Miracle-dz/UniFAR-FTUs huggingface dataset:
Hybrid Stem Cell States: Insights Into the Relationship Between Mammary Development and Breast Cancer Using Single-Cell Transcriptomics

Similarities between stem cells and cancer cells have implicated mammary stem cells in breast carcinogenesis. Recent evidence suggests that normal breast stem cells exist in multiple phenotypic states: epithelial, mesenchymal, and hybrid epithelial/mesenchymal (E/M). Hybrid E/M cells in particular have been implicated in breast cancer metastasis and poor prognosis. Mounting evidence also suggests that stem cell phenotypes change throughout the life course, for example, through embryonic development and pregnancy. The goal of this study was to use single cell RNA-sequencing to quantify cell state distributions of the normal mammary (NM) gland throughout developmental stages and when perturbed into a stem-like state in vitro using conditional reprogramming (CR). Using machine learning based dataset alignment, we integrate multiple mammary gland single cell RNA-seq datasets from human and mouse, along with bulk RNA-seq data from breast tumors in the Cancer Genome Atlas (TCGA), to interrogate hybrid stem cell states in the normal mammary gland and cancer. CR of human mammary cells induces an expanded stem cell state, characterized by increased expression of embryonic stem cell associated genes. Alignment to a mouse single-cell transcriptome atlas spanning mammary gland development from in utero to adulthood revealed that NM cells align to adult mouse cells and CR cells align across the pseudotime trajectory with a stem-like population aligning to the embryonic mouse cells. Three hybrid populations emerge after CR that are rare in NM: KRT18+/KRT14+ (hybrid luminal/basal), EPCAM+/VIM+ (hybrid E/M), and a quadruple positive population, expressing all four markers. Pseudotime analysis and alignment to the mouse developmental trajectory revealed that E/M hybrids are the most developmentally immature. Analyses of single cell mouse mammary RNA-seq throughout pregnancy show that during gestation, there is an enrichment of hybrid E/M cells, suggesting that these cells play an important role in mammary morphogenesis during lactation. Finally, pseudotime analysis and alignment of TCGA breast cancer expression data revealed that breast cancer subtypes express distinct developmental signatures, with basal tumors representing the most “developmentally immature” phenotype. These results highlight phenotypic plasticity of normal mammary stem cells and provide insight into the relationship between hybrid cell populations, stemness, and cancer.
Differential Effects of Voclosporin and Tacrolimus on Insulin Secretion From Human Islets

Context The incidence of new onset diabetes after transplant (NODAT) has increased over the past decade, likely due to calcineurin inhibitor-based immunosuppressants, including tacrolimus (TAC) and cyclosporin (CsA). Voclosporin (VCS), a next generation calcineurin inhibitor is reported to cause fewer incidences of NODAT but the reason is unclear. Objective Whilst calcineurin signaling plays important roles in pancreatic β-cell survival, proliferation, and function, its effects on human β-cells remain understudied. In particular, we do not understand why some calcineurin inhibitors have more profound effects on the incidence of NODAT. Methods We compared the effects of TAC and VCS on the dynamics of insulin secretory function, programmed cell death rate, and the transcriptomic profile of human islets. We studied two clinically relevant doses of TAC (10 ng/ml, 30 ng/ml) and VCS (20 ng/ml, 60 ng/ml), meant to approximate the clinical trough and peak concentrations. Results TAC, but not VCS, caused a significant impairment of 15 mM glucose-stimulated and 30 mM KCl-stimulated insulin secretion. This points to molecular defects in the distal stages of exocytosis after voltage-gated Ca2+ entry. No significant effects on islet cell survival or total insulin content were identified. RNA sequencing showed that TAC significantly decreased the expression of 17 genes, including direct and indirect regulators of exocytosis (SYT16, TBC1D30, PCK1, SMOC1, SYT5, PDK4, and CREM), whereas VCS has less broad and milder effects on gene expression. Conclusions Clinically relevant doses of TAC, but not VCS, directly inhibit insulin secretion from human islets, likely via transcriptional control of exocytosis machinery.
Impact of Formate Supplementation on Body Weight and Plasma Amino Acids

Current nutritional recommendations are focused on energy, fat, carbohydrate, protein and vitamins. Less attention has been paid to the nutritional demand of one-carbon units for nucleotide and methionine synthesis. Here we investigate the impact of sodium formate supplementation as a nutritional intervention to increase the dietary intake of one-carbon units. A cohort of six female and six male mice received 125 mM sodium formate in the drinking water for three months. A control group of another six female and six male mice was also followed up for the same period of time. Tail vein blood samples were collected once a month and profiled with a haematology Analyser. At the end of the study blood and tissues were collected for metabolomics analysis and immune cell sorting. Formate supplementation has no significant physiological effect on male mice. Formate supplementation has no significant effect on the immune cell counts during the intervention or at the end of the study in either gender. In female mice however, the body weight and spleen wet-weight were significantly increased by formate supplementation, while the blood plasma levels of amino acids were decreased. Formate supplementation also increased the frequency of probiotic bacteria in the stools of female mice. We conclude that formate supplementation induces physiological changes in female mice.
Closure motif-mediated state changes of the HORMA protein ASY1

Meiotic HORMA domain-containing proteins (HORMADs) are key components of the chromosome axis and play an essential role in meiosis in many organisms. The meiotic HORMADs, including yeast Hop1, mouse HORMAD1 and HORMAD2, and Arabidopsis ASY1, assemble along chromosomes at early prophase and the closure motif at their C-termini has been hypothesized to be instrumental for this step by promoting HORMAD oligomerization. In late prophase, ASY1 and its homologs are progressively removed from synapsed chromosomes promoting chromosome synapsis and recombination. The conserved AAA+ ATPase PCH2/TRIP13 has been intensively studied for its role in removing HORMADs from synapsed chromosomes. In contrast, not much is known about how HORMADs are loaded onto chromosomes. Here, we reveal that the PCH2-mediated dissociation of the HORMA domain of ASY1 from its closure motif is important for the nuclear targeting and subsequent chromosomal loading of ASY1. This indicates that the promotion of ASY1 to an ‘unlocked’ state is a prerequisite for its nuclear localization and chromosomal assembly. Likewise, we find that the closure motif is also necessary for the removal of ASY1 by PCH2 later in prophase. Our work results in a grand unified model for PCH2 and HORMADs function in meiosis.
The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation

Cell fate decisions are essential for tissue development and homeostasis and can be induced by chemical and mechanical inputs. While the mechanisms by which chemical signals control cell fate have been well studied, how mechanical inputs impact cell fate decisions are not well understood. Here, we use the well-defined system of keratinocyte differentiation in the skin to define mechanisms by which mechanical inputs control cell fate. Although mechanical signals are known to induce epidermal differentiation, whether and how direct force transmission to the nucleus regulates cell fate decisions remains unknown. Here, we measure tension on the nucleus through the Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes using a molecular biosensor during keratinocyte differentiation. We show that undifferentiated epidermal stem cells display high tension on the LINC complex when integrins are engaged. During differentiation, tension decreases on the LINC complex and on A-type lamins, suggesting that the state of the nuclear lamina is altered during differentiation. LINC complex ablation in mice reveals that LINC complexes are required to repress epidermal differentiation in vitro and in vivo, suggesting that force transduction from engaged integrins to the nucleus plays a role in maintaining keratinocyte progenitors. This work reveals a direct mechanotransduction pathway capable of relaying adhesion-specific signals to regulate cell fate.
Disentangling listening effort and memory load beyond behavioural evidence: Pupillary response to listening effort during a concurrent memory task

Recent research has demonstrated that pupillometry is a robust measure for quantifying listening effort. However, pupillary responses in listening situations where multiple cognitive functions are engaged and sustained over a period of time remain hard to interpret. This limits our conceptualisation and understanding of listening effort in realistic situations, because rarely in everyday life are people challenged by one task at a time. Therefore, the purpose of this experiment was to reveal the dynamics of listening effort in a sustained listening condition using a word repeat and recall task. Words were presented in quiet and speech-shaped noise at different signal-to-noise ratios (SNR). Participants were presented with lists of 10 words, and required to repeat each word after its presentation. At the end of the list, participants either recalled as many words as possible or moved on to the next list. Simultaneously, their pupil dilation was recorded throughout the whole experiment. When only word repeating was required, peak pupil dilation (PPD) was bigger in 0dB versus other conditions; whereas when recall was required, PPD showed no difference among SNR levels and PPD in 0dB was smaller than repeat-only condition. Baseline pupil diameter and PPD followed different growth patterns across the 10 serial positions in conditions requiring recall: baseline pupil diameter built up progressively and plateaued in the later positions (but shot up at the onset of recall, i.e. the end of the list); PPD decreased at a pace quicker than in repeat-only condition. The current findings concur with the recent literature in showing that additional cognitive load during a speech intelligibility task could disturb the well-established relation between pupillary response and listening effort. Both the magnitude and temporal pattern of task-evoked pupillary response differ greatly in complex listening conditions, urging for more listening effort studies in complex and realistic listening situations.
Ustilago maydis Serves as a Novel Production Host for the Synthesis of Plant and Fungal Sesquiterpenoids

Sesquiterpenoids are important secondary metabolites with various pharma- and nutraceutical properties. In particular, higher basidiomycetes possess a versatile biosynthetic repertoire for these bioactive compounds. To date, only a few microbial production systems for fungal sesquiterpenoids have been established. Here, we introduce Ustilago maydis as a novel production host. This model fungus is a close relative of higher basidiomycetes. It offers the advantage of metabolic compatibility and potential tolerance for substances toxic to other microorganisms. We successfully implemented a heterologous pathway to produce the carotenoid lycopene that served as a straightforward read-out for precursor pathway engineering. Overexpressing genes encoding enzymes of the mevalonate pathway resulted in increased lycopene levels. Verifying the subcellular localisation of the relevant enzymes revealed that initial metabolic reactions might take place in peroxisomes: despite the absence of a canonical peroxisomal targeting sequence, acetyl-CoA C-acetyltransferase Aat1 localised to peroxisomes. By expressing the plant (+)-valencene synthase CnVS and the basidiomycete sesquiterpenoid synthase Cop6, we succeeded in producing (+)-valencene and α-cuprenene, respectively. Importantly, the fungal compound yielded about tenfold higher titres in comparison to the plant substance. This proof of principle demonstrates that U. maydis can serve as promising novel chassis for the production of terpenoids.
SH3KBP1 scaffolds endoplasmic reticulum and controls skeletal myofibers architecture and integrity

The building block of skeletal muscle is the multinucleated muscle fiber, formed by the fusion of hundreds of mononucleated precursor cells, myoblasts. In the normal course of muscle fiber development or regeneration, myonuclei are actively positioned throughout muscular development and adopt special localization in mature fibers: regular spacing along muscle fibers periphery, raising the notion of MyoNuclear Domains (MNDs). There is now growing support for a direct connection between myonuclear positioning and normal function of muscles, but how myonuclei affects muscle function remains poorly characterized. To identify new factors regulating forces applied on myonuclei in muscles fibers, we performed a siRNA screen and identified SH3KBP1 as a new factor controlling myonuclear positioning in early phases of myofibers formation. Depletion of SH3KBP1 induces a reset of MNDs establishment in mature fibers reflected by a dramatic reduction in pairwise distance between myonuclei. We show that SH3KBP1 scaffolds Endoplasmic Reticulum (ER) in myotubes that in turn controls myonuclei velocity and localization and thus myonuclear domains settings. Additionally, we show that in later phases of muscle maturation, SH3KBP1 contributes to the formation and maintenance of Sarcoplasmic Reticulum (SR) and Transverse-tubules (T-tubules). We also demonstrate that in muscle fibers, GTPase dynamin-2 (DNM2) binds to SH3 domains of SH3KBP1. Interestingly, we observed that Sh3kbp1 mRNA is up regulated in a mouse model harboring the most frequent mutation for Autosomal Dominant CentroNuclear Myopathy (AD-CNM): Dnm2+/R465W. SH3KBP1 thus appears as a compensation mechanism in this CNM model since its depletion contributes to an increase of CNM-like phenotypes (reduction of muscle fibers Cross-section Areas (CSA) and increase in slow fibers content). Altogether our results identify SH3KBP1 as a new regulator of myonuclear domains establishment in the early phase of muscle fibers formation through ER scaffolding and later in myofibers integrity through T-tubules scaffolding/maintenance. Summary Myonuclei are actively positioned throughout muscular development. Guiraud, Christin, Couturier et al show that SH3KBP1 scaffolds the ER through Calnexin interaction and controls myonuclei motion during early steps of muscle fibers formation. Besides SH3KBP1 participates in cell fusion and T-tubules formation/maintenance in mature skeletal muscle fibers and contributes to slow-down CNM-like phenotypes.
Crystal Structure of the Werner’s Syndrome Helicase

Werner syndrome helicase (WRN) plays important roles in multiple pathways of DNA repair and the maintenance of genome integrity. Recently, loss of WRN was identified as a strong synthetic lethal interaction for microsatellite instable (MSI) cancers making WRN a promising drug target. Yet, structural information for the helicase domain is lacking, which prevents structure-based design of drug molecules. In this study, we show that ATP binding and hydrolysis in the helicase domain are required for genome integrity and viability of MSI cancer cells. We then determined the crystal structure of an ADP bound form of the WRN helicase core at 2.2 Å resolution. The structure features an atypical mode of nucleotide binding with extensive contacts formed by motif VI, which in turn defines the relative positioning of the two RecA like domains. The structure features a novel additional β-hairpin in the second RecA and an unusual helical hairpin in the Zn2+ binding domain, and modelling DNA substrates based on existing RecQ DNA complexes suggests roles for these features in the binding of alternative DNA structures. We have further analysed possible interfaces formed from the interactions between the HRDC domain and the helicase core by NMR. Together, this study will facilitate the structure-based design of inhibitors against WRN helicase.
Mallophaga from Apteryx, and their Significance; with a Note on the Genus Rallicola

Through the courtesy of Dr Hans Gadow I have had the opportunity of examining a number of skins of five species of Apteryx in the collection of the Cambridge University Museum. From these I have collected at least three species of Mallophaga, which I find to belong to the genus Rallicola. These are described below, with some discussion of the genus to which they belong, and of the significance of the occurrence of that genus upon Apteryx, from which no Mallophaga have previously been recorded.
Characterization of macrophages in the tail of the anuran tadpole

Macrophage-like cells in the tail mesenchyme of bullfrog tadpoles were isolated and characterized. These cells actively ingested latex beads into their perinuclear cytoplasm and thyroid hormone was essential for their survival in vitro. The requirement for this hormone is a unique property of these cells since other types of tail cells, such as epidermal cells and mesenchymal fibroblasts, are unable to survive in the presence of the hormone. The unusual characteristics of the cells suggest that they are indeed phagocytic macrophages and that they play a crucial role in the thyroid hormone-dependent breakdown of the tail.
Características histoquímicas, morfométricas y metabólicas del músculo Gluteus medius de equinos entrenados para competencias de salto

Se obtuvieron biopsias a 3 y 6 cm de profundidad del musculo de 7 yeguas y 3 machos castrados de 4 anos de edad, los cuales habian realizado previamente un programa de adiestramiento para salto por 6 meses. Las muestras fueron analizadas por las tecnicas mATPasa miofibrilar, para determinar la composicion fibrilar, y NADH-TR para determinar la capacidad oxidativa. La actividad metabolica de las fibras se determino por la actividad de las enzimas CS, HAD, LDH Y CK. Los promedios obtenidos para la composicion fibrilar fueron de 14, 28, 41 y 17% a 3 cm y 25, 30, 34 Y 10% a 6 cm de profundidad para las fibras tipo I, IIA, IIB oxidativas y IIB no oxidativas respectivamente. Al analizar el factor profundidad de la biopsia se encontraron diferencias estadisticamente significativas, especificamente para las fibras tipo I y IIB, mientras que las tipo IIA mantuvieron valores similares. El tamano de las fibras fue de 2191±624, 3466±840 y 5578±1439 mm² a los 3 cm de profundidad y 243, 3929 y 5558 mm² a los 6 cm de profundidad para las fibras tipo I, IIA Y IIB, respectivamente. El diametro menor fue de 19; 24 Y 30 a 3 cm de profundidad y 20, 25 Y 30 para las fibras tipo I, IIA y IIB a 6 cm de profundidad. La actividad de las enzimas CS, HAD, LDH Y CK fueron 817, 243, 1, 2 y 1, 8 U/mg de proteinas a 3 cm de profundidad y 1479, 542, 1, 58 y 3, 30 a 6 cm de profundidad, existiendo diferencias significativas para las enzimas CS y HAD entre las dos profundidades durante el periodo de adiestramiento. Los resultados indican que el adiestramiento produce cambios en la composicion fibrilar y capacidad metabolica del musculo Gluteus medius en equinos preparados para competencias de salto.
Characterization of experimental Cryptosporidium parvum infection in IFN-γ knockout mice

Severe cryptosporidial infections were produced in gamma interferon (IFN-γ) knockout mice. Mean oocyst shedding increased from 332 to 30717 oocysts/100 μl of faecal suspension between day 4 and 9 after administration of 1×105 oocysts/mouse. No significant differences in oocyst shedding were observed in mice after being inoculated with 1×105, 1×104 or 1×103 oocysts/mouse (P>0·05). Infected mouse weights decreased an average 3–4 g before death or euthanization. Histological studies revealed heavy parasite colonization in small intestinal epithelium (approximately 250 organisms/high-power field at ×400). Mesenteric lymph nodes in infected mice were markedly enlarged compared to controls (P<0·05). Both CD4+ and CD8+ T cell populations increased in spleens of infected mice while the B cell population increased in mesenteric lymph nodes from infected mice. No significant proliferation was observed when pooled lymphocytes from infected mice were exposed to C. parvum antigens in vitro. Addition of recombinant mouse IFN-γ did not restore antigen responsiveness. While lymphoproliferative responses to specific antigen were not significant in the short period following infection, this mouse model provides unique features to study the characteristics of acute infection and the immune response against C. parvum.
Challenges in Pharmacogenomic Testing

Since the International Human Genome Sequencing Consortium declared that the Human Genome Project had been completed in 2003, pharmacogenomic testing gained acceleration, especially in translational cancer research and clinical trials investigating existing and newly developed drugs. Th e enthusiasm increased in the scientiﬁ c and clinical community since the promise of personalized medicine seemed to be an achievable goal in the near future. Currently, the term genetic testing is synonymously used with pharmacogenetic testing. While genetic testing refers more to inherited genetic variations associated with a disease phenotype (mainly diagnostic), pharmacogenetic testing mainly describes genetic variations associated with response to a drug (diagnostic and therapeutic). Recent epigenetic studies indicated that the diﬀ erential methylation patterns of drug metabolizing enzymes could inﬂ uence response to treatment; therefore we suggest the use of the term pharmacogenomic testing instead of pharmacogenetic testing to account for both genetic and epigenetic testing and other possible future mechanisms regulating the human genome. Owing to the recent advances in pharmacogenomic testing and the increased knowledge about the inter-individual diﬀ erences in drug metabolism and disposition, the concept of “one size ﬁ ts all” became questionable. Th is is more obvious in chemotherapeutic agents showing a narrow therapeutic window and adverse events in a signiﬁ cant number of cancer patients. To date, despite the progress and outstanding advance in genetic and genomic molecular technology, the era of the promised personalized medicine is delayed by sev-eral challenges. Th ese challenges are at the analytical, clinical, industrial, regulatory, marketing and ﬁ nancial levels. Facing these challenges fall under the jurisdiction of many agencies including governmental (e.g., Food and Drug Administration, FDA), non-governmental (e.g., patients advocacy groups), academic (e.g., universities and research centers) and industrial (e.g., pharmaceutical companies) organizations.
Growth promotion of groundnut by IAA producing rhizobacteria Bacillus licheniformis MML2501

B. licheniformis MML2501 which was isolated from groundnut rhizosphere soil showed increased populations on spermozphere colonisation and significantly increased the seed germination and other growth parameters in groundnut under in vitro conditions. B. licheniformis MML2501 did not solubilise phosphate but produced indole acetic acid (IAA), with a maximum of 23 μg/ml under optimised conditions such as pH 7.0, temperature 35°C, tryphtophan at a concentration of 16 mM and at 200 rpm shaken conditions. The production of IAA by B. licheniformis MML2501 was further confirmed by TLC and HPLC analyses, in which the Rf value and retention time of 0.66 and 4.1 min respectively, match with that of the authentic IAA. Seed treatment of B. licheniformis MML2501 in groundnut showed a significant increase in seed germination, other growth parameters and yield parameters under potted plant experiments.