Tissue Development Research Articles

In silico assessment of biocompatibility and toxicity: molecular docking and dynamics simulation of PMMA-based dental materials for interim prosthetic restorations

Aim: This study aimed to comprehensively assess the biocompatibility and toxicity profiles of poly(methyl methacrylate) (PMMA) and its monomeric unit, methyl methacrylate (MMA), crucial components in dental materials for interim prosthetic restorations. Methodology: Molecular docking was employed to predict the binding affinities, energetics, and steric features of MMA and PMMA with selected receptors involved in bone metabolism and tissue development, including RANKL, Fibronectin, BMP9, NOTCH2, and other related receptors. The HADDOCK standalone version was utilized for docking calculations, employing a Lamarckian genetic algorithm to explore the conformational space of ligand-receptor interactions. Furthermore, molecular dynamics (MD) simulations over 100 nanoseconds were conducted using the GROMACS package to evaluate dynamic actions and structural stability. The LigandScout was utilized for pharmacophore modeling, which employs a shape-based screening approach to identify potential ligand binding sites on protein targets. Results: The molecular docking studies elucidated promising interactions between PMMA and MMA with key biomolecular targets relevant to dental applications. MD simulation results provided strong evidence supporting the structural stability of PMMA complexes over time. Pharmacophore modeling highlighted the significance of carbonyl and hydroxyl groups as pharmacophoric features, indicating compounds with favorable biocompatibility profiles. Conclusion: This study underscores the potential of PMMA in dental applications, emphasizing its structural stability, molecular interactions, and safety considerations. These findings lay a foundation for future advancements in dental biomaterials, guiding the design and optimization of materials for enhanced biocompatibility. Future directions include experimental validation of computational findings and the development of PMMA-based dental materials with improved biocompatibility and clinical performance.Graphical

Three-Dimensional Printing in Breast Reconstruction: Current and Promising Applications.

Three-dimensional (3D) printing is dramatically improving breast reconstruction by offering customized and precise interventions at various stages of the surgical process. In preoperative planning, 3D imaging techniques, such as computer-aided design, allow the creation of detailed breast models for surgical simulation, optimizing surgical outcomes and reducing complications. During surgery, 3D printing makes it possible to customize implants and precisely shape autologous tissue flaps with customized molds and scaffolds. This not only improves the aesthetic appearance, but also conforms to the patient's natural anatomy. In addition, 3D printed scaffolds facilitate tissue engineering, potentially favoring the development and integration of autologous adipose tissue, thus avoiding implant-related complications. Postoperatively, 3D imaging allows an accurate assessment of breast volume and symmetry, which is crucial in assessing the success of reconstruction. The technology is also a key educational tool, enhancing surgeon training through realistic anatomical models and surgical simulations. As the field evolves, the integration of 3D printing with emerging technologies such as biodegradable materials and advanced imaging promises to further refine breast reconstruction techniques and outcomes. This study aims to explore the various applications of 3D printing in breast reconstruction, addressing current challenges and future opportunities.

A gain-of-function mutation in BnaIAA13 disrupts vascular tissue and lateral root development in Brassica napus.

Rapeseed (Brassica napus) is an important oilseed crop worldwide. Plant vascular tissues are responsible for material transport and provide mechanical support. The lateral roots (LRs) absorb sufficient water and nutrients. The genetic basis of vascular tissues and LRs development in rapeseed remains unknown. This study characterized an EMS-mutagenized rapeseed mutant, T16, which showed dwarf stature, reduced LRs, and leaf wilting. Scanning electron microscopy observations showed that the internode-cell shortened. Observations of the tissue sections revealed defects in the development of vascular bundles in the stems and petioles. Genetic analysis revealed that the phenotypes of T16 were controlled by a single semi-dominant nuclear gene. Map-based cloning and genetic complementarity confirmed that BnaA03.IAA13 is the functional gene, a G-to-A mutation in second exon changed the glycine at the 79th position to glutamic acid, disrupting the conserved degron motif VGWPP. Transcriptome analysis in roots and stems showed that auxin and cytokinin signaling pathways were disordered in T16. Evolutionary analysis showed that AUXIN/INDOLE-3-ACETIC ACID was conserved during plant evolution. The heterozygote of T16 significantly reduced the plant height while maintaining other agronomic traits. Our findings provide novel insights into the regulatory mechanisms of vascular tissues and LRs development, and provide a new germplasm resource for rapeseed breeding.

Improved Genome Annotation of Brassica oleracea Highlights the Importance of Alternative Splicing

Brassica oleracea has been developed into many important crops, including cabbage, kale, cauliflower, broccoli and so on. The genome and gene annotation of cabbage (cultivar JZS), a representative morphotype of B. oleracea, has been widely used as a common reference in biological research. Although its genome assembly has been updated twice, the current gene annotation still lacks information on untranslated regions (UTRs) and alternative splicing (AS). Here, we constructed a high-quality gene annotation (JZSv3) using a full-length transcriptome acquired by nanopore sequencing, yielding a total of 59 452 genes and 75 684 transcripts. Additionally, we re-analyzed the previously reported transcriptome data related to the development of different tissues and cold response using JZSv3 as a reference, and found that 3 843 out of 11 908 differentially expressed genes (DEGs) underwent AS during the development of different tissues and 309 out of 903 cold-related genes underwent AS in response to cold stress. Meanwhile, we also identified many AS genes, including BolLHCB5 and BolHSP70, that displayed distinct expression patterns within variant transcripts of the same gene, highlighting the importance of JZSv3 as a pivotal reference for AS analysis. Overall, JZSv3 provides a valuable resource for exploring gene function, especially for obtaining a deeper understanding of AS regulation mechanisms.

An Exploration of Organoid Technology: Present Advancements, Applications, and Obstacles.

Organoids are in vitro models that exhibit a three-dimensional structure and effectively replicate the structural and physiological features of human organs. The capacity to research complex biological processes and disorders in a controlled setting is laid out by these miniature organ-like structures. This work examines the potential applications of organoid technology, as well as the challenges and future directions associated with its implementation. It aims to emphasize the pivotal role of organoids in disease modeling, drug discovery, developmental biology, precision medicine, and fundamental research. The manuscript was put together by conducting a comprehensive literature review, which involved an in-depth evaluation of globally renowned scientific research databases. The field of organoids has generated significant attention due to its potential applications in tissue development and disease modelling, as well as its implications for personalised medicine, drug screening, and cell-based therapies. The utilisation of organoids has proven to be effective in the examination of various conditions, encompassing genetic disorders, cancer, neurodevelopmental disorders, and infectious diseases. The exploration of the wider uses of organoids is still in its early phases. Research shall be conducted to integrate 3D organoid systems as alternatives for current models, potentially improving both fundamental and clinical studies in the future.

Cellular and molecular mechanisms of development and regeneration.

Regeneration involves a highly coordinated interplay of intricate cellular processes, enabling living organisms to renew and repair themselves, from individual cells to entire ecosystems. Further, regeneration offers profound insights into developmental biology, tissue engineering and regenerative medicine. The Cellular and Molecular Mechanisms of Development and Regeneration (CMMDR) 2024 conference, which took place at the Shiv Nadar Institute of Eminence and University (India), gathered together an international array of researchers studying a wide variety of organisms across both plant and animal kingdoms. In this short Meeting Review, we highlight some of the outstanding research presented at this conference and draw together some of the common themes that emerged.

Disparity of gene expression in coronary artery disease: insights from MEIS1, HIRA, and Myocardin.

Coronary artery disease (CAD) in young adults can have devastating consequences. The cardiac developmental gene MEIS1 plays important roles in vascular networks and heart development. This gene effects on the regeneration capacity of the heart. Considering role of MEIS1 in cardiac tissue development and the progression of myocardial infarction this study investigated the expression levels of the MEIS1, HIRA, and Myocardin genes in premature CAD patients compared to healthy subjects and evaluated the relationships between these genes and possible inflammatory factors. The study conducted a case-control design involving 35 CAD patients and 35 healthy individuals. Peripheral blood mononuclear cells (PBMCs) were collected, and gene expression analysis was performed using real-time PCR. Compared with control group, the number of PBMCs in the CAD group exhibited greater MEIS1 and HIRA gene expression, with fold changes of 2.45 and 3.6. The expression of MEIS1 exhibited a negative correlation with IL-10 (r= -0.312) expression and positive correlation with Interleukin (IL)-6 (r = 0.415) and tumor necrosis factor (TNF)-α (r = 0.534) gene expression. Moreover, there was an inverse correlation between the gene expression of HIRA and that of IL-10 (r= -0.326), and a positive correlation was revealed between the expression of this gene and that of the IL-6 (r = 0.453) and TNF-α (r = 0.572) genes. This research demonstrated a disparity in expression levels of MEIS1, HIRA, and Myocardin, between CAD and healthy subjects. The results showed that, MEIS1 and HIRA play significant roles in regulating the synthesis of proinflammatory cytokines, namely, TNF-α and IL-6.

Enhancing maturity in 3D kidney micro-tissues through clonogenic cell combinations and endothelial integration.

As an advance laboratory model, three-dimensional (3D) organoid culture has recently been recruited to study development, physiology and abnormality of kidney tissue. Micro-tissues derived from primary renal cells are composed of 3D epithelial structures representing the main characteristics of original tissue. In this research, we presented a simple method to isolate mouse renal clonogenic mesenchymal (MLCs) and epithelial-like cells (ELCs). Then we have done a full characterization of MLCs using flow cytometry for surface markers which showed that more than 93% of cells expressed these markers (Cd44, Cd73 and Cd105). Epithelial and stem/progenitor cell markers characterization also performed for ELC cells and upregulating of these markers observed while mesenchymal markers expression levels were not significantly increased in ELCs. Each of these cells were cultured either alone (ME) or in combination with human umbilical vein endothelial cells (HUVECs) (MEH; with an approximate ratio of 10:5:2) to generate more mature kidney structures. Analysis of 3D MEH renal micro-tissues (MEHRMs) indicated a significant increase in renal-specific gene expression including Aqp1 (proximal tubule), Cdh1 (distal tubule), Umod (loop of Henle), Wt1, Podxl and Nphs1 (podocyte markers), compared to those groups without endothelial cells, suggesting greater maturity of the former tissue. Furthermore, ex ovo transplantation showed greater maturation in the constructed 3D kidney.

Effect of LncRNA LOC106505926 on myogenesis and Lipogenesis of porcine primary cells

BackgroundSkeletal muscle development and fat deposition have important effects on meat quality. The study of regulating skeletal muscle development and fat deposition is of great significance in improving the quality of carcass and meat. In the present study, whole transcriptome sequencing (including RNA-Seq and miRNA-Seq) was performed on the longissimus dorsi muscle (LDM) of Jinfen White pigs at 1, 90, and 180 days of age.ResultsThe results showed that a total of 245 differentially expressed miRNAs were screened in any two comparisons, which may be involved in the regulation of myogenesis. Among them, compared with 1-day-old group, miR-22-5p was significantly up-regulated in 90-day-old group and 180-day-old group. Functional studies demonstrated that miR-22-5p inhibited the proliferation and differentiation of porcine skeletal muscle satellite cells (PSCs). Pearson correlation coefficient analysis showed that long non-coding RNA (lncRNA) LOC106505926 and CXXC5 gene had strong negative correlations with miR-22-5p. The LOC106505926 and CXXC5 were proven to promote the proliferation and differentiation of PSCs, as opposed to miR-22-5p. In terms of mechanism, LOC106505926 functions as a molecular sponge of miR-22-5p to modulate the expression of CXXC5, thereby inhibits the differentiation of PSCs. In addition, LOC106505926 regulates the differentiation of porcine preadipocytes through direct binding with FASN.ConclusionsCollectively, our results highlight the multifaceted regulatory role of LOC106505926 in controlling skeletal muscle and adipose tissue development in pigs and provide new targets for improving the quality of livestock products by regulating skeletal muscle development and fat deposition.

Myogenesis of Porcine Muscle Satellite Cells by Extracellular Matrix From Fibrotic Adipose Tissue-Derived Mesenchymal Stem Cells

Cultivated meat relies on the production of muscle tissue using cell culture techniques. Myogenesis is a cellular differentiation process in which muscle satellite cells (SC) transform into myoblasts, or myotubes. The extracellular matrix (ECM) plays a pivotal role in meat quality, myogenesis, SC proliferation, and muscle tissue development to replicate meat texture. Because fibrotic differentiation and ECM are involved in the myogenic process, we aimed to induce myogenesis in SC using fibrotic differentiation of adipose tissue-derived mesenchymal stem cells (ADSC). ADSC, fibroblasts (FC), and SC were isolated from the back fat, ear margin tissues, and femur muscle tissues, respectively, of a 1-year-old Berkshire piglet. ADSC were treated with connective tissue growth factor (CTGF; 0, 12.5, 25, or 50 ng/mL) for 72 h to induce fibrotic differentiation. Conditioned media from differentiated ADSC and FC were prepared and labeled CA-CM and F-CM, respectively. SC were exposed to CA-CM or F-CM to assess their effects on myogenesis. The results demonstrated α-smooth muscle actin and collagen I as the optimal markers for assessing fibrotic differentiation in ADSC. CTGF significantly elevated both mRNA and protein expression of α-smooth muscle actin and collagen I (P < 0.05), suggesting that CTGF acts as an inducer of fibrotic differentiation in ADSC. Moreover, CTGF elevated the expression of ECM components (laminin, fibronectin, and collagen I) in ADSC (P < 0.05). mRNA expression of myogenesis-related genes (MyoG, MyoD, Myf5, and Myf6) increased in SC exposed to both CA-CM and F-CM (P < 0.05). Our data revealed that fibrotic differentiation of ADSC increased ECM production, and ECM-containing conditioned medium induced myogenesis in SC. These findings indicate that ECM-containing conditioned medium is a good option for enhancing myogenesis in SC, with potential implications for cultivated meat production.

Non-canonical Wnt pathway expression in the developing mouse and human retina

The non-canonical Wnt pathway is an evolutionarily conserved pathway essential for tissue patterning and development across species and tissues. In mammals, this pathway plays a role in neuronal migration, dendritogenesis, axon growth, and synapse formation. However, its role in development and synaptogenesis of the human retina remains less established. In order to address this knowledge gap, we analyzed publicly available single-cell RNA sequencing (scRNAseq) datasets for mouse retina, human retina, and human retinal organoids over multiple developmental time points during outer retinal maturation. We identified ligands, receptors, and mediator genes with a putative role in retinal development, including those with novel or species-specific expression, and validated this expression using fluorescence in situ hybridization (FISH). By quantifying outer nuclear layer (ONL) versus inner nuclear layer (INL) expression, we provide evidence for the differential expression of certain non-canonical Wnt signaling components in the developing mouse and human retina during outer plexiform layer (OPL) development. Importantly, we identified distinct expression patterns of mouse and human FZD3 and WNT10A, as well as previously undescribed expression, such as for mouse Wnt2b in Chat+ starburst amacrine cells. Human retinal organoids largely recapitulated the human non-canonical Wnt pathway expression. Together, this work provides the basis for further study of non-canonical Wnt signaling in mouse and human retinal development and synaptogenesis.

Immunohistochemical properties of embryonic telocytes in a myogenic microenvironment.

Telocytes are a unique interstitial cell type that functions in adulthood and embryogenesis. They have characteristic immunohistochemical phenotypes while acquiring different immunohistochemical properties related to the organ microenvironment. The present study aims to investigate the immunohistochemical features of embryonic telocytes during myogenesis and describe their morphology using light microscopy and TEM. Telocytes represent a major cellular constituent in the interstitial elements. They had distinguished telopodes and podoms and formed a 3D interstitial network in the developing muscles. They formed heterocellular contact with myoblasts and nascent myotubes. Telocytes also had distinctive secretory activity. Telocytes identified by CD34. They also express CD68 and MMP-9 to facilitate the development of new tissues. Expression of CD21 by telocytes may reveal their function in immune defense. They also express VEGF, which regulates angiogenesis. In conclusion, the distribution and immunological properties of telocytes in the myogenic tissue indicate that telocytes provide biological and structural support in the development of the myogenic tissue architecture and organization.

How Much Do You Fuse? A Comparison of Cell Fusion Assays in a Breast Cancer Model.

Cell fusion is a biological process that is crucial for the development and homeostasis of different tissues, but it is also pathophysiologically associated with tumor progression and malignancy. The investigation of cell fusion processes is difficult because there is no standardized marker. Many studies therefore use different systems to observe and quantify cell fusion in vitro and in vivo. The comparability of the results must be critically questioned, because both the experimental procedure and the assays differ between studies. The comparability of the fluorescence-based fluorescence double reporter (FDR) and dual split protein (DSP) assay was investigated as part of this study, in which general conditions were kept largely constant. In order to be able to induce both a high and a low cell fusion rate, M13SV1 breast epithelial cells were modified with regard to the expression level of the fusogenic protein Syncytin-1 and its receptor ASCT2 and were co-cultivated for 72 h with different breast cancer cell lines. A high number of fused cells was found in co-cultures with Syncytin-1-overexpressing M13SV1 cells, but differences between the assays were also observed. This shows that the quantification of cell fusion events in particular is highly dependent on the assay selected, but the influence of fusogenic proteins can be visualized very well.

Current state of in situ gene expression studiesin animal tissues

Morphological studies of farm animals are most often conducted using simple preparation and staining techniques. The study of the processes of embryogenesis, postembryonic features of the development of organs and tissues, as well as the effect of various substances remains to be elucidated not only using histochemical and immunohistochemical staining methods, but also using RNA in situ hybridization and transcriptome in situ sequencing. Aspects of many cellular and tissue processes for cattle, pigs and chickens in the context of comparative physiology have not yet been studied. The high productivity of farm animals is associated with the intensive functioning of all organs and systems of the body. The influence of the agricultural management of farm animals and its consequences on the development of an organism in ontogeny deserve a separate area of research from the point of in situ gene expression. Despite rapid development of transcriptome sequencing technologies, which result in the discovery of new candidate genes for many processes, RNA in situ hybridization remains the gold standard for their validation. This review briefly presents modern techniques and their modifications for studying in situ gene expression. Transcriptome studies that have been implemented in cattle, pigs and chickens as model organisms include: in situ RNA hybridization using ZZ-probes, tyramide signal amplification, hybridization chain reaction, digoxigenin-labeled probes, RT-PCR, single cell transcriptome sequencing, in situ transciptome sequencing. This paper is a review of the results of studies on cattle, pigs and chickens. The results of research in this area are relevant for understanding the features of adaptation mechanisms at the transcriptomic level in highly productive animals under industrial conditions in order to search for new markers of valuable agricultural traits. It should be noted that in literature there are very few studies using RNA in situ hybridization, despite the availability and simplicity of the method.

Unraveling Divergent Transcriptomic Profiles: A Comparative Single-Cell RNA Sequencing Study of Epithelium, Gingiva, and Periodontal Ligament Tissues.

The periodontium comprising periodontal ligament (PDL), gingiva, and epithelium play crucial roles in maintaining tooth integrity and function. Understanding tissue cellular composition and gene expression is crucial for illuminating periodontal pathophysiology. This study aimed to identify tissue-specific markers via scRNA-Seq. Primary human PDL, gingiva, and epithelium tissues (n = 7) were subjected to cell hashing and sorting. scRNA-Seq library preparation using 10× Genomics protocol and Illumina sequencing was conducted. The analysis was performed using Cellranger (v3.1.0), with downstream analysis via R packages Seurat (v5.0.1) and SCORPIUS (v1.0.9). Investigations identified eight distinct cellular clusters, revealing the ubiquitous presence of epithelial and gingival cells. PDL cells evolved in two clusters with numerical superiority. The other clusters showed varied predominance regarding gingival and epithelial cells or an equitable distribution of both. The cluster harboring most cells mainly consisted of PDL cells and was present in all donors. Some of the other clusters were also tissue-inherent, while the presence of others was environmentally influenced, revealing variability across donors. Two clusters exhibited genetic profiles associated with tissue development and cellular integrity, respectively, while all other clusters were distinguished by genes characteristic of immune responses. Developmental trajectory analysis uncovered that PDL cells may develop after epithelial and gingival cells, suggesting the inherent PDL cell-dominated cluster as a final developmental stage. This single-cell RNA sequencing study delineates the hierarchical organization of periodontal tissue development, identifies tissue-specific markers, and reveals the influence of environmental factors on cellular composition, advancing our understanding of periodontal biology and offering potential insights for therapeutic interventions.

Extent of foetal exposure to maternal elexacaftor/tezacaftor/ivacaftor during pregnancy.

Cystic fibrosis (CF) patients are living longer and healthier due to improved treatments, e.g. cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy elexacaftor/tezacaftor/ivacaftor (ETI), with treatment possibly occurring in pregnancy. The risk of ETI to foetuses remain unknown. Thus the effect of maternally administered ETI on foetal genetic and structural development was investigated. Pregnant Sprague Dawley rats were orally treated with ETI (6.7mg·kg-1·day-1 elexacaftor + 3.5mg·kg-1·day-1 tezacaftor + 25 mg·kg-1·day-1 ivacaftor) for 7days from E12 to E19. Tissue samples collected at E19 were analysed using histology and RNA sequencing. Histological changes and differentially expressed genes (DEG) were assessed. No overt structural abnormalities were found in foetal pancreas, liver, lung and small intestine after 7-day ETI exposure. Very few non-functionally associated DEG in foetal liver, lung and small intestine were identified using RNA-seq. 29 DEG were identified in thymus (27 up-regulated and two down-regulated) and most were functionally linked to each other. Gene ontology enrichment analysis revealed that multiple muscle-related terms were significantly enriched. Many more DEG were identified in cortex (44 up-regulated and four down-regulated) and a group of these were involved in central nervous system and brain development. Sub-chronic ETI treatment in late pregnancy does not appear to pose a significant risk to the genetic and structural development of many foetal tissues. However, significant gene changes in foetal thymic myoid cells and cortical neuronal development requires future follow-up studies to assess the risk to these organs.

Comparison of craniomaxillofacial morphology in children with unilateral cleft lip and palate treated with/without presurgical nasoalveolar molding - a retrospective study with a mean age of 5 years.

To three-dimensionally assess differences in craniomaxillofacial skeletal development in patients with operated unilateral cleft lip and palate (UCLP) treated with/without presurgical nasoalveolar molding (PNAM) with a mean age of 5 years. Cone-beam CT radiographs of 30 patients with UCLP who had undergone PNAM and 34 patients with UCLP who did not receive PNAM were analyzed. The data were stored in DICOM file format and were imported into the Dolphin Imaging program for 3D image reconstruction and landmark identification. 33 landmarks, 17 linear and three angular variables representing craniofacial morphology were analyzed and compared by using the Mann-Whitney U tests. The vast majority of linear variables and 3D coordinates of landmark points reflecting craniofacial skeletal symmetry were not significantly different between the two groups. In terms of craniofacial skeletal development, the PNAM group had a significantly smaller anterior nasal spine offset in the midsagittal plane and a greater maxillary length compared to the non-PNAM group. Evaluations performed in early childhood showed that treatment with/without PNAM in the neonatal period was not a major factor influencing craniomaxillofacial hard tissue development in patients with UCLP; moreover, PNAM treatment showed significant correction of skeletal deviation at the base of the nose. Follow-up in early childhood has shown that PNAM treatment administered during the neonatal stage does not impede maxillary development and has benefits in correcting nasal floor deviation. It is a viable option for improving nasal deformity in children with unilateral cleft lip and palate.

Alpha lipoic acid controls degeneration and ensures follicular development in ovine ovarian tissue cultured in vitro

This study aims to evaluate the effects of adding alpha lipoic acid (ALA) to the in vitro ovarian tissue culture medium, either fresh or after vitrification/warming. For this purpose, 10 ovaries from five adult sheep were used. Each pair of ovaries gave rise to 16 fragments and were randomly distributed into two groups: fresh (n = 8) and vitrified (n = 8). Two fresh fragments were fixed immediately and considered the control, while another six were cultured in vitro for 14 days in the absence; presence of a constant (100 μM/0–14 day) or dynamic (50 μM/day 0–7 and 100 μM/day 8–14) concentration of ALA. As for the vitrified fragments, two were fixed and the other six were cultured in vitro under the same conditions described for the fresh group. All the fragments were subjected to morphological evaluation, follicular development and stromal density (classical histology), DNA fragmentation (TUNEL), senescence (Sudan Black), fibrosis (Masson's Trichome), and endoplasmic reticulum stress (immunofluorescence). Measurements of the antioxidant capacity against the free radicals 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and estradiol (E2) levels in the culture medium was performed. The results showed that in the absence of ALA, in vitro culture of vitrified ovarian fragments showed a significant reduction (P < 0.05) in follicular morphology and increased the presence of senescence and tissue fibrosis (P < 0.05). Dynamic ALA maintained E2 levels unchanged (P > 0.05) until the end of vitrified ovarian tissue culture and controlled the levels of ABTS and DPPH radicals in fresh or vitrified cultures. Therefore, it is concluded that ALA should be added to the vitrified ovarian tissue in vitro culture medium to reduce the damage that leads to loss of ovarian function. To ensure steroidogenesis during in vitro culture, ALA should be added dynamically (different concentrations throughout culture).

De novo TLK1 and MDM1 mutations in a patient with a neurodevelopmental disorder and immunodeficiency

The Tousled-like kinases 1 and 2 (TLK1/TLK2) regulate DNA replication, repair and chromatin maintenance. TLK2 variants underlie the neurodevelopmental disorder (NDD) 'Intellectual Disability, Autosomal Dominant 57' (MRD57), characterized by intellectual disability and microcephaly. Several TLK1 variants have been reported in NDDs but their functional significance is unknown. A male patient presenting with ID, seizures, global developmental delay, hypothyroidism, and primary immunodeficiency was determined to have a heterozygous TLK1 variant (c.1435C>G, p.Q479E), as well as a mutation in MDM1 (c.1197dupT, p.K400∗). Cells expressing TLK1 p.Q479E exhibited reduced cytokine responses and elevated DNA damage, but not increased radiation sensitivity or DNA repair defects. The TLK1 p.Q479E variant impaired kinase activity but not proximal protein interactions. Our study provides the first functional characterization of NDD-associated TLK1 variants and suggests that, such as TLK2, TLK1 variants may impact development in multiple tissues and should be considered in the diagnosis of rare NDDs.

PPA1 promotes adipogenesis by regulating the stability of C/EBPs.

Adipogenesis significantly contributes to healthy adipose tissue expansion in obesity. Increasing adipocyte number or function to alleviate adipose tissue overload could serve as a therapeutic strategy for both lipodystrophy and obesity-related metabolic syndrome. Inorganic pyrophosphatase (PPA1) is an enzyme that catalyzes the hydrolysis of pyrophosphate (PPi) and is involved in many biochemical reactions, but its function in adipose tissue has not been studied previously. In this study, we demonstrated that adipose-specific PPA1 knockout (PPA1AKO) mice showed lipodystrophy and spontaneously developed hepatic steatosis and severe insulin resistance under normal chow diet feeding. PPA1 deficiency suppressed the differentiation of primary adipocyte precursors and 3T3-L1 cells. Notably, PPA1 overexpression can restore inhibited adipogenesis in preadipocytes isolated from db/db mice and type 2 diabetes patients. Mechanistic studies have revealed that PPA1 acts as a positive regulator of early adipocyte differentiation by promoting CCAAT/enhancer-binding proteinβ and δ (C/EBPβ and δ) protein stability. Moreover, the function of PPA1 in adipogenesis is independent of its PPi catalytic activity. Collectively, our in vivo and in vitro findings demonstrated that PPA1 is a novel critical upstream regulator of adipogenesis, controlling adipose tissue development and whole-body metabolic homeostasis.