Organoids From Mucinous Appendiceal Adenocarcinomas as High-Fidelity Models for Individual Therapy.

Background: Mucinous appendiceal adenocarcinoma (MAA) is a rare, heterogeneous disease. Patients with unrespectable mucinous appendiceal adenocarcinoma presenting with peritoneal spread are treated by intraperitoneal chemotherapy, hyperthermic intraperitoneal chemotherapy, systemic chemotherapy, or targeted therapy. However, there are no guidelines for efficacious drugs against mucinous appendiceal adenocarcinoma. Therefore, relevant high-fidelity models should be investigated to identify effective drugs for individual therapy. Methods: Surgical tumor specimens were obtained from a mucinous appendiceal adenocarcinoma patient. The tissue was digested and organoid culture was established. H&E and immunohistochemistry staining as well as DNA sequencing was performed on tissue and organoid. The pathological characteristics and gene mutations of the organoid were compared to those of the original tumor. Drug sensitivity tests were performed on organoid and the patient clinical responds to chemotherapy and targeted therapy was compared. Results: Organoids were successfully established and stably passaged. Pathological characteristics of organoids including H&E staining and expression of protein markers (CK20, CDX-2, STAB2, CD7, PAX8) were consistent to those of the original tumor. Moreover, the organoids carried the same gene mutations as the primary tumor. Sensitivity of the organoids to chemotherapeutic drugs and tyrosine kinase inhibitors included: 5-FU (IC50 43.95 µM), Oxaliplatin (IC50 23.49 µM), SN38 (IC50 1.02 µM), Apatinib (IC50 0.10 µM), Dasatinib (IC50 2.27 µM), Docetaxel (IC50 5.26 µM), Regorafenib (IC50 18.90 µM), and Everolimus (IC50 9.20 µM). The sensitivities of organoid to these drugs were comparable to those of the patient's clinical responses. Conclusion: The mucinous appendiceal adenocarcinoma organoid model which retained the characteristics of the primary tumor was successfully established. Combined organoid-based drug screening and high throughput sequencing provided a promising way for mucinous appendiceal adenocarcinoma treatment.

[Intra-articular radioulnar ligament reconstruction for chronic instability of distal radioulnar joint].

OBJECTIVE: To evaluate the effectiveness of intra-articular radioulnar ligament reconstruction in the treatment of chronic instability of the distal radioulnar joint. METHODS: Between January 2006 and June 2012, the intraarticular radioulnar ligament reconstruction was used to treat chronic instability of the distal radioulnar joint in 12 patients. Of 12 cases, 8 were males and 4 were females with an average age of 37.3 years (range, 22-54 years). The causes of injury were tumble in 9 patients, traffic accident in 2 patients, and sprain in 1 patient. The average time from initial injury to operation was 6.2 months (range, 2-13 months). The pain and function of the wrists were assessed with Patient-Rated Wrist Evaluation (PRWE) (27.5 +/- 4.7). Broadening of the distal radioulnar joint space was seen on the anteroposterior radiograph in all the patients. Lateral view showed dorsal instability in 10 patients and palmar instability in 2 patients. There was no radial fracture, ulnar fractures or degeneration of the distal radioulnar joint. RESULTS: All patients achieved primary healing of incision. No complication of deep infection or nerve injury occurred. They were followed up from 12 to 30 months (mean, 20.6 months). Pain and the clicking sound of the wrists disappeared; grip strength increased; the range of motion values of the wrist and forearm were restored. PRWE score was 5.8 +/- 2.1 at last follow-up, showing significant difference when compared with preoperative score (t = 14.215, P = 0.000). Imaging examination showed good appositions of the distal radioulnar joint, with no dislocation or subluxation. CONCLUSION: Intra-articular radioulnar ligament reconstruction can rebuild the anatomic stability of the distal radioulnar joint, which does not damage the adjacent structures of the joints and can obtain satisfactory function of the reconstructed joint. It is a good choice for chronic instability of the distal radioulnar joint without articular degeneration.

Three-dimensional finite element analysis of occipitocervical fixation using an anterior occiput-to-axis locking plate system: a pilot study.

BACKGROUND CONTEXT: Although there are many techniques for occipitocervical fixation, there have been no reports regarding occipitocervical fixation via the use of an anterior anatomical locking plate system. PURPOSE: The biomechanics of this new system were analyzed by a three-dimensional finite element to provide a theoretical basis for clinical application. STUDY DESIGN: This was a modeling study. PATIENT SAMPLE: We studied a 27-year-old healthy male volunteer in whom cervical disease was excluded via X-ray examination. OUTCOME MEASURES: The states of stress and strain of these two internal fixation devices were analyzed. METHODS: A three-dimensional finite element model of normal occiput-C2 was established based on the anatomical data from a Chinese population. An unstable model of occipital-cervical region was established by subtracting several unit structures from the normal model. An anterior occiput-to-axis locking titanium plate system was then applied and an anterior occiput-to-axis screw fixation was performed on the unstable model. Limitation of motion was performed on the surface of the fixed model, and physiological loads were imposed on the surface of the skull base. RESULTS: Under various loads from different directions, the peak values of displacement of the anterior occiput-to-axis locking titanium plate system decreased 15.5%, 12.5%, 14.4%, and 23.7%, respectively, under the loads of flexion, extension, lateral bending, and axial rotation. Compared with the anterior occiput-to-axis screw fixation, the peak values of stress of the anterior occiput-to-axis locking titanium plate system also decreased 3.9%, 2.9%, 9.7%, and 7.2%, respectively, under the loads of flexion, extension, lateral bending, and axial rotation. CONCLUSION: The anterior occiput-to-axis locking titanium plate system proved superior to the anterior occiput-to-axis screw system both in the stress distribution and fixation stability based on finite element analysis. It provides a new clinical option for anterior occipitocervical fixation.

[Surgical strategy for upper cervical vertebrae instability through the anterior approach].

OBJECTIVE: To explore the choice and effect of internal fixation in treating upper cervical vertebrae instability through anterior approach. METHODS: From March 2000 to September 2010,83 patients with upper cervical vertebrae instability were treated with internal fixation through anterior approach. There were 59 males and 24 females with a mean age of 42 years old (ranged, 20 to 68). Among these patients, 36 patients were treated with odontoid screw fixation, 16 patients with C1,2 transarticular screw fixation, 23 patients with C2,3 steel plate fixation, 5 patients with odontoid screw and transarticular screw fixation,2 patients with odontoid screw and C2.3 steel plate fixation, 1 patient with C1,2 transarticular screw and C2,3 steel plate fixation. RESULTS: One patient with completely cervical vertebrae cord injury died of pulmonary infection after C1,2 transarticular screw fixation. Other patients were followed up from 8 to 36 months with an average of 15 months. Upper cervical vertebrae stability were restored without vertebral artery and spinal cord injury. Thirty-six patients were treated with odontoid screw fixation and 5 patients were treated with screw combined with transarticular screw fixation obtained bone union in the dentations without bone graft. Among the 16 patients treated with C1,2 transarticular screw fixation, 13 patients obtained bone union after bone graft; 1 patient died of pulmonary infection after surgery; 1 patient with comminuted odontoid fracture of type II C and atlantoaxial anterior dislocation did not obtain bone union after bone graft,but the fibrous healing was strong enough to maintain the atlantoaixal joint stability; 1 patient with obsolete atlantoaxial anterior dislocation were re-treated with Brooks stainless steel wire fixation and bone graft through posterior approach, and finally obtained bone union. CONCLUSION: It could obtain satisfactory effects depending on the difference of cervical vertebrae instability to choose the correctly surgical method.

Evaluation of biomechanical properties of anterior atlantoaxial transarticular locking plate system using three-dimensional finite element analysis.

PURPOSE: To evaluate a new anterior atlantoaxial transarticular locking plate system using finite element analysis. METHODS: Thin-section spiral computed tomography was performed from occiput to C2 region. A finite element model of an unstable atlantoaxial joint, treated with an anterior atlantoaxial transarticular locking plate system, was compared with the simple anterior atlantoaxial transarticular screw system. Flexion, extension, lateral bending, and axial rotation were imposed on the model. Displacement of the atlantoaxial transarticular screw and stress at the screw-bone interface were observed for the two internal fixation systems. RESULTS: Screw displacement was less using the anterior atlantoaxial transarticular locking plate system compared to simple anterior atlantoaxial transarticular screw fixation under various conditions, and stability increased especially during flexion and extension. CONCLUSIONS: The anterior atlantoaxial transarticular locking plate system not only provided stronger fixation, but also decreased screw-bearing stress and screw-bone interface stress compared to simple anterior atlantoaxial transarticular screw fixation.

Fine mapping of fatness QTL on porcine chromosome X and analyses of three positional candidate genes.

BACKGROUND: Porcine chromosome X harbors four QTL strongly affecting backfat thickness (BFT), ham weight (HW), intramuscular fat content (IMF) and loin eye area (LEA). The confidence intervals (CI) of these QTL overlap and span more than 30 cM, or approximately 80 Mb. This study therefore attempts to fine map these QTL by joint analysis of two large-scale F2 populations (Large White × Meishan and White Duroc × Erhualian constructed by INRA and JXAU respectively) and furthermore, to determine whether these QTL are caused by mutations in three positional candidate genes (ACSL4, SERPINA7 and IRS4) involved in lipid biosynthesis. RESULTS: A female-specific linkage map with an average distance of 2 cM between markers in the initial QTL interval (SW2456-SW1943) was created and used here. The CI of QTL for BFT, HW and LEA were narrowed down to 6-7 cM, resulting from the joint analysis. For IMF, two linked QTL were revealed in the INRA population but not in the JXAU population, causing a wider CI (13 cM) for IMF QTL. Linkage analyses using two subsets of INRA F1 dam families demonstrate that the BFT and HW QTL were segregating in the Meishan pigs. Moreover, haplotype comparisons between these dams suggest that within the refined QTL region, the recombination coldspot (~34 Mb) flanked by markers MCSE3F14 and UMNP1218 is unlikely to contain QTL genes. Two SNPs in the ACSL4 gene were identified and showed significant association with BFT and HW, but they and the known polymorphisms in the other two genes are unlikely to be causal mutations. CONCLUSION: The candidate QTL regions have been greatly reduced and the QTL are most likely located downstream of the recombination coldspot. The segregation of SSCX QTL for BFT and HW within Meishan breed provides an opportunity for us to make effective use of Meishan chromosome X in crossbreeding. Further studies should attempt to identify the impact of additional DNA sequence (e.g. CNV) and expression variation in the three genes or their surrounding genes on these traits.

Recombinational landscape of porcine X chromosome and individual variation in female meiotic recombination associated with haplotypes of Chinese pigs.

BACKGROUND: Variations in recombination fraction (theta) among chromosomal regions, individuals and families have been observed and have an important impact on quantitative trait loci (QTL) mapping studies. Such variations on porcine chromosome X (SSC-X) and on other mammalian chromosome X are rarely explored. The emerging assembly of pig sequence provides exact physical location of many markers, facilitating the study of a fine-scale recombination landscape of the pig genome by comparing a clone-based physical map to a genetic map. Using large offspring of F1 females from two large-scale resource populations (Large White male symbol x Chinese Meishan female symbol, and White Duroc male symbol x Chinese Erhualian female symbol), we were able to evaluate the heterogeneity in theta for a specific interval among individual F1 females. RESULTS: Alignments between the cytogenetic map, radiation hybrid (RH) map, genetic maps and clone map of SSC-X with the physical map of human chromosome X (HSA-X) are presented. The most likely order of 60 markers on SSC-X is inferred. The average recombination rate across SSC-X is of approximately 1.27 cM/Mb. However, almost no recombination occurred in a large region of approximately 31 Mb extending from the centromere to Xq21, whereas in the surrounding regions and in the Xq telomeric region a recombination rate of 2.8-3.3 cM/Mb was observed, more than twice the chromosome-wide average rate. Significant differences in theta among F1 females within each population were observed for several chromosomal intervals. The largest variation was observed in both populations in the interval UMNP71-SW1943, or more precisely in the subinterval UMNP891-UMNP93. The individual variation in theta over this subinterval was found associated with F1 females' maternal haplotypes (Chinese pig haplotypes) and independent of paternal haplotype (European pig haplotypes). The theta between UMNP891 and UMNP93 for haplotype 1122 and 4311 differed by more than fourteen-fold (10.3% vs. 0.7%). CONCLUSIONS: This study reveals marked regional, individual and haplotype-specific differences in recombination rate on SSC-X. Lack of recombination in such a large region makes it impossible to narrow QTL interval using traditional fine-mapping approaches. The relationship between recombination variation and haplotype polymorphism is shown for the first time in pigs.

[Inhibition of vascular endothelial growth factor gene expression and proliferation of leukemia cells by RNA interference].

OBJECTIVE: To explore the feasibility of selective inhibiting VEGF expression using VEGF short hairpin RNA (shRNA) interference, and observe the effects of VEGF gene silencing on NB4 cells growth. METHODS: Three 19 bp reverse repeated motifs targeting exons 3, 4, 5 respectively of VEGF gene were synthesized and cloned into eukaryotic expression plasmid pGenesil-1 containing U6 shRNA promoter and termination signal of RNA polymerase. The recombinant plasmids pGenesil-VR1, pGenesil-VR2, pGenesil-VR3 and pGenesil-con (plasmid containing random DNA fragment) were transfected into NB4 cells respectively through lipofectamine reagent. The alteration of VEGF expression was examined by fluorescent real time RT-PCR and Western blot. The proliferation capacity of leukemia cells was measured by trypan blue exclusion, MTT assay, colony formation assay and cell cycles analysis. RESULTS: Recombinant plasmids containing three shRNAs and random fragment were successfully constructed and transfected into NB4 cells respectively by liposome-mediated gene transfer method. shRNA in pGenesil-VR3 cells knocked down the expression of VEGF mRNA and protein dramatically in a sequence-specific manner when compared with that of pGenesil-VR1, Genesil-VR2 and pGenesil-con. The NB4 cells transfected with pGenesil-VR3 (NB4-VR3) had a more significant decrease in proliferation ability than NB4 and that transfected with pGenesil-con (NB4-con). The colony forming efficiencies of NB4-VR3, NB4-con and NB4 cell were (13.3 +/- 3.8)%, (21.3 +/- 6.4)% and (24.5 +/- 5.2)%, respectively (P < 0.05). Higher G(1) and lower S proportion were found in cell cycle distribution in comparison with the control groups by FCM. CONCLUSIONS: The shRNA can efficiently suppress VEGF expression in NB4 cells. Selective VEGF gene silence can inhibit the malignant proliferation of leukemia cells.