Essential roles of the nucleolus during early embryonic development: a regulatory hub for chromatin organization.

The nucleolus is the most prominent liquid droplet-like membrane-less organelle in mammalian cells. Unlike the nucleolus in terminally differentiated somatic cells, those in totipotent cells, such as murine zygotes or two-cell embryos, have a unique nucleolar structure known as nucleolus precursor bodies (NPBs). Previously, it was widely accepted that NPBs in zygotes are simply passive repositories of materials that will be gradually used to construct a fully functional nucleolus after zygotic genome activation (ZGA). However, recent research studies have challenged this simplistic view and demonstrated that functions of the NPBs go beyond ribosome biogenesis. In this review, we provide a snapshot of the functions of NPBs in zygotes and early two-cell embryos in mice. We propose that these membrane-less organelles function as a regulatory hub for chromatin organization. On the one hand, NPBs provide the structural platform for centric and pericentric chromatin remodelling. On the other hand, the dynamic changes in nucleolar structure control the release of the pioneer factors (i.e. double homeobox (Dux)). It appears that during transition from totipotency to pluripotency, decline of totipotency and initiation of fully functional nucleolus formation are not independent events but are interconnected. Consequently, it is reasonable to hypothesize that dissecting more unknown functions of NPBs may shed more light on the enigmas of early embryonic development and may ultimately provide novel approaches to improve reprogramming efficiency.

LncRNA SNHG1 acts as a ceRNA for miR-216a-3p to regulate TMBIM6 expression in esophageal squamous cell carcinoma.

Background: The long noncoding RNA small nucleolar RNA host gene 1 (SNHG1) has been demonstrated to play a crucial role in the progression of esophageal squamous cell carcinoma (ESCC). The current study aims to explore the deeper molecular mechanisms of SNHG1 in ESCC. Methods: Fifty patients with ESCC were enrolled to assess overall survival. Quantitative real-time PCR was performed to measure the levels of SNHG1, miR-216a-3p, and TMBIM6 in ESCC cells. Functional assessments of SNHG1 on ESCC cells were conducted using CCK-8 assay, flow cytometry, and Transwell assays. Western blot was conducted to detect the protein levels of TMBIM6 and proapoptotic proteins (Calpain and Caspase-12). The interaction among SNHG1, miR-216a-3p, and TMBIM6 was assessed with luciferase reporter assays. Results: Our study revealed that SNHG1 was notably increased in both clinical ESCC samples and cellular lines. Upregulation of SNHG1 in ESCC tissues was indicative of poor overall survival. Functionally, SNHG1 knockdown significantly inhibited the proliferation, migration, and invasion while promoting apoptosis in ESCC cells. Mechanistically, SNHG1 functioned as a competing endogenous RNA by sequestering miR-216a-3p to modulate TMBIM6 levels in ESCC cells. Notably, inhibiting miR-216a-3p or restoring TMBIM6 reversed the inhibitory effect induced by SNHG1 knockdown in ESCC cells. Conclusions: We demonstrate for the first time that SNHG1 may act as a competing endogenous RNA and promote ESCC progression through the miR-216a-3p/TMBIM6 axis. This highlights the potential of SNHG1 as a target for ESCC treatment.

A ratiometric fluorescent probe with dual-targeting capability for heat shock imaging.

HSO3- is an important reactive sulfur species that maintains the normal physiological activities of living organisms and participates in a variety of redox homeostatic processes. It has been found that changes in HSO3- levels is closely related to the heat stroke phenomenon of the organism. Heat stroke causes damage to normal cells, which in turn causes damage to the body and even death. It is crucial to accurately monitor and track the physiological behavior of HSO3- during heat stroke. Herein, a ratiometric multifunctional fluorescent probe DRM-SO2 with dual-targeting ability to rapidly and precisely recognize HSO3- being constructed based on the FRET mechanism. DRM-SO2 has extra Large Stokes shift (216 nm), very high sensitivity (DL = 12.2 nM), fast response time and good specificity. When DRM-SO2 undergoes Michael addition with HSO3-, the fluorescence emission peak was blue-shifted from 616 nm to 472 nm, and a clear ratiometric signal appeared. The interaction between lysosomes and mitochondria in maintaining cellular homeostasis was investigated by the dual-targeting ability of the probe using HSO3- as a mediator. DRM-SO2 achieved successful targeting and real-time monitoring of exogenous and endogenous HSO3- in the cells. More importantly, imaging experiments in heat stroke mice revealed high HSO3- expression in intestinal tissues. This provides new ideas and research tools for early prevention of heat stroke-induced diseases such as intestinal injuries. In addition, the semi-quantitative monitoring experiments for paper-based visualization of HSO3- make the probe promising for the design of portable detectors.

Steatotic liver disease induced by TCPOBOP-activated hepatic constitutive androstane receptor: primary and secondary gene responses with links to disease progression.

Constitutive Androstane Receptor (CAR, Nr1i3), a liver nuclear receptor and xenobiotic sensor, induces drug, steroid and lipid metabolizing enzymes, stimulates liver hypertrophy and hyperplasia, and ultimately, hepatocellular carcinogenesis. The mechanisms linking early CAR responses to later disease development are poorly understood. Here we show that exposure of CD-1 mice to TCPOBOP, a halogenated xenochemical and selective CAR agonist ligand, induces pericentral steatosis marked by hepatic accumulation of cholesterol and neutral lipid, and elevated circulating alanine aminotransferase, indicating hepatocyte damage. TCPOBOP-induced steatosis was weaker in the pericentral region but stronger in the periportal region in females compared to males. Early (1-day) TCPOBOP transcriptional responses were enriched for CAR-bound primary response genes, and for lipogenesis and xenobiotic metabolism and oxidative stress protection pathways; late (2-wk) TCPOBOP responses included many CAR binding-independent secondary response genes, with enrichment for macrophage activation, immune response and cytokine and reactive oxygen species production. Late upstream regulators specific to TCPOBOP-exposed male liver were linked to pro-inflammatory responses and hepatocellular carcinoma progression. TCPOBOP administered weekly to male mice using a high corn oil vehicle activated carbohydrate-responsive transcription factor (MLXIPL)-regulated target genes, dysregulated mitochondrial respiratory and translation regulatory pathways, and induced more advanced liver pathology. Overall, TCPOBOP exposure recapitulates histological and gene expression changes characteristic of emerging steatotic liver disease, including secondary gene responses in liver non-parenchymal cells indicative of transition to a more advanced disease state. Upstream regulators of both the early and late TCPOBOP response genes include novel biomarkers for foreign chemical-induced metabolic dysfunction-associated steatotic liver disease.

Phylogenomic profiles of whole-genome duplications in Poaceae and landscape of differential duplicate retention and losses among major Poaceae lineages.

Poaceae members shared a whole-genome duplication called rho. However, little is known about the evolutionary pattern of the rho-derived duplicates among Poaceae lineages and implications in adaptive evolution. Here we present phylogenomic/phylotranscriptomic analyses of 363 grasses covering all 12 subfamilies and report nine previously unknown whole-genome duplications. Furthermore, duplications from a single whole-genome duplication were mapped to multiple nodes on the species phylogeny; a whole-genome duplication was likely shared by woody bamboos with possible gene flow from herbaceous bamboos; and recent paralogues of a tetraploid Oryza are implicated in tolerance of seawater submergence. Moreover, rho duplicates showing differential retention among subfamilies include those with functions in environmental adaptations or morphogenesis, including ACOT for aquatic environments (Oryzoideae), CK2ß for cold responses (Pooideae), SPIRAL1 for rapid cell elongation (Bambusoideae), and PAI1 for drought/cold responses (Panicoideae). This study presents a Poaceae whole-genome duplication profile with evidence for multiple evolutionary mechanisms that contribute to gene retention and losses.

Developmentally dependent reprogramming of the Arabidopsis floral transcriptome under sufficient and limited water availability.

BACKGROUND: Environmental stresses negatively impact reproductive development and yield. Drought stress, in particular, has been examined during Arabidopsis reproductive development at morphological and transcriptomic levels. However, drought-responsive transcriptomic changes at different points in reproductive development remain unclear. Additionally, an investigation of the entire transcriptome at various stages during flower development is of great interest. RESULTS: Here, we treat Arabidopsis plants with well-watered and moderately and severely limiting water amounts when the first flowers reach maturity and generate RNA-seq datasets for early, middle, and late phases during flower development at 5, 6, and 7 days following treatment. Under different drought conditions, flowers in different developmental phases display differential sets of drought-responsive genes (DTGs), including those that are enriched in different GO functional categories, such as transcriptional regulation and response to stresses (early phase), lipid storage (middle phase), and pollen and seed development and metabolic processes (late phase). Some gene families have different members induced at different floral phases, suggesting that similar biochemical functions are carried out by distinct members. Developmentally-regulated genes (DVGs) with differential expression among the three floral phases belong to GO terms that are similar between water conditions, such as development and reproduction, metabolism and transport, and signaling and stress response. However, for different water conditions, such similar GO terms correspond to either distinct gene families or different members of a gene family, suggesting that drought affects the expression of distinct families or family members during reproductive development. A further comparison among transcriptomes of tissues collected on different days after treatment identifies differential gene expression, suggesting age-related genes (ARGs) might reflect the changes in the overall plant physiology in addition to drought response and development. CONCLUSION: Together, our study provides new insights into global transcriptome reprogramming and candidate genes for drought response, flower development, aging and coordination among these complex biological processes.

The Role of ß3-Adrenergic Receptors in Cold-Induced Beige Adipocyte Production in Pigs.

After exposure to cold stress, animals enhance the production of beige adipocytes and expedite thermogenesis, leading to improved metabolic health. Although brown adipose tissue in rodents is primarily induced by ß3-adrenergic receptor (ADRB3) stimulation, the activation of major ß-adrenergic receptors (ADRBs) in pigs has been a topic of debate. To address this, we developed overexpression vectors for ADRB1, ADRB2, and ADRB3 and silenced the expression of these receptors to observe their effects on the adipogenic differentiation stages of porcine preadipocytes. Our investigation revealed that cold stress triggers the transformation of subcutaneous white adipose tissue to beige adipose tissue in pigs by modulating adrenergic receptor levels. Meanwhile, we found that ADRB3 promotes the transformation of white adipocytes into beige adipocytes. Notably, ADRB3 enhances the expression of beige adipose tissue marker genes, consequently influencing cellular respiration and metabolism by regulating lipolysis and mitochondrial expression. Therefore, ADRB3 may serve as a pivotal gene in animal husbandry and contribute to the improvement of cold intolerance in piglets.

Comparative Study on the Immunogenicity and Efficacy of Different Post-exposure Intramuscular Rabies Vaccination Regimens in China.

Objective: This study aimed to compare the current Essen rabies post-exposure immunization schedule (0-3-7-14-28) in China and the simple 4-dose schedule (0-3-7-14) newly recommended by the World Health Organization in terms of their safety, efficacy, and protection. Methods: Mice were vaccinated according to different immunization schedules, and blood was collected for detection of rabies virus neutralizing antibodies (RVNAs) on days 14, 21, 28, 35, and 120 after the first immunization. Additionally, different groups of mice were injected with lethal doses of the CVS-11 virus on day 0, subjected to different rabies immunization schedules, and assessed for morbidity and death status. In a clinical trial, 185 rabies-exposed individuals were selected for post-exposure vaccination according to the Essen schedule, and blood was collected for RVNAs detection on days 28 and 42 after the first immunization. Results: A statistically significant difference in RVNAs between mice in the Essen and 0-3-7-14 schedule groups was observed on the 35th day ( P < 0.05). The groups 0-3-7-14, 0-3-7-21, and 0-3-7-28 showed no statistically significant difference ( P > 0.05) in RVNAs levels at any time point. The post-exposure immune protective test showed that the survival rate of mice in the control group was 20%, whereas that in the immunization groups was 40%. In the clinical trial, the RVNAs positive conversion rates on days 28 (14 days after 4 doses) and 42 (14 days after 5 doses) were both 100%, and no significant difference in RVNAs levels was observed ( P > 0.05). Conclusion: The simple 4-dose schedule can produce sufficient RVNAs levels, with no significant effect of a delayed fourth vaccine dose (14-28 d) on the immunization potential.

The plasma-soluble CSF1R level is a promising prognostic indicator for pediatric Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare hematologic neoplasm characterized by the clonal proliferation of Langerhans-like cells. Colony-stimulating factor 1 receptor (CSF1R) is a membrane-bound receptor that is highly expressed in LCH cells and tumor-associated macrophages. In this study, a soluble form of CSF1R protein (sCSF1R) was identified by plasma proteome profiling, and its role in evaluating LCH prognosis was explored. We prospectively measured plasma sCSF1R levels in 104 LCH patients and 10 healthy children using ELISA. Plasma sCSF1R levels were greater in LCH patients than in healthy controls (p < .001) and significantly differed among the three disease extents, with the highest level in MS RO+ LCH patients (p < .001). Accordingly, immunofluorescence showed the highest level of membrane-bound CSF1R in MS RO+ patients. Furthermore, the plasma sCSF1R concentration at diagnosis could efficiently predict the prognosis of LCH patients treated with standard first-line treatment (AUC = 0.782, p < .001). Notably, dynamic monitoring of sCSF1R levels could predict relapse early in patients receiving BRAF inhibitor treatment. In vitro drug sensitivity data showed that sCSF1R increased resistance to Ara-C in THP-1 cells expressing ectopic BRAF-V600E. Overall, the plasma sCSF1R level at diagnosis and during follow-up is of great clinical importance in pediatric LCH patients.

Updating mRNA variants of the human RSK4 gene and their expression in different stressed situations.

We determined RNA spectrum of the human RSK4 (hRSK4) gene (also called RPS6KA6) and identified 29 novel mRNA variants derived from alternative splicing, which, plus the NCBI-documented ones and the five we reported previously, totaled 50 hRSK4 RNAs that, by our bioinformatics analyses, encode 35 hRSK4 protein isoforms of 35-762 amino acids. Many of the mRNAs are bicistronic or tricistronic for hRSK4. The NCBI-normalized NM_014496.5 and the protein it encodes are designated herein as the Wt-1 mRNA and protein, respectively, whereas the NM_001330512.1 and the long protein it encodes are designated as the Wt-2 mRNA and protein, respectively. Many of the mRNA variants responded differently to different situations of stress, including serum starvation, a febrile temperature, treatment with ethanol or ethanol-extracted clove buds (an herbal medicine), whereas the same stressed situation often caused quite different alterations among different mRNA variants in different cell lines. Mosifloxacin, an antibiotics and also a functional inhibitor of hRSK4, could inhibit the expression of certain hRSK4 mRNA variants. The hRSK4 gene likely uses alternative splicing as a handy tool to adapt to different stressed situations, and the mRNA and protein multiplicities may partly explain the incongruous literature on its expression and comports.

Design and Implementation of a Four-Unit Array Piezoelectric Bionic MEMS Vector Hydrophone.

High-performance vector hydrophones have been gaining attention for underwater target-monitoring applications. Nevertheless, there exists the mutual constraint between sensitivity and bandwidth of a single hydrophone. To solve this problem, a four-unit array piezoelectric bionic MEMS vector hydrophone (FPVH) was developed in this paper, which has a cross-beam and a bionic fish-lateral-line-nerve-cell-cilia unit array structure. Simulation analysis and optimization in the design of the bionic microstructure have been performed by COMSOL 6.1 software to determine the structure dimensions and the lead zirconate titanate (PZT) thin film distribution. The FPVH was manufactured using MEMS technology and tested in a standing wave bucket. The results indicate that the FPVH has a sensitivity of up to -167.93 dB@1000 Hz (0 dB = 1 V/µPa), which is 12 dB higher than that of the one-unit piezoelectric MEMS vector hydrophone (OPVH). Additionally, the working bandwidth of the FPVH reaches 20 Hz~1200 Hz, exhibiting a good cosine curve with an 8-shape. This work paves a new way for the development of multi-unit piezoelectric vector hydrophones for underwater acoustic detectors.

Six versus four or five cycles of first-line etoposide and platinum-based chemotherapy combined with thoracic radiotherapy in patients with limited-stage small-cell lung cancer: A propensity score-matched analysis of a prospective randomized trial.

OBJECTIVES: The recommended treatment for limited-stage small-cell lung cancer (LS-SCLC) is a combination of thoracic radiotherapy (TRT) and etoposide plus cisplatin (EP) chemotherapy, typically administered over 4-6 cycles. Nonetheless, the optimal duration of chemotherapy is still not determined. This study aimed to compare the outcomes of patients with LS-SCLC who received either 6 cycles or 4-5 cycles of EP chemotherapy combined with TRT. MATERIALS AND METHODS: In this retrospective analysis, we utilized data from our prior prospective trial to analyze the outcomes of 265 LS-SCLC patients who received 4-6 courses of EP combined with concurrent accelerated hyperfractionated TRT between 2002 and 2017. Patients were categorized into two groups depending on their number of chemotherapy cycles: 6 or 4-5 cycles. To assess overall survival (OS) and progression-free survival (PFS), we employed the Kaplan-Meier method after conducting propensity score matching (PSM). RESULTS: Among the 265 LS-SCLC patients, 60 (22.6%) received 6 cycles of EP chemotherapy, while 205 (77.4%) underwent 4-5 cycles. Following PSM (53 patients for each group), the patients in the 6 cycles group exhibited a significant improvement in OS and PFS in comparison to those in the 4-5 cycles group [median OS: 29.8 months (95% confidence interval [CI], 23.6-53.1 months) vs. 22.7 months (95% CI, 20.8-29.1 months), respectively, p = 0.019; median PFS: 17.9 months (95% CI, 13.7-30.5 months) vs. 12.0 months (95% CI, 9.8-14.2 months), respectively, p = 0.006]. The two-year and five-year OS rates were 60.38% and 29.87% in the 6 cycles group, whereas 47.17% and 15.72% in the 4-5 cycles group, respectively. CONCLUSION: Patients diagnosed with LS-SCLC who were treated with EP regimen chemotherapy combined with TRT exhibited notably enhanced survival when administered 6 cycles of chemotherapy, as compared to those who underwent only 4-5 cycles.

Prenatal transposition of great arteries diagnosis and management: a Chinese single-center study.

Objective: This study aimed to assess the diagnostic value of prenatal echocardiography for identifying transposition of the great arteries (TGA) during pregnancy and evaluating the associated outcomes. Methods: We conducted a retrospective analysis of 121 prenatally diagnosed patients with TGA at our hospital between January 2012 and September 2022. This analysis included prenatal ultrasound, prenatal screening, clinical management and follow-up procedures. Results: Among the 103 fetuses considered in the study, 90 (87.4%) were diagnosed with complete transposition of the great arteries (D-TGA), while 13 (12.6%) exhibited corrected transposition of the great arteries (CC-TGA). Diagnoses were distributed across the trimester, with 8 D-TGA and 2 CC-TGA patients identified in the first trimester, 68 D-TGA patients and 9 CC-TGA patients in the second trimester, and 14 D-TGA and 2 CC-TGA patients referred for diagnosis in the third trimester. Induction of labour was pursued for 76 D-TGA patients (84.4%) and 11 CC-TGA patients (84.6%), and 14 D-TGA patients (15.6%) and 2 CC-TGA patients (15.4%) continued pregnancy until delivery. Among the D-TGA patients, 9 fetuses (10.0%) underwent surgery, two of which were inadvertent fatality, while the remaining seven experienced positive outcomes. Additionally, seven TGA patients received palliative care, leading to four fatalities among D-TGA patients (5.2%), whereas 1 D-TGA patients and 2 CC-TGA patients survived. Conclusion: This study underscores the feasibility of achieving an accurate prenatal diagnosis of TGA during early pregnancy. The utility of prenatal ultrasound in the development of personalized perinatal plans and the application of multidisciplinary treatment during delivery are conducive.

Simultaneous Determination of Ibrutinib, Dihydroxydiol Ibrutinib, and Zanubrutinib in Human Plasma by Liquid Chromatography-Mass Spectrometry/Mass Spectrometry.

BACKGROUND: Ibrutinib and zanubrutinib are Bruton tyrosine kinase inhibitors used to treat mantle cell lymphoma, chronic lymphocytic leukemia, and small lymphocytic lymphoma. Dihydroxydiol ibrutinib (DHI) is an active metabolite of the drug. A liquid chromatography-tandem mass spectrometry method was developed to detect ibrutinib, DHI, and zanubrutinib in human plasma. METHODS: The method involved a protein precipitation step, followed by chromatographic separation using a gradient of 10 mM ammonium acetate (containing 0.1% formic acid)-acetonitrile. Ibrutinib-d5 was used as an internal standard. Analytes were separated within 6.5 minutes. The optimized multiple reaction monitoring transitions of m/z 441.1 → 304.2, 475.2 → 304.2, 472.2 → 455.2, and 446.2 → 309.2 were selected to inspect ibrutinib, DHI, zanubrutinib, and the internal standards in positive ion mode. RESULTS: The validated curve ranges included 0.200-800, 0.500-500, and 1.00-1000 ng/mL for ibrutinib, DHI, and zanubrutinib, respectively. The precisions of the lower limit of quantification of samples were below 15.5%, the precisions of the other level samples were below 11.4%, and the accuracies were between -8.6% and 8.4%. The matrix effect and extraction recovery of all compounds ranged between 97.6%-109.0% and 93.9%-105.2%, respectively. The selectivity, accuracy, precision, matrix effect, and extraction recovery results were acceptable according to international method validation guidelines. CONCLUSIONS: A simple and rapid method was developed and validated in this study. This method was used to analyze plasma concentrations of ibrutinib and zanubrutinib in patients with mantle cell lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma, or diffuse large B-cell lymphoma. The selected patients were aged between 44 and 74 years.

Comparison of postoperative analgesia and side effects in pediatric laparoscopic surgery with morphine and nalbuphine.

There is currently no consensus on the optimal perioperative pain management strategy involving specific opioids. This study aims to compare the postoperative analgesia, the associated side effects between nalbuphine and morphine in children undergoing laparoscopic surgery. One hundred ninety children were randomly assigned to nalbuphine (0.2 mg/kg) or morphine (0.2 mg/kg). Nalbuphine's analgesic effect was non-inferior to morphine, with similar total rescue analgesic consumption during PACU stay (0.03 ± 0.05mg vs. 0.04 ± 0.06 mg, p > 0.05). Nalbuphine group had a lower incidence of respiratory depression (RR ≤ 10/min) (4.8% vs. 38.6%, p < 0.001), PONV (2.4% vs. 18.1%, p = 0.002), and pruritus (0% vs. 16.9%, p < 0.001) than morphine. Additionally, nalbuphine showed a shorter laryngeal mask airway removal time (13.9 [12.7, 15.1]) compared with morphine (17.0 [15.1, 18.9], p = 0.011). Nalbuphine provides equipotent analgesia with significantly lower incidences of respiratory depression, PONV, and pruritus compared with morphine in pediatric laparoscopic surgery.

Corrigendum: Altered expression of inflammation-associated molecules in striatum: an implication for sensitivity to heavy ion radiations.

[This corrects the article DOI: 10.3389/fncel.2023.1252958.].

A meta­ and bioinformatics analysis of maspin expression levels influencing the prognosis of patients with breast cancer.

Maspin is a serine protease inhibitor that is encoded by the human SERPINB5 gene. As a tumor inhibitor, it can inhibit the growth of tumor cells, increase adhesion between tumor cells and inhibit tumor angiogenesis. In the present study, a meta- and bioinformatics analysis was performed through the PubMed and China National Knowledge Infrastructure databases including entries added until up to March 20, 2023. It was found that compared with normal breast tissue, maspin expression was downregulated in breast cancer tissue. Maspin expression was negatively associated with lymph node metastasis. According to Kaplan-Meier plotter, it was found that lower maspin expression was negatively associated with the overall and distant metastasis-free survival rate of patients with estrogen receptor-positive, luminal A and grade 2 breast cancer. High expression of maspin was also positively associated with the relapse-free survival rate of patients of the luminal A subtype. Low maspin expression was positively associated with the post-progression and distant metastasis-free survival rate of the progesterone receptor-negative subtype. According to the GEPIA database, SERPINB5 mRNA expression was higher in normal than breast cancer tissues and negatively correlated with the TNM stage. High expression of maspin was also positively associated with the overall survival rate. In the UALCAN database, it was found that the mRNA and promoter methylation levels of SERPINB5 were higher in normal than in breast cancer tissues. These findings suggest that the expression of maspin may serve as a potential marker to indicate the occurrence, subsequent progression and even prognosis of breast cancer.

HMOX1 as a therapeutic target associated with diabetic foot ulcers based on single-cell analysis and machine learning.

Diabetic foot ulcers (DFUs) are a serious chronic complication of diabetes mellitus and a leading cause of disability and death in diabetic patients. However, current treatments remain unsatisfactory. Although macrophages are associated with DFU, their exact role in this disease remains uncertain. This study sought to detect macrophage-related genes in DFU and identify possible therapeutic targets. Single-cell datasets (GSE223964) and RNA-seq datasets (GSM68183, GSE80178, GSE134431 and GSE147890) associated with DFU were retrieved from the gene expression omnibus (GEO) database for this study. Analysis of the provided single-cell data revealed the distribution of macrophage subpopulations in the DFU. Four independent RNA-seq datasets were merged into a single DFU cohort and further analysed using bioinformatics. This included differential expression (DEG) analysis, multiple machine learning algorithms to identify biomarkers and enrichment analysis. Finally, key results were validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western bolt. Finally, the findings were validated using RT-qPCR and western blot. We obtained 802 macrophage-related genes in single-cell analysis. Differential expression analysis yielded 743 DEGs. Thirty-seven macrophage-associated DEGs were identified by cross-analysis of marker genes with macrophage-associated DEGs. Thirty-seven intersections were screened and cross-analysed using four machine learning algorithms. Finally, HMOX1 was identified as a potentially valuable biomarker. HMOX1 was significantly associated with biological pathways such as the insulin signalling pathway. The results showed that HMOX1 was significantly overexpressed in DFU samples. In conclusion, the analytical results of this study identified HMOX1 as a potentially valuable biomarker associated with macrophages in DFU. The results of our analysis improve our understanding of the mechanism of macrophage action in this disease and may be useful in developing targeted therapies for DFU.

Induction of somatic cell haploidy by premature cell division.

Canonical mitotic and meiotic cell divisions commence with replicated chromosomes consisting of two sister chromatids. Here, we developed and explored a model of premature cell division, where nonreplicated, G0/G1-stage somatic cell nuclei are transplanted to the metaphase cytoplasm of mouse oocytes. Subsequent cell division generates daughter cells with reduced ploidy. Unexpectedly, genome sequencing analysis revealed proper segregation of homologous chromosomes, resulting in complete haploid genomes. We observed a high occurrence of somatic genome haploidization in nuclei from inbred genetic backgrounds but not in hybrids, emphasizing the importance of sequence homology between homologs. These findings suggest that premature cell division relies on mechanisms similar to meiosis I, where genome haploidization is facilitated by homologous chromosome interactions, recognition, and pairing. Unlike meiosis, no evidence of recombination between somatic cell homologs was detected. Our study offers an alternative in vitro gametogenesis approach by directly reprogramming diploid somatic cells into haploid oocytes.

Improved gene therapy for spinal muscular atrophy in mice using codon-optimized hSMN1 transgene and hSMN1 gene-derived promotor.

Physiological regulation of transgene expression is a major challenge in gene therapy. Onasemnogene abeparvovec (Zolgensma®) is an approved adeno-associated virus (AAV) vector gene therapy for infants with spinal muscular atrophy (SMA), however, adverse events have been observed in both animals and patients following treatment. The construct contains a native human survival motor neuron 1 (hSMN1) transgene driven by a strong, cytomegalovirus enhancer/chicken ß-actin (CMVen/CB) promoter providing high, ubiquitous tissue expression of SMN. We developed a second-generation AAV9 gene therapy expressing a codon-optimized hSMN1 transgene driven by a promoter derived from the native hSMN1 gene. This vector restored SMN expression close to physiological levels in the central nervous system and major systemic organs of a severe SMA mouse model. In a head-to-head comparison between the second-generation vector and a benchmark vector, identical in design to onasemnogene abeparvovec, the 2nd-generation vector showed better safety and improved efficacy in SMA mouse model.