Cytological Observation and RNA-Seq Analyses Reveal miR9564 and Its Target Associated with Pollen Sterility in Autotetraploid Rice.

Understanding the regulation of autotetraploid sterility is essential for harnessing the strong advantages in genomic buffer capacity, biodiversity, and heterosis of autotetraploid rice. miRNAs play crucial roles in fertility regulation, yet information about their reproductive roles and target genes in tetraploid rice remains limited. Here, we used three tetraploid lines, H1 (fertile), HF (fertile), and LF (sterile), to investigate cytological features and identify factors associated with autotetraploid sterility. LF showed abnormal meiosis, resulting in low pollen fertility and viability, ultimately leading to scarce fertilization and a low-seed setting compared to H1 and HF. RNA-seq revealed 30 miRNA-candidate target pairs related to autotetraploid pollen sterility. These pairs showed opposite expression patterns, with differential expression between fertile lines (H1 and HF) and the sterile line (LF). qRT-PCR confirmed that miR9564, miR528, and miR27874 were highly expressed in the anthers of H1 and HF but not in LF, while opposite results were obtained in their targets (ARPS, M2T, and OsRPC53). Haplotype and expression pattern analyses revealed that ARPS was specifically expressed in lines with the same haplotype of MIR9564 (the precursor of miR9564) as LF. Furthermore, the Dual-GFP assay verified that miR9564 inhibited the fluorescence signal of ARPS-GFP. The over-expression of ARPS significantly decreased the seed setting rate (59.10%) and pollen fertility (50.44%) of neo-tetraploid rice, suggesting that ARPS plays important roles in autotetraploid pollen sterility. This study provides insights into the cytological characteristic and miRNA expression profiles of tetraploid lines with different fertility, shedding light on the role of miRNAs in polyploid rice.

Seed development-related genes contribute to high yield heterosis in integrated utilization of elite autotetraploid and neo-tetraploid rice.

Introduction: Autotetraploid rice holds high resistance to abiotic stress and substantial promise for yield increase, but it could not be commercially used because of low fertility. Thus, our team developed neo-tetraploid rice with high fertility and hybrid vigor when crossed with indica autotetraploid rice. Despite these advances, the molecular mechanisms underlying this heterosis remain poorly understood. Methods: An elite indica autotetraploid rice line (HD11) was used to cross with neo-tetraploid rice, and 34 hybrids were obtained to evaluate agronomic traits related to yield. WE-CLSM, RNA-seq, and CRISPR/Cas9 were employed to observe endosperm structure and identify candidate genes from two represent hybrids. Results and discussion: These hybrids showed high seed setting and an approximately 55% increase in 1000-grain weight, some of which achieved grain yields comparable to those of the diploid rice variety. The endosperm observations indicated that the starch grains in the hybrids were more compact than those in paternal lines. A total of 119 seed heterosis related genes (SHRGs) with different expressions were identified, which might contribute to high 1000-grain weight heterosis in neo-tetraploid hybrids. Among them, 12 genes had been found to regulate grain weight formation, including OsFl3, ONAC023, OsNAC024, ONAC025, ONAC026, RAG2, FLO4, FLO11, OsISA1, OsNF-YB1, NF-YC12, and OsYUC9. Haplotype analyses of these 12 genes revealed the various effects on grain weight among different haplotypes. The hybrids could polymerize more dominant haplotypes of above grain weight regulators than any homozygous cultivar. Moreover, two SHRGs (OsFl3 and SHRG2) mutants displayed a significant reduction in 1000-grain weight and an increase in grain chalkiness, indicating that OsFl3 and SHRG2 positively regulate grain weight. Our research has identified a valuable indica autotetraploid germplasm for generating strong yield heterosis in combination with neo-tetraploid lines and gaining molecular insights into the regulatory processes of heterosis in tetraploid rice.

MORE FLORET1 controls anther development by negatively regulating key tapetal genes in both diploid and tetraploid rice.

Polyploid hybrid rice (Oryza sativa) has great potential for increasing yields. However, hybrid rice depends on male fertility and its regulation, which is less well studied in polyploid rice than in diploid rice. We previously identified an MYB transcription factor, MORE FLORET1 (MOF1), whose mutation causes male sterility in neo-tetraploid rice. MOF1 expression in anthers peaks at anther Stage 7 (S7) and progressively decreases to low levels at S10. However, it remains unclear how the dynamics of MOF1 expression contribute to male fertility. Here, we carefully examined anther development in both diploid and tetraploid mof1 rice mutants, as well as lines ectopically expressing MOF1 in a temporal manner. MOF1 mutations caused delayed degeneration of the tapetum and middle layer of anthers and aberrant pollen wall organization. Ectopic MOF1 expression at later stages of anther development led to retarded cytoplasmic reorganization of tapetal cells. In both cases, pollen grains were aborted and seed production was abolished, indicating that precise control of MOF1 expression is essential for male reproduction. We demonstrated that 5 key tapetal genes, CYP703A3 (CYTOCHROME P450 HYDROXYLASE 703A3), OsABCG26 (O. sativa ATP BINDING CASSETTE G26), PTC1 (PERSISTENT TAPETAL CELL1), PKS2 (POLYKETIDE SYNTHASE 2), and OsABCG15 (O. sativa ATP BINDING CASSETTE G15), exhibit expression patterns opposite to those of MOF1 and are negatively regulated by MOF1. Moreover, DNA affinity purification sequencing (DAP-seq), luciferase activity assays, and electrophoretic mobility shift assays indicated that MOF1 binds directly to the PKS2 promoter for transcriptional repression. Our results provide a mechanistic basis for the regulation of male reproduction by MOF1 in both diploid and tetraploid rice. This study will facilitate the development of polyploid male sterile lines, which are useful for breeding of polyploid hybrid rice.

All-in-one porous membrane enables full protection in guided bone regeneration.

The sophisticated hierarchical structure that precisely combines contradictory mechanical and biological characteristics is ideal for biomaterials, but it is challenging to achieve. Herein, we engineer a spatiotemporally hierarchical guided bone regeneration (GBR) membrane by rational bilayer integration of densely porous N-halamine functionalized bacterial cellulose nanonetwork facing the gingiva and loosely porous chitosan-hydroxyapatite composite micronetwork facing the alveolar bone. Our GBR membrane asymmetrically combine stiffness and flexibility, ingrowth barrier and ingrowth guiding, as well as anti-bacteria and cell-activation. The dense layer has a mechanically matched space maintenance capacity toward gingiva, continuously blocks fibroblasts, and prevents bacterial invasion with multiple mechanisms including release-killing, contact-killing, anti-adhesion, and nanopore-blocking; the loose layer is ultra-soft to conformally cover bone surfaces and defect cavity edges, enables ingrowth of osteogenesis-associated cells, and creates a favorable osteogenic microenvironment. As a result, our all-in-one porous membrane possesses full protective abilities in GBR.

Long-lasting renewable antibacterial porous polymeric coatings enable titanium biomaterials to prevent and treat peri-implant infection.

Peri-implant infection is one of the biggest threats to the success of dental implant. Existing coatings on titanium surfaces exhibit rapid decrease in antibacterial efficacy, which is difficult to promisingly prevent peri-implant infection. Herein, we report an N-halamine polymeric coating on titanium surface that simultaneously has long-lasting renewable antibacterial efficacy with good stability and biocompatibility. Our coating is powerfully biocidal against both main pathogenic bacteria of peri-implant infection and complex bacteria from peri-implantitis patients. More importantly, its antibacterial efficacy can persist for a long term (e.g., 12~16 weeks) in vitro, in animal model, and even in human oral cavity, which generally covers the whole formation process of osseointegrated interface. Furthermore, after consumption, it can regain its antibacterial ability by facile rechlorination, highlighting a valuable concept of renewable antibacterial coating in dental implant. These findings indicate an appealing application prospect for prevention and treatment of peri-implant infection.

Rac1 Regulates the Proliferation, Adhesion, Migration, and Differentiation of MDPC-23 Cells.

INTRODUCTION: Stem cells are responsible for replacing damaged pulp tissue; therefore, promoting their survival and inducing their adhesion to dentin are vital. As a member of the Rho family of guanosine triphosphatases, Rac1 is an important regulator of osteoblast functions. However, little is known about its role in regenerative endodontic procedures. The current study examined the role of Rac1 in the proliferation, migration, and odontoblastic differentiation of MDPC-23 cells. METHODS: MDPC-23 cells were transfected with small interfering RNA to knock down Rac1 expression, and then their proliferation, migration, adhesion, and odontoblastic differentiation were examined in vitro. RESULTS: MDPC-23 cells transfected with si-Rac1 exhibited the increased expression of several key odontogenic protein markers, including Dmp1, Dspp, Runx2, and alkaline phosphatase, as well as decreased proliferation and migration in vitro. The results suggest that Rac1 might regulate nuclear factor kappa B signaling in MDPC-23 cells. CONCLUSION: Rac1 may have vital roles in the proliferation, migration, adhesion, and odontoblastic differentiation of MDPC-23 cells.

Odontogenic cutaneous sinus tract associated with a mandibular second molar having a rare distolingual root: a case report.

INTRODUCTION: Odontogenic cutaneous sinus tracts are often misdiagnosed as lesions of non-odontogenic origin, leading to the treatment of patients with unnecessary and ineffective therapies. Sinus tracts of endodontic origin usually respond well to endodontic therapy. However, root canal treatment of mandibular molars with aberrant canal anatomy can be diagnostically and technically challenging. Herein we present a patient with a cutaneous odontogenic sinus tract in the right submandibular area. CASE REPORT: A 23-year-old Chinese female patient presented with a cutaneous odontogenic sinus tract that was initially misdiagnosed as a sebaceous cyst. The patient had undergone surgical excision and traditional Chinese medical therapy before endodontic consultation. With the aid of cone beam computed tomography (CBCT), it was confirmed that the causative factor of the cutaneous odontogenic sinus tract was chronic periapical periodontitis of the right mandibular second molar, which had a rare and curved distolingual root. The resolution of the sinus tract and apical healing was accomplished following nonsurgical root canal treatment. CONCLUSION: A dental aetiology must be included in the differential diagnosis of cutaneous sinus tracts in the neck and face. Elimination of odontogenic cutaneous sinus tract infection by endodontic therapy results in resolution of the sinus tract without surgical excision or systemic antibiotic therapy. This case report also indicates that CBCT imaging is useful for identifying the tooth involved, ascertaining the extent of surrounding bone destruction and accurately managing the aberrant canal morphology.

Effects of acid etching and adhesive treatments on host-derived cysteine cathepsin activity in dentin.

PURPOSE: To analyze the effects of different processes during bonding on endogenous cysteine cathepsin activity in dentin. MATERIALS AND METHODS: Dentin powder, prepared from extracted human third molars, was divided into 10 groups. Two lots of dentin powder were used to detect the effects of the procedure of protein extraction on endogenous cathepsin activity. The others were used to study effects of different acid-etching or adhesive treatments on enzyme activity. Concentrations of 37% phosphoric acid or 10% phosphoric acid, two etch-and-rinse adhesive systems, and two self-etching adhesive systems were used as dentin powder treatments. The untreated mineralized dentin powder was set as the control. After treatment, the proteins of each group were extracted. The total cathepsin activity in the extracts of each group was monitored with a fluorescence reader. RESULTS: In the control group, there were no significant differences in cathepsin activity between the protein extract before EDTA treatment and the protein extract after EDTA treatment (p > 0.05). The cathepsin activities of the three different extracts in the 37% phosphoric acid-treated group were different from each other (p < 0.05). The two acid-etching groups and two etch-and-rinse groups showed significant enzyme activity reduction vs the control group (p < 0.05). There were no significant differences between those four groups (p > 0.05). Treating the dentin powder with any of the two self-etching adhesives resulted in an increase in cathepsin activity (p < 0.05). CONCLUSIONS: The activity of cysteine cathepsins can be detected in dentin powder. Treatment with EDTA during protein extraction exerted an influence on cathepsin activity. Acid etching or etch-and-rinse adhesive systems may reduce the activity of endogenous cathepsins in dentin. Self-etching adhesive systems may increase the enzyme activity.

Control efficacy of an endophytic Bacillus amyloliquefaciens strain BZ6-1 against peanut bacterial Wilt, Ralstonia solanacearum.

We aimed to isolate and identify endophytic bacteria that might have efficacy against peanut bacterial wilt (BW) caused by Ralstonia solanacearum. Thirty-seven endophytic strains were isolated from healthy peanut plants in R. solanacearum-infested fields and eight showed antagonistic effects against R. solanacearum. Strain BZ6-1 with the highest antimicrobial activity was identified as Bacillus amyloliquefaciens based on morphology, biochemistry, and 16S rRNA analysis. Culture conditions of BZ6-1 were optimized using orthogonal test method and inhibitory zone diameter in dual culture plate assay reached 34.2 mm. Furthermore, main antimicrobial substances of surfactin and fengycin A homologues produced by BZ6-1 were analyzed by high performance liquid chromatography electrospray ionization tandem mass spectrometry. Finally, pot experiments were adopted to test the control efficiency of BZ6-1 against peanut BW. Disease incidence decreased significantly from 84.5% in the control to 12.1% with addition of 15 mL (10(8) cfu mL(-1)) culture broth for each seedling, suggesting the feasibility of strain BZ6-1 in the biological control of peanut plants BW.

[Effect of different molarity cathepsins specific inhibitor E-64 on dentin-resin bonding durability].

OBJECTIVE: To investigate, in vitro, the effect of cathepsins specific inhibitor N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide(E-64) on dental endogenous cathepsins and to find its most effective molarity to elevate dentin-resin bonding durability. METHODS: Fifty recently extracted human third molars were divided into five groups according to random number table, and treated with different molarity of E-64 as follow: 0, 2.5, 5.0, 10.0 and 20.0 µmol/L. The group 0 µmol/L was control group. Then 20 specimens of dentin-resin composite were fabricated in each group. Half of the specimens were tested after 24 h water storage(37 °C) and the other half were tested after 90 days water storage(37 °C) followed by 3000 cycles'thermocyling(5-55 °C) as aging treatment. Fractured specimens were analyzed using scanning electron microscopy(SEM). RESULTS: After 24 h water storage, no significant differences were found in micro-tensile bond strength(µTBS) of samples between different groups (P > 0.05). However, after ageing treatment, µTBS of the samples in group 2.5, 5.0, 10.0 and 20.0 µmol/L [(18.7 ± 2.7), (20.8 ± 3.4), (18.3 ± 2.8) and (19.1 ± 2.7) MPa] were significantly higher than that in group 0 µmol/L [(15.1 ± 3.0) MPa] (P < 0.05). Only in the group 5.0 µmol/L no significant difference was found between the original and the decreased value of µTBS(P > 0.05), while the µTBS in other groups decreased significantly after aging treatment(P < 0.05). Failure types were almost adhesive and mixed types. Collagens in hybrid layer were less degraded in the groups using E-64 after aging treatment than control group. CONCLUSIONS: E-64 was effective on inhibiting cathepsins activity in dentin, and induced less collagens degradation in smear layer for better dentin-resin bond durability.

Optimizing mixed culture of two acidophiles to improve copper recovery from printed circuit boards (PCBs).

In the previous research, the effects of different addition time and amount of printed circuit boards (PCBs) on cells growth and metals recovery in separated and mixed culture of Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans were investigated. This paper aimed to optimize mixed culture of both acidophiles for maximizing PCBs addition amounts and copper leaching percentage simultaneously. Initially, influences of inoculums ratio between two acidophiles on their cells growth were studied. Then, initial medium pH and concentrations of FeSO4 · 7H2O and elemental sulfur (S(0)) were optimized by response surface methodology (RSM) to improve copper recovery. Finally, multiple-point PCBs addition was tested to determine maximal amounts. Results showed that with initial inoculums ratio (Af:At) 1:2, pH 1.56, FeSO4 · 7H2O and S(0) at 16.88 and 5.44 g/L, and PCBs addition 28.8 g/L, copper recovery reached 92.6% after 240 h cultivation. It was indicated that copper could be efficiently leached out from increased PCBs addition amount and FeSO4 · 7H2O was remarkably reduced from 22.1 to 16.88 g/L.

[Effects of cleaners on the color stability of prosthesis silicone rubbers].

OBJECTIVE: To evaluate the effect of different cleaners on the color stability of two silicone rubbers used for maxillofacial prosthesis, and to provide recommendations for clinical use. METHODS: Thirty skin-color columniform specimens (12 mm diameter, 10 mm height) of two silicone rubber (A:A-2000; Z:ZY-1) were prepared, randomly divided into 6 groups according to the table of random number, and cleaned with the following solutions: isopropyl alcohol (I), three kinds of denture cleaners (P: Polident, S: Steradent, C: Cleansoft) and distilled water (D), simulating the total immersion time of 1 year (1, 15, 10, 3 and 10 min each time respectively). Control group was kept in dark place without treatment. The L(*), a(*), b(*) value were tested before and after immersion. Then color difference value was calculated. RESULTS: Color differences were different among groups. Color difference in group I (A: 2.15, Z: 2.00) were significantly greater than that in any other group. There were no significant differences between groups using denture cleaner P (A: 0.36, Z: 0.36), C (A: 0.42, Z: 0.37) and S (A: 0.33, Z: 0.38), and group D (A: 0.22, Z: 0.23). CONCLUSIONS: Isopropyl alcohol causes the most severe fading, and denture cleaners and distilled water cause obscure fading.

[Effect of concentration of chlorhexidine on bonding durability of dentine and resin].

OBJECTIVE: To evaluate the effect of concentration of chlorhexidine on bonding durability of dentine and resin. METHODS: Forty extracted third molars were randomly allocated into five groups, which include one control group and four test groups. Teeth dentin surfaces in each group were treated with one of the following solution, 1.5 microl water (control), 0.02%, 0.2%, 2% and 20% chlorhexidine. Single Bond 2 adhesive was applied to the dentin surface according to manufacturer's recommendations. Then 5 mm thickness of composite (Z250) was built up on the dentin of each molar. The teeth were sectioned into microtensile samples and subdivided into two subgroups, 16 samples each. Samples in subgroup II and I were tested after being stored in distilled water for 24 h and 6 month respectively. Each fractured sample was examined with SEM. RESULTS: No significant differences of 24 h bonding strength were found among the five groups. There were significant difference in bonding strength between 0.2%, 2% chlorhexidine groups [(24.68 ± 5.26) and (23.19 ± 5.26) MPa] and the control group [(19.10 ± 4.67) MPa] after 6 month (P = 0.007,0.045), and significant differences were also found between 0.2%, 2% and 0.02% chlorhexidine group [(19.01 ± 6.87) MPa, P = 0.006, 0.041). Most of the fractured modes were mixed or interface failures after 24 h of water storage, and the mixed failure increased after six month of water storage. In the 0.2%, 2% and 20% chlorhexidine treated group, most of the failure was found at the top of hybrid layer, while in the 0.02% group, it was found in the base part of hybrid layer. CONCLUSIONS: Chlorhexidine could increase the bonding durability of resin and dentine. Higher than 0.2%, concentration of chlorhexidine couldn't improve bonding durability.

Optimization of sodium dedecyl sulfate (SDS) addition coupled with adenosine triphosphate (ATP) regeneration for glutathione overproduction in high density cultivation of Candida utilis.

In the previous study, glutathione production was elevated by adding precursor amino acids and adenosine triphosphate (ATP) in high cell density cultivation of Candida utilis. Furthermore, in the present research, glutathione production was further improved by optimizing sodium dedecyl sulfate (SDS) addition coupled with ATP regeneration. Results indicated that with 2g/l ATP added at 60h and 0.8g/l SDS added repeatedly at 60, 63, 66 and 69h, final glutathione yield reached 2286mg/l after 72h cultivation. Moreover, by applying the novel strategies of regenerating ATP by feeding glucose at 6g/(lh) from 60 to 72h coupled with impulse SDS treatments, a total glutathione yield of 2485mg/l was achieved at 72h, which was 8.7% higher than with addition of ATP and SDS, suggesting application of the proposed strategies as being feasible for glutathione overproduction on industrial scales.

A new strategy to enhance glutathione production by multiple H2O2 induced oxidative stresses in Candida utilis.

Effect of H(2)O(2)-induced oxidative stress on glutathione (GSH) production in Candida utilis was investigated. Based on the results that H(2)O(2) can effectively stimulate GSH accumulation but inhibit cell growth simultaneously, a novel strategy of multiple H(2)O(2) stresses with different concentrations (1 mmol/L at 4h, 2 mmol/L at 8h, and 4 mmol/L at 12h) were developed to maximize GSH production. As a result, a maximal GSH yield of 218 mg/L was achieved and a corresponding intracellular GSH content was 2.15%, which were 54.6% and 58.1% higher than the control. By further applying this strategy to 7 L fermentor, GSH yield and intracellular GSH content were 328 mg/L and 2.30%. Moreover, increased activities of catalase (CAT) and GSH reductase (GR) indicated that GSH and CAT were directly involved in protecting cell against oxidative stress by H(2)O(2).