PHF12 regulates HDAC1 to promote tumorigenesis via EGFR/AKT signaling pathway in non-small cell lung cancer.

BACKGROUND: Lung cancer stands as the second most prevalent malignant neoplasm worldwide. Addressing the underlying mechanisms propelling the progression of non-small cell lung cancer is of paramount importance. In this study, we have elucidated the pivotal role of PHF12 in this context. MATERIALS AND METHODS: We harnessed clinical lung cancer tissue samples and non-small cell lung cancer cell lines to discern the expression pattern of PHF12. In vitro assays probing cell proliferation were conducted to substantiate the functional impact of PHF12. Furthermore, an in vivo Xenograft model was employed to dissect the role of PHF12. Employing ChIP assays and qRT-PCR, we delved into the intricate binding dynamics between PHF12 and HDAC1. Mechanistic insights into the PHF12-HDAC1 axis in lung cancer progression were pursued via RNA-seq and GSEA analyses. RESULTS: Notably, PHF12 exhibited a substantial upregulation within tumor tissue, concomitant with its correlation to HDAC1. The trilogy of cell proliferation assays, transwell assays, and the Xenograft model collectively underscored the promoting influence of PHF12 on lung cancer proliferation, both in vitro and in vivo. The ChIP assay unveiled the transcriptional regulatory role of PHF12 in governing HDAC1 expression. This correlation extended to both mRNA and protein levels. PHF12 promotes NSCLC progression through regulating HDCA1 expression. Intriguingly, the rescue of function within NSCLC cell lines post PHF12 knockdown was achievable through HDAC1 overexpression. Additionally, our findings unveiled the capacity of the PHF12-HDAC1 axis to activate the EGFR/AKT signaling pathway, thereby further corroborating its significance in lung cancer progression. CONCLUSION: Our study identified PHF12 as an oncogenic role in lung cancer proliferation and migration for the first time. PHF12 transcriptionally regulate HDAC1 and activate EGFR/AKT signaling pathway in NSCLC progression. PHF12 may serve as an important target in lung cancer therapy.

Population-based BRCA germline mutation screening in the Han Chinese identifies individuals at risk of BRCA mutation-related cancer: experience from a clinical diagnostic center from greater Shanghai area.

BACKGROUND: Deleterious BRCA1/2 (BRCA) mutation raises the risk for BRCA mutation-related malignancies, including breast, ovarian, prostate, and pancreatic cancer. Germline variation of BRCA exhibits substantial ethnical diversity. However, there is limited research on the Chinese Han population, constraining the development of strategies for BRCA mutation screening in this large ethnic group. METHODS: We profile the BRCA mutational spectrum, including single nucleotide variation, insertion/deletion, and large genomic rearrangements in 2,080 apparently healthy Chinese Han individuals and 522 patients with BRCA mutation-related cancer, to determine the BRCA genetic background of the Chinese Han population, especially of the East Han. Incident cancer events were monitored in 1,005 participants from the healthy group, comprising 11 BRCA pathogenic/likely pathogenic (PLP) variant carriers and 994 PLP-free individuals, including 3 LGR carriers. RESULTS: Healthy Chinese Han individuals demonstrated a distinct BRCA mutational spectrum compared to cancer patients, with a 0.53% (1 in 189) prevalence of pathogenic/likely pathogenic (PLP) variant, alongside a 3 in 2,080 occurrence of LGR. BRCA1 c. 5470_5477del demonstrated high prevalence (0.44%) in the North Han Chinese and penetrance for breast cancer. None of the 3 LGR carriers developed cancer during the follow-up. We calculated a relative risk of 135.55 (95% CI 25.07 to 732.88) for the development of BRCA mutation-related cancers in the BRCA PLP variant carriers (mean age 42.91 years, median follow-up 10 months) compared to PLP-free individuals (mean age 48.47 years, median follow-up 16 months). CONCLUSION: The unique BRCA mutational profile in the Chinese Han highlights the potential for standardized population-based BRCA variant screening to enhance BRCA mutation-related cancer prevention and treatment.

l-Proline and GelMA hydrogel complex：An efficient antifreeze system for cell cryopreservation.

Cryopreservation of biological samples is an important technology for expanding their applications in the biomedical field. However, the quality and functionality of samples after rewarming are limited by the toxicity of commonly used cryoprotectant agents (CPAs). Here, we developed a novel preservation system by combining the natural amino acid l-proline (L-Pro) with gelatin methacryloyl (GelMA) hydrogels. Compared with dimethyl sulfoxide (DMSO), L-Pro and GelMA demonstrated excellent biocompatibility when co-culturing with cells. Cryopreservation procedures were optimized using 3T3 as model cells. The results showed that rapid cooling was the most suitable cooling procedure for L-Pro and GelMA among the three cooling procedures. Co-culturing with cells for 3 h before cryopreservation, 6 % L-Pro +7 % GelMA had the highest survival rate, reaching up to 80 %. Differential Scanning Calorimetry (DSC) analysis showed that 6 % L-Pro + 7 % GelMA lowered the freezing point of the solution to -4.2 °C and increased the unfrozen water content to 20 %. To the best of our knowledge, this is the first report of cell cryopreservation using a combination of L-Pro and GelMA hydrogels, which provides a new strategy for improving cell cryopreservation.

Genetic Analysis Reveals Relationships Among Populations of Puccinia striiformis f. sp. tritici from Shaanxi-Gansu-Ningxia-Xinjiang of Northwestern and Sichuan-Yunnan of Southwestern China.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases worldwide. In China, wheat stripe rust generally occurs in the northwestern and southwestern regions; however, the genetic relationships of Pst populations between these regions are largely unclear. To determine the population structure and potential migration route in these regions, 235 isolates collected from Xinjiang (XJ), Gansu (GS), Ningxia (NX), Shaanxi (SX), Sichuan (SC), and Yunnan (YN) provinces in 2021 and 2022 were phenotyped using two sets of Pst differentials and genotyped using 20 competitive allele-specific PCR-single nucleotide polymorphism (KASP-SNP) markers. The phenotype tests indicated that CYR34, CYR32, and CYR33 were the predominant races with different occurrence frequencies in different regions and years. Genotypic analysis revealed that a total of 183 multilocus genotypes were identified, and the genetic diversity in the YN subpopulation was the highest. The genetic background in the SX subpopulation was similar to that in the GS and NX subpopulations, and the genetic background in the YN subpopulation was similar to that in the SC and SX subpopulations. A high level of gene flow (Nm) was found between the SX and GS, SX and NX, GS and NX, and SC and YN subpopulations, suggesting the migration of Pst among these regions, while a small amount of Nm existed between the SX and SC subpopulations. SC may serve as a bridge connecting Pst subpopulations between the northwestern provinces (SX, GS, and NX) and the southwestern provinces (SC and YN). With a relatively high genetic distance and low Nm values compared with other Pst subpopulations, XJ is considered a relatively independent epidemiological region in China. These results improved our current understanding of the wheat stripe rust epidemic in northwestern and southwestern regions of China.

Terahertz Sensing of L-Valine and L-Phenylalanine Solutions.

To detect and differentiate two essential amino acids (L-Valine and L-Phenylalanine) in the human body, a novel asymmetrically folded dual-aperture metal ring terahertz metasurface sensor was designed. A solvent mixture of water and glycerol with a volume ratio of 2:8 was proposed to reduce the absorption of terahertz waves by reducing the water content. A sample chamber with a controlled liquid thickness of 15 µm was fabricated. And a terahertz time-domain spectroscopy (THz-TDS) system, which is capable of horizontally positioning the samples, was assembled. The results of the sensing test revealed that as the concentration of valine solution varied from 0 to 20 mmol/L, the sensing resonance peak shifted from 1.39 THz to 1.58 THz with a concentration sensitivity of 9.98 GHz/mmol∗L-1. The resonance peak shift phenomenon in phenylalanine solution was less apparent. It is assumed that the coupling enhancement between the absorption peak position of solutes in the solution and the sensing peak position amplified the terahertz localized electric field resonance, which resulted in the increase in frequency shift. Therefore, it could be shown that the sensor has capabilities in performing the marker sensing detection of L-Valine.

Review of lipocalin-2-mediated effects in diabetic retinopathy.

BACKGROUND: Studies have uncovered LCN2 as a marker of inflammation strongly related to obesity, insulin resistance, and abnormal glucose metabolism in humans, and is involved in vascular diseases, inflammatory diseases, and neurological diseases. In recent years, studies have shown that elevated levels of LCN2 have a strong association with diabetic retinopathy (DR), but the pathogenesis is unknown. Here, we reviewed the relevant literature and compiled the pathogenesis associated with LCN2-induced DR. METHODS: We searched PubMed and Web of Science electronic databases using "lipocalin-2, diabetic retinopathy, retinal degeneration, diabetic microangiopathies, diabetic neuropathy and inflammation" as subject terms. RESULTS: In diabetic retinal neuropathy, LCN2 causes impaired retinal photoreceptor function and retinal neurons; in retinal microangiopathy, LCN2 induces apoptosis of retinal vascular endothelial cells and promotes angiogenesis; in retinal inflammation, increased secretion of LCN2 recruits inflammatory cells and induces pro-inflammatory cytokines. Moreover, LCN2 has the potential as a biomarker for DR. Recent studies have shown that retinal damage can be attenuated by silencing LCN2, which may be associated with the inhibition of caspase-1-mediated pyroptosis, and LCN2 may be a new target for the treatment of DR. CONCLUSIONS: In conclusion, LCN2, involved in the development of diabetic retinopathy, is a key factor in diabetic retinal microangiopathy, neurodegeneration, and retinal inflammation. LCN2 is likely to be a novel molecular target leading to DR, and a more in-depth study of the pathogenesis of DR caused by LCN2 may provide considerable benefits for clinical research and potential drug development.

The crystallization properties of antifreeze GelMA hydrogel and its application in cryopreservation of tissue-engineered skin constructs.

Gelatin methacrylate (GelMA) hydrogels are expected to be ideal skin tissue engineering dressings for a wide range of clinical treatments. Herein, we report the preparation of GelMA or antifreeze GelMA hydrogel sheets with different GelMA concentrations, crosslinking times, and cryoprotectant (CPA) concentrations. The crystallization properties of GelMA or antifreeze GelMA hydrogel sheets were studied by cryomicroscopy and differential scanning calorimetry (DSC). It was found that the growth of ice crystals was slower when GelMA hydrogel concentration was more than 7%. The 10% DMSO-7% GelMA hydrogel sheets crosslinked for 60 min showed no ice crystal formation and growth during cooling and warming. The DSC results showed that the vitrification temperature of the 10% DMSO-7% GelMA hydrogel sheet was -111°C. Furthermore, slow freezing and rapid freezing of fibroblast-laden GelMA or antifreeze GelMA hydrogel sheets, and tissue-engineered skin constructs were studied. The results showed no significant difference in cell survival between slow (88.8% ± 1.51) and rapid (89.2% ± 3.00) freezing of fibroblast-loaded 10% DMSO-7% GelMA hydrogel sheets, and significantly higher than that of 7% GelMA hydrogel sheets (33.4% ± 5.46). The cell viability was higher in tissue-engineered skin constructs after slow freezing (86.34% ± 1.45) than rapid freezing (72.74% ± 1.34). We believe that the combination of antifreeze hydrogels and tissue engineering will facilitate the cryopreservation of tissue engineering constructs.

Visualization of Ice Crystal Behavior in Mouse Oocytes During High-Speed Quench Cooling and Ice Inhibition by Antifreezing Hydrogels.

Oocyte vitrification has become a widely adopted method in clinical practice. However, the solidification behavior and its impact on oocytes during the ultrarapid cooling process remain poorly understood. In this study, we established a system and methodology to observe crystallization behavior in oocytes during quench cooling and warming. Subsequently, the threshold concentration of cryoprotective agents (CPAs) required for oocyte vitrification was determined through a visualization method. The results demonstrated that the ice front could not be observed in the image sequence when using 16.5% DMSO +16.5% EG during high-speed quench cooling (2821.58°C/min). Finally, oocytes were encapsulated with an antifreezing hydrogel (7.5% EG +7.5% DMSO +0.5% alginate) and subjected to high-speed quench cooling. No ice crystals appeared in the antifreezing hydrogel-encapsulated oocytes at a low concentration of osmotic CPA (2.4 M). This research opens up new possibilities for oocyte vitrification with a reduced concentration of CPA.

[Determination of adenosine content in heart tissue by ultra performance liquid chromatography-triple quadrupole mass spectrometry].

A method based on ultra performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS) was developed and validated for the rapid and accurate determination of adenosine (Ado) in cardiac tissues with high sensitivity and specificity. The samples were dissolved in 1 mL of ultrapure water containing 10 µmol/L 2-hydroxy-3-nonyladenine hydrochloride (EHNA) as a stabilizer, ground at low temperature for 2 min, and then ultrasonically extracted at 60 Hz in an ice-water bath for 40 min. Methanol and 5 mmol/L ammonium acetate solution were used as the mobile phases under a flow rate of 0.4 mL/min, a column temperature of 40 ℃ and an injection volume of 3 µL. The Ado in cardiac tissue was qualitatively and quantitatively analyzed by electrospray ionization (ESI) positive-ion-switching in multiple reaction monitoring (MRM) mode. A solvent standard curve and the external standard method were used for the accurate quantification of Ado. The results showed that the matrix effect of Ado in cardiac tissue was very low. A good linear relationship was obtained in the range of 0.1-160 ng/mL, and the correlation coefficient (r2) was 0.9930. The limits of detection (LOD) and quantification (LOQ) were 0.03 and 0.1 ng/mL, respectively. The spiked recoveries of Ado in murine cardiac tissue were 113.6%, 96.3%, and 102.9% at three spiked levels of low, medium, and high, respectively. The intra-day repeatability (RSDs) were 1.7%-8.4%, and the inter-day reproducibility (RSDs) were 2.6%-7.4%. Based on the correlation and consistency results, a positive bias was observed between the proposed UPLC-MS/MS method and the double-antibody sandwich method. Moreover, the Ado contents detected by these two methods were significantly positively correlated (P<0.0001). Cardiac tissue samples were collected from 17 mice and 17 rats and detected in our laboratory. The content ranges of Ado in the cardiac tissues of mice and rats determined by the developed UPLC-MS/MS method were 3.25-8.78 mg/kg and 10.24-15.19 mg/kg, respectively (average adenosine contents: 5.37 and 12.60 mg/kg, respectively). The developed method is simple, accurate, sensitive, and it is suitable for the determination of Ado in cardiac tissues. It also provides important technical support for cardiac clinical research and disease diagnosis.

[Fertility preservation through natural and artificial ovaries: a review].

Ovarian tissue cryopreservation (OTC) is currently the exclusive choice for preserving fertility in both young girls before reaching puberty and young women who require immediate chemotherapy. Ovarian tissue transplantation has proven to be effective in restoring hormonal cycles and fertility. However, in certain cancer cases, there is a potential risk of inadvertently reintroducing malignant cells when transplanting cryopreserved ovarian tissue. Therefore, the use of an artificial ovary as an innovative and complementary approach allows for the development of isolated follicles, facilitates oocyte maturation and ovulation, and can partially restore endocrine function. This paper presents a comprehensive overview of techniques used to preserve fertility in natural ovarian tissues, including slow freezing, vitrification and hydrogel encapsulation methods. Additionally, it reviews fertility preservation techniques for artificial ovarian tissues, such as strategies involving hydrogel-encapsulated follicle, scaffolding for constructing ovarian microtissues, and 3D printing engineering. Lastly, this article explores current challenges and difficulties encountered in preserving ovarian tissue fertility, while also anticipating future trends in development, making it a valuable reference for the implementation of ovarian tissue fertility preservation.

Application of photocrosslinked gelatin, alginate and dextran hydrogels in the in vitro culture of testicular tissue.

Testicular tissue culture in vitro is considered an important tool for the study of spermatogenesis and the treatment of male infertility. Although agarose hydrogel is commonly used in testicular tissue culture, the efficiency of spermatogenesis in vitro is limited. In this study, testicular tissues from adult mice were cultured using a gas-liquid interphase method based on agarose (Agarose), gelatin methacryloyl (GelMA), alginate methacryloyl (AlgMA), dextran methacryloyl (DexMA), and mixture GelMA-Agarose, AlgMA-Agarose, and DexMA-Agarose hydrogels, respectively, for 32 days in vitro. The integrity of the seminiferous tubules, the density and proportions of spermatogonia, spermatocytes, Sertoli cells, and testosterone concentrations were quantified and compared between groups. Properties of different hydrogels including compression modulus, Fourier Infrared Spectroscopy (FITR) spectra, pore size, water absorption, and water retention were tested to investigate how biochemical and physical properties of hydrogels affect the results of testicular tissue culture. The results indicate that testicular tissues cultured on AlgMA exhibited the highest seminiferous tubule integrity rate (0.835 ± 0.021), the presence of a high density of spermatocytes (2107.627 ± 232.082/mm2), and a high proportion of SOX9-positive well-preserved seminiferous tubules (0.473 ± 0.047) compared to all remaining experimental groups on day 32. This may be due to the high water content of AlgMA reducing the toxic effect of oxygen on testicular tissue. In the later period of culture, testicular tissues cultured on DexMA, not DexMA-Agarose, produced significantly more testosterone (18.093 ± 3.302 ng/mL) than the other groups, suggesting that DexMA is friendly to Leydig cells. Our study provides a new idea for the optimization of the gas-liquid interphase method for achieving in vitro spermatogenesis, facilitating the future achievement of efficient in vitro spermatogenesis in more species, including humans.

Fine mapping of QYrsv.swust-1BL for resistance to stripe rust in durum wheat Svevo.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a serious disease that affects wheat worldwide. There is a great need to develop cultivars with combinations of all-stage resistance (ASR) and adult-plant resistance (APR) genes for sustainable control of the disease. QYrsv.swust-1BL in the Italian durum wheat (Triticum turgidum ssp. durum) cultivar Svevo is effective against Pst races in China and Israel, and the gene has been previously mapped to the long arm of chromosome 1B. The gene is flanked by SNP (single nucleotide polymorphism) markers IWB5732 and IWB4839 (0.75 cM). In the present study, we used high-density 660K SNP array genotyping and the phenotypes of 137 recombinant inbred lines (RILs) to fine map the QYrsv.swust-1BL locus within a 1.066 Mb region in durum wheat Svevo (RefSeq Rel. 1.0) on chromosome arm 1BL. The identified 1.066 Mb region overlaps with a previously described map of Yr29/QYr.ucw-1BL, a stripe rust APR gene. Twenty-five candidate genes for QYrsv.swut-1BL were identified through comparing polymorphic genes within the 1.066 Mb region in the resistant cultivar. SNP markers were selected and converted to Kompetitive allele-specific polymerase chain reaction (KASP) markers. Five KASP markers based on SNP were validated in a F2 and F2:3 breeding population, providing further compelling evidence for the significant effects of QYrsv.swut-1BL. These markers should be useful in marker-assisted selection for incorporating Yr29/QYrsv.swust-1BL into new durum and common wheat cultivars for resistance to stripe rust.

Possible immune mechanisms of gut microbiota and its metabolites in the occurrence and development of immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by increased platelet destruction and impaired production, leading to an elevated bleeding tendency. Recent studies have demonstrated an important link between the gut microbiota and the onset and progression of several immune diseases in humans, emphasizing that gut microbiota-derived metabolites play a non-negligible role in autoimmune diseases. The gut microbiota and its metabolites, such as short-chain fatty acids, oxidized trimethylamine, tryptophan metabolites, secondary bile acids and lipopolysaccharides, can alter intestinal barrier permeability by modulating immune cell differentiation and cytokine secretion, which in turn affects the systemic immune function of the host. It is therefore reasonable to hypothesize that ecological dysregulation of the gut microbiota may be an entirely new factor in the triggering of ITP. This article reviews the potential immune-related mechanisms of the gut microbiota and representative metabolites in ITP, as well as the important influence of leaky gut on the development of ITP, with a view to enriching the theoretical system of ITP-related gut microecology and providing new ideas for the study of ITP.

Efficacy and safety of immune checkpoint inhibitors for advanced non-small cell lung cancer with leptomeningeal metastases harboring targetable mutations.

Background: Driver gene-positive non-small cell lung cancer (NSCLC) patients are prone to develop leptomeningeal metastasis (LM), leading to an extremely high mortality. The objective of this study was to assess the efficacy and safety of immune checkpoint inhibitors (ICIs) treatments for patients with NSCLC and LM harboring targetable mutations. Methods: We retrospectively collected records of patients with NSCLC harboring targetable mutations and prescribed ICIs following the diagnosis of LM at Huashan Hospital, Fudan University. In addition, we reviewed relevant literature and enrolled patients who met the inclusion criteria. Clinical characteristics were statistically analyzed, and the Kaplan-Meier method and log-rank test were employed to assess the median progression-free survival (mPFS) and median overall survival (mOS). Results: A total of 37 patients with NSCLC harboring targetable mutations who received ICIs after LM diagnosis were included. The median age of the enrolled patients was 54 years (range, 33-70 years), and 62.2% were female. Following ICI administration, the intracranial objective response rate (iORR) and intracranial disease control rate (iDCR) for all enrolled patients were 18.9% and 62.2%, respectively. The mPFS of all patients was 2.5 months [95% confidence interval (CI): 2.166-2.834 months] and the mOS was 5.8 months (95% CI: 5.087-6.513 months). Both univariate and multivariate analyses revealed a significant increase in mOS or individuals who had previously undergone cranial radiation therapy compared to those who had not. Furthermore, different histology molecular types were found to be potentially associated with survival time. Conclusions: Some patients with NSCLC harboring targetable gene mutations following LM diagnosis may benefit from ICI treatment with relatively good tolerance. However, further screening of the most suitable patient populations for ICIs is required.

Metabolic engineering of an industrial bacterium Zymomonas mobilis for anaerobic l-serine production.

Due to the complicated metabolic and regulatory networks of l-serine biosynthesis and degradation, microbial cell factories for l-serine production using non-model microorganisms have not been reported. In this study, a combination of synthetic biology and process optimization were applied in an ethanologenic bacterium Zymomonas mobilis for l-serine production. By blocking the degradation pathway while introducing an exporter EceamA from E. coli, l-serine titer in recombinant Z. mobilis was increased from 15.30 mg/L to 62.67 mg/L. It was further increased to 260.33 mg/L after enhancing the l-serine biosynthesis pathway. Then, 536.70 mg/L l-serine was achieved by removing feedback inhibition with a SerA mutant, and an elevated titer of 687.67 mg/L was further obtained through increasing serB copies while enhancing the precursors. Finally, 855.66 mg/L l-serine can be accumulated with the supplementation of the glutamate precursor. This work thus not only constructed an l-serine producer to help understand the bottlenecks limiting l-serine production in Z. mobilis for further improvement, but also provides guidance on engineering non-model microbes to produce biochemicals with complicated pathways such as amino acids or terpenoids.

Co-contamination and interactions of multiple mycotoxins and heavy metals in rice, maize, soybeans, and wheat flour marketed in Shanghai City.

Mycotoxins and heavy metals extensively contaminate grains and grain products, posing severe health risks. This work implements validated ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) methods to quantify the concentration of 12 mycotoxins and five heavy metals in rice, maize, soybeans, and wheat flour samples marketed in Shanghai. The mixed contamination characteristics were analyzed using correlation cluster analysis and co-contamination index, and the probabilities of all cross combinations of contaminations were analyzed using a self-designed JAVA language program. The results showed that grains and grain products were frequently contaminated with both mycotoxins and heavy metals, mostly with deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3-ADON), 15-acetyl-deoxynivalenol (15-ADON), ochratoxin A (OTA), aflatoxins, fumonisin B1 (FB1), fumonisin B2 (FB2), fumonisin B3 (FB3), arsenic (As), chromium (Cr) and cadmium (Cd). All the samples (100 %) were contaminated with two or more contaminants, and 77.3 % of the samples were co-contaminated with more than four contaminants. In cereals and cereal products, the following combinations were closely associated: (FB3 +3-ADON), (FB1 +As), (FB1 +FB2), (DON+FB1), (DON+Cd), (As+Cd), (DON+Cd+As), (FB1 +FB2 +As), and (DON+3-ADON+15-ADON). The results indicated that mycotoxins and heavy metals frequently co-occurred in Shanghai grains and grain products, and they provided primary data for safety assessments, early warnings, and regulatory measures on these contaminants to protect public health.

Prenatal prednisone exposure impacts liver development and function in fetal mice and its characteristics.

Prednisone, a widely used glucocorticoid drug in human and veterinary medicine, has been reported to cause developmental toxicity. However, systematic studies about the effect of prednisone on fetal liver development are still unclear. We investigated the potential effects of maternal exposure to clinically equivalent doses of prednisone during different gestational stages on cell proliferation and apoptosis, cell differentiation, glucose and lipid metabolism, and hematopoiesis in the liver of fetal mice, and explored the potential mechanisms. Results showed that prenatal prednisone exposure (PPE) could suppress cell proliferation, inhibit hepatocyte differentiation, and promote cholangiocyte differentiation in the fetal liver. Meanwhile, PPE could result in the enhancement of glyconeogenesis and bile acid synthesis and the inhibition of fatty acid ß-oxidation and hematopoiesis in the fetal liver. Further analysis found that PPE-induced alterations in liver development had obvious stage and sex differences. Overall, the alteration in fetal liver development and function induced by PPE was most pronounced during the whole pregnancy (GD0-18), and the males were relatively more affected than the females. Additionally, fetal hepatic insulin-like growth factor 1 (IGF1) signaling pathway was inhibited by PPE. In conclusion, PPE could impact fetal liver development and multiple functions, and these alterations might be partially related to the inhibition of IGF1 signaling pathway.

Generation of mouse testicular organoids with highly compartmentalized tubular lumen structure and their cryopreservation.

Testicular organoids have great potential for maintaining male fertility and even restoring male infertility. However, existing studies on generating organoids with testis-specific structure and function are scarce and come with many limitations. Research on cryopreservation of testicular organoids is even more limited, and inappropriate cryopreservation methods may result in the loss of properties in resuscitated or regenerated organoids, rendering them unsuitable for clinical or research needs. In this paper, we investigated the effects of mouse age and cell number on the self-aggregation of testicular cells into spheres in low-adsorption plates. Various media compositions, culture systems, and cell numbers were used to culture cell spheres for 14 days to form testicular organoids, and the self-organization of the organoids was assessed by histological and immunofluorescence staining. We determined the appropriate cryopreservation conditions for testicular cells, cell spheres, and tissues. Subsequently, organoids derived from cryopreserved testicular tissues, testicular cells, and testicular cell spheres were compared and evaluated by histological and immunofluorescence staining. The results indicate that testicular cell spheres consisting of 30 × 104 testicular cells from 2-week-old mice were able to form organoids highly similar to the luminal structure and cell distribution of natural mouse testicular tissues. This transformation occurred over 14 days of incubation in α-MEM medium containing 10 % knockout serum replacer (KSR) using an agarose hydrogel culture system. Additionally, the Sertoli cells were tightly connected to form a blood-testis barrier. The relative rates of tubular area, germ cells, Sertoli cells, and peritubular myoid cells were 36.985 % ±â€¯0.695, 13.347 % ±â€¯3.102, 47.570 % ±â€¯0.379, and 27.406 % ±â€¯1.832, respectively. The optimal cryopreservation protocol for primary testicular cells involved slow freezing with a cryoprotectant consisting of α-MEM with 10 % dimethyl sulfoxide (DMSO). Slow freezing with cryoprotectants containing 5 % DMSO and 5 % ethylene glycol (EG) was optimal for all different volumes of testicular cell spheres. Compared to testicular organoids generated from frozen testicular tissue and cell spheres, freezing testicular cells proved most effective in maintaining organoid differentiation characteristics and cell-cell interactions. The findings of this study contribute to a "universal" testicular organoid in vitro culture protocol with promising applications for fertility preservation and restoration in prepubertal cancer patients and adult infertile patients.

Dexamethasone induces developmental axon damage in the offspring hippocampus by activating miR-210-3p/miR-362-5p to target the aberrant expression of Sonic Hedgehog.

Given the extensive application of dexamethasone in both clinical settings and the livestock industry, human exposure to this drug can occur through various sources and pathways. Prior research has indicated that prenatal exposure to dexamethasone (PDE) heightens the risk of cognitive and emotional disorders in offspring. Axonal development impairment is a frequent pathological underpinning for neuronal dysfunction in these disorders, yet it remains unclear if it plays a role in the neural damage induced by PDE in the offspring. Through RNA-seq and bioinformatics analysis, we found that various signaling pathways related to nervous system development, including axonal development, were altered in the hippocampus of PDE offspring. Among them, the Sonic Hedgehog (SHH) signaling pathway was the most significantly altered and crucial for axonal development. By using miRNA-seq and targeting miRNAs and glucocorticoid receptor (GR) expression, we identified miR-210-3p and miR-362-5p, which can target and suppress SHH expression. Their abnormal high expression was associated with GR activation in PDE fetal rats. Further testing of PDE offspring rats and infant peripheral blood samples exposed to dexamethasone in utero showed that SHH expression was significantly decreased in peripheral blood mononuclear cells (PBMCs) and was positively correlated with SHH expression in the hippocampus and the expression of the axonal development marker growth-associated protein-43. In summary, PDE-induced hippocampal GR-miR-210-3p/miR-362-5p-SHH signaling axis changes lead to axonal developmental damage. SHH expression in PBMCs may reflect axonal developmental damage in PDE offspring and could serve as a warning marker for fetal axonal developmental damage.

Hydrogel encapsulation facilitates a low-concentration cryoprotectant for cryopreservation of mouse testicular tissue.

Cryopreserved testicular tissue offers a promising method to restore fertility in male infertility patients. Current protocols rely on high concentrations of penetrating cryoprotectants (pCPAs), such as dimethyl sulfoxide (DMSO), which necessitating complex washing procedures and posing risks of toxicity. Hydrogel encapsulation presents a non-toxic alternative for cellular cryopreservation. This study investigates the effects of various types, concentrations, and thicknesses of hydrogel encapsulation on the cryopreservation of mouse testicular tissue. Testicular tissues loaded with varying concentrations of DMSO were encapsulated in alginate or gelatin-methacryloyl (GelMA) hydrogels. We evaluated hydrogels as potential CPAs to reduce pCPA concentrations and determine optimal combinations for cryopreservation. Post-cryopreservation, tissues were cultured using organ culture methods to assess spermatogenesis progression. Cryomicroscopy and differential scanning calorimetry (DSC) were used to examine ice crystal formation, melting enthalpy, and non-freezing water content in different hydrogels during cooling. Results indicate that 3 % alginate or 5 % GelMA hydrogel with thin encapsulation optimally preserves mouse testicular tissue. Using 20 % DMSO in 5 % GelMA thin encapsulation showed comparable apoptosis rates, improved morphology, higher mitochondrial activity, and enhanced antioxidant capacity compared to conventional 30 % DMSO without encapsulation. This suggests that hydrogel encapsulation reduces pCPA concentration by 10 %, thereby mitigating toxic damage. Hydrogel encapsulation can reduce basement membrane shrinkage of testicular tissue during cryopreservation. Moreover, frozen tissues remained viable with preserved germ cells after being cultured for one week on alginate methacryloyl (AlgMA) hydrogel using the gas-liquid interphase method. Cryomicroscopy and DSC studies confirmed the hydrogel's ability to inhibit ice crystal growth. In conclusion, this study introduces novel strategies for male fertility preservation and advances cryopreservation technology for clinical applications in assisted reproduction.