Various Stages Of Differentiation Research Articles

Mitochondrial respiration of a primary mixed culture of neurons from hippocampus at various stages of differentiation

BACKGROUND: Primary mixed culture of neurons is often used to evaluate the molecular mechanisms underlying neurodegenerative disorders. However, isolating cells from the brain is accompanied by profound changes in cell morphology, behavior, and metabolic rate due to enzymatic disintegration and microenvironmental changes in cells. In this regard, the mitochondrial basal respiration of neurons should be considered as an indicator of the formation of functional activity of the cell.
 AIM: To evaluate mitochondrial respiration in a primary mixed culture of hippocampal neurons at various stages of differentiation.
 METHODS: This study included mice of line CD-1 and was conducted in two series: the first studied the primary mixed embryonic culture on the 18th day of gestation (E18) and the second used the postnatal culture on the 2nd day after birth (P2). A Seahorse XF HS Mini (Agilent, USA) was used to measure the functional parameters of cell metabolism. The oxygen consumption rate was calculated based on the results of the mitochondrial respiration profile of the primary mixed culture built during its differentiation.
 RESULTS: On the 5th day of neuronal differentiation in the culture, maximum mitochondrial respiration was established both in the hippocampal culture obtained from embryos on the 18th day of gestation and in the culture taken from mice on the 2nd day after birth. As cells differentiated in culture from days 2 to 11, the substrate oxidation rate increased almost twofold in the culture of hippocampal neurons obtained from embryos, which increased the metabolic potential.
 CONCLUSION: The study results prove that the hippocampus can be used to study the role of mitochondria in neurogenesis.

Chemokine receptor distribution on the surface of repolarizing T cells

T cells migrate constitutively with a polarized morphology, underpinned by signaling compartmentalization and discrete cytoskeletal organizations, giving rise to a dynamic and expansive leading edge, distinct from the stable and constricted uropod at the rear. In vivo, the motion and function of T cells at various stages of differentiation is highly directed by chemokine gradients. When cognate ligands bind chemokine receptors on their surface, T cells respond by reorientating their polarity axis and migrating toward the source of the chemokine signal. Despite the significance of such chemotactic repolarization to the accurate navigation and function of T cells, the precise signaling mechanisms that underlie it remain elusive. Notably, it remained unclear whether the distribution of chemokine receptors on the T cell surface is altered during repolarization. Here, we developed parallel cell-secreted and microfluidics-based chemokine gradient delivery methods and employed both fixed imaging and live lattice light-sheet microscopy to investigate the dynamics of chemokine receptor CCR5 on the surface of primary murine CD8+ T cells. Our findings show that, during constitutive migration, chemokine receptor distribution is largely isotropic on the T cell surface. However, upon exposure to a CCL3 gradient, surface chemokine receptor distributions exhibit a transient bias toward the uropod. The chemokine receptors then progressively redistribute from the uropod to cover the T cell surface uniformly. This study sheds new light on the dynamics of surface chemokine receptor distribution during T cell repolarization, advancing our understanding of the signaling of immune cells in the complex chemokine landscapes they navigate.

Single-Cell RNA Sequencing Reveals the Cellular Landscape of Longissimus Dorsi in a Newborn Suhuai Pig.

The introduction of single-cell RNA sequencing (scRNA-seq) technology has spurred additional advancements in analyzing the cellular composition of tissues. The longissimus dorsi (LD) in pigs serves as the primary skeletal muscle for studying meat quality in the pig industry. However, the single-cell profile of porcine LD is still in its infancy stage. In this study, we profiled the transcriptomes of 16,018 cells in the LD of a newborn Suhuai pig at single-cell resolution. Subsequently, we constructed a cellular atlas of the LD, identifying 11 distinct cell populations, including endothelial cells (24.39%), myotubes (18.82%), fibro-adipogenic progenitors (FAPs, 18.11%), satellite cells (16.74%), myoblasts (3.99%), myocytes (5.74%), Schwann cells (3.81%), smooth muscle cells (3.22%), dendritic cells (2.99%), pericytes (1.86%), and neutrophils (0.33%). CellChat was employed to deduce the cell-cell interactions by evaluating the gene expression of receptor-ligand pairs across different cell types. The results show that FAPs and pericytes are the primary signal contributors in LD. In addition, we delineated the developmental trajectory of myogenic cells and examined alterations in the expression of various marker genes and molecular events throughout various stages of differentiation. Moreover, we found that FAPs can be divided into three subclusters (NR2F2-FAPs, LPL-FAPs, and TNMD-FAPs) according to their biological functions, suggesting that the FAPs could be associated with the differentiation of tendon cell. Taken together, we constructed the cellular atlas and cell communication network in LD of a newborn Suhuai pig, and analyzed the developmental trajectory of myogenic cells and the heterogeneity of FAPs subpopulation cells. This enhances our comprehension of the molecular features involved in skeletal muscle development and the meat quality control in pigs.

Expression of Pfkfb isoenzymes during in vitro differentiation of mouse embryonic stem cells into insulin-producing cells.

Type 1 diabetes mellitus (T1DM) is caused by the autoimmune-mediated destruction of insulin-producing cells (IPCs) and still has no effective cure. Better understanding of the molecular mechanisms involved in the differentiation of embryonic stem cells (ESCs) into IPCs may help us improve the therapeutic strategies for treating T1DM. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (Pfkfb1-4) are key regulators of glucose metabolism. Although Pfkfb3 has been shown to be required for the growth of early differentiated mouse ESCs (mESCs), more studies are needed to further assess the roles of Pfkfb isoenzymes in embryonic development and differentiation, particularly into specific cell types. In this study, we aimed to elucidate the changes in the expression of Pfkfb isoenzymes on the differentiation of mESCs into IPCs. A 3-step protocol was used to differentiate R1 and J1 mESCs into IPCs. The changes in the gene expression of MafA, MafB, Ins2, and Nkx6.1 (IPC specific markers) and Pfkfb1-4 were analyzed using real-time quantitative polymerase chain reaction (qPCR). Insulin expression and secretion were determined by immunofluorescence (IF) staining and the enzyme linked immunosorbent assay (ELISA), respectively. Upon differentiation, the IPC specific markers in differentiated cells were upregulated. Continued differentiation was confirmed by the development of insulin-positive islet-like clusters that secreted insulin in response to glucose uptake. Expressions of the Pfkfb2 and Pfkfb3 isoenzymes were markedly increased in various stages of differentiation. These findings suggest that Pfkfb2 and Pfkfb3 may impact the differentiation of mESCs into IPCs and the regulation of the insulin response to glucose levels. This study also lays a foundation for researchers to further probe the roles of Pfkfb isoenzymes on the differentiation of mESCs into IPCs and may open new avenues for regenerative medicine.

Title: Single-Cell Transcriptome Analyses Further Elucidate the Mechanism of Action of Eed Inhibition on HbF Induction

Mutations in the gene encoding the hemoglobin β (HBβ) subunit lead to red blood cell disorders such as Sickle Cell Disease (SCD) and β-thalassemia. Hemoglobin γ subunit, a fetal counterpart of HBβ, can relieve the disease symptoms by forming a functional complex with hemoglobin a subunit, thereby compensating for the mutated HBβ. The expression of β-like hemoglobins is tightly regulated during development, with a switch from the fetal ortholog (γ) to the adult ortholog (β) shortly after birth. Maintenance of HBγ is therapeutically beneficial for SCD patients and is observed clinically in individuals with mutations for both SCD and hereditary persistent fetal hemoglobin (HPFH) who exhibit less severe or even no SCD-related symptoms. Polycomb repressive complex (PRC) proteins have been shown to regulate the expression of β-like hemoglobins. Our previous research has demonstrated inhibitors targeting the PRC2 protein embryonic ectoderm development (EED) upregulate fetal hemoglobin (HbF) expression, leading to the formation of HbF complexes in both human CD34 + HSC-derived erythroid cells and the Townes SCD mouse model. However, the functional mechanisms of PRC complexes in this regulatory process and their target genes remain to be clarified. In our study, we conducted single-cell transcriptome analyses on erythroid cells derived from CD34 + HSCs collected at various stages of differentiation. We identified several erythroid subpopulations, from early progenitors to maturing cells, within the CD34 + derived erythroid cell cultures. HbF + and HbF - cell populations were also identified using known markers. To explore the mechanisms underlying HbF upregulation by EED inhibition, we compared single-cell transcriptome profiles of samples treated with and without an EED inhibitor. Our findings reveal that EED inhibitor treatment consistently leads to increased HbF + populations across multiple donors without impacting erythroid cell differentiation. Differential gene expression analysis revealed sequential changes in the expression of previously identified HbF regulators during differentiation in the presence of EED inhibitor treatment. Through pseudotime analyses, we identified erythroid progenitor cells undergoing fate bifurcation, leading to an increase in the lineage of mature cells that express HBγ. Finally, by comparing the pseudotime trajectories of different samples, we identified genes whose expression changed along the differentiation trajectories in response to EED inhibitor treatment which influence cell fate. Overall, this study provides a deeper understanding of the relevant biological pathways that respond to EED inhibition and potential for the identification of novel molecular targets for HbF induction.

Modeling the crosstalk between malignant B cells and their microenvironment in B-cell lymphomas: challenges and opportunities.

B-cell lymphomas are a group of heterogeneous neoplasms resulting from the clonal expansion of mature B cells arrested at various stages of differentiation. Specifically, two lymphoma subtypes arise from germinal centers (GCs), namely follicular lymphoma (FL) and GC B-cell diffuse large B-cell lymphoma (GCB-DLBCL). In addition to recent advances in describing the genetic landscape of FL and GCB-DLBCL, tumor microenvironment (TME) has progressively emerged as a central determinant of early lymphomagenesis, subclonal evolution, and late progression/transformation. The lymphoma-supportive niche integrates a dynamic and coordinated network of immune and stromal cells defining microarchitecture and mechanical constraints and regulating tumor cell migration, survival, proliferation, and immune escape. Several questions are still unsolved regarding the interplay between lymphoma B cells and their TME, including the mechanisms supporting these bidirectional interactions, the impact of the kinetic and spatial heterogeneity of the tumor niche on B-cell heterogeneity, and how individual genetic alterations can trigger both B-cell intrinsic and B-cell extrinsic signals driving the reprogramming of non-malignant cells. Finally, it is not clear whether these interactions might promote resistance to treatment or, conversely, offer valuable therapeutic opportunities. A major challenge in addressing these questions is the lack of relevant models integrating tumor cells with specific genetic hits, non-malignant cells with adequate functional properties and organization, extracellular matrix, and biomechanical forces. We propose here an overview of the 3D in vitro models, xenograft approaches, and genetically-engineered mouse models recently developed to study GC B-cell lymphomas with a specific focus on the pros and cons of each strategy in understanding B-cell lymphomagenesis and evaluating new therapeutic strategies.

Megakaryocytes Direct CD4 + T Cell Responses Via Major Histocompatibility Complex Class II Antigen Presentation

Megakaryocytes (MKs) are rare, large, hematopoietic cells that canonically have the role of producing platelets. Advances in single-cell RNA-sequencing (scRNA-seq) have revealed a previously under-appreciated degree of MK heterogeneity suggesting that both murine and human MKs may perform functions beyond platelet production. Among other inflammatory molecules, these studies identified that select MK subsets express major histocompatibility complex (MHC II) antigen presentation-associated transcripts and display an upregulation of antigen processing pathways. Together, these findings suggest a potential immunoregulatory function of select MK subsets. However, functional validation substantiating these transcriptional signatures and the proposed MK immunoregulatory capacities has lagged, and the expression of MHC II in bone marrow MKs remains controversial. In our studies, we aimed to both quantify and functionally assess immunomodulatory bone marrow MKs. Using flow cytometry and high-resolution confocal imaging, we showed that MHC class II is expressed on ~20% of MKs at various stages of differentiation. Of the cell populations identified, MHC class II expression was observed in CD41+ and vWF+ platelet-biased hematopoietic stem cells (HSCs), c-Kit+CD150+CD41+ MK progenitors, immature CD41+ MKs and mature CD41+ CD42d+ MKs. Intriguingly, we also observed that MHC class II expression was dramatically upregulated, (2.5-fold increase in expression) in mature MKs and MK progenitors of aged mice. Additionally, MHCII expression was significantly correlated with vWF and CD41 expression in long-term HSCs, with 60-70% of MHCII+ HSCs co-expressing both of these markers. To further validate MHC class II expression in the MK lineage, we utilized two unique murine MHCII transgenic reporter models. The first was an MHCII-EGFP knock-in mouse in which endogenous class II product is replaced by GFP, and the second was a Cd74tdT reporter mouse in which the invariant chain (CD74) can be visualized via tdTomato upon MHC class II pathway activation. In both models, approximately 20-30% of MKs were MHCII positive, consistent with antibody staining in wild-type mice. To substantiate the immunoregulatory function of primary murine MKs, we assayed antigen presentation capacity and ability to stimulate T-cells in vitro. We observed that bone marrow derived MKs express basal levels of multiple co-stimulatory receptors including CD80, CD83, CD86, and CD40 in addition to MHC class II. We also observed significant upregulated of these receptors upon TLR stimulation (TLR3, TLR4 TLR9) in a dose dependent manner. Notably, co-culture of MKs with CD4+ T cells resulted in naive T cell activation and proliferation. To verify that these results extended to human MKs, we examined HLA class II expression in MKs differentiated from CD34+ cells obtained from human bone marrow. Comparable with the murine studies, we found that human MKs also upregulated class II pathway molecules, suggesting that human MKs may also engage with the adaptive immune system. Together, our results support the hypothesis that there is an immunomodulatory sub-population of MKs that can be distinguished by MHCII surface expression, engages in class II antigen presentation, and can activate CD4+ T cells to prime adaptive immune responses. Future studies will investigate the in vivo role of these immunomodulatory functions of MK and how they may be exploited to alter T cell differentiation in the context of inflammatory, infectious, or age-associated thrombotic diseases.

Mechanism of Neural Stem Cell-Derived Exosomal miR-9a-5p Overexpression Improving Survival and Neurogenesis in Ischemic Stroke Rats

As a momentous condition disease, ischemic stroke could lead to physical disability and death. Here, the protective effect of miRNA up-regulated in neural stem cells (NSCs) derived exosomes on ischemic stroke in rats and their molecular mechanisms were investigated to reveal the therapeutic target of exosomes and suggests new approaches to treat ischemic stroke. miRNAs differentially expressed in exosomes derived from NSCs at various differentiation stages were detected by high-throughput sequencing for miRNAs. The impacts of miR-9a-5p upregulation were assessed on the differentiation of NSCs. The effects of exosomes derived from normal NSCs and NSCs with up-regulated miR-9a-5p on cell survival and differentiation and AMPK activation were investigated in vitro and in vivo. The high-throughput sequencing analysis revealed that miR-9a-5p was differentially expressed in NSC-derived exosomes at various stages of differentiation. MiR-9a-5p upregulation in exosomes promoted cell differentiation of NSCs. Furthermore, it can sensitized the AMPK signaling pathway. Following deprivation/reperfusion of oxygen-glucose, the differentiation of NSCs was restored, and exosomes significantly reduced cell apoptosis. MiR-9a-5p exosomes reduced the blood-brain barrier permeability and the infarct volume of rats with ischemic stroke in vivo. Neural cell apoptosis was reduced, thus indicating that miR-9a-5p could inhibit the cell apoptosis in vivo. AMPK activation was induced and increased in the MACO/R rat with miR-9a-5p exosomes. MiR-9a-5p exosomes could promote AMPK phosphorylation, increase NSC survival and enhance cell differentiation; this could inhibit the progression of ischemic stroke by maintaining an adequate number of neural cells and promoting endogenous NSC differentiation.

MDB-14. MULTI-OMICS OF HUMAN MEDULLOBLASTOMAS AND GENETIC LINEAGE TRACING IN VIVO IDENTIFY REGULATORS OF STEM-CELL MAINTENANCE

Abstract Recent studies implicate the glutamatergic lineage of the rhombic lip (RL) as the origin of group 3/4 medulloblastomas (MBs). MB stem cells resemble stalled RL progenitors and targeting common pluripotency factors induces differentiated phenotypes. However, the genetic and epigenetic regulators of stemness in MB remain incompletely understood. We profiled tissues resected from 38 patients who underwent surgery for MB at UCSF via single-nucleus RNA sequencing (snRNA-seq). Of these, 13 were also subjected to single-cell assay for transposase-accessible chromatin by sequencing (scATAC-seq). These assays generated 560,509 and 71,276 cells respectively, after quality control. Motif and latent-space analyses were used to infer cell-type specific transcription-factor expression and binding, and associated cis-regulatory enhancers. Further, we performed single-cell genetic lineage tracing on xenografts obtained from two distinct group-3/4 patient-derived lines, engineered to express heritable barcodes. We trained a machine-learning classifier on public snRNA-seq of human RL and classified Nestin-high quiescent stem-like cells, Nestin-moderate/ASPM-high proliferating progenitor cells, and glutamatergic lineage cells at various stages of differentiation in our MB clinical specimens and xenografts. Integrated analysis of these datasets maps the transition from quiescent to proliferating stem-like MB cells to more differentiated cell types at the level of transcription-factor activity and binding. We found that both the expression and binding of epigenetic regulators SUZ12, EZH2, DNMT1, and BRD7/8 were high in stem cells, but attenuated during lineage commitment. This was also the case for genes known to maintain neural stem cells in the developing brain, e.g. LIN28, FOXO3, HES6, LHX1/2/4, NUP98. Comparative analysis of developmental and MB gene expression, with motif analysis, implicates a conserved embryonal mechanism for indefinite replicative division in MB stem-like cells. These studies provide a single-cell atlas of transcriptomic and epigenetic cellular states in human MB, a map of MB cellular hierarchical organization and transcription-factor regulation of stem-cell maintenance in vivo.

Клінічний випадок маніфестації первинного імунодефіциту з лімфоми Беркіта

Lymphoma is a group of neoplastic diseases characterized by the clonal proliferation of lymphoid cells, manifested by the response of various stages of differentiation of normal B-lymphocytes, less often T-lymphocytes or natural killer cells. The etiology of most lymphomas is unknown. Some of the proven causal-consequence factors are: viral infections and immunodeficiency conditions. Purpose - to draw the attention of non-immunologists to the peculiarities of detecting the primary immunodeficiency (PID) states and the problem of their late diagnosis based on the data of a clinical case. Clinical case. A clinical observation of severe non-genetically differentiated PID is given. Screening signs of PID in the form of recurrent infectious respiratory tract syndrome and frequent long-term use of antibacterial drugs are highlighted, which should alert doctors to clinical and immunological markers of primary immunodeficiency. The presence of Burkitt's lymphoma in the boy also does not exclude the possibility of the manifestation of an immunodeficiency state, both secondary to the drug treatment of lymphoma, and primary, which can manifest itself in puberty. Common variable immunodeficiency is one of those PIDs that "starts" with Burkitt's lymphoma. Conclusions. The problem of low detection of probable PID in children in Ukraine is urgent. Primary care physicians are the first to be able to partially solve this problem by examining the infectious history and laboratory screening tests. There is incomplete awareness of related medical specialties regarding the signs of PID in lymphoproliferative syndrome in children. This clinical case demonstrates signs of PID at an early age. Low detection is also associated with poor capacity and quality and/or lack of immunological laboratory testing, particularly in public facilities. The research was carried out in accordance with the principles of the Helsinki Declaration. The informed consent of the of the child’s parents was obtained for the research No conflict of interests was declared by the authors.

Genetic Analysis of Golden Eagles (Aquila chrysaetos) from the Mongol-Altai: A Hotspot of Diversity and Implications for Global Phylogeography

ABSTRACT The Golden Eagle (Aquila chrysaetos) is widely distributed in the northern Hemisphere and is thus an ideal candidate to explore large-scale biogeographic patterns. In recent years, Golden Eagle population genetics has gained considerable attention. However, the Eastern Palearctic region, a numerical stronghold for the species, is one of the least genetically studied regions within its geographic range. In West Mongolia, the Altai (Altay) Kazakhs collect wild Golden Eagle nestlings from eyries or trap juveniles and subadults on passage during seasonal movement and migration for traditional hunting, providing easy access for researchers to sample falconry eagles for population genetic analyses. We analyzed a 402-bp fragment of the mitochondrial control region and 14 nuclear microsatellite loci in combination with previously published genetic data to assess phylogeographic patterns, levels of genetic diversity, and fine-scale structuring of the Golden Eagle population within the Mongol-Altai Mountains. Golden Eagles in the Mongol-Altai Mountains exhibited overall high levels of genetic diversity. Mitochondrial DNA data across the species' geographic range reveal various stages of differentiation within the Holarctic clade: whereas divergence between the Western and Eastern Palearctic is relatively shallow, the Eastern Palearctic and Nearctic show a deeper divergence, although these geographic regions have not yet reached reciprocal monophyly. These patterns might reflect past climatic oscillations: Northern Europe may have been recolonized by Golden Eagles from the Eastern Palearctic region, and continental populations might still be connected by long-distance dispersers, but the Eastern Palearctic and Nearctic were likely connected via Beringia during the Quaternary and are today largely isolated from each other.

Asprosin Enhances Cytokine Production by a Co-Culture of Fully Differentiated Mature Adipocytes and Macrophages Leading to the Exacerbation of the Condition Typical of Obesity-Related Inflammation.

Asprosin, a fasting-induced, glucogenic, and orexigenic adipokine, has gained popularity in recent years as a potential target in the fight against obesity and its complications. However, the contribution of asprosin to the development of moderate obesity-related inflammation remains still unknown. The present study aimed to evaluate the effect of asprosin on the inflammatory activation of adipocyte-macrophage co-cultures at various stages of differentiation. The study was performed on co-cultures of the murine 3T3L1 adipocyte and the RAW264.7 macrophage cell lines treated with asprosin before, during, and after 3T3L1 cell differentiation, with or without lipopolysaccharide (LPS) stimulation. Cell viability, overall cell activity, and the expression and release of key inflammatory cytokines were analyzed. In the concentration range of 50-100 nM, asprosin increased the pro-inflammatory activity in the mature co-culture and enhanced the expression and release of tumor necrosis factor α (TNF-α), high-mobility group box protein 1 (HMGB1), and interleukin 6 (IL-6). Macrophage migration was also increased, which could be related to the upregulated expression and release of monocyte chemoattractant protein-1 (MCP-1) by the adipocytes. In summary, asprosin exerted a pro-inflammatory effect on the mature adipocyte-macrophage co-culture and may contribute to the spread of moderate obesity-associated inflammation. Nevertheless, further research is needed to fully elucidate this process.

Transcriptomic analysis of airway epithelial cells identifies an altered expression profile of T2 and non-T2 genes in chronic rhinosinusitis with nasal polyposis

Aberrant differentiation of basal epithelial cells is a hallmark feature of chronic rhinosinusitis with nasal polyposis (CRSwNP). We hypothesized that unbiased transcriptomic analysis of upper airway epithelial cells at various stages of differentiation would identify genes fundamental to CRSwNP.

Human iPSC-derived midbrain organoids functionally integrate into striatum circuits and restore motor function in a mouse model of Parkinson's disease.

Rationale: Parkinson's disease (PD) is a prevalent neurodegenerative disorder that is characterized by degeneration of dopaminergic neurons (DA) at the substantia nigra pas compacta (SNpc). Cell therapy has been proposed as a potential treatment option for PD, with the aim of replenishing the lost DA neurons and restoring motor function. Fetal ventral mesencephalon tissues (fVM) and stem cell-derived DA precursors cultured in 2-dimentional (2-D) culture conditions have shown promising therapeutic outcomes in animal models and clinical trials. Recently, human induced pluripotent stem cells (hiPSC)-derived human midbrain organoids (hMOs) cultured in 3-dimentional (3-D) culture conditions have emerged as a novel source of graft that combines the strengths of fVM tissues and 2-D DA cells. Methods: 3-D hMOs were induced from three distinct hiPSC lines. hMOs at various stages of differentiation were transplanted as tissue pieces into the striatum of naïve immunodeficient mouse brains, with the aim of identifying the most suitable stage of hMOs for cellular therapy. The hMOs at Day 15 were determined to be the most appropriate stage and were transplanted into a PD mouse model to assess cell survival, differentiation, and axonal innervation in vivo. Behavioral tests were conducted to evaluate functional restoration following hMO treatment and to compare the therapeutic effects between 2-D and 3-D cultures. Rabies virus were introduced to identify the host presynaptic input onto the transplanted cells. Results: hMOs showed a relatively homogeneous cell composition, mostly consisting of dopaminergic cells of midbrain lineage. Analysis conducted 12 weeks post-transplantation of day 15 hMOs revealed that 14.11% of the engrafted cells expressed TH+ and over 90% of these cells were co-labeled with GIRK2+, indicating the survival and maturation of A9 mDA neurons in the striatum of PD mice. Transplantation of hMOs led to a reversal of motor function and establishment of bidirectional connections with natural brain target regions, without any incidence of tumor formation or graft overgrowth. Conclusion: The findings of this study highlight the potential of hMOs as safe and efficacious donor graft sources for cell therapy to treat PD.

Reversal of splicing infidelity is a pre-activation step in B cell differentiation.

B cell activation and differentiation is central to the adaptive immune response. Changes in exon usage can have major impacts on cellular signaling and differentiation but have not been systematically explored in differentiating B cells. We analyzed exon usage and intron retention in RNA-Seq data from subsets of human B cells at various stages of differentiation, and in an in vitro laboratory model of B cell activation and differentiation (Epstein Barr virus infection). Blood naïve B cells were found to have an unusual splicing profile, with unannotated splicing events in over 30% of expressed genes. Splicing changed substantially upon naïve B cell entry into secondary lymphoid tissue and before activation, involving significant increases in exon commitment and reductions in intron retention. These changes preferentially involved short introns with weak splice sites and were likely mediated by an overall increase in splicing efficiency induced by the lymphoid environment. The majority of transcripts affected by splicing changes showed restoration of encoded conserved protein domains and/or reduced targeting to the nonsense-mediated decay pathway. Affected genes were enriched in functionally important immune cell activation pathways such as antigen-mediated signaling, cell cycle control and mRNA processing and splicing. Functional observations from donor B cell subsets in progressive states of differentiation and from timecourse experiments using the in vitro model suggest that these widespread changes in mRNA splicing play a role in preparing naïve B cells for the decisive step of antigen-mediated activation and differentiation.

Oocytes could rearrange immunoglobulin production to survive over adverse environmental stimuli.

Immunoglobulins are key humoral immune molecules produced and secreted by B lymphocytes at various stages of differentiation. No research has reported whether immunoglobulins are present in the non-proliferative female germ cells-oocytes-and whether they are functionally important for oocyte quality, self-protection, and survival. Herein, we found that IgG was present in the oocytes of immunodeficient mice; the IgG-VDJ regions were highly variable between different oocytes, and H3K27Ac bound and regulated the IgG promoter region. Next, IgG mRNA and protein levels increased in response to LPS, and this increment was mediated by CR2 on the oocyte membrane. Finally, we revealed three aspects of the functional relevance of oocyte IgG: first, oocytes could upregulate IgG to counteract the increased ROS level induced by CSF1; second, oocytes could upregulate IgG in response to injected virus ssRNA to maintain mitochondrial integrity; third, upon bacterial infection, oocytes could secrete IgG, subsequently encompassing the bacteria, thus increasing survival compared to somatic cells. This study reveals for the first time that the female germ cells, oocytes, can independently adjust intrinsic IgG production to survive in adverse environments.

Sinonasal lymphoma: A primer for otolaryngologists.

Sinonasal lymphomas are a rare entity that commonly present with nonspecific sinonasal symptoms and are often recognized immediately. Through this review, we aim to summarize important principles in diagnosis and treatment of sinonasal lymphomas, with the goal of disseminating the current knowledge of this under-recognized malignancy to otolaryngologists. Systemic review using PRISMA guidelines of foundational scholarly articles, guidelines, and trials were reviewed focusing on clinical characteristics of key sinonasal lymphoma subtypes, along with available treatments in the otolaryngology, medical oncology, and radiation oncology literature. Sinonasal lymphoma are derived from clonal proliferation of lymphocytes at various stages of differentiation, of which diffuse large B-cell lymphoma (DLBCL) and extranodal natural killer/T-cell lymphoma (ENKTL) are the most common. Diagnosis and staging require biopsy with immunohistochemistry in conjunction with imaging and laboratory studies. Treatment is ever evolving and currently includes multi-agent chemotherapy and/or radiation therapy. Otolaryngologists may be the first to recognize sinonasal lymphoma, which requires a comprehensive workup and a multidisciplinary team for treatment. Symptoms are nonspecific and similar to many sinonasal pathologies, and it is crucial for otolaryngologists to keep a broad differential. 5.

Amino acid metabolism in skeletal cells

Amino acid metabolism regulates essential cellular functions, not only by fueling protein synthesis, but also by supporting the biogenesis of nucleotides, redox factors and lipids. Amino acids are also involved in tricarboxylic acid cycle anaplerosis, epigenetic modifications, next to synthesis of neurotransmitters and hormones. As such, amino acids contribute to a broad range of cellular processes such as proliferation, matrix synthesis and intercellular communication, which are all critical for skeletal cell functioning. Here we summarize recent work elucidating how amino acid metabolism supports and regulates skeletal cell function during bone growth and homeostasis, as well as during skeletal disease. The most extensively studied amino acid is glutamine, and osteoblasts and chondrocytes rely heavily on this non-essential amino acid during for their functioning and differentiation. Regulated by lineage-specific transcription factors such as SOX9 and osteoanabolic agents such as parathyroid hormone or WNT, glutamine metabolism has a wide range of metabolic roles, as it fuels anabolic processes by producing nucleotides and non-essential amino acids, maintains redox balance by generating the antioxidant glutathione and regulates cell-specific gene expression via epigenetic mechanisms. We also describe how other amino acids affect skeletal cell functions, although further work is needed to fully understand their effect. The increasing number of studies using stable isotope labelling in several skeletal cell types at various stages of differentiation, together with conditional inactivation of amino acid transporters or enzymes in mouse models, will allow us to obtain a more complete picture of amino acid metabolism in skeletal cells.

Cell types as species: Exploring a metaphor.

The concept of “cell type,” though fundamental to cell biology, is controversial. Cells have historically been classified into types based on morphology, physiology, or location. More recently, single cell transcriptomic studies have revealed fine-scale differences among cells with similar gross phenotypes. Transcriptomic snapshots of cells at various stages of differentiation, and of cells under different physiological conditions, have shown that in many cases variation is more continuous than discrete, raising questions about the relationship between cell type and cell state. Some researchers have rejected the notion of fixed types altogether. Throughout the history of discussions on cell type, cell biologists have compared the problem of defining cell type with the interminable and often contentious debate over the definition of arguably the most important concept in systematics and evolutionary biology, “species.” In the last decades, systematics, like cell biology, has been transformed by the increasing availability of molecular data, and the fine-grained resolution of genetic relationships have generated new ideas about how that variation should be classified. There are numerous parallels between the two fields that make exploration of the “cell types as species” metaphor timely. These parallels begin with philosophy, with discussion of both cell types and species as being either individuals, groups, or something in between (e.g., homeostatic property clusters). In each field there are various different types of lineages that form trees or networks that can (and in some cases do) provide criteria for grouping. Developing and refining models for evolutionary divergence of species and for cell type differentiation are parallel goals of the two fields. The goal of this essay is to highlight such parallels with the hope of inspiring biologists in both fields to look for new solutions to similar problems outside of their own field.

Evaluation of Interleukin-6 Level Before Chemotherapy in Acute Lymphoblastic Leukemia L1 Standard-Risk and High-risk Patients

BACKGROUND: Acute lymphoblastic leukemia (ALL) is a malignancy originating from the clonal expansion of lymphoid progenitors that have undergone neoplastic transformation at various stages of differentiation. Interleukin-6 (IL-6) is a pleiotropic inflammatory cytokine produced by various cell types, including T-cells, macrophages, and stromal cells in response to tumor necrosis factor-alpha and interleukin-1. AIM: This study aims to evaluate serum levels of IL-6 before chemotherapy in standard-risk (SR) and high-risk (HR) ALL patients. METHODS: The research method was a cross-sectional study conducted on ALL patients who were treated at Dr. Wahidin Sudirohusodo hospital from April 2021 to June 2021. A total of 60 ALL patients were included in the study. Serum IL-6 specimens were examined before the patient received chemotherapy. RESULTS: The results showed that from 60 samples of ALL patients, 30 were SR and 30 were HR. Elevated levels of IL-6 before chemotherapy were found in both SR and HR ALL but were significantly higher in the HR group compared to the SR group with p value = 0.022. The cutoff point of 64.23 ng/mL for HRALL patients was obtained through ROC with a sensitivity of 63.3%, specificity of 63.3%, a positive predictive value of 63.3%, and a negative predictive value of 63.3%. Adjusted odds ratio are 2,983 with 95% confidence interval of 1.044–8.527. CONCLUSION: IL-6 levels among HR ALL were higher than SR. Elevated levels of IL-6 were found in both SR and HR ALL.