Production of Four-Gene (GTKO/hCD55/hTBM/hCD39)-Edited Donor Pigs and Kidney Xenotransplantation.

BACKGROUND: The number of multigene-modified donor pigs for xenotransplantation is increasing with the advent of gene-editing technologies. However, it remains unclear which gene combination is suitable for specific organ transplantation. METHODS: In this study, we utilized CRISPR/Cas9 gene editing technology, piggyBac transposon system, and somatic cell cloning to construct GTKO/hCD55/hTBM/hCD39 four-gene-edited cloned (GEC) pigs and performed kidney transplantation from pig to rhesus monkey to evaluate the effectiveness of these GEC pigs. RESULTS: First, 107 cell colonies were obtained through drug selection, of which seven were 4-GE colonies. Two colonies were selected for somatic cell nuclear transfer (SCNT), resulting in seven fetuses, of which four were GGTA1 biallelic knockout. Out of these four, two fetuses had higher expression of hCD55, hTBM, and hCD39. Therefore, these two fetuses were selected for two consecutive rounds of cloning, resulting in 97 live piglets. After phenotype identification, the GGTA1 gene of these pigs was inactivated, and hCD55, hTBM, and hCD39 were expressed in cells and multiple tissues. Furthermore, the numbers of monkey IgM and IgG binding to the peripheral blood mononuclear cells (PBMCs) of the 4-GEC pigs were markedly reduced. Moreover, 4-GEC porcine PBMCs had greater survival rates than those from wild-type pigs through complement-mediated cytolysis assays. In pig-to-monkey kidney xenotransplantation, the kidney xenograft successfully survived for 11 days. All physiological and biochemical indicators were normal, and no hyperacute rejection or coagulation abnormalities were found after transplantation. CONCLUSION: These results indicate that the GTKO/hCD55/hTBM/hCD39 four-gene modification effectively alleviates immune rejection, and the pig kidney can functionally support the recipient monkey's life.

Alantolactone Induced Apoptosis and DNA Damage of Cervical Cancer through ATM/CHK2 Signaling Pathway.

Traditional Chinese Medicine, known for its minimal side effects and significant clinical efficacy, has attracted considerable interest for its potential in cancer therapy. In particular, Inula helenium L. has demonstrated effectiveness in inhibiting a variety of cancers. This study focuses on alantolactone (ALT), a prominent compound from Inula helenium L., recognized for its anti-cancer capabilities across multiple cancer types. The primary objective of this study is to examine the influence of ALT on the proliferation, apoptosis, cell cycle, and tumor growth of cervical cancer (CC) cells, along with its associated signaling pathways. To determine protein expression alterations, Western blot analysis was conducted. Furthermore, an in vivo model was created by subcutaneously injecting HeLa cells into nude mice to assess the impact of ALT on cervical cancer. Our research thoroughly investigates the anti-tumor potential of ALT in the context of CC. ALT was found to inhibit cell proliferation and induce apoptosis in SiHa and HeLa cell lines, particularly targeting ataxia-telangiectasia mutated (ATM) proteins associated with DNA damage. The suppression of DNA damage and apoptosis induction when ATM was inhibited underscores the crucial role of the ATM/cell cycle checkpoint kinase 2 (CHK2) axis in ALT's anti-tumor effects. In vivo studies with a xenograft mouse model further validated ALT's effectiveness in reducing CC tumor growth and promoting apoptosis. This study offers new insights into how ALT combats CC, highlighting its promise as an effective anti-cervical cancer agent and providing hope for improved treatment outcomes for CC patients.

In vitro and in vivo development of interspecies Asian elephant embryos reconstructed with pig enucleated oocytes.

Interspecies somatic cell nuclear transfer (iSCNT) has an immense potential to rescue endangered animals and extinct species like mammoths. In this study, we successfully established an Asian elephant's fibroblast cell lines from ear tissues, performed iSCNT with porcine oocytes and evaluated the in vitro and in vivo development of reconstructed embryos. A total of 7780 elephant-pig iSCNT embryos were successfully reconstructed and showed in vitro development with cleavage rate, 4-cell, 8-cell and blastocyst rate of 73.01, 30.48, 5.64, and 4.73%, respectively. The total number of elephant-pig blastocyte cells and diameter of hatched blastocyte was 38.67 and 252.75 µm, respectively. Next, we designed species-specific markers targeting EDNRB, AGRP and TYR genes to verify the genome of reconstructed embryos with donor nucleus/species. The results indicated that 53.2, 60.8, and 60.8% of reconstructed embryos (n = 235) contained elephant genome at 1-cell, 2-cell and 4-cell stages, respectively. However, the percentages decreased to 32.3 and 32.7% at 8-cell and blastocyst stages, respectively. Furthermore, we also evaluated the in vivo development of elephant-pig iSCNT cloned embryos and transferred 2260 reconstructed embryos into two surrogate gilts that successfully became pregnant and a total of 11 (1 and 10) fetuses were surgically recovered after 17 and 19 days of gestation, respectively. The crown-rump length and width of elephant-pig cloned fetuses were smaller than the control group. Unfortunately, none of these fetuses contained elephant genomes, which suggested that elephant embryos failed to develop in vivo. In conclusion, we successfully obtained elephant-pig reconstructed embryos for the first time and these embryos are able to develop to blastocyst, but the in vivo developmental failure needs further investigated.

Faecalibacter macacae gen. nov., sp. nov., isolated from the faeces of Macaca assamensis.

Huge numbers of bacteria reside in the digestive tract of most animals. During an investigation into the bacterial diversity of primates, strain YIM 102668T was isolated. When neighbour-joining phylogenetic analysis based on 16S rRNA gene sequences was conducted, strain YIM 102668T formed a cluster within the family Flavobacteriaceae and in a lineage not associated with any known group of previously proposed genera. Closely related genera were Algoriella (94.8 %), Chishuiella (94.8 %), Empedobacter (highest 94.6 %), Moheibacter (90.9 %) and Weeksella (90.6 %). In addition, strain YIM 102668T contained MK-6 as the predominant respiratory quinone and iso-C15 : 0 as the major fatty acid. The major polar lipid was phosphatidylethanolamine and the genomic DNA G+C content was 30.6 mol%. These chemotaxonomic characterizations confirmed that strain YIM 102668T belonged to the family Flavobacteriaceae. Supported by the results of phylogenetic, phenotypic and chemotaxonomic analyses, we propose that strain YIM 102668T represents a novel genus, for which the name Faecalibacter macacae gen. nov., sp. nov. is proposed. The type strain is YIM 102668T (=KCTC 52109T=CCTCC AB 2016016T).

Functional and transcript analysis of a novel metal transporter gene EpNramp from a dark septate endophyte (Exophiala pisciphila).

Various metal transporters mediate sub-cellular sequestration of diverse metal ions, contribute to cellular metal tolerance, and control metal partitioning, particularly under conditions of high rates of metal influx into organisms. In the current study, a ubiquitous and evolutionary conserved metal transporter gene, homology to natural resistance associated macrophage protein (Nramp), was cloned from a metal-tolerant isolate of dark septate endophyte (DSE, Exophiala pisciphila), and its functional and transcript characterization were analyzed. The full-length Nramp gene from E. pisciphila (named EpNramp) was 1716 bp and expected to encode a polypeptide of 571 amino acid residues. EpNramp fused to green fluorescent protein suggested that EpNramp was a plasma membrane metal transporter, which was consistent with the results of bioinformatics analysis with 11 transmembrane domains. Yeast functional complementation revealed that EpNramp could complement the growth defect of Fe-uptake yeast mutant (fet3fet4 double mutant) by mediating the transport of Fe(2+). Expression of EpNramp increased Cd(2+) sensitivity and Cd(2+) accumulation in yeast. In addition, qPCR data revealed that E. pisciphila significantly down-regulated EpNramp expression with elevated Cd(2+) exposure. Altogether, EpNramp is a bivalent cation transporter localized in cell membrane, which is necessary for efficient translocation of both Fe and Cd, and its activities partly attributed to the tolerance of DSE to toxic and excessive Cd(2+) supplements.

[Analysis of sulfonamids and their metabolites in drinking water by high Performance liquid chromatography tandem mass spectrometry].

OBJECTIVE: To develop a comprehensive method for simultaneous analysis of sulfonamides and their metabolites in drinking water by high performance liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODS: Different solid-phase extraction columns were compared with respect to the recovery of target drugs from drinking water. The drinking water samples were adjusted to 3 by HCl and purified by a mix mode cation-ion exchange solid-phase extraction (SPE), following determination using LG-MS/MS. A total of 21 sulfonamides were separated by a C15 column (2.1 mm x 100 mm, 1.7 µm) and analyzed under positive ion mode with multi-reaction monitoring. The matrix-matched external standard calibration was used for quantification. RESULTS: The method quantification limits for 21 analytes were 0.03-0.63 ng/L with overall recoveries of 50.1%-114.9%, and the relative standard deviations less than 20%. The method was finally used to analyze sulfonamides in drinking water in Beijing, and 5 target compounds (sulfadiazine, sulfathiazole, sulfapyridine, trimethoprim and sulfamethazine) were detected at a concentration range of 0.08-32.54 ng/L. CONCLUSION: This method could be applied in simultaneous analysis of sulfonamides and their metabolites in drinking water samples.

SIM2: Its Prognostic Significance and Oncogenic Role in Endometrial Carcinoma.

Background: Endometrial carcinoma ranks as the second most widespread malignancy affecting the reproductive system in females. Effective prognostic biomarkers are required to further improve survival rates for patients. Single-minded homolog 2 (SIM2) is known to participate in neurogenesis as a transcription factor. However, the potential role of SIM2 in endometrial carcinoma remains elusive. Methods: Multiple public databases, including TIMER2.0, GEIPA2, UALCAN, LinkedOmics, BioGRID, DAVID and cBioPortal, were used to investigate SIM2 mRNA expression, SIM2-associated genes, PPI network, functional enrichment analysis, SIM2 gene alterations and methylation. The association between SIM2 expression and immune cell infiltrates was explored using GSVA. The effects of gene alterations and methylation on patient survival and CD8+T infiltration were examined using GSCA. Moreover, the prognostic potential of SIM2 was evaluated using COX regression, ROC curves and a nomogram model. Finally, the differential expression and function of SIM2 in UCEC were explored using qPCR, WB, CCK8 and Transwell assays. Results: Our findings revealed the heightened expression of SIM2 in endometrial carcinoma, and that its DNA methylation and CNV alterations were correlated with immune infiltration and patients' prognosis. Additionally, functional enrichment revealed the involvement of SIM2 in transcription regulation and signal transduction. Moreover, we performed cell-based experiments to corroborate the oncogenic function of SIM2 in facilitating cell proliferation, migration and invasion. Conclusion: Collectively, these results suggest that SIM2 holds promise as both a potential prognostic indicator and a viable treatment target for endometrial carcinoma.

Bruceine A inhibited breast cancer proliferation and metastasis by inducing autophagy via targeting PI3K-AKT signaling pathway.

Although there have been significant advances in cancer treatment, the urgent need to inhibit breast cancer metastasis remained unmet. Bruceine A (BA) is a natural compound extracted from Bruceae Fructus and has long been recognized to have antitumor effects with high safety and biocompatibility. However, the mechanisms and/or targets of BA for metastatic breast cancer treatment are still not fully elucidated. In this study, we systematically investigated the effects of BA on inhibition of breast cancer metastasis and its underlying mechanisms. We found that, in addition to its cytotoxic effects, BA significantly inhibited the invasion and migration capabilities of two types of breast cancer cell lines (MDA-MB-231 and MCF-7) while concurrently promoting apoptosis in these cells. Further mechanistic studies revealed that, by targeting the canonical PI3K-AKT signaling pathway, BA initiated autophagy of both types of breast cancer cell lines in vitro. In vivo results further confirmed the in vitro findings, manifested by shrinkage of size and weight of breast tumor as well as initiation of autophagy (indicated by upregulation of LC3I/II) through targeting PI3K-AKT pathway on mice model. These data collectively demonstrated the potential of BA in antimetastasis of breast cancer cells, suggesting its future clinical transformation in metastatic breast cancer therapy.

Unified and distinct cognitive control deficits in adolescents with cognitive disengagement syndrome and learning burnout.

Introduction: Cognitive disengagement syndrome (CDS) is a psychological disorder characterized by daydreaming, mental fogginess, and slow thinking, while learning burnout (LB) is characterized by a passive and inattentive attitude toward learning. These two disorders are closely related but can be challenging to differentiate from one another. The present study aimed to identify shared and distinct cognitive control deficits between CDS and LB. Methods: We recruited 136 adolescents (aged 14 to 17 years) from an initial screening of CDS and LB (N = 476) and divided them into four groups: CDS, LB, CDS + LB, and typically developing control. After a second screening, 129 adolescents completed two tasks to assess their attentional networks and cognitive control capacity (CCC). Results: Adolescents with high CDS symptoms (both CDS group and CDS+LB group) exhibited impaired disengaging effect of attention and lower CCC, indicating deficits in orienting attention and the upper limit of information processing for cognitive control specifically. Furthermore, support vector machine modeling identified CCC as the most significant parameter differentiating the CDS and LB groups. Discussion: Our findings suggest that while adolescents with high CDS and high LB symptoms have similar outward manifestations in the adolescent's school life, deficits in attention and cognitive control, particularly in the CCC, may distinguish between the two groups.

A retrospective study of female reproductive parameters in the Kunming dog.

The Kunming dog is the first and only working dog breed from China to be recognized worldwide. As a domestic working dog, its excellent working performance has been well established; however, its normal reproductive parameters are not well understood. Therefore, this study was conducted to document the main reproductive parameters of this purebred working dog in field breeding conditions. Data on 1004 heats (753 with mating) from 203 bitches between 2008 to 2014, were collected and analyzed. The pregnancy rate and whelping rate was 79.42% and 75.30%, respectively. Finally, for 567 litters (4298 puppies), the mean litter size was 7.19 ± 0.12 puppies (range 1-15). The mean gestation period and birth weight were approximately 61.64 ± 0.10 days and 407.25 ± 1.21 g. The mean sex ratio was 1.03 males to 1.00 female. Estrus occurred throughout the year with no significant differences between seasons and months (P > 0.05), which confirms that Kunming dogs are non-seasonal breeders; births occurred in every month of the year. Pregnant bitches exhibited significantly longer inter-estrus intervals than non-pregnant bitches (220.85 ± 2.05 vs. 180.19 ± 2.94 days, P < 0.05). Bitch parity influenced litter size, and the gestation length and birth weight of the puppies were negatively affected by litter size. This study helps elucidate the reproductive potential of this breed and provides reference values for reproductive performance in the Kunming dog.

Birth of puppies of predetermined sex after artificial insemination with a low number of sex-sorted, frozen-thawed spermatozoa in field conditions.

The aim of this study was to evaluate fertility and sex ratios after artificial insemination in dogs under field conditions. Semen was cryopreserved as unsorted (control) or was separated into X- and Y-chromosome-bearing sperm using a cell sorter. Sixty female dogs were inseminated with frozen-thawed spermatozoa of 100 × 106 unsorted (a dose in practice) and 4 × 106 sorted (X and Y group, respectively). A total of 20 dogs became pregnant and 126 puppies were born from the three groups. The percentage of parturition was similar for the X (5/20; 25.0%) and Y (4/20; 20.0%) group (P > 0.05), but lower than controls (11/20; 55.0%) (P < 0.05). Ultimately 28 out of the 32 puppies produced from X group were female (87.5%) and 19/22 (86.4%) puppies of Y group were male. In contrast, sex ratio (51.4% to 48.6%) in the control was significantly different from the X, Y group (P < 0.05). However, male and female puppies in the control had similar birth weights and weaning weights to those from the X and Y groups. This preliminary information indicated that normal puppies of predicted sex can be produced with low numbers of sorted cryopreserved dog spermatozoa at a farm level, making sperm-sexing technology potentially applicable for elite breeding units.

Multiplacenta derived stem cell/cytokine treatment increases survival time in a mouse model with radiation-induced bone marrow damage.

Nuclear Warfare and nuclear leakage can result in a large number of patients with radiation-induced bone marrow damage. Based on the fact that hematopoietic stem cells and hematopoietic growth factors are characterized as a novel strategy for therapy, the aim of this study was to explore a safe and routine stem cell/cytokine therapeutic strategy. Allogeneic multiplacenta derived hematopoietic and mesenchymal stem cells/cytokines were intraperitoneally injected into a moderate dose of total body irradiation-induced mouse bone marrow damage model a single time. Then, the mouse posttransplantation survival time, peripheral blood hemoglobin count, bone marrow architecture, and donor cell engraftment were assessed. Each mouse that received placenta-derived stem cells exhibited positive donor hematopoietic and mesenchymal stem cell engraftment both in the bone marrow and peripheral blood after transplantation. The peripheral blood hemoglobin count and survival time were greater in the group with the combined treatment of multiplacenta-derived stem cells and cytokines, compared with model-only controls (both P < 0.001). The blood smear mesenchymal/hematopoietic stem cell count was significantly higher in the combined treatment group than in the mice treated only with placenta-derived cells (28.08 ± 5.824 vs. 20.40 ± 5.989, P < 0.001; 7.74 ± 2.153 vs. 4.23 ± 1.608, P < 0.001, respectively). However, there was no marked change on the bone marrow pathology of any of the experimental mice after the transplantation. These results indicate that for radiation-induced bone marrow damage treatment, multiplacenta-derived stem cells and cytokines can increase the life span of model mice and delay but not abrogate the disease progression. Intraperitoneally transplanted stem cells can survive and engraft into the host body through the blood circulation. Improvement of peripheral blood hemoglobin levels, but not the bone marrow architecture response, probably explains the increase in survival time observed in this study.