A novel shark single-domain antibody targeting OGT as a tool for detection and intracellular localization.

Introduction: O-GlcNAcylation is a type of reversible post-translational modification on Ser/Thr residues of intracellular proteins in eukaryotic cells, which is generated by the sole O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). Thousands of proteins, that are involved in various physiological and pathological processes, have been found to be O-GlcNAcylated. However, due to the lack of favorable tools, studies of the O-GlcNAcylation and OGT were impeded. Immunoglobulin new antigen receptor (IgNAR) derived from shark is attractive to research tools, diagnosis and therapeutics. The variable domain of IgNARs (VNARs) have several advantages, such as small size, good stability, low-cost manufacture, and peculiar paratope structure. Methods: We obtained shark single domain antibodies targeting OGT by shark immunization, phage display library construction and panning. ELISA and BIACORE were used to assess the affinity of the antibodies to the antigen and three shark single-domain antibodies with high affinity were successfully screened. The three antibodies were assessed for intracellular function by flow cytometry and immunofluorescence co-localization. Results: In this study, three anti-OGT VNARs (2D9, 3F7 and 4G2) were obtained by phage display panning. The affinity values were measured using surface plasmon resonance (SPR) that 2D9, 3F7 and 4G2 bound to OGT with KD values of 35.5 nM, 53.4 nM and 89.7 nM, respectively. Then, the VNARs were biotinylated and used for the detection and localization of OGT by ELISA, flow cytometry and immunofluorescence. 2D9, 3F7 and 4G2 were exhibited the EC50 values of 102.1 nM, 40.75 nM and 120.7 nM respectively. VNAR 3F7 was predicted to bind the amino acid residues of Ser375, Phe377, Cys379 and Tyr 380 on OGT. Discussion: Our results show that shark single-domain antibodies targeting OGT can be used for in vitro detection and intracellular co-localization of OGT, providing a powerful tool for the study of OGT and O-GlcNAcylation.

Time taken for a primary tumor to metastasize to the brain and the overall survival of patients with brain metastasis: An analysis of outcomes and factors affecting survival.

BACKGROUND: Brain metastasis (BM) is one of the main causes of high morbidity and mortality in cancer patients. OBJECTIVE: To evaluate the factors that influenced the survival time of patients with primary cancer and survival time after BM. METHODS: Ninety patients with BM diagnosed by magnetic resonance imaging (MRI) were included in the study. We retrospectively analyzed the time to brain metastasis (TTB), overall survival time (OS1) and survival time after BM (OS2). The Kaplan-Meier method and Cox regression analysis were used for survival analyses. RESULTS: The median TTB was 12.0 (95% CI: 9.2-14.8 months), the median OS1 was 31.0 (95% CI: 25.8-35.2 months) and the median OS2 was 14.0 (95% CI: 10.9-17.1 months). Surgical excision of the primary tumor was an independent factor for a prolonged TTB (p< 0.000) and prolonged OS1 (p< 0.000). A single intracranial metastatic lesion was an independent protective factor for prolonged OS1 (p= 0.011) and prolonged OS2 (p= 0.050). TTB, OS1 and OS2 were analyzed with Gender (p< 0.000, < 0.000, and = 0.017, respectively). CONCLUSIONS: It suggests that TTB can be prolonged by primary tumor resection. Furthermore, women with a prolonged TTB and single intracranial metastasis are associated with high OS. These were helpful for the clinical treatment of BM patients before brain metastasis.

Delayed development of vacuoles and recanalization in the duodenum: a study in human fetuses to understand susceptibility to duodenal atresia/stenosis.

ObjectiveWe compared the cross-sectional areas of the duodenum to the distal small intestine during early gestation to determine if there is a difference in age for recanalization.MethodsSerial sagittal sections of six fetuses of gestational age (GA) 8-10 weeks were examined morphologically to compare the degree of recanalization of the duodenum with to the more distal small intestine.ResultsAt GA 8-9 weeks, the duodenum had more epithelial plugs and vacuoles with no or narrower spaces compared to the distal small bowel. Quantitative assessment at GA 10 weeks showed that the cross-sectional area of the duodenal cavity was significantly less than the distal small bowel.ConclusionThe development and recanalization of vacuoles in the duodenum occurs later than the jejunum and ileum may be involved in the more frequent development of atresia/stenosis of the duodenum compared to more distal gastrointestinal tract.

Gut lumen formation defect can cause intestinal atresia: evidence from histological studies of human embryos and intestinal atresia septum.

Intestinal atresia (IA), a common cause of neonatal intestinal obstruction, is a developmental defect, which disrupts the luminal continuity of the intestine. Here, we investigated (i) the process of lumen formation in human embryos; and (ii) how a defective lumen formation led to IA. We performed histological and histochemical study on 6-10 gestation week human embryos and on IA septal regions. To investigate the topology of embryonic intestine development, we conducted 3D reconstruction. We showed that a 6-7th gestation week embryonic gut has no lumen, but filled with mesenchyme cells and vacuoles of a monolayer of epithelial cells. A narrow gut lumen was formed by gestation week-9, the gut was filled with numerous vacuoles of different sizes, some vacuoles were merging with the developing embryonic gut wall. At gestation week-10, a prominent lumen was developed, only few vacuoles were present and were merging with the intestine wall. At IA septal regions, vacuoles were located in the submucous layer, covered by a single layer of epithelium without glandular structure, and surrounded with fibrous tissue. The mucosal epithelium was developed with lamina propria and basement membrane, but the submucosa and the longitudinal smooth muscle layers were not properly developed. Hence, the vacuoles in IA septum could represent a remnant of vacuoles of embryonic gut. In conclusion, the fusion of vacuoles with the developing intestine wall associates with the disappearance of vacuoles and gut lumen formation in human embryos, and perturbation of these developmental events could lead to IA.

WITHDRAWN: Immunohistochemical study of inflammatory responses in septa arising from type I intestinal atresia.

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Complete Chloroplast Genome Sequencing and Phylogenetic Analysis of Two Dracocephalum Plants.

Dracocephalum tanguticum and Dracocephalum moldavica are important herbs from Lamiaceae and have great medicinal value. We used the Illumina sequencing technology to sequence the complete chloroplast genome of D. tanguticum and D. moldavica and then conducted de novo assembly. The two chloroplast genomes have a typical quadripartite structure, with the gene's lengths of 82,221 bp and 81,450 bp, large single-copy region's (LSC) lengths of 82,221 bp and 81,450 bp, and small single-copy region's (SSC) lengths of 17,363 bp and 17,066 bp, inverted repeat region's (IR) lengths of 51,370 bp and 51,352 bp, respectively. The GC content of the two chloroplast genomes was 37.80% and 37.83%, respectively. The chloroplast genomes of the two plants encode 133 and 132 genes, respectively, among which there are 88 and 87 protein-coding genes, respectively, as well as 37 tRNA genes and 8 rRNA genes. Among them, the rps2 gene is unique to D. tanguticum, which is not found in D. moldavica. Through SSR analysis, we also found 6 mutation hotspot regions, which can be used as molecular markers for taxonomic studies. Phylogenetic analysis showed that Dracocephalum was more closely related to Mentha.