[Effects of prenatal nicotine exposure on enamel formation of offspring mice].

Objective: To investigate the effects of nicotine on the morphology, structure of offspring's dental germ, enamel organ and other dental tissues and the further potential epigenetic mechanisms by establishing prenatal nicotine exposure mouse model. Methods: Ten C57BL/6 pregnant mice were randomly divided into control group (physiological saline subcutaneous injection) and prenatal nicotine exposure (PNE) group (nicotine subcutaneous injection) by using a random number table. Postnatal day 0 (P0), postnatal day 14 (P14) and postnatal day 25 (P25) offspring mice were collected for subsequent experiments. The offspring mice were divided into offspring control group and offspring PNE group according to the maternal group respectively. Weights of P0 and P25 offspring mice were recorded. Micro-CT, scanning electron microscope (SEM) and Vickers hardness test were performed to analyze the related parameters of hard tissues including alveolar bones and mandibular incisors. Total RNAs were extracted from mandible tissues and the third generation of dental epithelial stem cells (DESC) in P25 mice. The relative expression levels of osteogenic and ameloblastic differentiation related genes were measured by real-time quantitative PCR (RT-qPCR). Immunohistochemical stainings of paraffin sections were then performed to observe the distribution and expression level of proliferating cell nuclear antigen (Pcna), amelogenin (Amelx), histone H3 trimethylated at lysine 27 (H3K27me3) and enhancer of zeste homolog 2 (Ezh2). Cell counting kit-8 (CCK-8) assays were used to detect the cell viabilities of DESCs after administrations of different concentrations of nicotine (0.01, 0.1, 1 mmol/L) and GSK126 (an inhibitor of histone methyltransferase Ezh2). Results: Compared with the control group, pregnant mice in PNE group were more likely to have adverse pregnancy outcomes, such as significantly lower offspring body weight [P0: offspring control (1.20±0.04) g, offspring PNE (0.99±0.02) g, P<0.001; P25: offspring control (15.26±1.70) g, offspring PNE (9.65±1.32) g, P<0.001] and increased stillbirths rate [offspring control (0), offspring PNE (46.40±9.30) %, P<0.001]. At P14 and P25, the distance parameters between the enamel mineralized deposits of mandibular incisors and the mesial surface of the first molar in offspring PNE group [P14: (-1 349±45) µm; P25: (-1 192±147) µm] was significantly decreased compared with the control group [P14: (-506±380) µm, P25: (504±198) µm] (P<0.05, P<0.001). The enamel column and enamel column stroma of incisors in offspring PNE group were blurred, arranged loosely and disorderly than those in the control group, while the microhardness of incisor enamel in offspring PNE group [(245.7±18.4) MPa] was significantly lower compared to the control group [(371.9±28.7) MPa] (P<0.001). HE staining showed disordered pre-ameloblast (Pre-Am) arrangement and delayed mineralization deposition point in offspring PNE group compared with the control group, while the length of transit-amplifying cell (TA) and Pre-Am region were prolonged as well. Immunohistochemical staining results displayed that the overall Pcna (P<0.05), H3K27me3 (P<0.01), Ezh2 (P<0.01) expression of labial cervical loop (LaCL) in PNE group were increased, while the positive signal of Amelx in ameloblast cytoplasm was impaired. In vitro, the addition of 1 mmol/L nicotine could significantly upregulate the expression level of Pcna (P<0.01) and downregulate the expression levels of B lymphoma Mo-MLV insertion region 1 (P<0.05), leucine rich repeats and immunoglobulin like domains 1 (P<0.05), Amelx (P<0.01). In addition, 1 mmol/L nicotine could also significantly enhance the proliferation activity of DESCs (P<0.001). Addition of 10 µmol/L GSK126, could rescue the proliferation activation effect of 1 mmol/L nicotine on DESCs. Conclusions: PNE may delay the process of enamel formation and lineage differentiation, leading to the abnormal proliferation of DESCs and changes of epigenetic modification state in H3K27me3, which affect the development of enamel in offspring mice,suggesting PNE might be one of risk environmental factor for tooth development.

[Treatment response of a two-dose regimen of dose-adjusted inotuzumab ozogamicin in relapsed/refractory B-cell acute lymphoblastic leukemia].

Objective: To observe the treatment response of a two-dose regimen of inotuzumab ozogamicin (inotuzumab), a monoclonal antibody targeting CD22, for patients with heavily treated relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL), including those failed or relapsed after chimeric antigen receptor (CAR) -T-cell therapy. Methods: Pediatric and adult patients who received two doses of inotuzumab and who were evaluated after inotuzumab treatment were included. Antibody infusions were performed between March 2020 and September 2022. All patients expressed CD22 antigen as detected by flow cytometry (>80% leukemic cells displaying CD22) before treatment. For adults, the maximum dosage per administration was 1 mg (with a total of two administrations). For children, the maximum dosage per administration was 0.85 mg/m(2) (no more than 1 mg/dose; total of two administrations). The total dosage administered to each patient was less than the standard dosage of 1.8 mg/m(2). Results: Twenty-one patients with R/R B-ALL were included, including five children (<18 years old) and sixteen adults. Seventeen patients presented with 5.0% -99.0% leukemic blasts in the bone marrow/peripheral blood or with extramedullary disease, and four patients were minimal residual disease (MRD) -positive. Fourteen patients underwent both CD19 and CD22 CAR-T-cell therapy, four underwent CD19 CAR-T-cell therapy, and three underwent blinatumomab therapy. Eleven patients underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). After inotuzumab treatment, 14 of 21 patients (66.7% ) achieved a complete response (CR, one was MRD-positive CR), and all four MRD-positive patients turned MRD-negative. Four of six patients who failed recent CD22 CAR-T-cell therapy achieved a CR after subsequent inotuzumab treatment. Seven patients (33.3% ) demonstrated no response. Grade 1-3 hepatotoxicity occurred in five patients (23.8% ), one child with no response experienced hepatic veno-occlusive disease (HVOD) during salvage transplantation and recovered completely. Conclusion: For patients with heavily treated R/R B-ALL, including those who had undergone allo-HSCT and CD19/CD22 CAR-T-cell therapy, the two-dose regimen of inotuzumab resulted in a CR rate of 66.7%, and the frequency of hepatotoxicity and HVOD was low.

[Relevance of renal cell carcinoma angiogenesis and tumor stem cells].

Objective: To investigate the relationship between tumor angiogenesis and renal cancer stem cells. Methods: The primary cell culture method was used to extract and isolate RCSCs, and then qRT-PCR and Western blot methods were used to detect the expression levels of the kidney cancer stem cell markers CD105 and Sox2 genes and proteins from different MVD kidney cancer tissues. Using TCGA database, analyze the correlation between tumor angiogenesis markers and tumor stem cell regulatory genes. Results: The stem cell markers CD105 and Sox2 genes in RCSCs derived from high MVD kidney cancer tissues were respectively increased by (2.34±1.77) times and (3.92±1.41) times (PCD105<0.01, PSox2<0.05)and protein levels were increased by (5.12±3.31) times and (4.90±3.30) times(PCD105<0.05, PSox2<0.01).Meanwhile,up to 30% of stem cell promoting stemness regulatory genes are positively correlated with angiogenesis genes CD31/PECAM1 and KDR, and 64 genes are also strongly positively correlated with CD31/PECAM1 and KDR genes. Conclusion: The high microvessel density of kidney cancer is strongly correlated with the existence of renal carcinoma stem cells.

[Effects of idiopathic hypoparathyroidism on tooth eruption of rats].

Objective: To explore the effects of reduced parathyroid function in early growth and development on tooth eruption and enamel development by establishing an animal model of idiopathic hypoparathyroidism (IHP) and to explore the mechanism of IHP affecting tooth eruption with a view to provide experimental basis for early diagnosis and clinical treatment of IHP. Methods: Forty-eight SD rats at postnatal day 7 were randomly and equally divided into sham operation group and IHP group. The bilateral parathyroidectomy (PTX) was performed by using carbon nanoparticles technique to establish an IHP rat model, while no parathyroids were removed in the sham operation group using the same technique. Serum was extracted after surgery, serum calcium, serum phosphorus, and serum parathyroid hormone (PTH) concentrations were detected in order to verify the success of the modeling. At postnatal day 14, day 25 and day 38 (P14, P25 and P38) the rats were sacrificed to collect the mandible samples (six from each group) and to analyze the volume of enamel, the height of the tooth eruption and the bone microarchitecture parameters of the root-oriented alveolar bone of mandibular third molar quantitatively by micro-CT scanning. Histological sections were prepared. The distribution and expression levels of osteoblast differentiation markers runt-related transcription factor 2 (RUNX2) and osterix (OSX) in the alveolar bone around the third molar were detected by immunohistochemical staining and the osteoclast activity was detected by tartrate-resistant acid phosphatase (TRAP) staining. After each of the third molars was isolated, the microhardness of the enamel was measure by using a microhardness tester and the enamel microstructure was photographed by using scanning electron microscope. Primary dental follicle stem cells were isolated from other six mandibulars from each group at P14 and cultured in vitro. The cell proliferation activity was tested by cell colony forming units detection. After induction of dental follicle stem cells into osteogenic differentiation, the degree of mineralization was detected by using alkaline phosphatase staining and alizarin red staining. The mRNA of mandibular tissues and dental follicle cells were extracted, the expression of genes related to osteoblasts and osteoclast differentiations and parathyroid hormone receptor 1 (PTH1R) were detected by real-time quantitative PCR. Results: Bilateral parathyroidectomy was successfully performed on rats with the help of carbon nanoparticles under stereomicroscope. After surgery, the serum calcium concentration reduced, the serum phosphorus concentration increased and the serum PTH concentration distinctly reduced (P<0.01). The volume of enamel [(4.58±0.24) mm3] and the microhardness [(167.76±21.86) MPa] in IHP group were significantly lower than that in sham operation group [(5.22±0.46) mm3, P<0.05; (223.92±10.94) MPa, P<0.01, respectively]. The eruption height of the mandibular third molar in the IHP group was respectively lower than that in the sham operation group (P<0.05). The bone volume over total volume and trabecular number of the root-oriented alveolar bone of the mandibular third molars in the IHP group were respectively lower than that in sham operation group (P<0.05). The expression levels of RUNX2 and OSX proteins in the root-oriented alveolar bone of the mandibular third molars in the IHP group respectively reduced, compared to that in sham operation group (P<0.05). Furthermore, the number of osteoclasts (3.86±1.07) in crown-oriented alveolar bone in the IHP group was respectively lower than that in sham operation group (6.43±1.27) (P<0.01). The proliferative activity of dental follicle stem cells in the IHP group respectively decreased (P<0.01). After the induction of osteogenic differentiation, the mineralization ability of dental follicle stem cells in the IHP group was weakened. In the mandibular tissues of IHP group, the expression levels of osteogenesis related genes such as RUNX2 and OSX and the the expression of PTH1R significantly reduced (P<0.05). The receptor activator of nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG) ratio reduced significantly (P<0.01) compared to those of sham operation group. Also in the dental follicle cells of IHP group, the expression levels of osteogenesis related genes such as RUNX2 and OSX, the RANKL/OPG ratio and the expression of PTH1R significantly decreased simultaneously compared to that in sham operation group (P<0.01). Conclusions: Under the condition of idiopathic hypoparathyroidism, the weakening of PTH/PTH1R signaling may reduce the proliferative activity of dental follicle stem cells, inhibit their regulation for osteoblast and osteoclast differentiations and functions, thereby interfere the bone remodeling of alveolar bone around the tooth germ during tooth eruption, which eventually leads to delayed tooth eruption.

Environmental conditions regulate the impact of plants on cloud formation.

The terrestrial vegetation emits large amounts of volatile organic compounds (VOC) into the atmosphere, which on oxidation produce secondary organic aerosol (SOA). By acting as cloud condensation nuclei (CCN), SOA influences cloud formation and climate. In a warming climate, changes in environmental factors can cause stresses to plants, inducing changes of the emitted VOC. These can modify particle size and composition. Here we report how induced emissions eventually affect CCN activity of SOA, a key parameter in cloud formation. For boreal forest tree species, insect infestation by aphids causes additional VOC emissions which modifies SOA composition thus hygroscopicity and CCN activity. Moderate heat increases the total amount of constitutive VOC, which has a minor effect on hygroscopicity, but affects CCN activity by increasing the particles' size. The coupling of plant stresses, VOC composition and CCN activity points to an important impact of induced plant emissions on cloud formation and climate.

Genome-wide identification, phylogenetic relationships, and expression analysis of the carotenoid cleavage oxygenase gene family in pepper.

Carotenoid cleavage oxygenases (CCOs) are a family of dioxygenases, which specifically catalyze the cleavage of conjugated double bonds in carotenoids and apocarotenoids in plants. In this study, genome-wide analysis of CCO genes in pepper plants was performed using bioinformatic methods. At least 11 members of the CCO gene family were identified in the pepper genome. Phylogenetic analysis showed that pepper and tomato CCO genes could be divided into two groups (CCDs and NCEDs). The CCD group included five sub-groups (CCD1, CCD4, CCD7, CCD8, and CCD-like). These results indicate that there is a close genetic relationship between the two species. Sequence analysis using the online tool, Multiple Expectation Maximization for Motif Elicitation (MEME), showed that the CCO proteins comprise multiple conserved motifs, with 20 to 41 amino acids. In addition, multiple cis-acting elements in the promoter of CCO genes were identified using the online tool PlantCARE, and were found to be involved in light responsiveness, plant hormone regulation, and biotic and abiotic stresses, suggesting potential roles of these proteins under different conditions. RNA-seq analysis revealed that the CCO genes exhibit distinct patterns of expression in the roots, stems, leaves, and fruit. These findings suggest that the CCO genes have important roles in the vegetative and reproductive development of pepper plants.

Establishment of an animal model of biliary ischemic stenosis with clamping in mice.

OBJECTIVE: The objective of this study was to explore a method to establish biliary ischemic stenosis in mice. METHODS: After the optimal time of biliary ischemia was determined, 20 Kunming mice were equally divided into 2 groups. In the experimental group a 0.4-cm length of common bile duct was clamped for 90 minutes with 2 micro-vessel clamps (width = 0.1 cm). The common bile duct was not clamped in the control group. Twenty-one days later, biliary tract visualization was performed in all mice. Blood samples were collected from the inferior vena cava to determine the serum levels of total bilirubin (TBIL) and alanine aminotransferase (ALT). Meanwhile, samples of the common bile duct and liver tissue were extracted for microscopic examination to observe morphological changes. RESULTS: In the experimental group, obvious dilatation of the common bile duct appeared over the clamp site. There was no dilatation of the common bile duct in the control group. Twenty-one days later, serum levels of TBIL and ALT were significantly higher among the experimental compared with the control group. Microscopic examination showed that the part of common bile duct at the clamp site was significantly expanded, with a smaller or occluded bile duct lumen necrotic mucosa with determination, and tubular wall with fibrosis and excrustation. A few dead liver cells and many inflammatory cells were observed in liver tissue samples. CONCLUSIONS: A biliary ischemic stenosis model was established using a clamping method in mice, which may provide a reliable technique for basic and clinical research into mechanisms of biliary ischemic stenosis after liver transplantation.

Molecular epidemiologic analysis of group A rotaviruses in adults and children with diarrhea in Wuhan city, China, 2000-2006.

To compare epidemiologic features and genetic characteristics of group A rotaviruses causing diarrhea in children and adults, a survey was conducted in Wuhan, China, during the period of Dec. 2000-May 2006. A total of 3839 stool specimens from diarrheal patients from eight hospitals were analyzed. Winter seasonality was observed for rotavirus diarrhea in both adults and children, showing overall rotavirus-positive rates of 9.0 and 23.9%, respectively. Throughout the study period, G3 was the most frequent G serotype in both adults and children (detection rates 86.2 and 87.8%, respectively), and was mostly associated with VP4 genotype P[8], VP 6 genotype II (subgroup II), and NSP4 genotype B. G3 rotaviruses were differentiated into eight electropherotypes, among which seven types were found in specimens from both adults and children. VP7 gene sequences of G3 rotaviruses from adults and children (6 and 4 strains, respectively), detected in different years and different hospitals, showed extremely high sequence identities (99-100%) to each other and to a few G3 rotavirus strains reported in Asia. However, lower sequence identities (82-96%) were observed to most of the human and animal G3 rotaviruses reported so far, including some Chinese strains. These findings indicate that in Wuhan, China, epidemic and genetic features of rotaviruses are similar in adults and children, and it has been suggested that G3 rotaviruses that might have originated from the same rotavirus were circulating among children and adults as prevailing viruses. In this study, two rotavirus strains, G9P[8] strain L169, derived from an adult, and G4P[6] strain R479, derived from a child, were isolated and genetically analyzed. The VP7 gene of L169 belongs to a major lineage of G9 rotaviruses that are globally widespread, but is distinct from G9 rotaviruses reported previously in China. The strain R479 had a VP7 gene which was divergent from most G4 human rotaviruses and showed an unusual dual subgroup specificity, I + II. The R479 VP6 gene does not belong to the main clusters of subgroup I and II rotaviruses phylogenetically, but is related to those of the porcine rotaviruses and some unusual human rotaviruses represented by the RMC321 strain isolated in eastern India.

Purification of avian circulating primordial germ cells by nycodenz density gradient centrifugation.

1. Primordial germ cells (PGCs) are the progenitor cells for gametes. In aves, PGCs show a unique migration pathway, that is, they circulate temporarily through the bloodstream during early development. 2. In this study we developed a method to purify circulating primordial germ cells (cPGCs) in quail and chicks by Nycodenz density gradient centrifugation. 3. The process consisted of primary and secondary purification. In primary purification, cPGCs were enriched at the interface of 8 and 12% Nycodenz fractions. In secondary purification, cPGCs were harvested from 8% Nycodenz fraction at a purity of 90% and from 10% Nycodenz fraction at a purity of 70%. The recovery rate of cPGCs was over 70%. 4. This method would facilitate research on cPGCs' culture and the production of transgenic birds using cPGCs.

[Study of the effects of using uterine stimulants on milk secretion during delivery].

The effects of two uterotonics, Oxytocin and Ergotocin, on postpartum lactation were observed and compared in this study. 30 puerperants were given Oxytocin and another 30 puerperants were given Ergotocin during delivery. The result showed that the time of lactation for the Oxytocin group was earlier than that of the Ergotocin group. The amount of lactation of the Oxytocin group was also much more than that of the Ergotocin group. This study indicated that the rate of breast-feeding may increase if the uterotonics are used properly during delivery.

Epitope analysis of an HLA-B27-derived synthetic peptide: a possible approach to analyzing HLA class I antigens.

A monoclonal antibody, F3H7, was generated by immunizing mice with a synthetic peptide corresponding to residues 63-84 of the B*2705 allele of the HLA-B27 antigens. The reactive epitope and the contact residues on the peptide were localized by ELISA using a large panel of overlapping peptides as well as peptides with substituted amino acids. Residues corresponding to R75, D77 and L78 on the HLA-B27 protein appeared to be critical. The clarity of these results indicate that this is a potentially useful approach to the study of HLA class I epitopes.