Hepatic stellate cells in zone 1 engage in capillarization rather than myofibroblast formation in murine liver fibrosis.

The combination of lineage tracing and immunohistochemistry has helped to identify subpopulations and fate of hepatic stellate cells (HSC) in murine liver. HSC are sinusoidal pericytes that act as myofibroblast precursors after liver injury. Single cell RNA sequencing approaches have recently helped to differentiate central and portal HSC. A specific Cre line to lineage trace portal HSC has not yet been described. We used three Cre lines (Lrat-Cre, PDGFRß-CreERT2 and SMMHC-CreERT2) known to label mesenchymal cells including HSC in combination with a tdTomato-expressing reporter. All three Cre lines labeled populations of HSC as well as smooth muscle cells (SMC). Using the SMMHC-CreERT2, we identified a subtype of HSC in the periportal area of the hepatic lobule (termed zone 1-HSC). We lineage traced tdTomato-expressing zone 1-HSC over 1 year, described fibrotic behavior in two fibrosis models and investigated their possible role during fibrosis. This HSC subtype resides in zone 1 under healthy conditions; however, zonation is disrupted in preclinical models of liver fibrosis (CCl4 and MASH). Zone 1-HSC do not transform into αSMA-expressing myofibroblasts. Rather, they participate in sinusoidal capillarization. We describe a novel subtype of HSC restricted to zone 1 under physiological conditions and its possible function after liver injury. In contrast to the accepted notion, this HSC subtype does not transform into αSMA-positive myofibroblasts; rather, zone 1-HSC adopt properties of capillary pericytes, thereby participating in sinusoidal capillarization.

Identification of Meibomian gland stem cell populations and mechanisms of aging.

Meibomian glands secrete lipid-rich meibum, which prevents tear evaporation. Aging-related Meibomian gland shrinkage may result in part from stem cell exhaustion and is associated with evaporative dry eye disease, a common condition lacking effective treatment. The identities and niche of Meibomian gland stem cells and the signals controlling their activity are poorly defined. Using snRNA-seq, in vivo lineage tracing, ex vivo live imaging, and genetic studies in mice, we identified markers for stem cell populations that maintain distinct regions of the gland and uncovered Hh signaling as a key regulator of stem cell proliferation. Consistent with this, human Meibomian gland carcinoma exhibited increased Hh signaling. Aged glands displayed decreased Hh and EGF signaling, deficient innervation, and loss of collagen I in niche fibroblasts, indicating that alterations in both glandular epithelial cells and their surrounding microenvironment contribute to age-related degeneration. These findings suggest new approaches to treat aging-associated Meibomian gland loss.

Anatomy, Histology, and Embryonic Origin of Adipose Tissue: Insights to Understand Adipose Tissue Homofunctionality in Regeneration and Therapies.

Preadipocytes are formed during the 14th and 16th weeks of gestation. White adipose tissue, in particular, is generated in specific areas and thereby assembles after birth, rapidly increasing following the propagation of adipoblasts, which are considered the preadipocyte cell precursors. The second trimester of gestation is a fundamental phase of adipogenesis, and in the third trimester, adipocytes, albeit small may be present within the main deposition areas. In the course of late gestation, adipose tissue develops in the foetus and promotes the synthesis of large amounts of uncoupling protein 1, in similar quantities relative to differentiated brown adipose tissue. In mammals, differentiation occurs in two functionally different types of adipose cells: white adipose cells resulting from lipid storage and brown adipose cells from increased metabolic energy consumption. During skeletogenesis, synovial joints develop through the condensation of mesenchymal cells, which forms an insertional layer of flattened cells that umlaut skeletal elements, by sharing the same origin in the development of synovium. Peri-articular fat pads possess structural similarity with body subcutaneous white adipose tissue; however, they exhibit a distinct metabolic function due to the micro-environmental cues in which they are embedded. Fat pads are an important component of the synovial joint and play a key role in the maintenance of joint homeostasis. They are also implicated in pathological states such as osteoarthritis.In this paper we explore the therapeutic potential of adipocyte tissue mesenchymal precursor-based stem cell therapy linking it back to the anatomic origin of adipose tissue.

Targeted next-generation sequencing of Mycobacterium tuberculosis from patient samples: lessons learned from high drug-resistant burden clinical settings in Bangladesh.

AbstractLack of appropriate early diagnostic tools for drug-resistant tuberculosis (DR-TB) and their incomplete drug susceptibility testing (DST) profiling is concerning for TB disease control. Existing methods, such as phenotypic DST (pDST), are time-consuming, while Xpert MTB/RIF (Xpert) and line probe assay (LPA) are limited to detecting resistance to few drugs. Targeted next-generation sequencing (tNGS) has been recently approved by WHO as an alternative approach for rapid and comprehensive DST. We aimed to investigate the performance and feasibility of tNGS for detecting DR-TB directly from clinical samples in Bangladesh. pDST, LPA and tNGS were performed among 264 sputum samples, either rifampicin-resistant (RR) or rifampicin-sensitive (RS) TB cases confirmed by Xpert assay. Resistotypes of tNGS were compared with pDST, LPA and composite reference standard (CRS, resistant if either pDST or LPA showed a resistant result). tNGS results revealed higher sensitivities for rifampicin (RIF) (99.3%), isoniazid (INH) (96.3%), fluoroquinolones (FQs) (94.4%), and aminoglycosides (AMGs) (100%) but comparatively lower for ethambutol (76.6%), streptomycin (68.7%), ethionamide (56.0%) and pyrazinamide (50.7%) when compared with pDST. The sensitivities of tNGS for INH, RIF, FQs and AMGs were 93.0%, 96.6%, 90.9%, and 100%, respectively and the specificities ranged from 91.3% to 100% when compared with CRS. This proof of concept study, conducted in a high-burden setting demonstrated that tNGS is a valuable tool for identifying DR-TB directly from the clinical specimens. Its feasibility in our laboratory suggests potential implementation and moving tNGS from research settings into clinical settings.

Prognostic value of a novel myeloid-to-lymphoid ratio biomarker in advanced gastric cancer.

BACKGROUND: Currently, immune checkpoint inhibitors (ICIs) have excellent performance in the clinical treatment of advanced gastric cancer (AGC). However, precisely selecting AGC patients who can benefit from immunotherapy is an urgent difficulty. In this study, we investigated the immunoprognostic role of myeloid-to-lymphocyte ratio (M:L) in AGC patients. METHODS: We collected information on 268 AGC patients who were hospitalized in the Department of Medical Oncology of PLA General Hospital from December 2014 to May 2021. The patients were divided into low M: L group (< 3.76) and high M:L group (≥ 3.76). Survival differences between different M: L level groups at baseline and after treatment were analyzed by methods such as Kaplan-Meier, Cox or Logistic regression model. RESULTS: Progression free survival (PFS) (5.8 months vs. 3.4 months, p = 0.001) and overall survival (OS) (14.1 months vs. 9.0 months, p = 0.001) were significantly longer in the low M:L group than in the high M:L group. After analyses of Cox regression modeling it was concluded that M:L was an independent prognostic factor for PFS (HR 1.371 95%CI 1.057-1.777 p = 0.017) and OS (HR 1.352 95%CI 1.003-1.824 p = 0.048), respectively. Subsequent subgroup analyses performed across immunotherapy lines, regimens, PD-1 inhibitor agents, and age groups revealed a poorer prognosis in the high M:L group. Notably, an increase in the value of M:L after treatment significantly increased the risk of poor prognosis. CONCLUSIONS: M:L ≥ 3.76 is associated with poor prognostic outcomes in AGC patients receiving immunotherapy and may be a predictive biomarker of prognosis. This result needs to be confirmed by larger prospective studies.

The cell fates of intermediate cell population in prostate development.

Organ development, regeneration and cancer initiation are typically influenced by the proliferation and lineage plasticity of tissue-specific stem cells. Prostate intermediate cells, which exhibit characteristics of both basal and luminal cells, are prevalent in pathological states and during organ development. However, the identity, fate and function of these intermediate cells in prostate development are not well understood. Through single-cell RNA-seq analysis on neonatal urogenital sinus tissue, we identified intermediate cells exhibiting stem cell potential. A notable decline in the population of intermediate cells was observed during prostate development. Prostate intermediate cells were specifically labeled in early and late postnatal development by the enhanced dual-recombinase-mediated genetic tracing systems. Our findings revealed that these cells possess significant stem cell capabilities as demonstrated in organoid formation and cell fate mapping assays. These intermediate cells also exhibited intrinsic bipotential properties, enabling them to differentiate into both basal and luminal cells. Additionally, we discovered a novel transition from intermediate cell expressing neuroendocrine markers to neuroendocrine cell during prostate development. This study highlights intermediate cells as a crucial stem cell population and enhances our understanding of their role in prostate development and the plasticity of prostate cancer lineage.

Baf155 controls hematopoietic differentiation and regeneration through chromatin priming.

Chromatin priming promotes cell-type-specific gene expression, lineage differentiation, and development. The mechanism of chromatin priming has not been fully understood. Here, we report that mouse hematopoietic stem and progenitor cells (HSPCs) lacking the Baf155 subunit of the BAF (BRG1/BRM-associated factor) chromatin remodeling complex produce a significantly reduced number of mature blood cells, leading to a failure of hematopoietic regeneration upon transplantation and 5-fluorouracil (5-FU) injury. Baf155-deficient HSPCs generate particularly fewer neutrophils, B cells, and CD8+ T cells at homeostasis, supporting a more immune-suppressive tumor microenvironment and enhanced tumor growth. Single-nucleus multiomics analysis reveals that Baf155-deficient HSPCs fail to establish accessible chromatin in selected regions that are enriched for putative enhancers and binding motifs of hematopoietic lineage transcription factors. Our study provides a fundamental mechanistic understanding of the role of Baf155 in hematopoietic lineage chromatin priming and the functional consequences of Baf155 deficiency in regeneration and tumor immunity.

The complete mitochondrial genome of Heptathela kimurai (Araneae: Heptathelidae).

Heptathela kimurai (Kishida, 1920) is a spider that belongs to the family Heptathelidae which is a basial lineage of spiders. The molecular information of ancestral species belonging to families like Heptathelidae is comparatively limited when compared to spider species from derived families. Here we present the complete mitochondrial genome sequence (mtDNA) of H. kimurai. The sequence was obtained using massively parallel sequencing technology. The circular genome was 14,224 bp in length, and the AT content was 69.53%. The H. kimurai mitochondrial genome contains 13 protein-coding genes (PCGs), 21 tRNA genes, and 2 rRNA genes. The majority of PCGs were found in the heavy strand.

Pan-Lineage Mycobacterium tuberculosis Reference Genome for Enhanced Molecular Diagnosis.

In Mycobacterium tuberculosis (MTB) control, whole genome sequencing-based molecular drug susceptibility testing (molDST-WGS) has emerged as a pivotal tool. However, the current reliance on a single-strain reference limits molDST-WGS's true potential. To address this, we introduce a new pan-lineage reference genome, "MtbRf". We assembled "unmapped" reads from 3,614 MTB genomes (751 L1; 881 L2; 1,700 L3; and 282 L4) into 35 shared, annotated contigs (54 CDSs). We constructed MtbRf through: 1) searching for contig homologs among genome database that precipitating results uniquely within Mycobacteria genus; 2) comparing genomes with H37Rv ("lift-over") to define 18 insertions; and 3) filling gaps in H37Rv with insertions. MtbRf adds 1.18% sequences to H37rv, salvaging >60% of previously unmapped reads. Transcriptomics confirmed gene-expression of new CDSs. The new variants provided a moderate DST predictive value (AUROC 0.60-0.75). MtbRf thus unveils previously hidden genomic information, and lays the foundation for lineage-specific molDST-WGS.

Overcoming post-transplant graft failure and adenovirus infection in a patient with FLT3-TKD-mutated mixed-phenotype acute leukemia: A case report.

Mixed-phenotype acute leukemia (MPAL) with FLT3-TKD mutations is a rare and challenging subtype of leukemia. Effective management strategies are crucial for improving patient outcomes. A 31-year-old man with FLT3-TKD-mutated MPAL achieved hematological remission through the JALSG ALL202-O protocol and gilteritinib, followed by cord blood transplantation (CBT). Post-transplant complications included adenovirus-induced hemorrhagic cystitis, managed with bladder irrigation and ribavirin, and engraftment failure, necessitating a second CBT on Day 35. Subsequent adenoviral conjunctivitis resolved with vidarabine. The patient achieved neutrophil engraftment by Day 76 and was discharged on Day 173 without relapse. This case highlights the importance of vigilant supportive care and tailored therapy in managing MPAL with FLT3 mutations, especially in the context of post-transplant complications.

Chimerism and mosaicism shape our physical constitution and impact medical conditions.

ABO blood group discrepancies in healthy individuals were caused by body-wide chimerism and mosaicism. They can be evaluated with new diagnostic options for disease-related cell clones that are typically associated with mosaicism. The observations raise the attention for sporadic mixed-field observations of any blood group antigen. Commentary on: Dauber et al. Body-wide chimerism and mosaicism are predominant causes of naturally occurring ABO discrepancies. Br J Haematol 2024 (Online ahead of print). doi:10.1111/bjh.19618.

Activated dormant stem cells recover spermatogenesis in chemoradiotherapy-induced infertility.

Male infertility is a recognized side effect of chemoradiotherapy. Extant spermatogonial stem cells (SSCs) may act as originators for any subsequent recovery. However, which type of SSCs, the mechanism by which they survive and resist toxicity, and how they act to restart spermatogenesis remain largely unknown. Here, we identify a small population of Set domain-containing protein 4 (Setd4)-expressing SSCs that occur in a relatively dormant state in the mouse seminiferous tubule. Extant beyond high-dose chemoradiotherapy, these cells then activate to recover spermatogenesis. Recovery fails when Setd4+ SSCs are deleted. Confirmed to be of fetal origin, these Setd4+ SSCs are shown to facilitate early testicular development and also contribute to steady-state spermatogenesis in adulthood. Upon activation, chromatin remodeling increases their genome-wide accessibility, enabling Notch1 and Aurora activation with corresponding silencing of p21 and p53. Here, Setd4+ SSCs are presented as the originators of both testicular development and spermatogenesis recovery in chemoradiotherapy-induced infertility.

Automated Cell Lineage Reconstruction using Label-Free 4D Microscopy.

Patterns of lineal descent play a critical role in the development of metazoan embryos. In eutelic organisms that generate a fixed number of somatic cells, invariance in the topology of their cell lineage provides a powerful opportunity to interrogate developmental events with empirical repeatability across individuals. Studies of embryonic development using the nematode Caenorhabditis elegans have been drivers of discovery. These studies have depended heavily on high throughput lineage tracing enabled by 4D fluorescence microscopy and robust computer vision pipelines. For a range of applications, computer-aided yet manual lineage tracing using 4D label-free microscopy remains an essential tool. Deep learning approaches to cell detection and tracking in fluorescence microscopy have advanced significantly in recent years, yet solutions for automating cell detection and tracking in 3D label-free imaging of dense tissues and embryos remain inaccessible. Here we describe embGAN, a deep learning pipeline that addresses the challenge of automated cell detection and tracking in label-free 3D time lapse imaging. embGAN requires no manual data annotation for training, learns robust detections that exhibits a high degree of scale invariance and generalizes well to images acquired in multiple labs on multiple instruments. We characterize embGAN's performance using lineage tracing in the C. elegans embryo as a benchmark. embGAN achieves near state-of-the-art performance in cell detection and tracking, enabling high-throughput studies of cell lineage without the need for fluorescent reporters or transgenics.

Structural changes shaping the Drosophila ellipsoid body ER-neurons during development and aging.

The ellipsoid body (EB) of the insect brain performs pivotal functions in controlling navigation. Input and output of the EB is provided by multiple classes of R-neurons (now referred to as ER-neurons) and columnar neurons which interact with each other in a stereotypical and spatially highly ordered manner. The developmental mechanisms that control the connectivity and topography of EB neurons are largely unknown. One indispensable prerequisite to unravel these mechanisms is to document in detail the sequence of events that shape EB neurons during their development. In this study, we analyzed the development of the Drosophila EB. In addition to globally following the ER-neuron and columnar neuron (sub)classes in the spatial context of their changing environment we performed a single cell analysis using the multi-color flip out (MCFO) system to analyze the developmental trajectory of ER-neurons at different pupal stages, young adults (4d) and aged adults (∼60d). We show that the EB develops as a merger of two distinct elements, a posterior and anterior EB primordium (prEBp and prEBa, respectively. ER-neurons belonging to different subclasses form growth cones and filopodia that associate with the prEBp and prEBa in a pattern that, from early pupal stages onward, foreshadows their mature structure. Filopodia of all ER-subclasses are initially much longer than the dendritic and terminal axonal branches they give rise to, and are pruned back during late pupal stages. Interestingly, extraneous branches, particularly significant in the dendritic domain, are a hallmark of ER-neuron structure in aged brains. Aging is also associated with a decline in synaptic connectivity from columnar neurons, as well as upregulation of presynaptic protein (Brp) in ER-neurons. Our findings advance the EB (and ER-neurons) as a favorable system to visualize and quantify the development and age-related decline of a complex neuronal circuitry.

Phylogenomics and pervasive genome-wide phylogenetic discordance among fin whales (Balaenoptera physalus).

Phylogenomics has the power to uncover complex phylogenetic scenarios across the genome. In most cases, no single topology is reflected across the entire genome as the phylogenetic signal differs among genomic regions due to processes, such as introgression and incomplete lineage sorting. Baleen whales are among the largest vertebrates on Earth with a high dispersal potential in a relatively unrestricted habitat, the oceans. The fin whale (Balaenoptera physalus) is one of the most enigmatic baleen whale species, currently divided into four subspecies. It has been a matter of debate whether phylogeographic patterns explain taxonomic variation in fin whales. Here we present a chromosome-level whole genome analysis of the phylogenetic relationships among fin whales from multiple ocean basins. First, we estimated concatenated and consensus phylogenies for both the mitochondrial and nuclear genomes. The consensus phylogenies based upon the autosomal genome uncovered monophyletic clades associated with each ocean basin, aligning with the current understanding of subspecies division. Nevertheless, discordances were detected in the phylogenies based on the Y chromosome, mitochondrial genome, autosomal genome and X chromosome. Furthermore, we detected signs of introgression and pervasive phylogenetic discordance across the autosomal genome. This complex phylogenetic scenario could be explained by a puzzle of introgressive events, not yet documented in fin whales. Similarly, incomplete lineage sorting and low phylogenetic signal could lead to such phylogenetic discordances. Our study reinforces the pitfalls of relying on concatenated or single locus phylogenies to determine taxonomic relationships below the species level by illustrating the underlying nuances which some phylogenetic approaches may fail to capture. We emphasize the significance of accurate taxonomic delineation in fin whales by exploring crucial information revealed through genome-wide assessments.

CRISPR Knock-Ins in Organoids to Track Tumor Cell Subpopulations.

The integration of CRISPR/Cas9 genome editing techniques with organoid technology has revolutionized the field of tumor modeling, enabling the creation of diverse tumor models with distinct mutational profiles. This protocol details the application of CRISPR knock-ins to engineer tumor organoids with reporter cassettes, which are regulated by endogenous promoters of specific genes of interest. This approach facilitates the precise fluorescent labeling, isolation, and subsequent manipulation of targeted tumor cell subpopulations. The utilization of these knock-in reporter cassettes not only allows the visualization and purification of specific tumor cell subsets but also enables conditional cell ablation and lineage tracing studies. In this chapter, we provide a comprehensive guide for the design, construction, delivery, and validation of CRISPR/Cas9 tools tailored for knock-in reporter cassette integration into specific marker genes of interest. By following this protocol, researchers can harness the potential of engineered tumor organoids to decipher intricate tumor heterogeneity, track metastatic trajectories, and unveil novel therapeutic vulnerabilities linked to specific tumor cell subpopulations.

Characterizing Rare Dormant and Cycling Lineages Using the Watermelon System.

Barcode-based lineage tracing approaches enable molecular characterization of clonal cell families. Barcodes that are expressed as mRNA can be used to deconvolve lineage identity from single-cell RNA sequencing transcriptional data. Here we describe the Watermelon system, which facilitates the simultaneous tracing of lineage, transcriptional, and proliferative state at a single cell level.

Transcriptome analysis and molecular characterization of novel small RNAs in Mycobacterium tuberculosis Lineage 1.

Mycobacterium tuberculosis (Mtb), the tuberculosis-causing agent, exhibits diverse genetic lineages, with known links to virulence. While genomic and transcriptomic variations between modern and ancient Mtb lineages have been explored, the role of small non-coding RNA (sRNA) in post-translational gene regulation remains largely uncharted. In this study, Mtb Lineage 1 (L1) Sabahan strains (n = 3) underwent sRNA sequencing, revealing 351 sRNAs, including 23 known sRNAs and 328 novel ones identified using ANNOgesic. Thirteen sRNAs were selected based on the best average cut-off value of 300, with RT-qPCR revealing significant expression differences for sRNA 1 (p = 0.0132) and sRNA 29 (p = 0.0012) between Mtb L1 and other lineages (L2 and L4, n = 3) (p > 0.05). Further characterization using RACE (rapid amplification of cDNA ends), followed by target prediction with TargetRNA3 unveils that sRNA 1 (55 base pairs) targets Rv0506, Rv0697, and Rv3590c, and sRNA 29 (86 base pairs) targets Rv33859c, Rv3345c, Rv0755c, Rv0107c, Rv1817, Rv2950c, Rv1181, Rv3610c, and Rv3296. Functional characterization with Mycobrowser reveals these targets involved in regulating intermediary metabolism and respiration, cell wall and cell processes, lipid metabolism, information pathways, and PE/PPE. In summary, two novel sRNAs, sRNA 1 and sRNA 29, exhibited differential expression between L1 and other lineages, with predicted roles in essential Mtb functions. These findings offer insights into Mtb regulatory mechanisms, holding promise for the development of improved tuberculosis treatment strategies in the future.

Defining context-dependent m6A RNA methylomes in Arabidopsis.

N6-Methyladenosine (m6A) prevalently occurs on cellular RNA across almost all kingdoms of life. It governs RNA fate and is essential for development and stress responses. However, the dynamic, context-dependent m6A methylomes across tissues and in response to various stimuli remain largely unknown in multicellular organisms. Here, we generate a comprehensive census that identifies m6A methylomes in 100 samples during development or following exposure to various external conditions in Arabidopsis thaliana. We demonstrate that m6A is a suitable biomarker to reflect the developmental lineage, and that various stimuli rapidly affect m6A methylomes that constitute the regulatory network required for an effective response to the stimuli. Integrative analyses of the census and its correlation with m6A regulators identify multiple layers of regulation on highly context-dependent m6A modification in response to diverse developmental and environmental stimuli, providing insights into m6A modification dynamics in the myriad contexts of multicellular organisms.