Functional expression of TRPA1 channel,TRPV1 channel and TMEM100 in human odontoblasts

TRPA1 and TRPV1 channels respond to external stimulation as pain mediators and form a complex with a transmembrane protein TMEM100 in some tissues. However, their expression and interaction in dental pulp is unclear. To investigate the functional co-expression of TRPA1 channel, TRPV1 channel and TMEM100 in human odontoblasts (HODs), immunohistochemistry, immunofluorescence staining and Western blot were used to study their co-localization and expression in both native HODs and cultured HOD-like cells. Calcium imaging was used to detect the functional interaction between TRPA1 and TRPV1 channels. Immunohistochemistry and multiple immunofluorescence staining of tooth slices showed positive expression of TRPA1 channel, TRPV1 channel and TMEM100 mainly in the cell bodies of HODs, and TRPA1 channel presented more obvious immunofluorescence in the cell processes than TRPV1 channel and TMEM100. HALO software analysis showed that TRPA1 and TRPV1 channels were positively expressed in most TMEM100⁺ HODs and these three proteins were strongly correlated in HODs (P < 0.01). The protein expression levels of TRPA1 channel, TRPV1 channel and TMEM100 in HODs showed no significant difference (P?>?0.05). Double immunofluorescence staining of cultured HOD-like cells visually demonstrated that TRPA1 and TRPV1 channel were both highly co-localized with TMEM100 with similar expressive intensity. Calcium imaging showed that there was a functional interaction between TRPA1 and TRPV1 channels in HOD-like cells, and TRPA1 channel might play a greater role in this interaction. Overall, we concluded that TRPA1 channel, TRPV1 channel and TMEM100 could be functionally co-expressed in HODs.

     

Fosmid library construction and end sequences analysis of the Pacific oyster,Crassostrea gigas

The Pacific oyster (Crassostrea gigas) is globally distributed and is one of the most commercially and ecologically important marine organisms. However, little is known about the genome of this species. In this study, a C. gigas fosmid library was constructed that contains 459,936 clones with an average insert size of approximately 40 kb, representing 22.34-fold haploid genome equivalents. End sequencing generated 90,240 fosmid end sequences (FESs) with an average length of 384.27 base pairs (bp), covering approximately 2.58% of the Pacific oyster genome. The FESs were subsequently assembled and annotated, resulting in 6332 sequences with predicted open reading frames≥300 and 1,189,100 bp repeats. Furthermore, a total of 3200 microsatellite repeats were identified, and dinucleotide repeats were found to occur most abundantly, with AG and AAT being the most abundant repeat class of dinucleotides and trinucleotides. We also found that the repeat number was generally negatively proportional to the repeat element length. Microsatellites composition between the transcribed sequences and genomic sequences was shown to be different. Point mutations of microsatellite were non-random and underwent strong selection stress. Overall, a comprehensive sequence resource for the Pacific oyster was created, including annotated transposable elements, tandem repeats, protein coding sequences and microsatellites. These initial findings will serve as resources for further in-depth studies of physical mapping, gene discovery, microsatellite marker developing and evolution studies.     

Smad7 attenuates TGF-β-mediated aging-related hypofunction of submandibular glands

Submandibular glands have essential functions in taste, mastication, swallowing, and digestion. Submandibular gland hypofunction is prevalent in the elderly, impairing the patients’ quality of life. Current clinical treatment strategies have not decelerated or reversed the pathological process of submandibular gland hypofunction. Therefore, novel restoration strategies should be explored. However, studies on the mechanism of aging-related submandibular gland hypofunction remain very limited. The role of the TGF-β/Smad pathway in fibrosis has been studied in other organs. Therefore, this study aimed to elucidate the role of TGF-β/Smad signaling in the aging-related submandibular gland hypofunction. The results showed that Smad7 knockout in mice decreased the salivary flow rate. H&E, Masson trichrome, and immunohistochemistry staining of MCP-1 and α-SMA showed that Smad7 knockout in mice resulted in lymphocytic infiltration, acinar cell atrophy, and interstitial fibrosis. The Western blotting of collagen I and III also confirmed extensive fibrosis. We then found that Smad7 depletion resulted in the TGF-β-mediated fibrosis via mir-21, mir-29, and np_5318, and NFκB-driven inflammation activation. This study confirmed the inhibitory role of Smad7 in the aging-related submandibular gland hypofunction. Therefore, it provided a promising treatment target for aging-related dysfunction and sialadenitis of submandibular gland.     

Decline of tactile acuity in aging: a study of body site, blood flow, and lifetime habits of smoking and physical activity

Tactile acuity of 60 older subjects (> or = 65 years) and 19 younger subjects (18-28 years) was assessed by two-point gap thresholds at the upper and lower surfaces of the forefinger, at the upper and lower surfaces of the feet, and at the volar surface of the forearm. The older subjects were assigned to one of four groups of 15 subjects each, depending on reported lifetime habits of physical activity and smoking: (1) active smokers, (2) active nonsmokers, (3) inactive smokers, and (4) inactive nonsmokers. Peripheral blood flow was assessed at the forefinger, foot, and forearm by means of laser-Doppler imaging and skin temperature recordings, under resting conditions and during and after a 5-min exposure to mild cooling (28 degrees C). Consistent with previous studies, tactile acuity thresholds in the foot and finger averaged about 80% higher in the older subjects than in the younger subjects, but only about 22% higher in the forearm. Although the upper surface of the fingertip was more sensitive than the lower surface in both younger and older subjects, the age-related decline in tactile acuity was nearly identical on both sides of the finger and foot. The latter finding refutes the hypothesis that the larger effect of aging in the extremities results from greater physical wear and tear on the contact surfaces of the hands and feet. Self-reported lifetime histories of physical activity and smoking were not significantly associated with measures of cutaneous blood flow or tactile thresholds. Possible reasons for this lack of association are discussed, including the inherent limitations of testing only healthy older subjects, and the concept of "successful aging".     

Clinical Significance of Cerebral Microbleeds Locations in CADASIL with R544C NOTCH3 Mutation

MethodsThis is a cohort study of patients who were diagnosed with genotype-confirmed R544C-mutation CADASIL. Primary neurologic symptoms were recorded. Symptomatic strokes were defined as transient ischemic attack, ischemic strokes and hemorrhagic strokes. CMBs were defined as focal areas of round signal loss on T2*-weighted gradient echo planar images with a diameter of less than 10 mm. The locations of CMBs were divided into lobar, basal ganglia, thalamus, brain stem and cerebellum. Multiple logistic regressions were performed to identify the epidemiologic or vascular risk factors associated with symptomatic stroke in patients with CADASIL.ResultsAmong total of 51 subjects in this cohort, CMBs were present in 20 of 32 patients (64.5%) in the symptomatic stroke-group and in 8 of 19 patients (42.1%) in the non-stroke group (p = 0.16). CMBs were observed more frequently in the basal ganglia (p<0.001) and the cerebellum (p<0.018) in the symptomatic stoke group compared to the non-stroke group. The mean number of CMBs was significantly higher in the symptomatic stroke group (15.4±18.0 lesions per patients with CMBs) versus those without symptomatic stroke (3.3±3.0 lesions per patients with CMBs) (p = 0.003). Hypertension was an independent risk factor for symptomatic stroke in CADASIL (p = 0.014). It was independently associated with CMBs locations as basal ganglia (p = 0.016), thalamus (p = 0.010), brainstem (p = 0.044), and cerebellum (p = 0.049). However, It was not independently associated with CMBs on lobar lesion (p = 0.152).ConclusionsIn this study hypertension was an independent predictor of CMBs presence in specific brain locations, as well as symptomatic stroke in the CADASIL patients. The distribution and burden of CMBs might be a clinically useful marker for the risk of symptomatic stroke. However, further prospective studies on the relationship between CMBs distribution and symptomatic stroke are required in order to support these preliminary findings.     

Pathogenic copy number variations are associated with foetal short femur length in a tertiary referral centre study

Shortened foetal femur length (FL) is a common abnormal phenotype that often causes anxiety in pregnant women, and standard clinical treatments remain unavailable. We investigated the clinical characteristics, genetic aetiology and obstetric pregnancy outcomes of foetuses with short FL and provided a reference for perinatal management of such cases. Chromosomal microarray analysis was used to analyse the copy number variations (CNV) in short FL foetuses. Of the 218 foetuses with short FL, 33 foetuses exhibited abnormal CNVs, including 19 with pathogenic CNVs and 14 with variations of uncertain clinical significance. Of the 19 foetuses with pathogenic CNVs, four had aneuploidy, 14 had deletions/duplications, and one had pathogenic uniparental diploidy. The 7q11.23 microdeletion was detected in three foetuses. The severity of short FL was not associated with the rate of pathogenic CNVs. The duration of short FL for the intrauterine ultrasound phenotype in foetuses carrying a pathogenic CNV was independent of the gestational age. Further, maternal age was not associated with the incidence of foetal pathogenic CNVs. Adverse pregnancy outcomes occurred in 77 cases, including termination of pregnancy in 63 cases, postnatal dwarfed foetuses with intellectual disability in 11 cases, and three deaths within 3 months of birth. Pathogenic CNVs closely related to foetal short FL were identified, among which the 7q11.23 microdeletion was highly associated with short FL development. This study provides a reference for the perinatal management of foetuses with short FL.     

<Emphasis Type="Italic">Multiple impairments in male reproduction 1 (mimr1)</Emphasis>, a novel male-sterile mutant of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>, shows several defects in male reproductive development

We have characterized a new male-sterile mutant in Arabidopsis that exhibits conditional sterility but has restored fertility when drought-stressed. This mutant, multiple impairments in male reproduction 1 (mimr1), shows pleiotropic defects in both vegetative and reproductive development. Examination with dissecting and scanning electron microscopes revealed that its pollen grains are not effectively released from the anther locule after dehiscence, and anther differentiation is defective. Growth of the style and stamen filaments are also abnormal. Histological analysis demonstrated that these phenomena are due not only to a noticeably reduced extension of the stamen but also greater elongation of the pistil. Genetic analysis indicated that mimr1 is a single locus recessive nuclear mutant. The mutation can be mapped to a locus strongly linked to a 1200-kb region on Chromosome 3. Meta-analysis of expression patterning presented several candidate genes in that region. No mutants with similar phenotypes have previously been reported, suggesting that mimr1 is a novel male-sterile locus. Characterization of MIMR1 will provide further insights into the molecular basis for the development of plant reproductive organs.     

The MET13 Methylenetetrahydrofolate Reductase Gene Is Essential for Infection-Related Morphogenesis in the Rice Blast Fungus Magnaporthe oryzae

Methylenetetrahydrofolate reductases (MTHFRs) play a key role in the biosynthesis of methionine in both prokaryotic and eukaryotic organisms. In this study, we report the identification of a novel T-DNA-tagged mutant WH672 in the rice blast fungus Magnaporthe oryzae, which was defective in vegetative growth, conidiation and pathogenicity. Analysis of the mutation confirmed a single T-DNA insertion upstream of MET13, which encodes a 626-amino-acid protein encoding a MTHFR. Targeted gene deletion of MET13 resulted in mutants that were non-pathogenic and significantly impaired in aerial growth and melanin pigmentation. All phenotypes associated with Δmet13 mutants could be overcome by addition of exogenous methionine. The M. oryzae genome contains a second predicted MTHFR-encoding gene, MET12. The deduced amino acid sequences of Met13 and Met12 share 32% identity. Interestingly, Δmet12 mutants produced significantly less conidia compared with the isogenic wild-type strain and grew very poorly in the absence of methionine, but were fully pathogenic. Deletion of both genes resulted in Δmet13Δmet12 mutants that showed similar phenotypes to single Δmet13 mutants. Taken together, we conclude that the MTHFR gene, MET13, is essential for infection-related morphogenesis by the rice blast fungus M. oryzae.