[Comparison of single incision robot-assisted laparoscopic radical prostatectomy with and without extraperitoneal special channel device].

Objective: To compare the clinical effects of single-incision robot-assisted laparoscopic radical prostatectomy (RARP) with and without extraperitoneal special channel device. Methods: The clinical data of 70 patients who had undergone RARP in the Robotic Minimally Invasive Surgery Center of Sichuan Provincial People's Hospital from September 2020 to February 2021 were analyzed retrospectively, including 29 cases who were operated on without special channel device (group A) and 41 cases with special channel device (group B). All operations were performed by robot-assisted single-incision retrograde bladder neck exfoliation via extraperitoneal approach in patients by the same operator. The operation time, intraoperative blood loss, the bladder neck urethral anastomosis time, postoperative hospital stay, postoperative exhaust time, positive rate of incisal margin, indwelling time of urinary catheter, retention rate of postoperative erectile function, satisfaction rate of immediate postoperative urine control, positive rate of postoperative lymph node pathology, incision length, treatment cost and the rate of prostate specific antigen (PSA)lower than 0.2 µg/L at 6 weeks after operation were compared between the two groups. Results: All 70 cases were operated successfully. The difference of age[ (68.9±3.9) vs (69.4±5.4) years], preoperative PSA level[14.1(6.3, 19.8)vs13.7(5.8, 18.1)µg/L], prostate volume[44.8(30.7,172.6)vs 56.3(40.9,163.4)ml ] of the two groups was not statistically significant(all P>0.05). The difference of operation time [ (59.1±18.5) vs (59.6±18.0) min ], intraoperative blood loss [93(66,198)vs 95(68,203) ml ], bladder neck urethral anastomosis time [ (12.6±1.3) vs (13.7±2.8) min ], postoperative hospital stay [ (8.1±2.3) vs (9.1±1.3) d], postoperative exhaust time [ (1.4±0.6) vs (1.3±0.6) d], positive rate of incisal margin (20.7% vs 19.5%), indwelling time of the urinary catheter after operation [ (6.8±1.5) vs (7.1±2.0) d ], the retention rate of postoperative erectile function (31.0% vs 27.0%), the satisfaction rate of immediate postoperative urine control (79.3% vs 75.6%), the positive rate of postoperative lymph node pathology (17.2% vs 14.6%), the length of incision [ (5.1±0.5) vs (6.1±0.4) cm ], the rate of PSA lower than 0.2 µg/L at 6 weeks after operation (86.2% vs 83.0%) of the two groups was not statistically significant(all P>0.05). The operation cost of group A[(62 000±4 000) yuan]was lower than group B[(68 000±4 000) yuan] (P<0.05). Conclusion: Extraperitoneal non-special channel device single-incision RARP is safe and feasible.

[A novel tray for recording maxillomandibular relationship of edentulous patients based on dimensional surveying of complete dentures].

OBJECTIVE: To measure the dimensional data of complete dentures and to design a novel tray for recording maxillomandibular relationship of edentulous patients. METHODS: For the measurement, 100 pairs of complete dentures from the clinic were surveyed for the following parameters: a1, the distance between the middle fossa of the upper left and right first molars; a2, the anterior-posterior distance between the middle fossa of the upper first molars and the incisal edge; a3, the width of the upper denture; a4, the anterior-posterior length of the upper denture; a51, the height from the mesio-lingual cusp of the right upper first molar to the saddle surface; a52, the height from the central fossa of the right lower first molar to the saddle surface; a6, the height from the notch of the upper lip frenulum to the upper central incisor edge; a7, the least thickness of the labial saddle base in the upper central incisor region. Based on the data, the trays with different sizes were designed and fabricated, and the key parameters were: b1, the distance between the foramina of screw posts, b2, the anterior-posterior distance between the foramina of the screw posts and the incisal edge; b3, the width of the tray; b4, the anterior-posterior length of the tray; b51, the height of the posterior platform with the screw nut; b52, the height of the screw post; b6, the height of the anterior tray handle; b7, the thickness of the anterior tray handle. RESULTS: The minimum, average and maximum data for each parameter were (in millimeter): a1: 37.1, 44.5, and 59.6; a2: 22.6, 29.0, and 38.1; a3: 48.5, 58.2, and 76.6; a4: 37.4, 50.8, and 61.0; a51: 5.6, 9.5, and 14.7; a52: 3.8, 9.9, and 18.8; a6: 8.9, 16.6, and 24.7; a7: 1.2, 2.8, and 5.9. Based on the data, the trays in small, medium and large sizes were designed and fabricated. In clinical application, the putty silicone rubber impression material was used to reline the tray, meanwhile the posterior platform and anterior tray handle were set as the occlusal plane, then the screw posts were added and adjusted till the proper vertical dimension, after that, the putty silicone rubber impression material was added around the screw posts to record the horizontal maxillomandibular relationship, finally, the anterior surface of the tray handle was used to record the midline of the face and lower edge of the upper lip at rest and with smile. CONCLUSION: The dimensional data offered reference for the analysis of restoration space in edentulous patients. The tray designed and fabricated in this study may serve as a new tool for recording the maxillomandibular relationship.

[Detection of EDA gene mutation and phenotypic analysis in patients with hypohidrotic ectodermal dysplasia].

OBJECTIVE: To detect the ectodysplasin A (EDA) gene mutation in patients with hypohidro-tic ectodermal dysplasia (HED), and to analyze the distribution pattern of missing permanent teeth and the systemic manifestation of HED patients with EDA gene mutation. METHODS: Twelve HED families were enrolled from clinic for genetic history collection, systemic physical examination and oral examination. Peripheral blood or saliva samples were collected from the probands and the family members to extract genomic DNA. PCR amplification and Sanger sequencing were utilized to detect the EDA gene variations, which were compared with the normal sequence (NM_001399.5). The functional impact of EDA gene variants was then evaluated by functional prediction of mutation, conservation analysis and protein structure prediction. The pathogenicity of each EDA gene variation was assessed according to the stan-dards and guidelines of the American College of Medical Genetics and Genomics (ACMG). The systemic phenotype and missing permanent tooth sites of HED patients with EDA gene mutations were summarized, and the missing rate of each tooth position was analyzed and compared. RESULTS: Eight out of twelve HED families were identified to carry EDA gene mutations, including: c.164T>C(p.Leu55Pro); c.457C>T (p.Arg153Cys); c.466C>T(p.Arg156Cys); c. 584G>A(p.Gly195Glu); c.619delG(p.Gly207Profs*73); c.673C>T(p.Pro225Ser); c.676C>T(p.Gln226*) and c.905T>G(p.Phe302Cys). Among them, c.164T>C(p.Leu55Pro); c.619delG(p.Gly207Profs*73); c.673C>T(p.Pro225Ser); c.676C>T(p.Gln226*) and c.905T>G(p.Phe302Cys) were novel mutations. The HED patients with EDA gene mutations in this study were all male. Our results showed that the average number of missing permanent teeth was 13.86±4.49, the average number of missing permanent teeth in the upper jaw was 13.14±5.76, the missing rate was 73.02%. And in the lower jaw, the average number of missing permanent teeth was 14.57±3.05, the missing rate was 80.95%. There was no significant difference in the number of missing teeth between the left and right sides of the permanent dentition (P>0.05). Specifi-cally, the maxillary lateral incisors, the maxillary second premolars and the mandibular lateral incisors were more likely to be missing, while the maxillary central incisors, the maxillary and mandibular first molars had higher possibility of persistence. CONCLUSION: This study detected novel EDA gene pathogenic variants and summarized the distribution pattern of missing permanent teeth of HED patients, thus enriched the variation and phenotype spectrum of EDA gene, and provided new clinical evidence for genetic diagnosis and prenatal consultation.

[Detection and functional analysis of BMP2 gene mutation in patients with tooth agenesis].

OBJECTIVE: To screen for BMP2 mutation with functional impact in patients with congenital tooth agenesis and to make oral and skeletal phenotype record and functional analysis with in vitro experiments. METHODS: We enrolled eighteen patients with congenital tooth agenesis. The medical and dental history was collected,and clinical and dental examinations including the X-ray examination of oral-facial and skeletal bone were performed for the phenotypic analysis. Blood samples were collected to extract DNA and whole exome sequencing was conducted. The genes involved in oral-facial development and congenital skeletal diseases were investigated for mutation screening. The mutations with functional impact were then investigated. In one patient, the BMP2 mutation with putative functional impact was selected for functional analysis. Wild type and mutant BMP2 plasmids with green fluorescent protein (GFP) tag were constructed and transfected into HEK293T cells. Subcellular protein distribution was observed under laser scanning confocal microscope. The activation of downstream SMAD1/5/9 phosphorylation by BMP2 was detected by Western blotting to investigate the functional impact and genetic pathogenicity. RESULTS: BMP2 mutation NM_001200.3:c.393A>T (p.Arg131Ser), rs140417301 was detected in one patient with congenital tooth agenesis, while for other genes involved in oral-facial development and congenital skeletal diseases, no functionally significant mutation was found. The proband's parents didn't carry this mutation. The father had normal dentition, while the mother lacked one premolar, and both the parents showed normal palate and maxilla. The patient also had maxillary hypoplasia in both sagittal and coronal planes, palatal dysmorphology, and malocclusion, and was diagonsed with osteopenia after the X-ray examnination of his skeletal bone. Functional analysis showed this mutation had normal subcelluar localization but reduced phosphorylation of SMAD1/5/9 (reduction by 32%, 22%, and 27% in three independent replicates). Taken together with family co-segregation, this mutaion was considered as "likely pathogenic". CONCLUSION: BMP2 mutation c.393A>T (p. Arg131Ser) affects bone morphogenetic protein signaling activity, and may affect the number of teeth, growth of maxilla and palate, and bone mineral density.

[Analysis of edge morphology of partial veneers made by different processing techniques and materials].

OBJECTIVE: To compare the edge morphology of partial veneers made of different materials by slurry molding, heat-pressed and computer aided design/computer aided manufacturing (CAD/CAM) techniques. METHODS: Thirty premolars with smooth surface and intact enamel were selected and randomly divided into five groups, 6 specimens for each group. Group A were made from feldspathic porcelain (Noritake®) by slurry molding, while Group B were made from lithium disilicate glass ceramic (IPS E.max® Press) by heat-pressed. Group C/D/E were respectively made from feldspar porcelain block (VITA Mark II®), zirconia-reinforced glass ceramic (VITA Suprinity®) and hybrid ceramic with a ceramic-polymer network (VITA Enamic®) by CAD/CAM techniques. All the partial veneers luted with light-cured composite resin. Then the partial veneers were trimmed and polished to achieve the smooth finishing margin, clinical polishing sets were used according to the product descriptions. Scanning electron microscope (SEM) was used to observe the edge morphology of prostheses and the exposure of resin cements. RESULTS: The smooth surface and knife-like edge of the partial veneers could be obtained after bonding, trimming and polishing. The edges of Group A were slightly rough and the width of the exposed adhesive was (106.00±9.17) µm. In Group B, the edges were smoother than Group A, and the exposed wide adhesive strip was visible, which was (138.33±20.59) µm. In Group E, the edges were smooth too, and the width of exposed adhesive strip was (186.00±5.66) µm. The edges of Group C and Group D were rough and uneven, and the adhesive was rarely exposed, they were (50.67±7.51) µm and (65.67±17.90) µm. There were all significant differences between two groups, except Group C and Group D. CONCLUSION: After trimming and polishing in accordance with clinical procedures, the expected knife-like edge can be obtained in all groups. The width of the exposed resin adhesive of each group is different, the order: Mark II/Suprinity < Noritake < E.max Press < Enamic. The edge morphology of partial veneers in different processing technic and materials are different.

[Oral hygiene maintenance of locator attachments implant overdentures in edentulous population: A longitudinal study].

OBJECTIVE: To investigate the oral hygiene status of edentulous patients with locator attachments implant overdentures (IOD) and to analyze the relationship among daily hygiene behavior, oral hygiene status and peri-implant diseases. METHODS: Edentulous patients who received IOD treatment with locator attachments from January 2012 to May 2016 were recruited. Clinical and radiographic examinations were conducted to assess the peri-implant tissue status. Modified plaque index (mPLI), sulcus bleeding index (SBI), gingival index (GI), and probing depth (PD) were recorded and peri-implant marginal bone loss (MBL) was measured using paralleling projection technique. Patients' peri-implant oral hygiene maintainence habits were investigated. The correlation between peri-implant diseases and oral hygiene status and behaviors was analyzed. RESULTS: Fifty patients (125 implants) with an average follow-up time of 22 months (6-54 months) were enrolled. The mean values of mPLI, SBI, and GI were 1.4±1.2, 0.8±0.7, and 0.7± 0.6, respectively. Average PD was (2.2±0.7) mm. Mesial and distal maginal bone resorptions were (1.1±1.1) mm and (0.9±0.9) mm, respectively. The prevalance of mucositis and peri-implantitis of the implants were 49.6% and 0. The prevelance of mucositis in the patients with poor oral hygiene (mPLI≥2) was 11.9 times as much as that of those with adequate oral hygiene (mPLI<1). The patients who performed oral hygiene procedure on attachments at least twice a day achieved much lower mPLI scores than those who cleaned less than twice a day. CONCLUSION: Oral hygiene condition in the group of patients with implant overdentures was poor, and it contributed to increased risk of peri-implant mucositis. The prevelance of musositis of the paitients with poor oral hygiene was 11.9 times as much as that of those with proper oral hygiene. Patients wearing IOD should pay more attention to the hygiene of the attachments.

[Establishment of Cas9 stably expressed human hepatocellular carcinoma and cholangiocarcinoma cell lines].

Objective: To facilitate using the CRISPR/Cas9 gene editing system in human liver and gallbladder cancer cells, we established Cas9 stably expressed human liver and gallbladder cancer cell lines, and validated the gene editing activity of Cas9. Methods: Human liver cancer cell lines (Huh7, PLC/PRF/5, HepG2, Hep3b, SK-HEP-1 and Li-7), human cholangiocarcinoma cells (RBE) and human gallbladder cancer cells (GBC-SD) were infected with 3 Cas9-expressing lentivirus vectors (pLv-EF1α-Cas9-Flag-Neo, pLv-EF1α-Cas9-Flag-Puro, Cas9m1.1), respectively, and Cas9 stably expressed colonies were screened and selected. We extracted the genomic DNA and protein, validated the stable expression of Cas9 by using genomic polymerase chain reaction (PCR) and western blot. Three of cell lines were further infected with Lv-EF1α-mCherry. Then mCherry positive cells were sorted by flow cytometry and infected with designed guide RNA (gRNA) vectors which targeted mCherry gene. Subsequently the gene editing activity of Cas9 was detected by genomic PCR, fluorescence microscopic observation and flow cytometry analysis. Results: One hundred Cas9-expressing human liver and gallbladder cancer cell lines were selected. Among them, 35 cell lines expressed Cas9-Neo, 25 expressed Cas9-puro, and 40 expressed mutant Cas9 (mCas9). We also established 3 cell lines with stable expression of mCherry (Huh7-mCas9-M, PLC/PRF/5-Cas9-M and SK-HEP-1-Cas9-M). The results of genomic PCR and sequencing showed that by lentiviral infection with 2 types of designed gRNA, the long fragment deletion of mCherry gene was found in these 3 cell lines. Moreover, mCherry(-)EGFP(+) cells infected with 2 types of gRNA were observed by fluorescence microscope. The results of flow cytometry showed that mCherry(-)EGFP(+) cells accounted from 0.3% to 93.6%. Conclusion: We successfully establish 100 human liver and gallbladder cancer cell lines with stable expression of Cas9 protein and validate their activities of gene editing.

[Genetic variants analysis and histological observation of teeth in a patient with hereditary opalescent dentin].

OBJECTIVE: To analyze the clinical characteristics and the genetic cause of a Chinese patient with hereditary opalescent dentin, and to make an observation of the histologic and elemental features of the affected teeth. METHODS: We enrolled a patient affected with hereditary opalescent dentin. The medical history was collected and clinical examinations were performed for the phenotypic analyses. The blood sample was collected for DNA extraction and PCRs of the coding sequence of DSPP were done for sanger sequencing. The teeth samples were collected for histological evaluation and elemental analysis. RESULTS: The patient showed typical clinical manifestations of opalescent dentin and had enamel dysplasia and skeletal class III malocclusion. Several polymorphisms (c.727G>A, c.897A>G, c.2053_2054ins18bp, c.2548G>A, c.2645_2646ins9bp, c.2706T>C, c.2878A>G, c.3004A>G, c.3069_3086del18bp, c.3249A>C, c.3264T>C, c.3266_3400del135bp, c.3418A>G, c.3454G>A, c.3461_3462ins18bp, c.3606C>T) but no pathogenic mutations were identified in DSPP. The histological analyses of the patient's teeth showed characteristic abnormalities that were significantly different from normal teeth. The dentin tubules of the affected teeth were decreased in number and sparsed in arrangement, while in the control teeth, they were more regular. The enamel-dentin junction of the affected teeth was abnormal in its less scallopped outline compared with the control teeth under the scanning electronic microscopy. The Mg proportion of the patient's teeth (0.615 0%±0.261 6%) was lower than that of the control teeth (1.283 3%±0.322 1%), the P value was 0.040. The Ca proportion was the higher compared with the control teeth (34.865 0%±0.388 9% vs. 29.221 7%±2.248 4%), the P value was 0.015. The Ca/P ration of the patient's teeth was 1.981 2±0.019 3, which was higher than that of control teeth (1.775 9±0.111 6), the P value was 0.049. The differences of Mg, Ca proportion and Ca/P ration between the affected teeth and the control teeth were significant. The C and O proportion of the patient's teeth were lower and the P proportion was higher compared with the control teeth, however, the differences were not significant. CONCLUSION: Our study of clinical manifestation analysis, genetic variants sequencing and histological observation has enlarged the phenotypic spectrum of hereditary opalescent dentin, and the genetic and histological results would contribute to further studies.

[Influence of implants prepared by selective laser melting on early bone healing].

OBJECTIVE: To evaluate the influence of the rough surface of dental implants prepared by selective laser melting (SLM) on early bone healing around titanium implants. METHODS: A total of sixteen titanium implants were involved in our research, of which eight implants were prepared by SLM (TIXOS Cylindrical, Leader-Novaxa, Milan, Italy; 3.3 mm×10 mm, internal hex) and the other eight were sandblasted, large-grit and acid-etched (SLA) implants (IMPLUS Cylindrical, Leader-Novaxa, Milan, Italy; 3.3 mm×10 mm, internal hex). All of the dental implants were inserted into the healed extraction sockets of the mandible of two adult male Beagle dogs. Half of the dental implants were designed to be healed beneath the mucosa and the other half were intended to be healed transgingivally and were immediately loaded by acrylic resin bridge restoration. Three types of tetracycline fluorescent labels, namely calcein blue, alizarin complexone and calcein, were administered into the veins of the Beagle dogs 2, 4, and 8 weeks after implant placement respectively for fluorescent evaluation of newly formed bone peri-implant. Both Beagle dogs were euthanized 12 weeks after implant insertion and the mandible block specimens containing the titanium implants and surrounding bone and soft tissue of each dog were carefully sectioned and dissected. A total of 16 hard tissue slices were obtained and stained with toluidine blue for microscopic examination and histomorphometric measurements. Histological observation was made for each slice under light microscope and laser scanning confocal microscope (LSCM). Comparison on new bone formation around titanium implants of each group was made and mineral apposition rate (MAR) was calculated for each group. RESULTS: Dental implants prepared by selective laser melting had achieved satisfying osseointegration to surrounding bone tissue after the healing period of 12 weeks. Newly formed bone tissue was observed creeping on the highly porous surface of the SLM implant and growing into the pores of surface structure. Higher MAR values were shown for SLM implants compared with SLA implants (P<0.01). CONCLUSION: Dental implants prepared by selective laser melting could promote early bone healing and improve mineral apposition rate.

[Evaluation of the fabrication deviation of a kind of milling digital implant surgical guides].

OBJECTIVE: To evaluate the deviation of digital implant surgical guides during fabrication process in the Organical Dental Implant (ODI) system. METHODS: This study included two parts. The first part was the in vitro study. A resin block with a diagnostic template was used for the planning. After cone beam computed tomography (CBCT) scanning, a surgical guide with eight implants was virtually designed using the ODI system. The guide was milled by a 5-axial numerical controlled milling machine, and an optical scanning was taken to digitalize the guide to a standard tessellation language (STL) form. The STL data were then imported into an ODI software and registered with the original design. The deviation of the sleeves between the design and the STL was measured in the ODI software and set as the golden standard. Then the ODI examination table was used to measure the deviation of the guide during fabrication. Examiners A and B measured 10 times separately. The reliability and the validity of the examination table was calculated. The second part was the in vivo study: The deviation during fabrication of 12 guides designed and fabricated by the ODI system were measured using the examination table. RESULTS: The standard deviation of the deviation measured using the examination table by examiners A and B were all below 0.40 mm (for the shell reference points) and 0.71 degree (for the angles). No significant difference was found between the two examiners for any implant sites. The result of the examination table was larger than that of the software for the shell reference point (t-test, P<0.05), but no significant difference was found for the angle deviation (t-test, P>0.05). The 45 implants positions in the 12 guides for the in vivo study were examined using the examination table. The deviations at the shell reference points were (1.06±0.29) mm (0.42-1.75 mm), and at the implant tip were (1.12±0.48) mm (0.41-2.44 mm). The angle deviations were (1.42±0.70) degree (0.29-2.96 degree). CONCLUSION: Deviation is unavoidable during the fabrication process of the guides. The examination table of the ODI system is a reliable and valid tool to measure the deviation during fabrication of the ODI guides. More studies should be designed to research the relationship between the fabrication deviation and the implant insertion deviation.

Spin-Orbit Coupling Controlled J=3/2 Electronic Ground State in 5d

Entanglement of spin and orbital degrees of freedom drives the formation of novel quantum and topological physical states. Here we report resonant inelastic x-ray scattering measurements of the transition metal oxides Ca_{3}LiOsO_{6} and Ba_{2}YOsO_{6}, which reveals a dramatic spitting of the t_{2g} manifold. We invoke an intermediate coupling approach that incorporates both spin-orbit coupling and electron-electron interactions on an even footing and reveal that the ground state of 5d^{3}-based compounds, which has remained elusive in previously applied models, is a novel spin-orbit entangled J=3/2 electronic ground state. This work reveals the hidden diversity of spin-orbit controlled ground states in 5d systems and introduces a new arena in the search for spin-orbit controlled phases of matter.

The complete mitochondrial genome of Orthocoelium streptocoelium (Digenea: Paramphistomidae) for comparison with other digeneans.

Orthocoelium streptocoelium is a common paramphistome species parasitizing the rumen and/or reticulum of small ruminants, leading to significant losses. This study first determined the complete mitochondrial (mt) genome of O. streptocoelium. The complete mt genome of O. streptocoelium was amplified, sequenced, assembled, analysed and then compared with those of other digeneans. The entire mt genome of O. streptocoelium is 13,800 bp in length, which is smaller than those of other digeneans except for Opisthorchis viverrini. This mt genome contains 12 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and two non-coding regions. The arrangement of the O. streptocoelium mt genome is the same as those of other digeneans except for Schistosoma haematobium and Schistosoma spindale. Phylogenetic analyses based on concatenated amino acid sequences of the 12 protein-coding genes representing 16 digeneans were conducted to assess the relationship of O. streptocoelium with other digeneans. The result indicated that O. streptocoelium is closely related to Paramphistomum cervi and Fischoederius elongates, which is in accordance with their relationships by taxonomy. This complete mt genome of O. streptocoelium enriched the mitochondrial genome data of paramphistomes and provided important molecular markers for diagnostics and studies of population variation, epidemiology, ecology and evolution of O. streptocoelium and other digeneans.

[Influence of the interforaminal arch form of edentulous mandibles on design of "All-on-4": preliminary research based on conebeam computed tomography].

OBJECTIVE: To classify the interforaminal arch form of edentulous mandibles by measuring the anterior-posterior distance (A-P distance) of implants planned to be inserted in "All-on-4" protocol using conebeam computed tomography (CBCT) data, and to investigate the influence of the arch form on the "All-on-4" distally tilted implants. METHODS: Seventy-four CBCT images of edentulous mandibles were collected, including 35 males and 39 females respectively. "All-on-4"implant supported fixed prostheses were designed for these patients based on the CBCT data. The A-P distance was measured in the plane which crossed bilateral mental foramens and was parallel to the occlusal plane. The interforaminal arch form of edentulous mandibles were classified according to the A-P distance. The radian of the jaw arch 7.5 mm mesially to the mental foramen was measured bilaterally, and its correlation with the A-P distance was studied. RESULTS: The average A-P distance of implant supported fixed prostheses planned in the interforaminal region was (8.5±1.5) mm (minimum 4.5 mm, maximum 11.8 mm). In the study, 12.2% of the subjects' mandibles were classified as square arch form with A-P distances ≤7 mm, 54.0% were classified as ovoid with A-P distances >7 mm and ≤9 mm, 33.8% were classified as tapered with A-P distances >9 mm. Bilaterally, 148 results of the radian of the jaw arch 7.5 mm mesially to the mental foramen were obtained, and the average radian was 15.9°±5.5° (minimum 5.6°, maximum 35.2°). The radian and the A-P distance showed a negative correlation with statistical significance. CONCLUSION: In this research, the ovoid arch form was the most common type in edentulous mandibles, followed by tapered arch form. The square arch form showed the lowest percentage. As the arch form went squarer, the A-P distance became shorter, the radian of the jaw arch mesially to the mental foramen went greater, and the bone width that distally tilted implants need became bigger. The interforaminal arch form of the edentulous mandible should be analyzed before an implant supported fixed restoration is designed in the interforaminal region. The angle of inclination of distal implants should be reasonable. The bone width of the distal implant site must be adequate. The square arch form contributes negatively to the structure of implant supported fixed prostheses with distal cantilever design.

[Specific cytotoxicity of a novel HER2-based chimeric antigen receptor modified T lymphocytes against HER2-positive tumor cells].

Objective: To construct the third generation chimeric antigen receptor based on a novel humanized anti-HER2 H1-2 scFv, and to investigate the specific cytotoxicity of H1-2 CAR modified T lymphocytes(CAR-T) against HER2(+) tumor cells. Method: The expression cassette of the third generation CAR gene and anti-HER2 H1-2 scFv were constructed and cloned into lentivirus transfer plasmid, and then the third generation H1-2 CAR was transduced into human T lymphocytes using lentivirus.Enzyme linked immunosorbent assay was used to detect the expression of cytokines IL2, and LDH release assay was used to detect the cytotoxic effect of the H1-2 CAR-T.Finally, NOD/SCID mice and HER2(+) breast cancer cell line SKBR3 were used to detect the anti-tumor effect of H1-2 CAR-T in vivo. Results: The third generation H1-2 CAR was successfully constructed.H1-2 CAR-T secreted high dose of IL2 after confrontation with HER2(+) breast cancer cells.In vitro, the cytolytic rate of H1-2 CAR-T on high expression HER2(+) tumor cells was significantly higher than that in low expression HER2 or non-expression HER2 tumor cells. At the efficacy to target ratio of 20, the cytolytic rate of H1-2 CAR-T against breast cancer cell SK-BR-3 could reach (90.1±2.8)%, while the cytolytic rate of H1-2 CAR-T against HER2(-) breast cancer cell MDA-MB-231 was only (13.5±4.7)%. In the mouse xenograft tumor model, H1-2 CAR-T cells inhibited breast cancer growth in vivo.At the end of the experiments, the average tumor weight in the H1-2 CAR-T cell treatment group was (0.7±0.1) g, the non-transfected T cell therapeutic group was (1.2±0.2) g, and the PBS group was (1.2±0.2) g. There was significant difference between the H1-2 CAR-T therapeutic group and the non-transfected T cell therapeutic group (P<0.05). However, there was no significant difference between the non-transfected T cell therapeutic group and the PBS treatment group (P>0.05). Conclusion: The HER2-sepcific H1-2 CAR-T cells specifically kill HER2 positive cells, and further studies on CAR-T cells for the treatment of HER2(+) cancers are useful.

[Establishment and validation of human cancer cell lines with stable Cas9 expression].

Objective: To establish human cancer cell strains with stable Cas9 expression, and to validate the gene editing activity of Cas9 for simple gene editing in future study. Methods: Fifteen cancer cell lines of different tissue origins were infected with pLv-EF1α-Cas9-Flag-Neo or pLv-EF1α-Cas9-Flag-Puro by lentivirus and clone selection was employed to screen Cas9 stably expressed cancer cell lines. Afterward designed guide RNA vectors targeting TSC22 gene were transiently transfected into 3 of cell lines, and subsequently the gene editing activity of Cas9 was evaluated by genomic PCR, sequencing and Western blot. Results: Sixty-nine human cancer cell strains with stable Cas9 expression from different cancers were established, and by transient transfection with designed guide RNA, long fragment deletion was detected in TSC22 gene. Conclusions: Sixty-nine human cancer cell strains are successfully established with stable expression of Cas9 protein and gene editing activity. These cell strains may be employed in large-scale drug screening, screening of new drug targets and gene function investigation.

[Association of the expression of leptin and leptin receptor with bone metastasis in pulmonary adenocarcinoma].

Objective: To explore the association of expression of leptin and leptin receptor (LR) with bone metastasis of pulmonary adenocarcinoma. Methods: One hundred and sixteen pulmonary adenocarcinoma patients who had complete clinicopathological data and definite pathological diagnosis in our hospital from January 2008 to January 2010 were selected. They were divided into the metastasis (n= 58) and non-metastasis (control, n=58) groups. The expressions of leptin and LR were identified by immunohistochemistry. The differences between expressions of leptin and LR in primary pulmonary adenocarcinoma tissues and metastasis, and between the groups with and without bone metastasis were analyzed. We also analyzed the correlation of leptin and LR expressed in primary adenocarcinoma and bone metastatic tissues, and the relationship between their expression levels and bone metastasis free survival (BMFS). Results: Among 58 patients of the metastasis group, the cases of high, moderate and low expressions of leptin were 36, 15 and 7, respectively, and the cases of high, moderate and low expressions of LR were 32, 17 and 9, respectively. Among the 58 patients of control group, the cases of high, moderate and low expressions of leptin were 19, 24 and 15, respectively, and those of LR were 17, 16 and 25, respectively. The expressions of leptin and LR in primary pulmonary adenocarcinoma tissues of metastasis group were significantly different from those of the control group (P=0.006, P=0.002, respectively). The expressions of leptin and LR in primary pulmonary adenocarcinoma tissues of the bone metastasis group were also significantly different from those of the non-bone metastasis group (P=0.029, P=0.032, respectively). The high/moderate expression rates of leptin and LR in the bone-metastatic tissues reached 91.4% (32/35) and 88.6% (31/35), respectively. The results showed that the expressions of leptin and LR in primary pulmonary adenocarcinoma tissues were positively related with their expressions in bone metastatic tissue (r = 0.612). The median bone metastasis free survival (BMFS) of the bone metastasis groups with high, moderate and low expressions of leptin were 14, 21 and 47 months, respectively, and the median BMFS of high, moderate and low expressions of LR in the bone metastasis group were 13, 19 and 27 months, respectively. The expressions of leptin and LR in pulmonary adenocarcinoma were significantly associated with BMFS (P<0.001, P=0.006, respectively). Conclusions: The expressions of leptin and LR are significantly up-regulated in primary pulmonary adenocarcinoma tissues and bone metastatic tissues, and are negatively correlated with BMFS. These two molecules may be used as effective predictors of bone metastasis in pulmonary adenocarcinoma.

[Distribution and content of transforming growth factor-ß1 and vascular endothelial growth factor in each layer of concentrated growth factors].

OBJECTIVE: To investigate the distribution and content of transforming growth factor-ß1 (TGF-ß1) and vascular endothelial growth factor (VEGF) in concentrated growth factors (CGF) gel, and to clarify the difference among different layers of CGF. METHODS: Venous blood samples were collected from 6 healthy volunteers to prepare CGF. The distribution, integrated optical density (IOD) and average optical density (AOD) of TGF-ß1 and VEGF in CGF gel and red blood cell (RBC) layer were measured using immunohistochemistry. The concentrations of TGF-ß1 and VEGF in the supernatant serum at baseline and the CGF releasate after 1 day were evaluated with enzyme-linked immunosorbent assays. RESULTS: Abundant TGF-ß1 and VEGF were concentrated in CGF gel. However, only a little could be found in polykaryocytes and sporadic platelets in RBC layer. Platelets and leukocytes were concentrated in between the two layers with high expression of TGF-ß1. The concentrations of TGF-ß1 and VEGF in the CGF releasate(55 236.78±3 686.34), (610.99±148.81) ng/L were significantly higher than those in the supernatant serum (20 710.20±4 523.14), (335.20±51.69) ng/L (P<0.001). CONCLUSION: CGF contains high quantities of TGF-ß1 that can promote new bone formation and tissue healing. We suggest that CGF gel should be used right after being prepared. Supernatant serum and the area between CGF gel and RBC layer could also be mixed with bone substitute materials.

[EDA mutation screening and phenotype analysis in patients with tooth agenesis].

OBJECTIVE: To screen the ectodysplasin A (EDA) gene mutation in the patients with non-syndromic tooth agenesis and ectodermal dysplasia, and to analyze the phenotype of missing teeth pattern in these two groups of patients. METHODS: In the study, 174 patients with tooth agenesis (143: non-syndromic, 31: ectodermal dysplasia) and 451 health control volunteers were enrolled from the clinic, and the genome DNA was extracted from either peripheral blood or oral mucosal swab. The coding region of EDA gene was then amplified by PCR, sequenced and blasted to online NCBI database. The missing teeth were recorded for all patients, and the missing teeth from patients with EDA mutation were compared among the different dentition sites. RESULTS: 33 patients were identified with EDA mutation. In the non-syndromic patients, 13/143(9.09%) were identified with EDA mutation, while in patients with ectodermal dysplasia, 20/31(64.52%) were found with EDA mutation. Ten novel EDA mutations were identified (c.769G>C[p.G257R ],c.936C>G[p.I312M],c.223G>A[p.E75K], .1166C>T[p.P389L],c.133G>C[p.G45R],c.1109G>A[p.E370K],c.914G>T[p.S305I], c.916C>T[p.Q306X],c.602G>T[p.G201V],c.88-89insG[p.A30GfsX69]). For each dentition site there was no statistic difference in the number of missing teeth between the left and right sides, so the number from both sides were combined later in the analysis. In the patients with EDA mutation, the non-syndromic patients had fewer missing teeth (15.9±6.4 missing teeth for each, 207/364 in total) than the patients with ectodermal dysplasia (23.9±4.3, 478/560). In the non-syndromic patients with EDA mutation, the maxillay central incisors and first molars were less affected, with the same missing rate as 19.2% (5/26). While the mandibular central incisors (with a missing rate of 76.9%, 20/26), the maxillary lateral incisors (the missing rate: 88.5%, 23/26), the mandibular lateral incisors (the missing rate: 80.8%, 21/26), and the maxillary first premolars (the missing rate: 80.8%, 21/26) were more likely to be missing. In the ectodermal dysplasia patients with EDA mutation, only maxillary central incisors (the missing rate: 60%, 24/40), maxillary canines (the missing rate: 70%, 28/40), mandibular canines (the missing rate: 67.5%, 27/40), maxillary first molars (the missing rate: 65%, 26/40) and mandibular first molars (the missing rate: 72.5%, 29/40) had higher possibility of persistence. Teeth at other dentition sites were more likely to be affected (the minimum missing rate: 87.5%, 35/40). CONCLUSION: The findings would help to reveal the EDA gene and its function in ectodermal organogenesis.

[EDA mutation screening and phenotype analysis in patients with tooth agenesis].

OBJECTIVE: To screen the ectodysplasin A (EDA) gene mutation in the patients with non-syndromic tooth agenesis and ectodermal dysplasia, and to analyze the phenotype of missing teeth pattern in these two groups of patients. METHODS: In the study, 174 patients with tooth agenesis (143: non-syndromic, 31: ectodermal dysplasia) and 451 health control volunteers were enrolled from the clinic, and the genome DNA was extracted from either peripheral blood or oral mucosal swab. The coding region of EDA gene was then amplified by PCR, sequenced and blasted to online NCBI database. The missing teeth were recorded for all patients, and the missing teeth from patients with EDA mutation were compared among the different dentition sites. RESULTS: 33 patients were identified with EDA mutation. In the non-syndromic patients, 13/143(9.09%) were identified with EDA mutation, while in patients with ectodermal dysplasia, 20/31(64.52%) were found with EDA mutation. Ten novel EDA mutations were identified (c.769G>C[p.G257R ],c.936C>G[p.I312M],c.223G>A[p.E75K], .1166C>T[p.P389L],c.133G>C[p.G45R],c.1109G>A[p.E370K],c.914G>T[p.S305I], c.916C>T[p.Q306X],c.602G>T[p.G201V],c.88-89insG[p.A30GfsX69]). For each dentition site there was no statistic difference in the number of missing teeth between the left and right sides, so the number from both sides were combined later in the analysis. In the patients with EDA mutation, the non-syndromic patients had fewer missing teeth (15.9±6.4 missing teeth for each, 207/364 in total) than the patients with ectodermal dysplasia (23.9±4.3, 478/560). In the non-syndromic patients with EDA mutation, the maxillay central incisors and first molars were less affected, with the same missing rate as 19.2% (5/26). While the mandibular central incisors (with a missing rate of 76.9%, 20/26), the maxillary lateral incisors (the missing rate: 88.5%, 23/26), the mandibular lateral incisors (the missing rate: 80.8%, 21/26), and the maxillary first premolars (the missing rate: 80.8%, 21/26) were more likely to be missing. In the ectodermal dysplasia patients with EDA mutation, only maxillary central incisors (the missing rate: 60%, 24/40), maxillary canines (the missing rate: 70%, 28/40), mandibular canines (the missing rate: 67.5%, 27/40), maxillary first molars (the missing rate: 65%, 26/40) and mandibular first molars (the missing rate: 72.5%, 29/40) had higher possibility of persistence. Teeth at other dentition sites were more likely to be affected (the minimum missing rate: 87.5%, 35/40). CONCLUSION: The findings would help to reveal the EDA gene and its function in ectodermal organogenesis.

Endocrine resistance in breast cancer.

Selective estrogen receptor modulators (SERMs) are synthetic molecules which bind to estrogen receptors (ER) and can modulate their transcriptional capabilities in different ways in diverse estrogen target tissues. Unfortunately, the use of resistant therapy is associated with acquired resistance. Several molecular mechanisms have been proposed to be responsible for endocrine resistance in breast cancer, including MIR-451, FGF and FGFR, ADAM12, fibronectin and other soluble stromal factors, PELP1-KDM1, HER2, NOTCH, δEF1, mTOR, AKT/mTOR, Pi3K/AKT, Pi3K/AKT/mTOR, NFκB, LMTK3, IGF1R, cyclin E2, IRF1, Tab2, and SRC-1. Further research is needed to know more about endocrine resistance.