To simulate the ischemia-reperfusion injury in vivo, hypoxia/reoxygenation injury model was established in vitro and primary cultured neonatal rat cardiomyocytes were underwent hypoxia with hydrosulfite (Na2S2O4) for 1 h followed by 1 h reoxygenation.
Materials and Methods:
Determination the cell viability by MTT colorimetric assay. We use kit to detect the activity of lactate dehydrogenase (LDH), Na+-K+-ATPase and Ca2+-ATPase. Do research on the effect which ferulic acid and its drug-containing plasma have to self-discipline, conductivity, action potential duration and other electrophysiological phenomena of myocardial cells by direct observation using a microscope and recording method of intracellular action potential.
The experimental datum showed that both can reduce the damage hydrosulfite to myocardial cell damage and improve myocardial viability, reduce the amount of LDH leak, increase activity of Na+-K+-ATPase, Ca2+-ATPase, and increase APA (Action potential amplitude), Vmax (Maximum rate of depolarization) and MPD (Maximum potential diastolic).
Taken together, therefore, we can get the conclusion that ferulic acid drug-containing plasma has better protective effect injured myocardial cell than ferulic acid.
ATPase; drug-containing plasma; electrophysiological phenomena; ferulic acid; hypoxia/reoxygenation; LDH; primary cultured neonatal rat cardiomyocytes; sodium hydrosulfite
Electroacupuncture (EA) is effective in relieving pain in patients with postherpetic neuralgia (PHN). However, the mechanism underlying the therapeutic effect of EA in PHN is still unclear. Systemic injection of resiniferatoxin (RTX), an ultrapotent analog of TRPV1 agonist, in adult rats can reproduce the clinical symptoms of PHN by ablating TRPV1-expressing sensory neurons. In this study, we determined the beneficial effect of EA and the potential mechanisms in this rat model of PHN.
PHN was induced in rats by a single injection of RTX. Thermal hyperalgesia was tested with a radiant heat stimulus, and mechanical allodynia was quantified with von Frey filaments. TRPV1 receptors were shown by using immunofluorescence labeling. The ultrastructural changes of the sciatic nerve were assessed by electron microscopic examination. The sprouting of myelinated primary afferent terminals into the spinal dorsal horn was mapped by using the transganglionic tracer cholera toxin B-subunit (CTB).
RTX injection diminished thermal sensitivity and gradually induced tactile allodynia within 3 weeks. EA applied to GB30 and GB34 at 2 and 15 Hz, but not 100 Hz, significantly increased the thermal sensitivity 4 weeks after treatment and decreased the tactile allodynia 2 weeks after treatment in RTX-treated rats. EA treatment at 2 and 15 Hz recovered the loss of TRPV1-positive dorsal root ganglion neurons and their central terminals of afferent fibers in the spinal superficial dorsal horn of RTX-treated rats. Moreover, EA significantly reduced the loss of unmyelinated fibers and the damage of the myelinated nerve fibers of RTX-treated rats. Furthermore, EA at 2 and 15 Hz inhibited the sprouting of myelinated primary afferent terminals into the spinal lamina II of RTX-treated rats.
EA treatment improves thermal perception by recovering TRPV1-positive sensory neurons and nerve terminals damaged by RTX. EA Also reduces RTX-induced tactile allodynia by attenuating the damage of myelinated afferent nerves and their abnormal sprouting into the spinal lamina II. Our study provides new information about the mechanisms of the therapeutic actions of EA in the treatment of PHN.
Neuropathic pain; Postherpetic neuralgia; Capsaicin; Acupuncture; Analgesia; Allodynia; TRPV1; Axonal sprouting
Bat flies of the family Nycteribiidae are known for their extreme morphological and physiological traits specialized for ectoparasitic blood-feeding lifestyle on bats, including lack of wings, reduced head and eyes, adenotrophic viviparity with a highly developed uterus and milk glands, as well as association with endosymbiotic bacteria. We investigated Japanese nycteribiid bat flies representing 4 genera, 8 species and 27 populations for their bacterial endosymbionts. From all the nycteribiid species examined, a distinct clade of gammaproteobacteria was consistently detected, which was allied to endosymbionts of other insects such as Riesia spp. of primate lice and Arsenophonus spp. of diverse insects. In adult insects, the endosymbiont was localized in specific bacteriocytes in the abdomen, suggesting an intimate host–symbiont association. In adult females, the endosymbiont was also found in the cavity of milk gland tubules, which suggests uterine vertical transmission of the endosymbiont to larvae through milk gland secretion. In adult females of Penicillidia jenynsii, we discovered a previously unknown type of symbiotic organ in the Nycteribiidae: a pair of large bacteriomes located inside the swellings on the fifth abdominal ventral plate. The endosymbiont genes consistently exhibited adenine/thymine biased nucleotide compositions and accelerated rates of molecular evolution. The endosymbiont genome was estimated to be highly reduced, ∼0.76 Mb in size. The endosymbiont phylogeny perfectly mirrored the host insect phylogeny, indicating strict vertical transmission and host–symbiont co-speciation in the evolutionary course of the Nycteribiidae. The designation ‘Candidatus Aschnera chinzeii' is proposed for the endosymbiont clade.
endosymbiont; nycteribiid bat fly; vertical transmission; adenotrophic viviparity; co-evolution
Current evidence suggests high serum uric acid may increase the risk of type 2 diabetes, but the association is still uncertain. The aim of the study was to evaluate the association between serum uric acid and future risk of type 2 diabetes by conducting a meta-analysis of prospective cohort studies.
Design and Methods
We conducted a systematic literature search of the PubMed database through April 2012. Prospective cohort studies were included in meta-analysis that reported the multivariate adjusted relative risks (RRs) and the corresponding 95% confidence intervals (CIs) for the association between serum uric acid and risk of type 2 diabetes. We used both fix-effects and random-effects models to calculate the overall effect estimate. The heterogeneity across studies was tested by both Q statistic and I2 statistic. Begg’s funnel plot and Egger’s regression test were used to assess the potential publication bias.
We retrieved 7 eligible articles derived from 8 prospective cohort studies, involving a total of 32016 participants and 2930 incident type 2 diabetes. The combined RR of developing type 2 diabetes for the highest category of serum uric acid level compared with the lowest was 1.56(95% CI, 1.39–1.76). Dose-response analysis showed the risk of type 2 diabetes was increased by 6% per 1 mg/dl increment in serum uric acid level (RR 1.06, 95% CI: 1.04–1.07). The result from each subgroup showed a significant association between serum uric acid and risk of type 2 diabetes. In sensitive analysis, the combined RR was consistent every time omitting any one study. Little evidence of heterogeneity and publication bias was observed.
Our meta-analysis of prospective cohort studies provided strong evidence that high level of serum uric acid is independent of other established risk factors, especially metabolic syndrome components, for developing type 2 diabetes in middle-aged and older people.
We investigated seed bugs of the genus Nysius (Insecta: Hemiptera: Lygaeidae) for their symbiotic bacteria. From all the samples representing 4 species, 18 populations and 281 individuals, specific bacterial 16S rRNA gene sequences were consistently identified, which formed a distinct clade in the Gammaproteobacteria. In situ hybridization showed that the bacterium was endocellularly localized in a pair of large bacteriomes that were amorphous in shape, deep red in color, and in association with gonads. In the ovary of adult females, the endosymbiont was also localized in the ‘infection zone' in the middle of each germarium and in the ‘symbiont ball' at the anterior pole of each oocyte, indicating vertical transmission of the endosymbiont through the ovarial passage. Phylogenetic analyses based on bacterial 16S rRNA, groEL and gyrB genes consistently supported a coherent monophyly of the Nysius endosymbionts. The possibility of a sister relationship to ‘Candidatus Kleidoceria schneideri', the bacteriome-associated endosymbiont of a lygaeid bug Kleidocerys resedae, was statistically rejected, indicating independent evolutionary origins of the endosymbionts in the Lygaeidae. The endosymbiont genes consistently exhibited AT-biased nucleotide compositions and accelerated rates of molecular evolution, and the endosymbiont genome was only 0.6 Mb in size. The endosymbiont phylogeny was congruent with the host insect phylogeny, suggesting strict vertical transmission and host–symbiont co-speciation over evolutionary time. Based on these results, we discuss the evolution of bacteriomes and endosymbionts in the Heteroptera, most members of which are associated with gut symbiotic bacteria. The designation ‘Candidatus Schneideria nysicola' is proposed for the endosymbiont clade.
stinkbug; Nysius; Lygaeidae; bacteriome; endosymbiont
AIM: To assess the diagnostic value of using magnifying chromoendoscopy combined with immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and p53 in the detection of gastric precancerous lesions.
METHODS: Ninety-five patients who were treated for abdominal discomfort, abdominal pain, bloating, and acid reflux at our hospital from January 2010 to December 2011 were included in the study. An ordinary gastroscopic procedure was initially performed to select the lesions. All subjects underwent magnifying chromoendoscopy to observe morphological changes of gastric pits. Biopsies were then taken from each area of interest and sent for pathological examination and detection of PCNA and p53 expression by immunohistochemistry. An immunoreactivity score for each lesion was calculated. Based on immunoreactivity scores, immunohistochemical staining was then considered.
RESULTS: Compared to intestinal metaplasia, gastric pits were more diverse in size, more irregular in shape, and more disorderly in arrangement in moderate and severe dysplasia. PCNA and p53 expression was significantly higher in precancerous lesions (intestinal metaplasia and dysplasia) than in chronic gastritis. PCNA expression showed an upward trend in types A-F pits. The number of cases that showed strong PCNA positivity increased significantly with an increase in the severity of lesions. Rank sum test for independent samples showed that p53 expression was significantly higher in types E and F pits than in types A-D pits (H = 33.068, P = 0.000). Rank sum test for independent samples showed that PCNA expression was significantly higher in types E and F pits than in types A-D pits (H = 31.791, P = 0.001).
CONCLUSION: The presence of types E and F pits, in which p53 and PCNA are highly expressed, is highly suggestive of the occurrence of early cancer, and patients developing these changes should be closely followed.
Magnifying chromoendoscopy; Gastric precancerous lesions; p53; Proliferating cell nuclear antigen; Early gastric cancer
Objective: Dracorhodin perchlorate (DP) was a synthetic analogue of the antimicrobial anthocyanin red pigment dracorhodin. It was reported that DP could induce apoptosis in human prostate cancer, human gastric tumor cells and human melanoma, but the cytotoxic effect of DP on human breast cancer was not investigated. This study would investigate whether DP was a candidate chemical of anti-human breast cancer.
Methods: The MTT assay reflected the number of viable cells through measuring the activity of cellular enzymes. Phase contrast microscopy visualized cell morphology. Fluorescence microscopy detected nuclear fragmentation after Hoechst 33258 staining. Flowcytometric analysis of Annexin V-PI staining and Rodamine 123 staining was used to detect cell apoptosis and mitochondrial membrane potential (MMP). Real time PCR detected mRNA level. Western blot examined protein expression.
Results: DP dose and time-dependently inhibited the growth of MCF-7 cells. DP inhibited MCF-7 cell growth through apoptosis. DP regulated the expression of Bcl-2 and Bax, which were mitochondrial pathway proteins, to decrease MMP, and DP promoted the transcription of Bax and inhibited Bcl-2. Apoptosis-inducing factor (AIF) and cytochrome c which localized in mitochondrial in physiological condition were released into cytoplasm when MMP was decreased. DP activated caspase-9, which was the downstream of mitochondrial pathway. Therefore DP decreased MMP to release AIF and cytochrome c into cytoplasm, further activating caspase 9, lastly led to apoptosis.
Conclusion: Therefore DP was a candidate for anti-breast cancer, DP induced apoptosis of MCF-7 through mitochondrial pathway.
dracorhodin perchlorate; apoptosis; mitochondrial pathway
Here we report a novel clade of secondary endosymbionts associated with insects and other arthropods. Seed bugs of the genus Nysius (Hemiptera: Lygaeidae) harbor the primary gammaproteobacterial symbiont Schneideria nysicola within a pair of bacteriomes in the abdomen. Our survey of Nysius species for their facultative bacterial associates consistently yielded a novel type of alphaproteobacterial 16S rRNA gene sequence in addition to those of Wolbachia. Diagnostic PCR survey of 343 individuals representing 24 populations of four Nysius species revealed overall detection rates of the alphaproteobacteria at 77.6% in Nysius plebeius, 87.7% in Nysius sp. 1, 81.0% in Nysius sp. 2, and 100% in Nysius expressus. Further survey of diverse stinkbugs representing 24 families, 191 species, and 582 individuals detected the alphaproteobacteria from an additional 12 species representing six families. Molecular phylogenetic analysis showed that the alphaproteobacteria from the stinkbugs form a distinct and coherent monophyletic group in the order Rickettsiales together with several uncharacterized endosymbionts from fleas and ticks. The alphaproteobacterial symbiont clade was allied to bacterial clades such as the endosymbionts of acanthamoebae, the endosymbionts of cnidarians, and Midichloria spp., the mitochondrion-associated endosymbionts of ticks. In situ hybridization and electron microscopy identified small filamentous bacterial cells in various tissues of N. plebeius, including the bacteriome and ovary. The concentrated localization of the symbiont cells at the anterior pole of oocytes indicated its vertical transmission route through host insect generations. The designation “Candidatus Lariskella arthropodarum” is proposed for the endosymbiont clade.
Patients with os odontoideum always present instability in atlantoaxial joint and need atlantoaxial fixation. C2 pedicle or laminar screws fixation has proven to be efficient and reliable for atlantoaxial instability. However, os odontoideum is a congenital or developmental disease, featured with anomalous bony anatomies. The anatomic measurements and guidelines for C2 pedicle screw placement in general population tends to differ with those of os odontoideum patients, for whom C2 pedicle screws are often needed. The option and techniques of C2 fixation are still challenging and yet to be fully explored.
Material and methods
We recruited 29 adult patients with os odontoideum and measured the dimension of C2 pedicle and lamina for each patient to examine how well do they match with the screws anatomically. In order to access the intra-observer reliability and inter-observer repeatability of the measurements, the intraclass correlation coefficient (ICC) was also calculated.
The results for reliability of the CT measurements showed excellent intraobserver (ICC = 0.95 and 0.96) and interobserver correlation coefficient (ICC = 0.93). The diameter and length of C2 pedicle were found to be 6.06 ± 1.37 and 24.05 ± 2.54 mm, while the corresponding figures of C2 laminar were 6.95 ± 0.82 and 25.60 ± 2.18 mm, respectively. In the measurements, all 29 cases had suitable diameter (larger than 5.5 mm) for C2 laminar screw (the laminar diameters ranged from 5.52 to 8.82 mm). In C2 pedicle measurements, the diameters of the 29 cases were from 3.50 to 9.86 mm, while 20 pedicles (34.5%) in 14 cases were less than 5.5 mm in diameter. Six had bilateral small pedicles where the diameter was less than 5.5 mm.
Anatomically, we found laminar screw is a better match in comparison with pedicle screw for C2 fixation in os odontoideum. The options for C2 fixation should be made based on careful preoperative imaging and thorough consideration. Preoperative reconstructive CT scan can offer great assistance for the choice of fixation in os odontoideum by revealing the anatomy of the C2 pedicles in detail.
Os odontoideum pedicle screw; Laminar screw; C2 fixation
Mechanically Interlocked molecules, such as catenanes and rotaxanes, are of great interest due to their fascinating structures and potential applications, while such molecules have been mainly restricted to comprising components of interlocked rings or polygons. The constructions of infinite polycatenanes and polyrotaxanes by discrete cages remain great challenge, and only two infinite polycatenanes fabricated by discrete cages have been reported so far, while the structures of polyrotaxanes and polypseudo-rotaxanes fabricated by discrete build units have not been documented to date. Herein we report the first example of a two-dimensional (2D) polypseudo-rotaxane fabricated by stool-like build units, the second example of a one-dimensional (1D) polycatenane, and the second example of a three-dimensional (3D) polycatenane, which were assemblied by discrete tetrahedral cages. The pores of dehydrated 3D polycatenane are dynamic, and display size-dependent adsorption/desorption behaviors of alcohols.
Classical swine fever virus (CSFV) can evade the immune response and establish chronic infection under natural and experimental conditions. Some genes related to antigen processing and presentation and to cytokine regulation are known to be involved in this response, but the precise mechanism through which each gene responds to CSFV infection remains unclear.
In this study, the amplification standard curve and corresponding linear regression equations for the genes SLA-2, TAP1, SLA-DR, Ii, CD40, CD80, CD86, IFN-α, and IFN-β were established successfully. Real-time RT-PCR was used to quantify the immune response gene transcription in PK-15 cells post CSFV infection. Results showed that: (1) immune response genes were generally down-regulated as a result of CSFV infection, and (2) the expression of SLA-2, SLA-DR, Ii and CD80 was significantly decreased (p<0.001).
We conclude that in vitro infection with CSFV inhibits the transcription of host immune response genes. These findings may facilitate the development of effective strategies for controlling CSF.
Classical swine fever virus (CSFV); Immune response genes; Real-time RT-PCR
This study explored the extent to which trait aggression is associated with suicidal behavior in a nationwide school-based sample of adolescents.
A nationwide sample of 14,537 high school students in urban areas of China was recruited. Information concerning suicide ideation, plans, attempts, trait aggression and other risk factors was collected by a self-reported questionnaire. Multivariate regression analyses were employed to predict suicidal behavior.
Approximately 18.5% of students reported suicide ideation, 8.7% reported suicide plans, and 4.1% reported attempts during the past one year. Hostility and trait anger had a significant positive association with suicidal ideation. Hostility and physical aggression were positively related to suicide plans. Hostility had a positive correlation with suicide attempts, while trait anger was inversely associated with suicide attempts.
This study suggests that hostility, physical aggression and trait anger may be able to be used to predict suicidal behavior among adolescents. Suicide prevention programs should target at attenuating the severity of hostility, anger and physical aggression. But teachers and parents should also give close attention to students with low trait anger.
Introduction. Posttraumatic stress disorder (PTSD) is accompanied by poor general psychological status (GPS). In the present study, we investigated the effects of a Chinese herbal formula on GPS in earthquake survivors with PTSD. Methods. A randomized, double-blind, placebo-controlled trial compared a Chinese herbal formula, Xiao-Tan-Jie-Yu-Fang (XTJYF), to placebo in 2008 Sichuan earthquake survivors with PTSD. Patients were randomized into XTJYF (n = 123) and placebo (n = 122) groups. Baseline-to-end-point score changes in the three global indices of the Symptom Checklist-90-Revised (SCL-90-R) and rates of response in the SCL global severity index (GSI) were the primary endpoints. A subanalysis of the nine SCL factors and the sleep quality score were secondary endpoints. Results and Discussion. Compared to placebo, the XTJYF group was significantly improved in all three SCL global indices (P = 0.001~0.028). More patients in the XTJYF group reported “much improved” than the placebo group (P = 0.001). The XTJYF group performed significantly better than control in five out of nine SCL factors (somatization, obsessive-compulsive behavior, depression, anxiety, and hostility (P = 0.001~0.036)), and in sleep quality score (P < 0.001). XTJYF produced no serious adverse events. These findings suggest that XTJYF may be an effective and safe treatment option for improving GPS in patients with PTSD.
In the title compound, C14H9ClI2N2O2, the dihedral angle between the benzene rings is 65.9 (2)° and an intramolecular O—H⋯N hydrogen bond generates an S(6) ring. The molecule has an E conformation about the C=N bond. In the crystal, molecules are linked into C(4) chains propagating in  by N—H⋯O hydrogen bonds.
The stinkbug Parastrachia japonensis (Hemiptera: Parastrachiidae) is known for its prolonged prereproductive nonfeeding period, maternal care of eggs in an underground nest, and maternal collection and provisioning of food (fruits) for nymphs. A previous study suggested that a bacterial symbiont is involved in uric acid recycling in this insect during the nonfeeding period, but the identity of this symbiont has not been determined. Here we characterized a novel bacterial symbiont obtained from P. japonensis. Molecular phylogenetic analyses based on 16S rRNA, gyrB, and groEL gene sequences consistently indicated that this symbiont constituted a distinct lineage in the Gammaproteobacteria that has no close relatives but is allied with gut symbionts of acanthosomatid and plataspid stinkbugs, as well as with endocellular symbionts of sharpshooters, tsetse flies, and aphids. The symbiont genes had a remarkably AT-biased nucleotide composition and exhibited significantly accelerated molecular evolution. The symbiont genome was extremely reduced; its size was estimated to be 0.85 Mb. These results suggest that there has been an intimate host-symbiont association over evolutionary time. The symbiont was localized in swollen crypts in a posterior part of the midgut, which was a specialized symbiotic organ. The possibility that the symbiont is involved in uric acid recycling is discussed. The designation “Candidatus Benitsuchiphilus tojoi” is proposed for the symbiont.
A microbial fuel cell (MFC) was inoculated with a random transposon insertion mutant library of Shewanella oneidensis MR-1 and operated with lactate as the sole fuel to select for mutants that preferentially grew in it. Agar plate cultivation of the resultant MFC enrichment culture detected an increased number of colonies exhibiting rough morphology. One such isolate, strain 4A, generated 50% more current in an MFC than wild-type MR-1. Determination of the transposon insertion site in strain 4A followed by deletion and complementation experiments revealed that the SO3177 gene, encoding a putative formyltransferase and situated in a cell surface polysaccharide biosynthesis gene cluster, was responsible for the increased current. Transmission electron microscopy showed that a layered structure at the cell surface, stainable with ruthenium red, was impaired in the SO3177 mutant (ΔSO3177), confirming that SO3177 is involved in the biosynthesis of cell surface polysaccharides. Compared to the wild type, ΔSO3177 cells preferentially attached to graphite felt anodes in MFCs, while physicochemical analyses revealed that the cell surface of ΔSO3177 was more hydrophobic. These results demonstrate that cell surface polysaccharides affect not only the cell adhesion to graphite anodes but also the current generation in MFCs.
Symbiotic associations with midgut bacteria have been commonly found in diverse phytophagous heteropteran groups, where microbiological characterization of the symbiotic bacteria has been restricted to the stinkbug families Acanthosomatidae, Plataspidae, Pentatomidae, Alydidae, and Pyrrhocoridae. Here we investigated the midgut bacterial symbiont of Cantao ocellatus, a stinkbug of the family Scutelleridae. A specific gammaproteobacterium was consistently identified from the insects of different geographic origins. The bacterium was detected in all 116 insects collected from 9 natural host populations. Phylogenetic analyses revealed that the bacterium constitutes a distinct lineage in the Gammaproteobacteria, not closely related to gut symbionts of other stinkbugs. Diagnostic PCR and in situ hybridization demonstrated that the bacterium is extracellularly located in the midgut 4th section with crypts. Electron microscopy of the crypts revealed a peculiar histological configuration at the host-symbiont interface. Egg sterilization experiments confirmed that the bacterium is vertically transmitted to stinkbug nymphs via egg surface contamination. In addition to the gut symbiont, some individuals of C. ocellatus harbored another bacterial symbiont in their gonads, which was closely related to Sodalis glossinidius, the secondary endosymbiont of tsetse flies. Biological aspects of the primary gut symbiont and the secondary Sodalis-allied symbiont are discussed.
Here we investigated the bacterial endosymbionts of weevils of the genus Curculio. From all four species of Curculio weevils examined, a novel group of bacterial gene sequences were consistently identified. Molecular phylogenetic analyses demonstrated that the sequences formed a distinct clade in the Gammaproteobacteria, which was not related to previously known groups of weevil endosymbionts such as Nardonella spp. and Sodalis-allied symbionts. In situ hybridization revealed that the bacterium was intracellularly harbored in a bacteriome associated with larval midgut. In adult females, the bacterium was localized in the germalia at the tip of each overiole, suggesting vertical transmission via ovarial passage. Diagnostic PCR surveys detected high prevalence of the bacterial infection in natural host populations. Electron microscopy identified the reduced cell wall of the bacterial cells, and the bacterial genes exhibited AT-biased nucleotide composition and accelerated molecular evolution, which are suggestive of a long-lasting endosymbiotic association. On the basis of these results, we conclude that the novel endocellular bacteria represent the primary symbiont of Curculio weevils and proposed the designation “Candidatus Curculioniphilus buchneri.” In addition to “Ca. Curculioniphilus,” we identified Sodalis-allied gammaproteobacterial endosymbionts from the chestnut weevil, Curculio sikkimensis, which exhibited partial infection frequencies in host insect populations and neither AT-biased nucleotide composition nor accelerated molecular evolution. We suggest that such Sodalis-allied secondary symbionts in weevils might provide a potential source for symbiont replacements, as has occurred in an ancestor of Sitophilus grain weevils.
Host-symbiont co-speciation and reductive genome evolution have been commonly observed among obligate endocellular insect symbionts, while such examples have rarely been identified among extracellular ones, the only case reported being from gut symbiotic bacteria of stinkbugs of the family Plataspidae. Considering that gut symbiotic communities are vulnerable to invasion of foreign microbes, gut symbiotic associations have been thought to be evolutionarily not stable. Stinkbugs of the family Acanthosomatidae harbor a bacterial symbiont in the midgut crypts, the lumen of which is completely sealed off from the midgut main tract, thereby retaining the symbiont in the isolated cryptic cavities. We investigated histological, ecological, phylogenetic, and genomic aspects of the unique gut symbiosis of the acanthosomatid stinkbugs.
Phylogenetic analyses showed that the acanthosomatid symbionts constitute a distinct clade in the γ-Proteobacteria, whose sister groups are the obligate endocellular symbionts of aphids Buchnera and the obligate gut symbionts of plataspid stinkbugs Ishikawaella. In addition to the midgut crypts, the symbionts were located in a pair of peculiar lubricating organs associated with the female ovipositor, by which the symbionts are vertically transmitted via egg surface contamination. The symbionts were detected not from ovaries but from deposited eggs, and surface sterilization of eggs resulted in symbiont-free hatchlings. The symbiont-free insects suffered retarded growth, high mortality, and abnormal morphology, suggesting important biological roles of the symbiont for the host insects. The symbiont phylogeny was generally concordant with the host phylogeny, indicating host-symbiont co-speciation over evolutionary time despite the extracellular association. Meanwhile, some local host-symbiont phylogenetic discrepancies were found, suggesting occasional horizontal symbiont transfers across the host lineages. The symbionts exhibited AT-biased nucleotide composition, accelerated molecular evolution, and reduced genome size, as has been observed in obligate endocellular insect symbionts.
Comprehensive studies of the acanthosomatid bacterial symbiosis provide new insights into the genomic evolution of extracellular symbiotic bacteria: host-symbiont co-speciation and drastic genome reduction can occur not only in endocellular symbiotic associations but also in extracellular ones. We suggest that many more such cases might be discovered in future surveys.
In situ detection of methanogens within the family Methanobacteriaceae is sometimes known to be unsuccessful due to the difficulty in permeability of oligonucleotide probes. Pseudomurein endoisopeptidase (Pei), a lytic enzyme that specifically acts on their cell walls, was applied prior to 16S rRNA-targeting fluorescence in situ hybridization (FISH). For this purpose, pure cultured methanogens within this family, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, and Methanothermobacter thermautotrophicus together with a Methanothermobacter thermautotrophicus-containing syntrophic acetate-oxidizing coculture, endosymbiotic Methanobrevibacter methanogens within an anaerobic ciliate, and an upflow anaerobic sludge blanket (UASB) granule were examined. Even without the Pei treatment, Methanobacterium bryantii and Methanothermobacter thermautotrophicus cells are relatively well hybridized with oligonucleotide probes. However, almost none of the cells of Methanobrevibacter ruminantium, Methanosphaera stadtmanae, cocultured Methanothermobacter thermautotrophicus, and the endosymbiotic methanogens and the cells within UASB granule were hybridized. Pei treatment was able to increase the probe hybridization ratio in every specimen, particularly in the specimen that had shown little hybridization. Interestingly, the hybridizing signal intensity of Methanothermobacter thermautotrophicus cells in coculture with an acetate-oxidizing H2-producing syntroph was significantly improved by Pei pretreatment, whereas the probe was well hybridized with the cells of pure culture of the same strain. We found that the difference is attributed to the differences in cell wall thicknesses between the two culture conditions. These results indicate that Pei treatment is effective for FISH analysis of methanogens that show impermeability to the probe.
Our objective was to develop an oligonucleotide DNA microarray (OMA) for genome-wide microRNA profiling and use this method to find miRNAs, which control organic development especially for nervous system.
Materials and methods
Eighteen organic samples included cerebrum and spinal cord samples from two aborted human fetuses. One was 12 gestational weeks old (G12w) and the other was 24 gestational weeks old (G24w). Global miRNA expression patterns of different organs were investigated using OMA and Northern blot.
The OMA revealed that 72–83% of miRNAs were expressed in human fetal organs. A series of microRNAs were found specifically and higher-expressed in the human fetal nervous system and confirmed consistently by Northern blot, which may play a critical role in nervous system development.
MicroRNA; Oligonucleotide microarray; Expression profile; Nervous system development
The Japanese common broad-headed bugs Riptortus clavatus and Leptocorisa chinensis possess a number of crypts in the posterior region of the midgut, whose lumen contains a copious amount of bacterial cells. We characterized the gut symbiotic bacteria by using molecular phylogenetic analysis, light and electron microscopy, in situ hybridization, and PCR-based detection techniques. Restriction fragment length polymorphism analysis of 16S rRNA gene clones suggested that a single bacterium dominated the microbiota in the crypts of the both bug species. The predominant 16S rRNA gene sequences obtained from different individuals and species of the bugs were not identical but were very similar to each other. Homology searches in the DNA databases revealed that the sequences showed the highest levels of similarity (96% to 99%) to the sequences of Burkholderia spp. belonging to the β subdivision of the class Proteobacteria. In situ hybridization with specific oligonucleotide probes confirmed the localization of the Burkholderia symbiont in the lumen of the midgut crypts. Electron microscopy showed that the lumen of the crypts was filled with rod-shaped bacteria of a single morphotype. Molecular phylogenetic analysis demonstrated that the Burkholderia symbionts of the bugs formed a well-defined monophyletic group, although the group also contained several environmental Burkholderia strains. The phylogenetic relationship of the Burkholderia symbionts did not reflect the relationship of the host bug species at all. The sequences from R. clavatus and the sequences from L. chinensis did not form clades but were intermingled in the phylogeny, suggesting that horizontal transmission of the symbiont might have occasionally occurred between populations and species of the bugs.
In natural populations of the pea aphid Acyrthosiphon pisum, a facultative bacterial symbiont of the genus Rickettsia has been detected at considerable infection frequencies worldwide. We investigated the effects of the Rickettsia symbiont on the host aphid and also on the coexisting essential symbiont Buchnera. In situ hybridization revealed that the Rickettsia symbiont was specifically localized in two types of host cells specialized for endosymbiosis: secondary mycetocytes and sheath cells. Electron microscopy identified bacterial rods, about 2 μm long and 0.5 μm thick, in sheath cells of Rickettsia-infected aphids. Virus-like particles were sometimes observed in association with the bacterial cells. By an antibiotic treatment, we generated Rickettsia-infected and Rickettsia-eliminated aphid strains with an identical genetic background. Comparison of these strains revealed that Rickettsia infection negatively affected some components of the host fitness. Quantitative PCR analysis of the bacterial population dynamics identified a remarkable interaction between the coexisting symbionts: Buchnera population was significantly suppressed in the presence of Rickettsia, particularly at the young adult stage, when the aphid most actively reproduces. On the basis of these results, we discussed the possible mechanisms that enable the prevalence of Rickettsia infection in natural host populations in spite of the negative fitness effects observed in the laboratory.
Adult-onset type II citrullinemia (CTLN2) is an autosomal recessive disease caused by mutations in SLC25A13, the gene encoding the mitochondrial aspartate/glutamate carrier citrin. The absence of citrin leads to a liver-specific, quantitative decrease of argininosuccinate synthetase (ASS), causing hyperammonemia and citrullinemia. To investigate the physiological role of citrin and the development of CTLN2, an Slc25a13-knockout (also known as Ctrn-deficient) mouse model was created. The resulting Ctrn−/− mice were devoid of Slc25a13 mRNA and citrin protein. Liver mitochondrial assays revealed markedly decreased activities in aspartate transport and the malate-aspartate shuttle. Liver perfusion also demonstrated deficits in ureogenesis from ammonia, gluconeogenesis from lactate, and an increase in the lactate-to-pyruvate ratio within hepatocytes. Surprisingly, Ctrn−/− mice up to 1 year of age failed to show CTLN2-like symptoms due to normal hepatic ASS activity. Serological measures of glucose, amino acid, and ammonia metabolism also showed no significant alterations. Nitrogen-loading treatments produced only minor changes in the hepatic ammonia and amino acid levels. These results suggest that citrin deficiency alone may not be sufficient to produce a CTLN2-like phenotype in mice. These observations are compatible, however, with the variable age of onset, incomplete penetrance, and strong ethnic bias seen in CTLN2 where additional environmental and/or genetic triggers are now suspected.
Foraging, defense and waste disposal are essential for sustaining social insect colonies. Hence, their nest generally has an open structure, wherein specialized castes called workers and soldiers perform these tasks. However, some social aphids form completely closed galls, wherein hundreds to thousands of insects grow and reproduce for several months in isolation. Why these social aphids are not drowned by accumulated honeydew has been an enigma. Here we report a sophisticated biological solution to the waste problem in the closed system: the gall inner surface is specialized for absorbing water, whereby honeydew is promptly removed via the plant vascular system. The water-absorbing closed galls have evolved at least twice independently among social aphids. The plant-mediated waste removal, which entails insect's manipulation of plant morphogenesis and physiology, comprises a previously unknown mechanism of nest cleaning, which can be regarded as ‘extended phenotype' and ‘indirect social behavior' of the social aphids.
Some social aphids have evolved to live inside completely closed galls, which presents a waste disposal problem of the honeydew that collects inside the gall. Here, Kutsukake et al. show that the gall inner surface is specialized for absorbing water, removing honeydew via the plant vascular system.