Spatial transcriptomics reveals gene interactions and signaling pathway dynamics in rat embryos with anorectal malformation.

Anorectal malformation (ARM) is a prevalent early pregnancy digestive tract anomaly. The intricate anatomy of the embryonic cloaca region makes it challenging for traditional high-throughput sequencing methods to capture location-specific information. Spatial transcriptomics was used to sequence libraries of frozen sections from embryonic rats at gestational days (GD) 14 to 16, covering both normal and ARM cases. Bioinformatics analyses and predictions were performed using methods such as WGCNA, GSEA, and PROGENy. Immunofluorescence staining was used to verify gene expression levels. Gene expression data was obtained with anatomical annotations of clusters, focusing on the cloaca region's location-specific traits. WGCNA revealed gene modules linked to normal and ARM cloacal anatomy development, with cooperation between modules on GD14 and GD15. Differential gene expression profiles and functional enrichment were presented. Notably, protein levels of Pcsk9, Hmgb2, and Sod1 were found to be downregulated in the GD15 ARM hindgut. The PROGENy algorithm predicted the activity and interplay of common signaling pathways in embryonic sections, highlighting their synergistic and complementary effects. A competing endogenous RNA (ceRNA) regulatory network was constructed from whole transcriptome data. Spatial transcriptomics provided location-specific cloaca region gene expression. Diverse bioinformatics analyses deepened our understanding of ARM's molecular interactions, guiding future research and providing insights into gene regulation in ARM development.

CircJag1 promotes apoptosis of ethylene thiourea-exposed anorectal malformations through sponging miR-137-3p by regulating Sox9 and suppressing Wnt/ß-catenin pathway during the hindgut development of rat embryos.

Anorectal malformations (ARMs) are common birth defects involving congenital structural anomalies of the gastrointestinal tract. As an important component of non-coding RNAs, circular RNAs (circRNAs) widely participate in the digestive system development; however, the specific molecular mechanism of their involvement in ARM occurrence remains obscure. Herein, we generated rat models of ARMs induced by ethylene thiourea. A novel circRNA (circJag1) was screened and identified by RNA-Seq, which is remarkably upregulated in hindgut tissues of ARM rat embryos. In vivo experiments, colocation analysis via fluorescence in situ hybridization, and immunofluorescence further demonstrated that the disordered circJag1/miR-137-3p/Sox9 expression caused a spatiotemporal imbalance in the urorectal septum (URS) of ARMs. In vitro, functional assays confirmed that circJag1 upregulation resulted in the degradation of nuclear ß-catenin, C-myc, and Cyclin D1 in rat intestinal epithelial cells, as well as the promotion of apoptosis and suppression of cell proliferation. Mechanistically, dual-luciferase reporter assay and RNA immunoprecipitation assay indicated that circJag1 acted as a miR-137-3p sponge, thereby inhibiting its repressive effect on its target Sox9. Further experiments showed that a loss of Sox9 abolished the circJag1-mediated increase in apoptosis. In conclusion, aberrantly high circJag1 expression promotes epithelial apoptosis by suppressing the canonical Wnt/ß-catenin pathway via the miR-137-3p/Sox9 axis, which leads to fusion failure of the URS and cloacal membrane, and eventually contributed to ARMs. Our achievements might boost the comprehension of ARM pathogenesis and could provide a novel candidate target for the development of therapies for ARMs to complement surgical treatment.

Is enema reduction in pediatric intussusception with a history of over 48 h safe: A retrospective cohort study.

BACKGROUND: Intussusception is one of the most common acute abdominal diseases in children. Enema reduction is the first-line treatment for intussusception in good condition. Clinically, a history of disease over 48 h is usually listed as a contraindication for enema reduction. However, with the development of clinical experience and therapy, an increasing number of cases have shown that the prolongation of the clinical course of intussusception in children is not an absolute contraindication for enema treatment. This study aimed to analyze the safety and efficacy of enema reduction in children with a history of disease longer than 48 h. METHODS: We conducted a retrospective matched-pair cohort study of pediatric patients with acute intussusception between 2017 and 2021. All patients were treated with ultrasound-guided hydrostatic enema reduction. According to the length of history, the cases were classified into two groups: history <48 h (<48 h group) and history greater than or equal to 48 h (≧48 h group). We generated a 1:1 matched-pair cohort matched for sex, age, admission time, main symptoms, and concentric circle size on ultrasound. Clinical outcomes were compared between the two groups, including success, recurrence, and perforation rates. RESULTS: From January 2016 to November 2021, 2701 patients with intussusception were admitted to the Shengjing Hospital of China Medical University. A total of 494 cases were included in the ≧48 h group, and 494 cases with a history of <48 h were selected for matched comparison in the <48 h group. The success rates of the ≧48 h and <48 h groups were 98.18% vs. 97.37% (p = 0.388), and the recurrence rates were 13.36% vs. 11.94% (p = 0.635), showing no difference according to the length of history. The perforation rate was 0.61% vs. 0%, respectively, with no significant difference (p = 0.247).The comparison of the different history groups showed that in patients with bloody stools, the length of history had no significant effect on the enema reduction outcome(94.90% vs. 86.76%, p = 0.064). CONCLUSIONS: Ultrasound-guided hydrostatic enema reduction is safe and effective for pediatric idiopathic intussusception with a history of ≧48 h.

Upregulation of PPPDE1 contributes to anorectal malformations via the mitochondrial apoptosis pathway during hindgut development in rats.

Congenital anorectal malformations (ARMs) are among the most prominent deformities of the gastrointestinal tract; however, their precise aetiology remains obscure. Immunohistochemistry demonstrated that, in the ARM group, the PPPDE1-positive cells were widely distributed in the hindgut epithelial tissue from GD13 to GD16. Immunofluorescence revealed that most TUNEL-, Bax-, and Cytochrome C (Cyt C)-positive cells overlapped with PPPDE1-positive cells in the urorectal septum (URS). Western blotting and quantitative real-time RT-PCR revealed that PPPDE1 levels were significantly higher in the ARM group from GD13 to GD14 (p < 0.05). IEC-6 cells were transfected with PPPDE1 overexpression plasmid/NC (negative control) or si-PPPDE1/si-NC. Flow cytometry analysis and CCK-8 assay (used to detect apoptosis and proliferation, respectively), as well as western blotting, showed that the levels of PPPDE1 were positively correlated with the pro-apoptotic molecules Bax and Cyt C. Accordingly, aberrantly high expression of PPPDE1 caused a spatiotemporal imbalance in foetal rats with ARMs during hindgut development. Therefore, the upregulation of PPPDE1 may promote epithelial apoptosis and reduce proliferation in the hindgut via the mitochondrial apoptotic pathway. This could affect the fusion of the URS and cloacal membrane, ultimately inhibiting the hindgut development and resulting in ARMs.

[Sensitivity Comparison Experiment of Four Testing Methods for Helicobacter pylori].

Objective: To measure with standard microbiology methods the sensitivity of 4 commonly used testing methods for Helicobacter pylori (Hp) and to conduct a comparative study of the correlations and differences across the 4 methods. Methods: With the Hp standard strain (SS1) as the reference, colony forming units (CFU) as the units of quantitative analysis for detection performance, and gradient dilution of SS1 suspension as the simulation sample, we measured the sensitivity of 4 Hp testing methods, including bacterial culture, rapid urease test, antigen test, and quantitative fluorescent PCR. CFU values at different concentrations corresponding to the 4 commonly used Hp testing methods were documented and the correlations and differences were analyzed accordingly. Results: The sensitivity of Hp bacterial culture, rapid urease test, antigen test and quantitative fluorescent PCR was 2.0×10 CFU/mL, 2.0×10 5 CFU/mL, 2.0×10 5 CFU/mL, and 2.0×10 2 CFU/mL, respectively. Conclusion: The testing turnover time and sensitivity of different laboratory methods for Hp testing varied significantly. The quantitative fluorescent PCR and bacterial culture both showed relatively high sensitivity, but bacterial culture has complicated operation procedures and is too time-consuming. The rapid urease test and antigen test both were simple and quick to perform, but showed low sensitivity. For clinical and laboratory testing of Hp, appropriate testing method that can identify the corresponding changes of Hp should be selected according to the actual testing purpose.

Association of air temperature with pediatric intussusception in northeastern China: A 10-year retrospective study.

OBJECTIVE: The aim of this study was to determine whether an association existed between intussusception and air temperature. METHODS: A retrospective study was performed between March 2006 and February 2016 to determine the relationship between pediatric primary intussusception (PPI) and air temperature. Information from hospital records of 5922 cases of PPI and Mean daily temperatures of Shenyang were obtained. Pearson correlation analysis was used to examine the association between monthly PPI cases and monthly mean temperature. Factorial analysis-of-variance was used to examine differences in the numbers of seasonal PPI cases during different seasons. RESULTS: Monthly PPI cases fluctuated throughout the year, with a peak in June, and a trough in February. Pearson correlation analysis showed that monthly PPI cases was associated with the monthly mean temperature (p < 0.01). Factorial analysis-of-variance showed there was significant difference in the numbers of seasonal PPI cases during different seasons. Multiple comparison showed a significant difference in seasonal PPI cases between spring and summer, spring and winter, summer and autumn, summer and winter, autumn and winter (p < 0.01). CONCLUSIONS: Monthly PPI cases were positively associated with monthly mean temperature in Shenyang. The incidence of intussusception shows a seasonal trend, with a peak in summer (May to July).

Superficial CD34-Positive Fibroblastic Tumor on the Chest Wall of an 8-Year-Old Girl: A Case Report and Literature Review.

This study reports a case of superficial CD34-positive fibroblastic tumor (SCPFT) in a child and analyze the major known clinicopathological features of SCPFT and other skin mesenchymal tumors, contributing to an accurate diagnosis of this rare disease. We summarize the clinicopathologic features of an 8-year-old girl who was diagnosed with SCPFT and 46 previously reported SCPFT cases. Post-operative histopathologic examination of the current case showed the tumor lesion was well-circumscribed; tumor cells were spindled-to-polygonal with a fascicular pattern; most nuclei displayed hyperchromasia and low mitotic rate; intranuclear pseudoinclusions could be found; and abundant eosinophilic cytoplasm and partial myxoid stroma were observed. Immunohistochemistry revealed strong and diffuse CD34-positivity, vimentin staining positively but no S-100, SMA, NSE, CD31, desmin, cytokeratin, STAT6, ß-catenin, MDM2, or ERG expression. The Ki-67 and CD68 labeling indexes were approximately 1%. There were no rearrangements of PDGFB or PRDM10 tested by FISH. After surgical resection, the patient had no signs of recurrence or metastasis at a 6-month follow-up. The present case is the first that describes SCPFT in children and has significant clinical implications. SCPFT should be differentiated from other skin mesenchymal tumors. The presented compilation of all so far published SCPFT cases will help in diagnosing successfully SCPFT and increasing awareness of this tumor to guide clinical practice.

[Preparation and Release Properties of Helicobacter pylori Recombinant Protein PLGA Microspheres and PLGA-Chitosan Microspheres].

OBJECTIVE: To preparethe poly lactic-co-glycolic acid (PLGA) microspheres and PLGA-chitosan microspheres containing Helicobacter pylori recombinant protein, namely the BIB protein, and to explore their optimal preparation parameters and in vitro release performance in gastric and intestinal fluids. METHODS: Double emulsions (water-in-oil-in-water, or W1/O/W2) solvent evaporation method was used to prepare the BIB-PLGA microspheres and the BIB-PLGA-chitosan microspheres. Univariate analysis was done to study the impact of the water-to-oil ratio (W1/O), PLGA mass fraction and PVA concentration on the morphology, particle size, polydispersity index (PDI), encapsulation efficiency (EE), and drug loading (DL) so as to identify the optimal parameters. Bicinchoninic acid (BCA) assay was used to determine the protein concentration and the release efficiency of BIB. RESULTS: The optimal preparation parameters identified in the study were as follows: W1/O at 1∶2, PLGA mass fraction at 5%, and PVA mass fraction at 0.2%. The BIB-PLGA microspheres were found to be (2.11±0.08) µm in particle size, 0.35±0.18 in PDI, (78.20±1.73)% in EE and (10.58±0.23)% in DL. The BIB-PLGA-chitosan microspheres were (2.28±0.52) µm in particle size, 0.39±0.54 in PDI, and (78.87±1.30)% and (15.50±0.25)% in EE and DL, respectively. Both BIB-PLGA microspheres and BIB-PLGA-chitosan microspheres showed slow-release property in gastric and intestinal fluids in vitro, with BIB-PLGA-chitosan microspheres showing better slow-release performance. CONCLUSION: The BIB-PLGA microspheres and BIB-PLGA-chitosan microspheres prepared with the double emulsions solvent evaporation method showed high DL and EE, controllable particle sizes, dispersive appearance, and slow-release property in gastric and intestinal fluids in vitro.

Zinc excess increases cellular demand for iron and decreases tolerance to copper in Escherichia coli.

Transition metals serve as an important class of micronutrients that are indispensable for bacterial physiology but are cytotoxic when they are in excess. Bacteria have developed exquisite homeostatic systems to control the uptake, storage, and efflux of each of biological metals and maintain a thermodynamically balanced metal quota. However, whether the pathways that control the homeostasis of different biological metals cross-talk and render cross-resistance or sensitivity in the host-pathogen interface remains largely unknown. Here, we report that zinc (Zn) excess perturbs iron (Fe) and copper (Cu) homeostasis in Escherichia coli, resulting in increased Fe and decreased Cu levels in the cell. Gene expression analysis revealed that Zn excess transiently up-regulates Fe-uptake genes and down-regulates Fe-storage genes and thereby increases the cellular Fe quota. In vitro and in vivo protein-DNA binding assays revealed that the elevated intracellular Fe poisons the primary Cu detoxification transcription regulator CueR, resulting in dysregulation of its target genes copA and cueO and activation of the secondary Cu detoxification system CusSR-cusCFBA Supplementation with the Fe chelator 2,2'-dipyridyl (DIP) or with the reducing agent GSH abolished the induction of cusCFBA during Zn excess. Consistent with the importance of this metal homeostatic network in cell physiology, combined metal treatment, including simultaneously overloading cells with both Zn (0.25 mm) and Cu (0.25 mm) and sequestering Fe with DIP (50 µm), substantially inhibited E. coli growth. These results advance our understanding of bacterial metallobiology and may inform the development of metal-based antimicrobial regimens to manage infectious diseases.

Activity-Based Sensing of Ascorbate by Using Copper-Mediated Oxidative Bond Cleavage.

Ascorbate is an important biological reductant and enzyme cofactor. Although direct detection through ascorbate-mediated reduction is possible, this approach suffers from poor selectivity due to the wide range of cellular reducing agents. To overcome this limitation, we leverage reduction potential of ascorbate to mediate a copper-mediated oxidative bond cleavage of ether-caged fluorophores. The copper(II) complexes supported by a {bis(2-pyridylmethyl)}benzylamine or a {bis(2-pyridylmethyl)}(2-methoxybenzyl)amine ligand were identified as an ascorbate responsive unit and their reaction with ascorbate yields a copper-based oxidant that enables rapid benzylic oxidation and the release of an ether-caged dye (coumarin or fluorescein). The copper-mediated bond cleavage is specific to ascorbate and the trigger can be readily derivatized for tuning photophysical properties of the probes. The probes were successfully applied for the fluorometric detection of ascorbate in commercial food samples, human plasma, and serum, and within live cells by using confocal microscopy and flow cytometry.

Paeonol reverses promoting effect of the HOTAIR/miR-124/Notch1 axis on renal interstitial fibrosis in a rat model.

Renal interstitial fibrosis (RIF) is a common manifestation of inflammatory and noninflammatory renal diseases, which correlates to renal excretory dysfunction. Recently, the long noncoding RNAs (lncRNAs) have been demonstrated to be involved in the development of various renal diseases. Here, we aim to determine whether paeonol (PAE) affects RIF with involvement of the lncRNA HOX transcript antisense intergenic RNA (HOTAIR)/microRNA-124 (miR-124)/Notch1 axis. RIF rat models were established by performing unilateral ureteral occlusion (UUO), in which interactions between HOTAIR, Notch1, and miR-124 were determined. To identify the roles of PAE and HOTAIR in RIF, rats were injected with HOTAIR or PAE. Subsequently, to further investigate the underlying mechanism of PAE in RIF, epithelial to mesenchymal transition (EMT)- and migration-related genes in NRK-49F cells were measured. Next, rats were further treated with IMR-1 (inhibitor of the Notch1/Jagged1 signaling pathway) to determine how PAE influences the Notch1/Jagged1 signaling pathway. HOTAIR interacted with miR-124, and miR-124 directly targeted Notch1, and HOTAIR was observed to be upregulated in RIF rats. PAE was found to decrease HOTAIR and Notch1 expression but to increase the miR-124 expression in RIF rats. PAE inhibited EMT and migration of NRK-49F cells facilitated by HOTAIR. HOTAIR activated the Notch1/Jagged1 signaling pathway by downregulating miR-124, while PAE reversed these effects of HOTAIR on the Notch1/Jagged1 signaling pathway. Overall, our study demonstrates the contributory effect of lncRNA HOTAIR on RIF by activating the Notch1/Jagged1 signaling pathway via inhibition of miR-124, whereas administration of PAE can alleviate the effects of HOTAIR on RIF.

LncRNA HOTAIR promotes renal interstitial fibrosis by regulating Notch1 pathway via the modulation of miR-124.

AIM: To understand the mechanism of long non-coding RNA (LncRNA) HOTAIR on renal interstitial fibrosis (RIF) by regulating Notch1 pathway via the modulation of miR-124. METHODS: Unilateral ureteral occlusion (UUO) was used to construct the RIF rat model. HK-2 cells induced by TGF-ß1 were used for the in vitro experiment, which were divided into five groups: Vehicle, TGF-ß1, si-HOTAIR+TGF-ß1, miR-124 inhibitor+TGF-ß1, and si-HOTAIR+miR-124 inhibitor+TGF-ß1 groups. Quantitative real-time PCR (qRT-PCR) and Western blot were performed to detect the expression of HOTAIR, miR-124, Notch1- and epithelial-to-mesenchymal transition (EMT)-related proteins. RESULTS: Significant elevated HOTAIR and reduced miR-124 were presented in UUO rats and TGF-ß1-induced HK-2 cells in a time-dependent manner, with the increased Jagged1 (JAG1), Notch1, NICD, α-SMA and FN, as well as the decreased E-cadherin (all P < 0.05). Compared with the TGF-ß1 group, cells in the si-HOTAIR+TGF-ß1 group were remarkably declined in cell proliferation and the protein expressions of JAG1, Notch1, NICD, α-SMA, and FN, but dramatically higher in E-cadherin expression (all P < 0.05). However, in comparison with the si-HOTAIR+TGF-ß1 group, cells in the si-HOTAIR+miR-124 inhibitor+TGF-ß1 group were apparently improved in proliferation and the protein expression of JAG1, Notch1, NICD, α-SMA, and FN, but substantially reduced in the level of E-cadherin protein (all P < 0.05). CONCLUSION: Silencing lncRNA HOTAIR can up-regulate miR-124 to block Notch1 pathway, and thereby alleviating EMT and RIF, indicating HOTAIR as a potential target for RIF treatment.

Salinomycin-loaded lipid-polymer nanoparticles with anti-CD20 aptamers selectively suppress human CD20+ melanoma stem cells.

Melanoma is the deadliest type of skin cancer. CD20+ melanoma stem cells (CSCs) are pivotal for metastasis and initiation of melanoma. Therefore, selective elimination of CD20+ melanoma CSCs represents an effective treatment to eradicate melanoma. Salinomycin has emerged as an effective drug toward various CSCs. Due to its poor solubility, its therapeutic efficacy against melanoma CSCs has never been evaluated. In order to target CD20+ melanoma CSCs, we designed salinomycin-loaded lipid-polymer nanoparticles with anti-CD20 aptamers (CD20-SA-NPs). Using a single-step nanoprecipitation method, salinomycin-loaded lipid-polymer nanoparticles (SA-NPs) were prepared, then CD20-SA-NPs were obtained through conjugation of thiolated anti-CD20 aptamers to SA-NPs via a maleimide-thiol reaction. CD20-SA-NPs displayed a small size of 96.3 nm, encapsulation efficiency higher than 60% and sustained drug release ability. The uptake of CD20-SA-NPs by CD20+ melanoma CSCs was significantly higher than that of SA-NPs and salinomycin, leading to greatly enhanced cytotoxic effects in vitro, thus the IC50 values of CD20-SA-NPs were reduced to 5.7 and 2.6 µg/mL in A375 CD+20 cells and WM266-4 CD+ cells, respectively. CD20-SA-NPs showed a selective cytotoxicity toward CD20+ melanoma CSCs, as evidenced by the best therapeutic efficacy in suppressing the formation of tumor spheres and the proportion of CD20+ cells in melanoma cell lines. In mice bearing melanoma xenografts, administration of CD20-SA-NPs (salinomycin 5 mg·kg-1·d-1, iv, for 60 d) showed a superior efficacy in inhibition of melanoma growth compared with SA-NPs and salinomycin. In conclusion, CD20 is a superior target for delivering drugs to melanoma CSCs. CD20-SA-NPs display effective delivery of salinomycin to CD20+ melanoma CSCs and represent a promising treatment for melanoma.

Clinical and Ultrasonographic Features of Secondary Intussusception in Children.

OBJECTIVES: The aim of this study was to review the ultrasonographic features of secondary intussusception (SI) in children and assess the value of ultrasound in the diagnosis of pediatric SI. METHODS: The authors performed a retrospective analysis on the ultrasound findings of 1977 cases of primary intussusception (PI) and 37 cases of SI in children. The SI cases were diagnosed by ultrasonography and confirmed by laparotomy or histopathologic diagnosis. The clinical and ultrasonographic features were analyzed and compared between these two groups. RESULTS: The age, no flatus or defecation, position, diameter and length of intussusception, the presence of free intraperitoneal liquid, and intestinal dialation at the proximal end present, all contributed to the differentiation between PI and SI (all P < 0.05). Ultrasound was able to demonstrate the pathological lead point (PLP) shadows in all of the 37 SI cases, either in the cervical part or intussusceptum of the intussusception. Among the 37 SI patients, 21 cases (56.8 %) were accurately categorized with lesions, including intestinal polyps, cystic intestinal duplication, intestinal wall lymphoma, and a small part of Meckel's diverticulum. CONCLUSIONS: Ultrasound can be used as a feasible and effective method to discriminate PI from SI. Once the PLP is detected, a definite diagnosis can be made. KEY POINTS: • The clinical and ultrasonographic features were compared between SI and PI. • The age, location, diameter and length of intussusception, and intestinal dilation were distinguishing features. • The causes of SI were found to be polyps, intestinal duplication, lymphoma, and Meckel's diverticulum. • Ultrasound can be used as an important method to diagnose SI. • Demonstration and confirmation of PLP are vital to diagnosing SI.

The expression analysis of Bmpr1a and Bmp2 during hindgut development in rat embryos with anorectal malformations.

The aim of this study was to determine Bmpr1a and Bmp2 expression patterns during anorectal development in normal and anorectal malformation (ARM) embryos with a view to establishing the possible role of Bmpr1a and Bmp2 in ARM pathogenesis. ARM was induced with ethylenethiourea on the 10th gestational day (GD10) in rat embryos. The embryos were harvested by Cesarean deliveries. The expression of Bmpr1a and Bmp2 was evaluated in normal rat embryos (n=213) and ARM embryos (n=236) from GD14 to GD16. Immunohistochemical staining revealed, in normal embryos, that Bmpr1a and Bmp2 was mainly expressed on the epithelium of the urorectal septum (URS) and the cloacal membrane (CM) on GD14 and GD15. When the rectum separated from the urogenital sinus (UGS) on GD16, Bmpr1a- and Bmp2-immunolabeled cells were observed on the anorectal epithelium. In ARM embryos, the epithelium of the hindgut and URS demonstrated faint immunostaining for Bmpr1a and Bmp2. Analyses by Western blot and Real-time PCR revealed that Bmpr1a and Bmp2 protein and mRNA expression were significantly decreased in the ARM hindgut compared with normal hindgut on GD14 and GD15 (P<0.05). In ARM embryos, an imbalance in the spatiotemporal expression of Bmpr1a and Bmp2 was noted during anorectal morphogenesis from GD14 to GD16. Therefore, downregulation of Bmpr1a and Bmp2 at the time of cloacal separation into the primitive rectum and UGS might be related to the development of ARM.

A simplified system for generating recombinant E3-deleted canine adenovirus-2.

Canine adenovirus type 2 (CAV-2) has been used extensively as a vector for studying gene therapy and vaccine applications. We describe a simple strategy for generating a replication-competent recombinant CAV-2 using a backbone vector and a shuttle vector. The backbone plasmid containing the full-length CAV-2 genome was constructed by homologous recombination in Escherichia coli strain BJ5183. The shuttle plasmid, which has a deletion of 1478 bp in the nonessential E3 viral genome region, was generated by subcloning a fusion fragment containing the flanking sequences of the CAV-2 E3 region and expression cassette sequences from pcDNA3.1(+) into modified pUC18. To determine system effectiveness, a gene for enhanced green fluorescent protein (EGFP) was inserted into the shuttle plasmid and cloned into the backbone plasmid using two unique NruI and SalI sites. Transfection of Madin-Darby canine kidney (MDCK) cells with the recombinant adenovirus genome containing the EGFP expression cassette resulted in infectious viral particles. This strategy provides a solid foundation for developing candidate vaccines using CAV-2 as a delivery vector.

Temporal and spatial expression of caudal-type homeobox gene-2 during hindgut development in rat embryos with ethylenethiourea-induced anorectal malformations.

The main aim of this study was to determine Cdx2 expression patterns during anorectal development in normal and anorectal malformation (ARM) embryos with a view to establishing the possible role of Cdx2 in ARM pathogenesis. ARM was induced with ethylenethiourea on the 10th gestational day (GD10) in rat embryos, and Cesarean deliveries were performed to harvest the embryos. The temporal and spatial expression of Cdx2 was evaluated in normal rat embryos (n = 303) and ARM embryos (n = 321) from GD13 to GD16. Immunohistochemical staining revealed that, in normal embryos, Cdx2 was mainly expressed on the epithelium of the urorectal septum (URS) and the hindgut on GD13. On GD14, Cdx2-immunopositive cells were extensively detected on the URS, hindgut, and cloacal membrane. On GD15, increased immunopositive tissue staining on the anal membrane was evident. In ARM embryos, the epithelium of the cloaca, URS, and anorectum were negative or faintly immunostaining for Cdx2. Analyses by Western blot and real-time reverse transcription plus the polymerase chain reaction revealed that, in the normal group, Cdx2 protein and mRNA expression showed time-dependent changes in the developing hindgut from GD13 to GD16. Upon the URS division of the cloaca into the primitive rectum and urogenital sinus (UGS) on GD15, Cdx2 expression began to decrease. Moreover, the Cdx2 expression level in the ARM group from GD13 to GD14 was significantly lower than that in the normal group (P < 0.05). In ARM embryos, an imbalance in the spatiotemporal expression of Cdx2 was noted during anorectal morphogenesis from GD13 to GD16. Downregulation of Cdx2 at the time of cloacal separation into the primitive rectum and UGS might thus be related to the development of ARM.

Spatiotemporal expression of Cdx4 in the developing anorectum of rat embryos with ethylenethiourea-induced anorectal malformations.

PURPOSE: The aim of this study was to determine the expression of Cdx4 (caudal-type homeobox gene-4) during anorectal development in normal and ethylenethiourea (ETU)-induced anorectal malformation (ARM) embryos with a view to establishing the possible role of Cdx4 in ARM pathogenesis. MATERIALS AND METHODS: ARM was induced by ETU on the 10th gestational day (GD10) in rat embryos. Cesarean deliveries were then performed to harvest the embryos. Spatiotemporal expression of Cdx4 was evaluated in normal rat embryos (n = 354) and ARM embryos (n = 378) from GD13 to GD16. RESULTS: Immunohistochemical staining and immunofluorescence revealed that, in normal embryos, Cdx4 expression was extensively detected on the epithelium of the cloaca on GD13. On GD14, the Cdx4-positive cells were intensively detected on the hindgut. On GD15, the anal membrane was constantly immunoreactive to Cdx4. On GD16, Cdx4-labeled cells were observed on the epithelium of the anus. In the ARM embryos, the epithelium of the cloaca, urorectal septum (URS) and anorectum was negative or faint for Cdx4. In the normal embryo group, Cdx4 protein and mRNA expression showed time-dependent changes in the developing hindgut from GD13 to GD16 on Western blot and real-time reverse transcription plus polymerase chain reaction. Once the URS divided the cloaca into the primitive rectum and urogenital sinus (UGS) on GD15, Cdx4 expression began to decrease. In addition, the expression level of Cdx4 in the ARM group from GD13 to GD15 was significantly lower than that in the normal group (p < 0.05). CONCLUSIONS: In ARM embryos, an imbalance in the spatiotemporal expression of Cdx4 was noted during anorectal morphogenesis from GD13 to GD16. This suggests that ETU may cause downregulation of Cdx4 expression. Downregulation of Cdx4 at the time of cloacal separation into the primitive rectum and UGS might thus be related to the development of ARM.

Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights.

Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset.

Glycosyl-modified diporphyrins for in vitro and in vivo fluorescence imaging.

The application of probes for optical imaging is becoming popular as they have high safety and good biocompatibility. We prepared two kinds of glycosyl-modified diporphyrins, and their potentials as fluorescent probes were tested for the first time. After preparation of the glycosyl-modified porphyrin monomers, Ag-promoted coupling of the monomers was used to obtain glucose-modified porphyrin dimer (GPD) and lactose-modified porphyrin dimer (LPD). The strong interaction between the two porphyrin rings achieves red-shifted emission, and thus circumvents autofluorescence and light-scattering in biological samples. Although the glycosylation improves solubility, it also yielded selective attachment to cell membranes, and to chorions of early developmental-stage zebrafish. Patch-clamp experiments revealed the biocompatibility and low toxicity of GPD and LPD. Moreover, an in vivo imaging experiment provided direct evidence that zebrafish chorion contains sugar-binding proteins. The modification and derivatization make porphyrins potential bioimaging probes for specific optical imaging.