Lineage Determination Research Articles

Mechanosensitive Differentiation of Human iPS Cell-Derived Podocytes.

Stem cell fate decisions, including proliferation, differentiation, morphological changes, and viability, are impacted by microenvironmental cues such as physical and biochemical signals. However, the specific impact of matrix elasticity on kidney cell development and function remains less understood due to the lack of models that can closely recapitulate human kidney biology. An established protocol to differentiate podocytes from human-induced pluripotent stem (iPS) cells provides a promising avenue to elucidate the role of matrix elasticity in kidney tissue development and lineage determination. In this study, we synthesized polyacrylamide hydrogels with different stiffnesses and investigated their ability to promote podocyte differentiation and biomolecular characteristics. We found that 3 kPa and 10 kPa hydrogels significantly support the adhesion, differentiation, and viability of podocytes. Differentiating podocytes on a more compliant (0.7 kPa) hydrogel resulted in significant cell loss and detachment. Further investigation of the mechanosensitive proteins yes-associated protein (YAP) and synaptopodin revealed nuanced molecular distinctions in cellular responses to matrix elasticity that may otherwise be overlooked if morphology and cell spreading alone were used as the primary metric for selecting matrices for podocyte differentiation. Specifically, hydrogels with kidney-like rigidities outperformed traditional tissue culture plates at modulating the molecular-level expression of active mechanosensitive proteins critical for podocyte health and function. These findings could guide the development of physiologically relevant platforms for kidney tissue engineering, disease modeling, and mechanistic studies of organ physiology and pathophysiology. Such advances are critical for realizing the full potential of in vitro platforms in accurately predicting human biological responses.

OrganogenesisDB: A Comprehensive Database Exploring the Cell-Type Identities and Gene Expression Dynamics during Organogenesis.

Organogenesis,the phase ofembryonic developmentthat starts at the end ofgastrulationand continues untilbirth is the critical process for understanding cellular differentiation and maturation during organ development. The rapid development of single-cell transcriptomics technology has led to many novel discoveries in understanding organogenesis while also accumulating a large quantity of data. To fill this gap, OrganogenesisDB (http://organogenesisdb.com/), which is a comprehensive database dedicated to exploring cell-type identification and gene expression dynamics during organogenesis, is developed. OrganogenesisDB contains single-cell RNA sequencing data for more than 1.4 million cells from 49 published datasets spanning various developmental stages. Additionally, 3324 cell markers are manually curated for 1120 cell types across 9 human organs and 4 mouse organs. OrganogenesisDB leverages various analysis tools to assist users in annotating and understanding cell types at different developmental stages and helps in mining and presenting genes that exhibit specific patterns and play key regulatory roles during cell maturation and differentiation. This work provides a critical resource and useful tool for deciphering cell lineage determination and uncovering the mechanisms underlying organogenesis.

MiR-1915-3p regulates megakaryocytic and erythroid differentiation by targeting SOCS4

BackgroundProper control of the lineage bias of megakaryocytic and erythroid progenitor cells (MEPs) is of significant importance, the disorder of which will lead to abnormalities in the number and function of platelets and erythrocytes. Unfortunately, the signaling pathways regulating MEP differentiation largely remain to be elucidated. This study aimed to analyze the role and the underlying molecular mechanism of miR-1915-3p in megakaryocytic and erythroid differentiation.MethodsWe utilized miRNA mimics and miRNA sponge to alter the expression of miR-1915-3p in megakaryocytic and/or erythroid potential cells; siRNA and overexpression plasmid to change the expression of SOCS4, a potential target of miR-1915-3p. The expression of relevant surface markers was detected by flow cytometry. We scanned for miR-1915-3p target genes by mRNA expression profiling and bioinformatic analysis, and confirmed the targeting by dual-luciferase reporter assay, western blot and gain- and lost-of-function studies. One-way ANOVA and t-test were used to analyze the statistical significance.ResultsIn this study, overexpression or knockdown of miR-1915-3p inhibited or promoted erythroid differentiation, respectively. Accordingly, we scanned for miR-1915-3p target genes and confirmed that SOCS4 is one of the direct targets of miR-1915-3p. An attentive examination of the endogenous expression of SOCS4 during megakaryocytic and erythroid differentiation suggested the involvement of SOCS4 in erythroid/megakaryocytic lineage determination. SOCS4 knockdown lessened erythroid surface markers expression, as well as improved megakaryocytic differentiation, similar to the effects of miR-1915-3p overexpression. While SOCS4 overexpression resulted in reversed effects. SOCS4 overexpression in miR-1915-3p upregulated cells rescued the effect of miR-1915-3p.ConclusionsmiR-1915-3p acts as a negative regulator of erythropoiesis, and positively in thrombopoiesis. SOCS4 is one of the key mediators of miR-1915-3p during the differentiation of MEPs.

Abstract We029: Common and Divergent Cellular Etiologies Underlying Hypoplastic Left Heart Syndrome and Hypoplastic Right Heart Syndrome

Single ventricle heart defects are the most severe forms of congenital heart defects and are classified based on the affected ventricles: hypoplastic left heart syndrome (HLHS) and hypoplastic right heart syndrome (HRHS). Cellular etiologies underlying differential left and right ventricular hypoplasia are unknown and remain intractable without an experimental model to probe common and divergent cellular etiologies between HLHS and HRHS. In this study, we leveraged patient-specific induced pluripotent stem cells (iPSCs) and single-cell transcriptomics to interrogate distinct etiologies of HLHS and HRHS. Both HLHS and HRHS iPSC-derived cardiomyocytes (iPSC-CMs) exhibit a reduced proliferation capacity compared to sex-matched family controls under both static and cyclic stretch conditions. Biological pathways related to cell cycle progression, DNA replication, and cell proliferation are downregulated in both HLHS and HRHS iPSC-CMs, suggesting an intrinsic cardiac proliferation deficiency. Single-cell transcriptomics indicate that differentiation of cardiac mesoderm towards second heart field (SHF) progenitors are compromised in both HLHS and HRHS. However, differentiation of epicardial progenitors is enhanced at the expense of SHF progenitors in HLHS, whereas first heart field (FHF) progenitors are prevalent in HRHS. Trajectory inference analysis uncovers distinct cell lineage determination pathways in FHF and SHF progenitors between HLHS and HRHS. Moreover, HLHS iPSC-CMs exhibit reduced mitochondrial activities whereas HRHS iPSC-CMs show enhanced mitochondrial respiration and ATP production compared to controls. In sum, these common and divergent cellular etiologies of HLHS and HRHS may underlie ventricular hypoplasia in the left side and right side of the heart.

Abstract We091: Maternal hyperglycemia disrupts human cardiac cell lineage differentiation

Maternal diabetes is associated with a 4-fold increased risk of offspring developing congenital heart defects (CHDs). It remains unknown how maternal diabetes interferes with cardiac cell lineage determination and leads to malformations in the heart. In this study, we aim to elucidate the cellular mechanisms by which maternal hyperglycemia causes high risk of CHDs in newborns. Here we leverage an in vitro hyperglycemic model using human induced pluripotent stem cells (iPSCs), which could recapture cardiac differentiation and cell lineage commitment during embryonic heart development. We collected differentiating cells at D5 (cardiac mesoderm), D10 (cardiac progenitors), and D14 (early cardiomyocytes) during cardiac differentiation under normal and hyperglycemic conditions, and performed single-cell transcriptomic analysis. We found that hyperglycemia significantly impedes cardiac differentiation of human iPSCs as robust cardiac differentiation is rarely observed in multiple iPSC lines (n=10) under hyperglycemia. At the cellular level, hyperglycemia interferes with cardiac differentiation of human iPSCs in response to WNT signaling activation, which is manifested by reduced number of cardiac mesoderm (MESP1+ PDGFRA+) cells at D5 of differentiation. In contrast, neural differentiation is enhanced under hyperglycemia, with a high proportion of neural cell lineage (SOX2+ PAX6+). At D10, differentiated neural cell lineage dominates the cell population under hyperglycemia at the expense of cardiac progenitors and early cardiomyocytes. At D14, the prevalence of neural lineage persist whereas early cardiomyocytes only accounts for a small portion of cell population under hyperglycemia. Moreover, we treated D30 iPSC-derived cardiomyocytes (iPSC-CMs) with high glucose concentration (25 mM) for 7 days and found that iPSC-CMs show reduced mitochondria respiration and ATP production, but elevated apoptosis and reactive oxygen species (ROS) generation. Together, our data suggest that maternal hyperglycemia could interrupt human embryonic heart morphogenesis through overriding WNT-medicated cardiac differentiation and promoting neural cell lineage determination by default.

Investigating InDels in YAP and TAZ genes and their impact on growth characteristics in goats

Abstract. Yes-associated protein (YAP) and a transcriptional co-activator with PDZ-binding motif (TAZ) genes are crucial for regulating the size of mammalian tissues and organs as well as for many biological processes such as bone formation, cell lineage determination, tissue regeneration, and cell proliferation. The purpose of this study was to characterize the YAP and TAZ gene polymorphisms in 266 Guanzhong Dairy Goats and 299 Shanbei White Cashmere Goats and to explore their potential relationship with growth characteristics such as body weight and body length. After genotyping and using PCR amplification and Sanger sequencing to find polymorphisms in the YAP and TAZ genes, five InDels loci were found in the goat YAP gene and three InDels loci in the TAZ gene. The findings of the association analysis demonstrated that the goats' body weight, height, cannon circumference, chest depth, chest breadth, and chest circumference were all substantially influenced by five InDels loci in the YAP gene (p<0.05). Goat body height, trunk breadth, trunk length, body length, and body weight were all substantially impacted by three InDels loci in the TAZ gene (p<0.05). In conclusion, eight InDels loci of goat YAP and TAZ were found in this study, and their impacts on goat phenotype were disclosed. These results might offer fresh avenues for boosting goat molecular breeding.

PATH-02. THE IMPACT OF EVOLVING DIAGNOSTIC METHODOLOGY ON THE CLASSIFICATION AND OUTCOME OF CENTRAL NERVOUS SYSTEM TUMOURS IN CHILDHOOD: A RETROSPECTIVE COHORT STUDY

Abstract BACKGROUND Central nervous system tumours are the second most common childhood tumours and the largest cause of childhood cancer death in Australia. Tumours arise from many cell types within the developing and mature CNS, leading to a wide spectrum of clinical behaviour despite overlapping histological appearances. Emerging molecular testing methods allow more precise determination of cell lineage as well as genetic and epigenetic changes driving tumour development. Particularly since 2016, molecular findings have been incorporated into international classification schema, potentially impacting the frequency of individual diagnoses and their observed prognosis. METHODS We performed a retrospective cohort study including patients with newly diagnosed CNS tumours at Royal Children’s Hospital, Melbourne between 2011 and 2020 (total 473). The first and last 100 eligible patients in the data set were reviewed for diagnosis, survival and recorded use of molecular testing. Reviewed patients were assigned to one of two cohorts, Cohort 1 2011-2013 (pre-2016 WHO revision) or Cohort 2 2018-2020 (post-2016 WHO revision). RESULTS The frequency of diagnoses on biopsy rather than imaging alone was stable (81% Cohort 1 vs 83% Cohort 2). Molecular testing utilization increased markedly over time (10 molecular investigations in Cohort 1 vs 348 in Cohort 2). The incidence of astrocytic and oligodendroglial diagnoses was stable. The incidence of embryonal and neuronal/ mixed glial neuronal diagnoses increased in Cohort 2, and the incidence of ependymal diagnoses decreased (p=0.0122). There were no event free or overall survival differences between the whole of Cohorts 1 and 2, with numbers of individual diagnoses too small for meaningful comparisons over time. CONCLUSIONS Access to molecular testing has impacted on the pattern of reported diagnoses of central nervous system tumours in children. Although outcomes have not changed, it is hoped that over time, improved molecular understanding will lead to more effective therapy selection.

MDB-26. GENE REGULATORY NETWORK LANDSCAPE OF GROUP 3/4 MEDULLOBLASTOMA

Abstract Intra-tumor heterogeneity in brain cancers contributes to therapy resistance and relapse through positive selection of resistant tumor cell populations. Resolving the molecular mechanisms driving intra-tumor heterogeneity will uncover tumor-specific vulnerabilities and advance effective treatment strategies. Group 3/4 medulloblastomas, the most common embryonal cerebellar tumor subgroup, are highly heterogeneous tumors with high frequency of relapse which are almost certainly fatal. Here, we generated and analyzed a comprehensive single-cell multi-omic Group 3/4 medulloblastoma atlas, comprising 355,295 cells from 38 tumor patients representing eight Group 3/4 molecular subtypes. We identified transcription factor gene regulatory networks driving intra- and inter-tumor heterogeneity in Group 3/4 medulloblastoma, and determined the common regulatory framework that drives this heterogeneity. We show that Group 3/4 tumor diversity stems from differential enrichment of cell-states along four molecular identity axes: photoreceptor, MYC, precursor, and unipolar brush cell-like. Further, we show that intermediate subtype I/VII tumors exhibit a unique biology where a tumor exhibits dual Group 3 and Group 4-like tumor cell-states in a single tumor, and for a subset of these tumors, we identified a potential role of PAX6 in driving the observed dual trajectory. Altogether, our study demonstrates how oncogenic events together with key lineage determinants hijack normal developmental programs and drive Group 3/4 tumor identity away from the normal cerebellar unipolar brush cell-lineage.

Regulation of mesenchymal stem cell differentiation by autophagy.

Autophagy, a process that isolates intracellular components and fuses them with lysosomes for degradation, plays an important cytoprotective role by eliminating harmful intracellular substances and maintaining cellular homeostasis. Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the capacity for self-renewal that can give rise to a subset of tissues and therefore have potential in regenerative medicine. However, a variety of variables influence the biological activity of MSCs following their proliferation and transplantation in vitro. The regulation of autophagy in MSCs represents a possible mechanism that influences MSC differentiation properties under the right microenvironment, affecting their regenerative and therapeutic potential. However, a deeper understanding of exactly how autophagy is mobilized to function as well as clarifying the mechanisms by which autophagy promotes MSCs differentiation is still needed. Here, we review the current literature on the complex link between MSCs differentiation and autophagy induced by various extracellular or intracellular stimuli and the molecular targets that influence MSCs lineage determination, which may highlight the potential regulation of autophagy on MSCs' therapeutic capacity, and provide a broader perspective on the clinical application of MSCs in the treatment of a wide range of diseases.

Sequencing analysis of SARS-CoV-2 cases in Slovenian long-term care facilities to support outbreak control.

Residents of long-term care facilities (LTCFs) are at high risk of morbidity and mortality due to COVID-19, especially when new variants of concern (VOC) emerge. To provide intradisciplinary data in order to tailor public health interventions during future epidemics, available epidemiologic and genomic data from Slovenian LTCFs during the initial phases of the COVID-19 pandemic was analyzed. The first part of the study included SARS-CoV-2 reverse-transcription Real-Time PCR (rtRT-PCR) positive LTCF residents, from 21 facilities with COVID-19 outbreaks occurring in October 2020. The second part of the study included SARS-CoV-2 rtRT-PCR positive LTCF residents and staff between January and April 2021, when VOC Alpha emerged in Slovenia. Next-generation sequencing (NGS) was used to acquire SARS-CoV-2 genomes, and lineage determination. In-depth phylogenetic and mutational profile analysis were performed and coupled with available field epidemiological data to assess the dynamics of SARS-CoV-2 introduction and transmission. 370/498 SARS-CoV-2 positive residents as well as 558/699 SARS-CoV-2 positive residents and 301/358 staff were successfully sequenced in the first and second part of the study, respectively. In October 2020, COVID-19 outbreaks in the 21 LTCFs were caused by intra-facility transmission as well as multiple independent SARS-CoV-2 introductions. The Alpha variant was confirmed in the first LTCF resident approximately 1.5 months after the first Alpha case was identified in Slovenia. The data also showed a slower replacement of existing variants by Alpha in residents compared to staff and the general population. Multiple SARS CoV-2 introductions as well as intra-facility spreading impacted disease transmission in Slovenian LTCFs. Timely implementation of control measures aimed at limiting new introductions while controlling in-facility transmission are of paramount importance, especially as new VOCs emerge. Sequencing, in conjunction with epidemiological data, can facilitate the determination of the need for future improvements in control measures to protect LTCF residents from COVID-19 or other respiratory infections.

Species Delimitation of Some Melanargia Species (Lepidoptera, Nymphalidae, Satyrinae) in The Southeastern Anatolia Region Based On The mtCOI Gene

Among the Palearctic species, butterflies of the genus Melanargia are known for their black and white wing patterns. The morphological character polarization of this genus is full of varying combinations of subgenus, species complex, and subspecies status. Its taxonomy is open to debate, especially in species and subspecies categories, with definitions mostly based on wing color. In recent years, cryptic species, phenotypically masked species, and species with intense intraspecific variation have been identified through the determination of lineages under the leadership of molecular systematics. The mtCOI gene, which is especially described as a species signature, is an important DNA barcode used for Lepidoptera. In the presented study, the mtCOI gene sequence of the populations of Melanargia larissa, M.grumi, M.hylata, M.syriaca, and M.russiae species in the southeastern Anatolia region was determined for the first time. To determine the boundaries of these species, gene characterization and genetic distances were carried out according to the Kimura-2 Parameter, and putative species analyses were carried out by the ABGD method. Trees were constructed with Maximum likelihood and Bayesian inference algorithms to determine the phylogenetic relationships between species of the genus. In light of these analyses, it has been shown that the genetic distance of morphological species M. larissa, M. grumi, M.hylata, and M. syriaca is not at the species level and that M.larissa maintains its species status according to the principle of priority. In addition, the M.russiae population presented in this study forms a monophyletic clade with other populations of the same species in the phylogenetic tree, proving that this taxon is a stable species.

Small-molecule α-lipoic acid targets ELK1 to balance human neutrophil and erythrocyte differentiation

BackgroundErythroid and myeloid differentiation disorders are commonly occurred in leukemia. Given that the relationship between erythroid and myeloid lineages is still unclear. To find the co-regulators in erythroid and myeloid differentiation might help to find new target for therapy of myeloid leukemia. In hematopoiesis, ALA (alpha lipoic acid) is reported to inhibit neutrophil lineage determination by targeting transcription factor ELK1 in granulocyte-monocyte progenitors via splicing factor SF3B1. However, further exploration is needed to determine whether ELK1 is a common regulatory factor for erythroid and myeloid differentiation.MethodsIn vitro culture of isolated CD34+, CMPs (common myeloid progenitors) and CD34+ CD371– HSPCs (hematopoietic stem progenitor cells) were performed to assay the differentiation potential of monocytes, neutrophils, and erythrocytes. Overexpression lentivirus of long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 transduced CD34+ HSPCs were transplanted into NSG mice to assay the human lymphocyte and myeloid differentiation differences 3 months after transplantation. Knocking down of SRSF11, which was high expressed in CD371+GMPs (granulocyte-monocyte progenitors), upregulated by ALA and binding to ELK1-RNA splicing site, was performed to analyze the function in erythroid differentiation derived from CD34+ CD123mid CD38+ CD371– HPCs (hematopoietic progenitor cells). RNA sequencing of L-ELK1 and S-ELK1 overexpressed CD34+ CD123mid CD38+ CD371– HPCs were performed to assay the signals changed by ELK1.ResultsHere, we presented new evidence that ALA promoted erythroid differentiation by targeting the transcription factor ELK1 in CD34+ CD371– hematopoietic stem progenitor cells (HSPCs). Overexpression of either the long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 inhibited erythroid-cell differentiation, but knockdown of ELK1 did not affect erythroid-cell differentiation. RNAseq analysis of CD34+ CD123mid CD38+ CD371– HPCs showed that L-ELK1 upregulated the expression of genes related to neutrophil activity, phosphorylation, and hypoxia signals, while S-ELK1 mainly regulated hypoxia-related signals. However, most of the genes that were upregulated by L-ELK1 were only moderately upregulated by S-ELK1, which might be due to a lack of serum response factor interaction and regulation domains in S-ELK1 compared to L-ELK1. In summary, the differentiation of neutrophils and erythrocytes might need to rely on the dose of L-ELK1 and S-ELK1 to achieve precise regulation via RNA splicing signals at early lineage commitment.ConclusionsALA and ELK1 are found to regulate both human granulopoiesis and erythropoiesis via RNA spliceosome, and ALA-ELK1 signal might be the target of human leukemia therapy.

Abstract 1707: YAP-driven epigenetic reprogramming in head and neck cancer

Abstract The ability to measure tumor evolution is difficult due to a lack of model systems able to capture tumor initiation, development, and invasion. We have developed a Dox-inducible spatiotemporally controlled HPV- and YAP-driven model of head and neck squamous cell carcinoma (HNSCC) with a well-defined cancer stem cell population. In order to better understand the epigenetic reprogramming associated with tumorigenesis we have systematically characterized the chromatin accessibility, histone landscape and YAP binding sites in this model. Cell lines were generated from tongue epithelia of normal and Dox-induced tumor-containing mice. Gene expression was measured using RNA-seq and chromatin accessibility was measured using ATAC-seq. CUT&Tag for H3K27me3, H3K27ac, and YAP was used to measure epigenetic reprogramming and YAP-DNA interactions. Differential gene expression shows that genes involved in hormone signaling, glutathione-related metabolism, fatty acid metabolism and p53 signaling were highly enriched in the normal tongue suggesting a change in metabolism upon tumorigenesis.Differential ATAC-seq peaks with higher binding levels in the normal tongue showed enrichment for p53 motifs as well as homebox motifs including Otx2, GSC and CRX suggesting p53 signaling is lost upon carcinogenesis and lineage determination is altered. Motifs for TEAD, AP-1, and KLF5 were enriched in the tumor-specific ATAC-seq peaks showing that YAP is involved in chromatin accessibility changes. CUT&Tag for YAP shows clear YAP binding near canonical YAP target genes including Axl, Ccn1, Ccn2 and Birc3. YAP peaks with higher binding in the normal tongue cell line were enriched for RUNX2 and p63 motifs and peaks higher in the tumor contained KLF5 motifs showing YAP is directly involved in the reprogramming of lineage determination. Multi-omics integration shows YAP-driven chromatin remodeling is associated with differential expression of genes involved in keratin production, EGFR and ER signaling. Direct epigenetic characterization of YAP involvement in tumor initiation is key to understanding the mechanisms of tumorigenesis in head and neck cancer. This work shows that YAP directly binds and affects the expression of genes involved in lineage determination and growth factor signaling to induce cancer progression and generate a cancer stem cell population. Citation Format: Adam Officer, Farhoud Faraji, Sydney Ramirez, Alon Goren, Pablo Tamayo, J. Silvio Gutkind. YAP-driven epigenetic reprogramming in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1707.

A let-7 microRNA-RALB axis links the immune properties of iPSC-derived megakaryocytes with platelet producibility

We recently achieved the first-in-human transfusion of induced pluripotent stem cell-derived platelets (iPSC-PLTs) as an alternative to standard transfusions, which are dependent on donors and therefore variable in supply. However, heterogeneity characterized by thrombopoiesis-biased or immune-biased megakaryocytes (MKs) continues to pose a bottleneck against the standardization of iPSC-PLT manufacturing. To address this problem, here we employ microRNA (miRNA) switch biotechnology to distinguish subpopulations of imMKCLs, the MK cell lines producing iPSC-PLTs. Upon miRNA switch-based screening, we find imMKCLs with lower let-7 activity exhibit an immune-skewed transcriptional signature. Notably, the low activity of let-7a-5p results in the upregulation of RAS like proto-oncogene B (RALB) expression, which is crucial for the lineage determination of immune-biased imMKCL subpopulations and leads to the activation of interferon-dependent signaling. The dysregulation of immune properties/subpopulations, along with the secretion of inflammatory cytokines, contributes to a decline in the quality of the whole imMKCL population.

Utilizing Science and Maqāṣid al-Sharī’ah in Resolving Contemporary Issues of Islamic Family Law

Contemporary ijtihad is required in the formulation of new formulas, such as legal protection of the rights of children outside marriage whose biological father is known. Reformulation of Jurisprudence as an analysis of Islamic law must be able to answer socio-cultural problems in the modern era, such as proof of the use of DNA as genealogy. An adaptive formulation of Jurisprudence on contemporary cases by combining science and Maqaṣid al-Shari'ah is analyzed in this article. The resolution of Islamic law by referring to the opinions of the Four Madhhabs of Jurisprudence needs to be approached or juxtaposed with science. A correct understanding of the values contained in the sharia and the application of Maqaṣid al-Shari'ah are indispensable in contemporary ijtihad so that the face of Jurisprudence can dialect with the times. So that the determination of lineage by DNA can be used as a legal reference that is adaptive to science and in accordance with the great objectives of Maqaṣid al-Shari'ah.

Improved Neural Inductivity of Size-Controlled 3D Human Embryonic Stem Cells Using Magnetic Nanoparticles.

Background: To improve the efficiency of neural development from human embryonic stem cells, human embryoid body (hEB) generation is vital through 3-dimensional formation. However, conventional approaches still have limitations: long-term cultivation and laborious steps for lineage determination. Methods: In this study, we controlled the size of hEBs for ectodermal lineage specification using cell-penetrating magnetic nanoparticles (MNPs), which resulted in reduced time required for initial neural induction. The magnetized cells were applied to concentrated magnetic force for magnet-derived multicellular organization. The uniformly sized hEBs were differentiated in neural induction medium (NIM) and suspended condition. This neurally induced MNP-hEBs were compared with other groups. Results: As a result, the uniformly sized MNP-hEBs in NIM showed significantly improved neural inductivity through morphological analysis and expression of neural markers. Signaling pathways of the accelerated neural induction were detected via expression of representative proteins; Wnt signaling, dopaminergic neuronal pathway, intercellular communications, and mechanotransduction. Consequently, we could shorten the time necessary for early neurogenesis, thereby enhancing the neural induction efficiency. Conclusion: Overall, this study suggests not only the importance of size regulation of hEBs at initial differentiation stage but also the efficacy of MNP-based neural induction method and stimulations for enhanced neural tissue regeneration.

Exploring the intricate cross-talk between clonal expansion and the bone marrow niche

Haematopoietic stem cells (HSCs) reside within an intricate network of cells in the bone marrow (BM) niche. HSC crosstalk with niche compartments influences lineage determination and blood cell production, while independent niche interactions are essential for the maintenance of HSC quiescence. How different niche components influence the genetic diversity of HSCs represents an expanding field of investigation. As such, we will summarise the current knowledge of the contribution to the Darwinian evolution of mutant HSCs of both haematopoietic and non-haematopoietic cells residing in the BM. In this review, we will disentangle how somatic evolution associates with the niche at two stages: from (1) the stage of preleukaemic HSC expansion and clonal haematopoiesis (CH) to (2) leukaemia-initiating cells (LICs) and the development of myeloid malignancies with acute myeloid leukaemia (AML) being the most prevalent. We will finally describe current challenges such as limitations in models used in the field or the difficulty in studying specific genetic clones in isolation.

Utilizing cost-effective portable equipment to enhance COVID-19 variant tracking both on-site and at a large scale.

Despite optimistic predictions on the eventual end of COVID-19 (Coronavirus Disease 2019), caution is necessary regarding the emergence of new variants to sustain a positive outlook and effectively address any potential future outbreaks. However, ongoing efforts to track COVID-19 variants are concentrated in developed countries and unique social practices and remote habitats of indigenous peoples present additional challenges. By combining small-sized equipment that is easily accessible and inexpensive, we performed SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) whole genome sequencing and measured the sample-to-answer time and accuracy of this portable variant tracking tool. Our portable design determined the variant of SARS-CoV-2 in an infected individual within 9 hours and 15 minutes without external power or internet connection, surpassing the speed of previous portable tools. It took only 16 minutes to complete sequencing run, whole genome assembly, and lineage determination using a single standalone laptop. We then demonstrated the capability to produce 289 SARS-CoV-2 whole genome sequences in a single portable sequencing run, representing a significant improvement over an existing throughput of 96 sequences per run. We verified the accuracy of portable sequencing by comparison with two other independent sequencing methods. We showed that our high-throughput data consistently represented the circulating variants in Los Angeles, United States, when compared with publicly available sequences. Our scheme is designed to be flexible, rapid, and accurate, making it a valuable tool for large-scale surveillance operations in low- and middle-income countries as well as targeted surveys for vulnerable populations in remote locations.IMPORTANCEThere have been significant efforts to track COVID-19 (Coronavirus Disease 2019) variants, accumulating over 15 million SARS-CoV-2 sequences as of 2023. However, the distribution of global survey data is highly skewed, with nearly half of all countries having inadequate or low levels of genomic surveillance. In addition, indigenous peoples face more severe threats from COVID-19, due to their generally remote residence and unique social practices. Cost-effective portable sequencing tools have been used to investigate Ebola and Zika outbreaks. However, these tools have a sample-to-answer time of around 24 hours and require an internet connection for data transfer to an off-site cloud server. In our study, we rapidly determined COVID-19 variants using only small and inexpensive equipment, with a completion time of 9 hours and 15 minutes. Furthermore, we produced 289 near-full-length SARS-CoV-2 genome sequences from a single portable Nanopore sequencing run, representing a threefold increase in throughput compared with existing Nanopore sequencing methods.

The regulation and differentiation of regulatory T cells and their dysfunction in autoimmune diseases.

The discovery of FOXP3+ regulatory T (Treg)cells as a distinct cell lineage with a central role in regulating immune responses provided a deeper understanding of self-tolerance. The transcription factor FOXP3 serves a key role in Treg cell lineage determination and maintenance, but is not sufficient to enable the full potential of Treg cell suppression, indicating that other factors orchestrate the fine-tuning of Treg cell function. Moreover, FOXP3-independent mechanisms have recently been shown to contribute to Treg cell dysfunction. FOXP3 mutations in humans cause lethal fulminant systemic autoinflammation (IPEX syndrome). However, it remains unclear to what degree Treg cell dysfunction is contributing to the pathophysiology of common autoimmune diseases. In this Review, we discuss the origins of Treg cells in the periphery and the multilayered mechanisms by which Treg cells are induced, as well as the FOXP3-dependent and FOXP3-independent cellular programmes that maintain the suppressive function of Treg cells in humans and mice. Further, we examine evidence for Treg cell dysfunction in the context of common autoimmune diseases such as multiple sclerosis, inflammatory bowel disease, systemic lupus erythematosus and rheumatoid arthritis.

Systematic SARS-CoV-2 S-gene sequencing in wastewater samples enables early lineage detection and uncovers rare mutations in Portugal

As the COVID-19 pandemic reached its peak, many countries implemented genomic surveillance systems to track the evolution and transmission of SARS-CoV-2. Transition from the pandemic to the endemic phase prioritized alternative testing strategies to maintain effective epidemic surveillance at the population level, with less intensive sequencing efforts. One such promising approach was Wastewater-Based Surveillance (WBS), which offers non-invasive, cost-effective means for analysing virus trends at the sewershed level. From 2020 onwards, wastewater has been recognized as an instrumental source of information for public health, with national and international authorities exploring options to implement national wastewater surveillance systems and increasingly relying on WBS as early warning of potential pathogen outbreaks. In Portugal, several pioneer projects joined the academia, water utilities and Public Administration around WBS.To validate WBS as an effective genomic surveillance strategy, it is crucial to collect long term performance data. In this work, we present one year of systematic SARS-CoV-2 wastewater surveillance in Portugal, representing 35 % of the mainland population. We employed two complementary methods for lineage determination - allelic discrimination by RT-PCR and S-gene sequencing. This combination allowed us to monitor variant evolution in near-real-time and identify low-frequency mutations.Over the course of this year-long study, spanning from May 2022 to April 2023, we successfully tracked the dominant Omicron sub-lineages, their progression and evolution, which aligned with concurrent clinical surveillance data. Our results underscore the effectiveness of WBS as a tracking system for virus variants, with the ability to unveil mutations undetected via massive sequencing of clinical samples from Portugal, demonstrating the ability of WBS to uncover new mutations and detect rare genetic variants.Our findings emphasize that knowledge of the genetic diversity of SARS-CoV-2 at the population level can be extended far beyond via the combination of routine clinical genomic surveillance with wastewater sequencing and genotyping.