin and eosin staining of the synovial tissue showed different levels of neutrophil infiltration in all groups except for the normal control group. Immunofluorescence staining of periprosthetic tissue and cancellous bone showed the most positive cells expressing human beta-defensin-3 and the largest mean optical density in the periprosthetic joint infection group, followed by the aseptic loosening group, the spacer treatment group, and the normal control group, with a significant difference in comparison between the periprosthetic joint infection group and the other 3 groups (P<.01). White blood cell count, erythrocyte sedimentation rate, and C-reactive protein level were highest in the periprosthetic joint infection group, whereas no difference was found between the other 3 groups. Taken together, high levels of human beta-defensin-3 protein expression were found in the periprosthetic tissue and cancellous bone of patients with periprosthetic joint infection and aseptic loosening, but there are differential expressions of human beta-defensin-3, and this may provide a new marker for the differential diagnosis of infection and loosening of the artificial joint.

Characterization and expression profiling of cucumber kinesin genes during early fruit development: revealing the roles of kinesins in exponential cell production and enlargement in cucumber fruit.

Rapid cell division and expansion in early fruit development are important phases for cucumber fruit yield and quality. Kinesin proteins are microtubule-based motors responsible for modulating cell division and enlargement. In this work, the candidate kinesin genes involved in rapid cell division and expansion during cucumber fruit development were investigated. The morphological and cellular changes during early fruit development were compared in four cucumber genotypes with varied fruit size. The correlation between the expression profiles of cucumber kinesin genes and cellular changes in fruit was investigated. Finally, the biochemical characteristics and subcellular localizations of three candidate kinesins were studied. The results clarified the morphological and cellular changes during early cucumber fruit development. This study found that CsKF2-CsKF6 were positively correlated with rapid cell production; CsKF1 and CsKF7 showed a strongly positive correlation with rapid cell expansion. The results also indicated that CsKF1 localized to the plasma membrane of fast-expanding fruit cells, that CsKF2 might play a role in fruit chloroplast division, and that CsKF3 is involved in the function or formation of phragmoplasts in fruit telophase cells. The results strongly suggest that specific fruit-enriched kinesins are specialized in their functions in rapid cell division and expansion during cucumber fruit development.

The expression profiling of the lipoxygenase (LOX) family genes during fruit development, abiotic stress and hormonal treatments in cucumber (Cucumis sativus L.).

Lipoxygenases (LOXs) are non-haem iron-containing dioxygenases that catalyse oxygenation of polyunsaturated fatty acids and lipids to initiate the formation of a group of biologically active compounds called oxylipins. Plant oxylipins play important and diverse functions in the cells. In the current study, expression analysis during cucumber development using semi-quantitative RT-PCR revealed that 13 of 23 CsLOX genes were detectable, and were tissue specific or preferential accumulation. In total, 12 genes were found to be differentially expressed during fruit development and have different patterns of expression in exocarp, endocarp and pulp at day 5 after anthesis. The expression analysis of these 12 cucumber LOX genes in response to abiotic stresses and plant growth regulator treatments revealed their differential transcript in response to more than one treatment, indicating their diverse functions in abiotic stress and hormone responses. Results suggest that in cucumber the expanded LOX genes may play more diverse roles in life cycle and comprehensive data generated will be helpful in conducting functional genomic studies to understand their precise roles in cucumber fruit development and stress responses.

Comparison of the effects of human ß-defensin 3, vancomycin, and clindamycin on Staphylococcus aureus biofilm formation.

Despite improvements in surgical techniques and implant design in orthopedic surgery, implantation-associated infections are still a challenging problem for surgeons. In 2006, trace quantities of human ß-defensin 3 (hBD-3) were found in human bone tissue and bone cells. Human ß-defensin 3 is a 45-amino-acid peptide that is considered the most promising class of defensin antimicrobial peptides and may help in the prevention and treatment of implantation-associated infections. Studies of the effectiveness of hBD-3 against Staphylococcus aureus showed that hBD-3 was more potent at low concentrations than other antibiotics. The effect of hBD-3 on S aureus biofilms has not been reported. We studied the effect of hBD-3, vancomycin, and clindamycin on S aureus biofilms and on the survival of the bacteria in the biofilms.Staphylococcus aureus biofilms were examined with confocal scanning laser microscopy. Staining with LIVE/DEAD BacLight viability stain (Molecular Probes Europe BV, Leiden, The Netherlands) differentiated between live and dead bacteria within the biofilms, and extracellular polymeric substances (slime) from the biofilms was evaluated after staining with calcofluor white (Sigma Chemical Company, Rocky Hill, New Jersey). Human ß-defensin 3 and clindamycin reduced the S aureus biofilm area. Human ß-defensin 3 was significantly more effective against bacteria from the S aureus biofilms than was clindamycin. Vancomycin did not reduce the S aureus biofilm area.

Foreign body embolus to ophthalmic artery following penetrating trauma of the neck.

Migration of metallic foreign body into the cerebral circulation is rarely seen. Most of the cases reported were due to gunshot wounds and shotgun wounds to the neck and face. When the foreign body is near the great vessel, it must be removed immediately or will cause complications. This study reported a case of delayed metallic foreign body embolus to the ophthalmic artery resulting from an injury to the right neck, which arose from the presence of metallic emboli to the cerebral circulation.

Antibiotic-impregnated cement spacer versus antibiotic irrigating metal spacer for infection management after THA.

We prospectively compared the effects of an antibiotic-impregnated cement spacer and an antibiotic irrigating metal spacer in the treatment of infections after total hip arthroplasty (THA) to determine a better method for 2-stage revision of infected THA. We used a uniform protocol of a 3-month spacer interval and specific local and systemic antibiotic therapies. The clinical outcome was determined by assessing operation time, blood loss during spacer implantation, hospital stay and time in bed, and joint function. The patients were followed for a mean 61.4 months (range, 24-94 months). Antibiotic-impregnated cement spacers had a higher rate of infection control than antibiotic irrigating metal spacers (96.2% vs 76%, respectively; P<.01), with no recurrence of infections or implant loosening. Patients receiving antibiotic-impregnated cement spacers had a shorter operation time (2.42±0.65 vs 3.34±0.36 hours metal spacers; P<.01), less blood loss (1085.48±279.49 vs 1964.78±469.23 mL metal spacers; P<.01), a shorter hospital stay (24.53±4.72 days vs 51.36±3.46 days metal spacers; P<.01), and better function before the second step of the 2-stage revision (Harris Hip Score, 88.16±6.94 vs 79.54±6.48 metal spacers; P<.05), and they were free from long-term irrigation. Compared with the antibiotic irrigating metal spacer, the antibiotic-impregnated cement spacer is characterized by higher infection control rate, better joint function recovery before the second operation, shorter operation time, and less blood loss, with no continuous irrigation required.

Preparation, release profiles and antibacterial properties of vancomycin-loaded Ca-P coating titanium alloy plate.

The aim of the study was to explore the feasibility of the Ca-P coating titanium alloy plate to be used as the vancomycin drug-delivery system by biomimetic coating technology. Through the X-ray diffraction study, the main components of the coatings were identified as octocalcium phosphate. The in vitro vancomycin release, bacteriostasis activity to Staphylococcus aureus (S. aureus), the scanning electron microscope (SEM) image and osteoblast adhesion and proliferation test of vancomycin-loaded Ca-P coating plate were evaluated. The bacteriostatic activity of the vancomycin-loaded Ca-P coating plate showed a continuous drug release and had an inhibitory effect on the growth of the S. aureus. In vitro osteoblast culture results showed that the Ca-P coating plate loaded with or without the vancomycin both obviously promoted the osteoblast attachment. It was suggested that the vancomycin-loaded Ca-P coating may be compounded in the surface of the internal fixators to reduce the incidence of the implant-associated infection.

Efficacy of a norvancomycin-loaded, PDLLA-coated plate in preventing early infection of rabbit tibia fracture.

Despite improvements in surgical techniques and implant designs in orthopedic surgery, implantation-associated infections are still a challenging problem for surgeons. The goal of this study was to evaluate the efficacy of a norvancomycin-loaded, PDLLA-coated stainless steel plate vs an uncoated stainless steel plate in a rabbit model (n=50). The norvancomycin was delivered from a biodegradable poly(D,L-lactide) (PDLLA) coating of a stainless steel plate. Intraoperatively, rabbit tibia fractures were contaminated with Staphylococcus aureus (10(5) colony forming units) after plate implantation. The implants were either uncoated or coated with PDLLA and norvancomycin. In vivo drug release profiles showed that the norvancomycin release rate was decreased by increasing the time. The norvancomycin concentration in the tissue around the plate was higher than the minimum inhibitory concentration on the 14th day after implantation surgery. The animals were followed up for 28 days. Radiographic examinations were performed, and C-reactive protein and erythrocyte sedimentation rate were determined. Infection was evaluated by histological, microbiological, and radiological analysis. Eight of 25 rabbits (32%) implanted with the norvancomycin-loaded, PDLLA-coated plates were infected. Twenty-three of 25 rabbits (92%) implanted with the uncoated plates were infected (P<.05). The norvancomycin-loaded, PDLLA-coated plate may be used to treat open fractures to reduce the incidence of early infection.

Progesterone attenuates neurological behavioral deficits of experimental autoimmune encephalomyelitis through remyelination with nucleus-sublocalized Olig1 protein.

Multiple sclerosis (MS) is the most common demyelination disease of central nervous system (CNS). The deterioration of the disease is characterized by the axonal loss with defective remyelination. Progesterone can promote the remyelination, but whether it exerts beneficial effect on treatment of MS still remains unclear. Olig1 protein is a key regulator in the remyelination, when the intracellular sublocalization plays an import role too. We observed the effect of progesterone on experimental autoimmune encephalomyelitis (EAE) in rats by injecting the progesterone after the neurological behavioral deficits were shown up. The results showed no continuous increase of the nervous function score from day 10 after injection (p<0.05). Electron microscopy and LFB staining found prominent increase of OD value of normal myelin in the brain from day 6 after injection (p<0.05). Olig1 protein was localized almost completely in the cytoplasm of Olig1-positive cells from normal rats' brain. In EAE rats, the Olig1 protein has been translocated to the nucleus of 32.17% of Olig1-positive cells, which was increased to 68.52% after injection with progesterone at day 6 after injection (p<0.01). The results indicate that the progesterone is beneficial to attenuating neurological behavioral deficits, for it can promote more successful remyelination of EAE with aid of the nucleus-sublocalized Olig1 protein.

Crystal structure of the neurotrophin-3 and p75NTR symmetrical complex.

Neurotrophins (NTs) are important regulators for the survival, differentiation and maintenance of different peripheral and central neurons. NTs bind to two distinct classes of glycosylated receptor: the p75 neurotrophin receptor (p75(NTR)) and tyrosine kinase receptors (Trks). Whereas p75(NTR) binds to all NTs, the Trk subtypes are specific for each NT. The question of whether NTs stimulate p75(NTR) by inducing receptor homodimerization is still under debate. Here we report the 2.6-A resolution crystal structure of neurotrophin-3 (NT-3) complexed to the ectodomain of glycosylated p75(NTR). In contrast to the previously reported asymmetric complex structure, which contains a dimer of nerve growth factor (NGF) bound to a single ectodomain of deglycosylated p75(NTR) (ref. 3), we show that NT-3 forms a central homodimer around which two glycosylated p75(NTR) molecules bind symmetrically. Symmetrical binding occurs along the NT-3 interfaces, resulting in a 2:2 ligand-receptor cluster. A comparison of the symmetrical and asymmetric structures reveals significant differences in ligand-receptor interactions and p75(NTR) conformations. Biochemical experiments indicate that both NT-3 and NGF bind to p75(NTR) with 2:2 stoichiometry in solution, whereas the 2:1 complexes are the result of artificial deglycosylation. We therefore propose that the symmetrical 2:2 complex reflects a native state of p75(NTR) activation at the cell surface. These results provide a model for NTs-p75(NTR) recognition and signal generation, as well as insights into coordination between p75(NTR) and Trks.

[Study on high mobility group-1 protein in patients with multiple trauma].

OBJECTIVE: To study the change in serum high mobility group-1 (HMG-1) protein in patients with multiple trauma and determine its relationship to severity of trauma and multiple organ dysfunction syndrome (MODS). METHODS: In 33 cases, HMG-1 levels were determined on 1, 3, 7 days after trauma respectively. The values of injury severity score (ISS), acute physiology and chronic health evaluation II (APACHE II) and systemic inflammatory response syndrome (SIRS) score were determined. Meanwhile, the occurrence of organ dysfunction was noted. RESULTS: The level of serum HMG-1 in patients with multiple trauma was much higher than that of normal controls, and also in those with organ dysfunction. HMG-1 and APACHE II were positively correlated (r=0.495, P=0.016). The level of serum HMG-1 and APACHE II value were much higher in patients with organ dysfunction than those of normal organ function, but there was no significant difference in SIRS score between the two groups (P=0.105). CONCLUSION: Serum HMG-1 increases in patients with trauma and is positively correlated with severity of trauma. Serial determination of HMG-1 is helpful to discover clinical infection earlier. HMG-1 can be used as a warning indicator of the onset of MODS.

Experimental study and clinical observation of minimum-contact plate in long bone fracture.

OBJECTIVE: To study the mechanical properties and the clinical results of minimum-contact plate in the treatment of fractures. METHODS: Four-point bending and torsion tests were conducted to compare the new minimum contact plate (MCP) with dynamic compression plate (DCP) and limited contact dynamic compression plate (LC-DCP). The fracture healing time and growth of bony callus were observed in 29 cases of long bone fracture fixed with MCP to evaluate the advantages of this kind of plate. RESULTS: The 29 patients who underwent MCP were followed up from 6 to 14 months. The average healing time for femoral, tibial and humeral fractures was 12, 13 and 10 weeks, respectively. All fractures were healed. No displacement of fracture, screw pullout, deformation or break of the plate were found. CONCLUSIONS: The bending stiffness of MCP is significantly greater than that of DCP and LC-DCP (P<0.05). MCP can protect the periosteal blood supply against avascular osseous necrosis and accelerate bone healing. It is a kind of reliable and effective plate in treatment of fractures.