The aerial parts of land plants are covered with cuticular waxes that limit non-stomatal water loss and gaseous exchange, and protect plants from ultraviolet radiation and pathogen attack. This is the first report on the characterization and genetic mapping of a novel dominant glossy mutant (BnaA.GL) in Brassica napus.
Transmission electron microscopy revealed that the cuticle ultrastructure of GL mutant leaf and stem were altered dramatically compared with that of wide type (WT). Scanning electron microscopy corroborated the reduction of wax on the leaf and stem surface. A cuticular wax analysis of the GL mutant leaves further confirmed the drastic decrease in the total wax content, and a wax compositional analysis revealed an increase in aldehydes but a severe decrease in alkanes, ketones and secondary alcohols. These results suggested a likely blockage of the decarbonylation step in the wax biosynthesis pathway. Genetic mapping narrowed the location of the BnaA.GL gene to the end of A9 chromosome. A single-nucleotide polymorphism (SNP) chip assay in combination with bulk segregant analysis (BSA) also located SNPs in the same region. Two SNPs, two single sequence repeat (SSR) markers and one IP marker were located on the flanking region of the BnaA.GL gene at a distance of 0.6 cM. A gene homologous to ECERIFERUM1 (CER1) was located in the mapped region. A cDNA microarray chip assay revealed coordinated down regulation of genes encoding enzymes of the cuticular wax biosynthetic pathway in the glossy mutant, with BnCER1 being one of the most severely suppressed genes.
Our results indicated that surface wax biosynthesis is broadly affected in the glossy mutant due to the suppression of the BnCER1 and other wax-related genes. These findings offer novel clues for elucidating the molecular basis of the glossy phenotype.
Brassica napus; Glossy mutant; Genetic mapping; Wax biosynthesis; Microarray assays; Candidate genes
Mutations in the GJB2 gene are responsible for up to 50% of cases of non-syndromic recessive hearing loss, with c.35delG, c.167delT and c.235delC being the predominant mutations in many world populations. However, a large number of rare mutations in this gene may also contribute to hearing loss. The aim of the present study was to conduct a clinical and molecular characterization of a Chinese family with non-syndromic hearing loss. Sequence analysis of the GJB2 gene led to the identification of a novel compound heterozygous mutation c.257C>G (p.T86R)/c.605ins46 in two profoundly deaf siblings whose hearing parents were each heterozygous, either for the c.257C>G (paternal) or for the c.605ins46 (maternal) mutations. Both c.257C>G and c.605ins46 are rare GJB2 mutations that have previously been reported to segregate with autosomal recessive hearing loss exclusively in East Asian populations. To study the pathogenic effect of the compound heterozygous mutation, a three-dimensional model was constructed and Anolea mean force potential energy was predicted for a bioinformatic structural analysis. HEK293 cells were used to study the pathogenic effect of mutant connexin 26 proteins. The results suggested that the c.257C>G (p.T86R)/c.605ins46 mutations in the GJB2 gene provides a novel molecular explanation for the role of the GJB2 gene in hearing loss.
GJB2; hearing loss; prelingual; gene mutation
Drinking water reservoir plays a vital role in the security of urban water supply, yet little is known about microbial community diversity harbored in the sediment of this oligotrophic freshwater environmental ecosystem. In the present study, integrating community level physiological profiles (CLPPs), nested polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and clone sequence technologies, we examined the sediment urease and protease activities, bacterial community functional diversity, genetic diversity of bacterial and fungal communities in sediments from six sampling sites of Zhou cun drinking water reservoir, eastern China. The results showed that sediment urease activity was markedly distinct along the sites, ranged from 2.48 to 11.81 mg NH3-N/(g·24h). The highest average well color development (AWCD) was found in site C, indicating the highest metabolic activity of heterotrophic bacterial community. Principal component analysis (PCA) revealed tremendous differences in the functional (metabolic) diversity patterns of the sediment bacterial communities from different sites. Meanwhile, DGGE fingerprints also indicated spatial changes of genetic diversity of sediment bacterial and fungal communities. The sequence BLAST analysis of all the sediment samples found that Comamonas sp. was the dominant bacterial species harbored in site A. Alternaria alternate, Allomyces macrogynus and Rhizophydium sp. were most commonly detected fungal species in sediments of the Zhou cun drinking water reservoir. The results from this work provide new insights about the heterogeneity of sediment microbial community metabolic activity and genetic diversity in the oligotrophic drinking water reservoir.
Senescence is a recognized mechanism of cardiovascular diseases; however, its contribution to myocardial fibrosis and rupture after infarction and the underlying mechanisms remain unclear. Here we showed that senescent cardiac fibroblasts markedly accumulated in heart after myocardial infarction. The expression of key senescence regulators, especially p53, was significantly up-regulated in the infarcted heart or hypoxia-treated fibroblasts. Furthermore, knockdown of endogenous p53 by siRNA in fibroblasts markedly reduced hypoxia-induced cell senescence, cytokine expression but increased collagen expression, whereas increased expression of p53 protein by adenovirus infection had opposite effects. Consistent with in vitro results in cardiac fibroblasts, p53 deficiency in vivo significantly decreased the accumulation of senescent fibroblasts, the infiltration of macrophages and matrix metalloproteinases, but enhanced collagen deposition after myocardial infarction. In conclusion, these results suggest that the p53-mediated fibroblast senescence limits cardiac collagen production, and inhibition of p53 activity could represent a novel therapeutic target to increase reparative fibrosis and to prevent heart rupture after myocardial infarction.
Rationale: Acute lung injury is a common complication after severe trauma, which predisposes patients to multiple organ failure. This syndrome largely accounts for the late mortality that arises and despite many theories, the pathological mechanism is not fully understood. Discovery of histone-induced toxicity in mice presents a new dimension for elucidating the underlying pathophysiology.
Objectives: To investigate the pathological roles of circulating histones in trauma-induced lung injury.
Methods: Circulating histone levels in patients with severe trauma were determined and correlated with respiratory failure and Sequential Organ Failure Assessment (SOFA) scores. Their cause–effect relationship was studied using cells and mouse models.
Measurements and Main Results: In a cohort of 52 patients with severe nonthoracic blunt trauma, circulating histones surged immediately after trauma to levels that were toxic to cultured endothelial cells. The high levels were significantly associated with the incidence of acute lung injury and SOFA scores, as well as markers of endothelial damage and coagulation activation. In in vitro systems, histones damaged endothelial cells, stimulated cytokine release, and induced neutrophil extracellular trap formation and myeloperoxidase release. Cellular toxicity resulted from their direct membrane interaction and resultant calcium influx. In mouse models, cytokines and markers for endothelial damage and coagulation activation significantly increased immediately after trauma or histone infusion. Pathological examinations showed that lungs were the predominantly affected organ with edema, hemorrhage, microvascular thrombosis, and neutrophil congestion. An anti-histone antibody could reduce these changes and protect mice from histone-induced lethality.
Conclusions: This study elucidates a new mechanism for acute lung injury after severe trauma and proposes that circulating histones are viable therapeutic targets for improving survival outcomes in patients.
lung; trauma; histones
Inflammatory myofibroblastic tumor of the temporal bone is an unusual but distinct disease entity. The most common presenting symptoms are otalgia, otorrhea, hearing loss, facial palsy, and vertigo. We describe here what we believe to be the first reported case of a patient presenting with persistent pulsatile tinnitus. The clinical features, radiological and histopathologic findings, and treatment outcomes of the patient are presented.
A 59-year-old woman of Chinese Han origin presented with complaints of left-sided pulsatile tinnitus and progressive hearing loss for several years. Clinical evaluations revealed a reddish mass behind the intact tympanic membrane, and a moderately severe conductive hearing loss in the left ear. The computed tomographic imaging of the temporal bone demonstrated a slightly ill-defined left middle ear soft tissue mass involving the posterior portion of the mesotympanum and epitympanum, and the mastoid antrum. The patient underwent surgical excision of the lesion which subsequently resolved her symptoms. Postoperative pathology was consistent with an inflammatory myofibroblastic tumor.
An inflammatory myofibroblastic tumor of the temporal bone can present clinically with pulsatile tinnitus and masquerade as venous hum or vascular tumors of the middle ear; therefore, it should be included in the differential diagnosis of pulsatile tinnitus.
Inflammatory Myofibroblastic Tumor; Middle Ear; Pulsatile Tinnitus; Temporal Bone
Background: The connection between gut microbiota and metabolism and its role in the pathogenesis of diabetes are increasingly recognized. The objective of this study was to quantitatively measure Bifidobacterium and Lactobacillus species, members of commensal bacteria found in human gut, in type 2 diabetic patients (T2D) patients from Southern China.
Methods: Fifty patients with T2D and thirty control individuals of similar body mass index (BMI) were recruited from Southern China. T2D and control subjects were confirmed with both oral glucose tolerance test (OGTT) and HbA1c measurements. Bifidobacterium and Lactobacillus species in feces were measured by real-time quantitative PCR. Data were analyzed with STATA 11.0 statistical software.
Results: In comparison to control subjects T2D patients had significantly more total Lactobacillus (+18%), L. bugaricus (+13%), L. rhamnosum (+37%) and L. acidophilus (+48%) (P < 0.05). In contrast, T2D patients had less amounts of total Bifidobacteria (−7%) and B. adolescentis (−12%) (P < 0.05). Cluster analysis showed that gut microbiota pattern of T2D patients is characterized by greater numbers of L. rhamnosus and L. acidophillus, together with lesser numbers of B. adolescentis (P < 0.05).
Conclusion: The gut microflora in T2D patients is characterized by greater numbers of Lactobacillus and lesser numbers of Bifidobacterium species.
microbiota; microflora; metabolic diseases; gut
Inflammatory pseudotumor (IP) is a clinically aggressive but histologically benign condition of unknown cause. Its appearance in the temporal bone is uncommon. We present clinical, radiological, and histopathologic findings of three cases originating in the temporal bone. In the first case, a simultaneous IP of the temporal bone and parotid gland was found with histopathologic confirmation. In the second case, an enlarged cervical node, which was also believed to be related to IP, was observed accompanied with the temporal lesion. While the third case presented with chronic suppurative otitis media. Two of them were treated by surgery alone with complete resolve of the diseases. Another one underwent tympanomastoidectomy in combination with oral steroids, radiation, and chemotherapy, but the IP still recurred. A comprehensive review of the literature on clinical features of the temporal pseudotumor was conducted.
Toxoplasma gondii infections are prevalent in a wide range of mammalian hosts including humans. Infection in pregnant women may cause the transmission of parasite to the fetus that makes serious problems. IgM antibodies against Toxoplasma (Toxo-IgM) have been believed to be significant indicators for both recently acquired and congenital toxoplasmosis. So far, however, there has not been any recognized protein of T. gondii that specifically reacts to IgM antibodies. Here, an antigen exclusively for detection of IgM antibodies screened by two-dimensional electrophoresis and mass spectrometry has been reported. The study identified 13 Toxoplasma proteins probed by IgG antibodies and one (rhpotry protein 2 [ROP2]) by IgM antibodies with human sera of Toxo-IgM–-IgG+ and -IgM+-IgG–, respectively, which had been prescreened by Toxo-IgM and -IgG commercial kits from the suspected cases. Following cloning, expression, and purification of the fragment of ROP2186–533, an enzyme-linked immunosorbent assay with rROP2186–533 to measure IgM and IgG antibodies was developed. As a result, 100%(48/48) of sera with Toxo-IgM+-IgG–showed positive Toxo-IgM but none of them (0%) showed positive Toxo-IgG when rROP2186–533 was used as antigen. Neither Toxo-IgG nor Toxo-IgM antibodies were found when tested with 59 sera of Toxo-IgM–-IgG+. These results indicate that rROP2186–533 could be used as an antigen that specifically capture Toxo-IgM antibodies and may have a high potential in the serological diagnosis of both acute acquired and congenital toxoplasmosis.
The aim of this study was to investigate the clinicopathologic characteristics, therapy methods, and prognosis of male breast cancer.
Patients and Methods
We retrospectively analyzed the clinicopathological characteristics, recurrence or metastasis, and survival information of 87 male breast cancer patients. Statistical analysis included the Kaplan-Meier method to analyze survivals, log-rank to compare curves between groups, and Cox regression for multivariate prognostic analysis. A p value of <0.05 was considered statistically significant.
5-year disease free survival (DFS) and 5-year overall survival (OS) were 66.3 and 77.0%, respectively. Monofactorial analysis showed tumor size, stage, lymph node involvement, and adjuvant chemotherapy to be prognostic factors with regard to 5-year DFS and 5-year OS. Multivariate Cox regression analysis showed tumor size, stage, and adjuvant chemotherapy to be independent prognostic factors with regard to 5-year DFS and 5-year OS.
Male breast cancer has a lower incidence rate and poor prognosis. Invasive ductal carcinoma is the main pathologic type. Operation-based combined therapy is the standard care for these patients. Tumor size, stage, and adjuvant chemotherapy are independent prognostic factors. More emphasis should be placed on early diagnosis and early therapy, and adjuvant chemotherapy may improve survival.
Male breast cancer; Clinical characteristics; Therapy; Prognosis
Toxoplasma gondii infection in pregnant women may result in abortion or in fetal teratogenesis; however, the underlying mechanisms are still unclear. In this paper, based on a murine model, we showed that maternal infection with RH strain T. gondii tachyzoites induced elevated production of reactive oxygen species (ROS), local oxidative stress, and subsequent apoptosis of placental trophoblasts. PCR array analysis of 84 oxidative stress-related genes demonstrated that 27 genes were upregulated at least 2-fold and that 9 genes were downregulated at least 2-fold in the T. gondii infection group compared with levels in the control group. The expression of NADPH oxidase 1 (Nox1) and glutathione peroxidase 6 (Gpx6) increased significantly, about 25-fold. The levels of malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) increased significantly with T. gondii infection, and levels of glutathione (GSH) decreased rapidly. T. gondii infection increased the early expression of endoplasmic reticulum stress (ERS) markers, followed by cleavage of caspase-12, activation of ASK1/JNK, and increased apoptosis of trophoblasts, both in vivo and in vitro. The apoptosis of trophoblasts, the activation of caspase-12 and the ASK1/JNK pathway, and the production of peroxides were dramatically inhibited by pretreatment with N-acetylcysteine (NAC). The upregulation of Nox1 was contact dependent and preceded the increase in levels of ERS markers and the activation of the proapoptosis cascade. Thus, we concluded that apoptosis in placental trophoblasts was initiated predominantly by ROS-mediated ERS via activation of caspase-12, CHOP, and the JNK pathway in acute T. gondii infection. Elevated ROS production is the central event in T. gondii-induced apoptosis of placental trophoblasts.
Mitochondrial fusion protein mutations are a cause of inherited neuropathies such as Charcot–Marie–Tooth disease and dominant optic atrophy. Previously we reported that the fusion protein optic atrophy 1 (OPA1) is decreased in heart failure.
Methods and Results
We investigated cardiac function, mitochondrial function, and mtDNA stability in a mouse model of the disease with OPA1 mutation. The homozygous mutation is embryonic lethal. Heterozygous OPA+/− mice exhibit reduced mtDNA copy number and decreased expression of nuclear antioxidant genes at 3 to 4 months. Although initial cardiac function was normal, at 12 months the OPA1+/− mouse hearts had decreased fractional shortening, cardiac output, and myocyte contraction. This coincided with the onset of blindness. In addition to small fragmented mitochondria, aged OPA1+/− mice had impaired cardiac mitochondrial function compared with wild-type littermates.
OPA1 mutation leads to deficiency in antioxidant transcripts, increased reactive oxygen species, mitochondrial dysfunction, and late-onset cardiomyopathy.
cardiomyopathy; mitochondrial fusion; mtDNA; OPA1; ROS
HSP40, also known as DnaJ, is one of the subfamilies of the heat shock protein family. DnaJ/Hsp40 proteins act as co-chaperones by binding to the chaperone Hsp70 through their J domain and stimulating ATP hydrolysis to aid protein translation, folding, unfolding, translocation and degradation. They are implicated in various human diseases, including neurodegenerative disorders and cancer. In the present study, we cloned and identified a new gene, DnaJ (HSP40) homolog, subfamily C, member 25 (DNAJC25), which is localized to the cytoplasm. Real-time PCR revealed that the expression of DNAJC25 is particularly high in the liver and is down-regulated in hepatocellular carcinoma (HCC) compared with adjacent normal tissues. The overexpression of DNAJC25 led to an inhibition of colony growth both in quantity and size. Flow cytometry analysis indicated that DNAJC25 also significantly increased cell apoptosis. Our data, therefore, indicate that DNAJC25 plays an important role in hepatocellular carcinogenesis, and should be further studied as a potential tumor suppressor candidate.
heat shock protein; downregulated; hepatocellular carcinoma; apoptosis; tumor suppressor
When embedded in adjacent distractors, a target becomes more difficult to perceive. The neural mechanism for this ubiquitous visual crowding effect remains unresolved [1, 2]. Stimuli presented on opposite sides of the vertical meridian initially project to different hemispheres, whereas stimuli with the same spatial distance but presented to one side of the vertical meridian project to the same hemisphere. Dissociation between visual spatial distance and cortical distance can also be found in V2 and V3 (quadrant representations of the visual hemifield) along the horizontal meridian. In the current study, we observed a strong crowding effect from spatially adjacent distractors with either Gabor or letter targets presented near the vertical or horizontal meridian. Interestingly, for a target presented near the vertical meridian, a distractor from the same side of the meridian (cortically near) had a significantly stronger crowding effect compared with an equidistant distractor presented on the opposite side (cortically remote). No such meridian modulation was observed across the horizontal meridian. These results constrain the cortical locus of the crowding effect to a stage in which left and right visual spaces are represented discontinuously but the upper and lower visual fields are represented continuously, likely beyond the early retinotopic areas.
This fMRI study shows that there is cortical reorganization of the early visual cortex in both age-related and juvenile-onset macular degeneration, but it is incomplete and task dependent.
Activity in regions of the visual cortex corresponding to central scotomas in subjects with macular degeneration (MD) is considered evidence for functional reorganization in the brain. Three unresolved issues related to cortical activity in subjects with MD were addressed: Is the cortical response to stimuli presented to the preferred retinal locus (PRL) different from other retinal loci at the same eccentricity? What effect does the role of age of onset and etiology of MD have on cortical responses? How do functional responses in an MD subject's visual cortex vary for task and stimulus conditions?
Eight MD subjects—four with age-related onset (AMD) and four with juvenile onset (JMD)—and two age-matched normal vision controls, participated in three testing conditions while undergoing functional magnetic resonance imaging (fMRI). First, subjects viewed a small stimulus presented at the PRL compared with a non-PRL control location to investigate the role of the PRL. Second, they viewed a full-field flickering checkerboard compared with a small stimulus in the original fovea to investigate brain activation with passive viewing. Third, they performed a one-back task with scene images to investigate brain activation with active viewing.
A small stimulus at the PRL generated more extensive cortical activation than at a non-PRL location, but neither yielded activation in the foveal cortical projection. Both passive and active viewing of full-field stimuli left a silent zone at the posterior pole of the occipital cortex, implying a lack of complete cortical reorganization. The silent zone was smaller in the task requiring active viewing compared with the task requiring passive viewing, especially in JMD subjects.
The PRL for MD subjects has more extensive cortical representation than a retinal region with matched eccentricity. There is evidence for incomplete functional reorganization of early visual cortex in both JMD and AMD. Functional reorganization is more prominent in JMD. Feedback signals, possibly associated with attention, play an important role in the reorganization.
Background and Aims
The polygalacturonase (PG) gene family has been found to be enriched in pollen of several species; however, little is currently known about the function of the PG gene in pollen development. To investigate the exact role that the PG gene has played in pollen development and about this family in general, one putative PG gene, Brassica campestris Male Fertility 9 (BcMF9), was isolated from Chinese cabbage (Brassica campestris ssp. chinensis, syn. B. rapa ssp. chinensis) and characterized.
RT-PCR, northern blotting and in situ hybridization were used to analyse the expression pattern of BcMF9, and antisense RNA technology was applied to study the function of this gene.
BcMF9 is expressed in particular in the tapetum and microspore during the late stages of pollen development. Antisense RNA transgenic plants that displayed decreased expression of BcMF9 showed pollen morphological defects that resulted in reduced pollen germination efficiency. Transmission electron microscopy revealed that the homogeneous pectic exintine layer of pollen facing the exterior was over-developed and predominantly occupied the intine, reversing the normal proportional distribution of the internal endintine layer and the external exintine in transgenic pollen. Inhibition of BcMF9 also resulted in break-up of the previously formed tectum and baculae from the beginning of the binucleate stage, as a result of premature degradation of tapetum.
Several lines of evidence, including patterns of BcMF9 expression and phenotypic defects, suggest a sporophytic role in exine patterning, and a gametophytic mode of action of BcMF9 in intine formation. BcMF9 might act as a co-ordinator in the late stages of tapetum degeneration, and subsequently in the regulation of wall material secretion and, in turn, exine formation. BcMF9 might also play a role in intine formation, possibly via regulation of the dynamic metabolism of pectin.
Brassica campestris; Chinese cabbage; exine; intine; PG; pollen wall; polygalacturonase; BcMF9
Globally, about 20% of cultivated land is now affected by salinity. Salt tolerance is a trait of importance to all crops in saline soils. Previous efforts to improve salt tolerance in crop plants have met with only limited success. Bacteria of the genus Deinococcus are known for their ability to survive highly stressful conditions, and therefore possess a unique pool of genes conferring extreme resistance. In Deinococcus radiodurans, the irrE gene encodes a global regulator responsible for extreme radioresistance.
Using plate assays, we showed that IrrE protected E. coli cells against salt shock and other abiotic stresses such as oxidative, osmotic and thermal shocks. Comparative proteomic analysis revealed that IrrE functions as a switch to regulate different sets of proteins such as stress responsive proteins, protein kinases, glycerol-degrading enzymes, detoxification proteins, and growth-related proteins in E. coli. We also used quantitative RT-PCR to investigate expression of nine selected stress-responsive genes in transgenic and wild-type Brassica napus plants. Transgenic B. napus plants expressing the IrrE protein can tolerate 350 mM NaCl, a concentration that inhibits the growth of almost all crop plants.
Expression of IrrE, a global regulator for extreme radiation resistance in D. radiodurans, confers significantly enhanced salt tolerance in both E. coli and B. napus. We thus propose that the irrE gene might be used as a potentially promising transgene to improve abiotic stress tolerances in crop plants.
Brassica campestris Male Fertility 2 (BcMF2) is a putative polygalacturonase (PG) gene previously isolated from the flower bud of Chinese cabbage (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis). This gene was found to be expressed specifically in tapetum and pollen after the tetrad stage of anther development. Antisense RNA technology was used to study the function of BcMF2 in Chinese cabbage. Scanning and transmission electron microscopy revealed that there were deformities in the transgenic mature pollen grains such as abnormal location of germinal furrows. In addition, the homogeneous pectic exintine layer facing the exterior seemed to be overdeveloped and predominantly occupied the intine, thus reversing the normal proportional distribution of the internal endintine layer and the external exintine layer. Since it is a continuation of the intine layer, the pollen tube wall could not grow normally. This resulted in the formation of a balloon-like swelling structure in the pollen tube tip in nearly 80% of the transgenic pollen grains. Premature degradation of tapetum was also found in these transgenic plants, which displayed decreased expression of the BcMF2 gene. BcMF2 might therefore encode a new PG with an important role in pollen wall development, possibly via regulation of pectin's dynamic metabolism.
Brassica campestris; Brassica rapa; Chinese cabbage; intine; PG; polygalacturonase; pollen wall