A intermediate (Aint) subgroup with warm anti-A1 antibody: A case report

A intermediate (Aint) subtypes exhibit characteristics intermediate between A1 and A2. Plasma from Aint individuals contains different enzyme, UDP-GalNAc: fucosylgalactoside-a-3-N-acetylgalactosaminyl transferase, which is different from the enzyme in A1 and A2 plasma. We encountered the case of a 54-year-old female (having pneumonia and chronic kidney disease) for pre-transfusion testing. On routine grouping, we encountered group discrepancies. On testing, anti-A gave 4+, anti-B-0, anti-A1 lectin-2+, anti-H lectin, and anti-AB antisera gave 4+ reactions. Reverse grouping gave 4+ with B cells, 2+ at room temperature with A cells, and 4+ and 1+ at 37°C and 4°C. Saliva inhibition studies showed A and H substances. It was typed as an Aint group with warm anti-A1 antibody. It’s the 1st time ever we encountered Aint case with a warm type anti-A1 antibody. Here, O group packed red cells are the suitable blood units to transfuse.

Molecular and Functional Characterization of Caveolae in Mixed Cultures of Human NT-2 Neurons and Astrocytes

Caveolae are plasma membrane invaginations that are enriched in cholesterol-binding proteins called caveolins. The presence of caveolae and caveolins in mixed cultures of human neurons and glia has not been investigated. Here, we sought to determine the presence of caveolae and caveolins in human NTera-2 (NT2/D1) cells, differentiated with retinoic acid into neuron-like (NT2/N) and astrocyte-like (NT2/A) cells. We found that while caveolin-3 mRNA levels remained relatively constant, caveolin-1 and -2 levels were upregulated in NT2/A and downregulated in NT2/N. No caveolin-1 immunoreactivity was detected in NT2/N. Electron microscopy revealed numerous flask-shaped invaginations (~86–102 nm in diameter) in the plasma membrane of NT2/A and NT2/N cells, while only few were detected in NT2/D1 cells. Immunoelectron microscopy localized caveolin-1 gold particles in the flask-shaped structures on plasmalemma and cytoplasmic vesicles of NT2/A cells. Furthermore, NT2/A endocytosed Alexa 488 conjugated-cholera toxin B subunit (CTX-B) through a caveolae- and clathrin-dependent pathway, whereas NT2/N endocytosed CTX-B through a caveolae-independent pathway. We have established that while NT2/A expressed functional caveolae, the molecular identity of the plasma membrane invaginations in NT2/N is unknown. The expression of caveolin proteins was differentially regulated in these cells. Taken together, our findings support the usefulness of the human NT2 model system to study the role of caveolins in neuron–glia communication, and their involvement in brain health and disease.

The Expression of Oct3/4A mRNA and Not Its Isoforms is Upregulated by the HPV16 E7 Oncoprotein

Oct3/4 a transcription factor is involved in maintaining the characteristics of cancer stem cells. Oct3/4 can be expressed differentially with respect to the progression of CC. In addition, Oct3/4 can give rise to three isoforms by alternative splicing of the mRNA Oct3/4A, Oct3/4B and Oct3/4B1. The aim of this study was to evaluate the mRNA expression from Oct3/4A, Oct3/4B and Oct3/4B1 in low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), cervical cancer (CC) samples, and measure the effect of the HPV16 E7 oncoprotein on the mRNA expression from Oct3/4 isoforms in the C-33 A cell line. The expression levels of Oct3/4A, Oct3/4B and Oct3/4B1 mRNA were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in patients with LSILs, HSILs and CC. Additionally, C-33 A cells that expressed the HPV16 E7 oncoprotein were established to evaluate the effect of E7 on the expression of Oct3/4 mRNA isoforms. Oct3/4A (p=0.02), Oct3/4B (p=0. 001) and Oct3/4B1 (p<0. 0001) expression is significantly higher in patients with LSIL, HSIL and CC than in woman with non-IL. In the C-33 A cell line, the expression of Oct3/4A mRNA in the presence of the E7 oncoprotein increased compared to that in nontransfected C-33 A cells. Oct3/4B and Oct3/4B1 mRNA were expressed at similar levels among the different groups. These data indicate that only the mRNA of Oct3/4A is upregulated by the HPV16 E7 oncoprotein.

Isolated Changes in Chromatin Accessibility and Enhancer-Promoter Contacts at the β-Globin Locus Distinguish Fetal Hemoglobin Producing F-Cells from a-Cells

Reversal of the developmental switch from fetal (HbF, α 2γ 2) to adult (HbA,α 2β 2) hemoglobin is an important therapeutic approach for sickle cell disease (SCD) and β-thalassemia. It has been noted since the 1950s that a small number of circulating red blood cells, called F-cells, produce elevated levels of HbF; these cells are resistant to sickling and are present in increased numbers in patients with SCD and those treated with pharmacological HbF inducers such as hydroxyurea. Because successful therapy for SCD requires increasing the number of F-cells, it is imperative to understand how these cells arise. This can potentially occur through a shift towards a global fetal-like program, selective variation in levels of known HbF silencers such as BCL11A or LRF, or through discrete epigenetic changes at the β-globin locus. We previously began to address this clinically important question using a novel experimental approach of sorting cultured primary human erythroblasts into HbF-high (F-cell) and HbF-low (A-cell) populations (Khandros et al, Blood 2020). We showed that surprisingly, F-cells from healthy donor primary erythroid cultures have minimal transcriptional differences with A-cells. Unexpectedly, this was also the case when comparing responders (F-cells) and non-responders (A-cells) to treatment with the HbF inducers pomalidomide and hydroxyurea, and there were no differences in the expression of known HbF regulators. We therefore hypothesize that HbF synthesis in F-cells is determined by epigenetic variation confined to the β-globin locus (and not by global changes in the cell fate or nuclear milieu). To test this hypothesis, we compared genome wide chromatin accessibility by Assay for Transposase-Accessible Chromatin (ATAC-seq) in differentiation stage-matched F- and A-cells from healthy donor primary erythroid cultures, treated with vehicle, hydroxyurea, or pomalidomide. We observed striking similarities between F- and A-cells: out of 83,295 peaks called across all conditions, a mere five regions of differential accessibility were found, all at the β-globin locus (at the promoters and 3' UTR regions of the HBG1 and HBG2 genes as well as the BGLT3 non-coding RNA and HBBP1 pseudogene). This remarkable similarity in the global chromatin landscape between A- and F-cells cements the notion that these cells are fundamentally the same in terms of developmental and differentiation states, and that local epigenetic variation at the β-globin locus underlies the differences in HbF production. We also found that the gains in ATAC signal at the HBG1/2 genes were the most pronounced in F-cells from pomalidomide treated cultures, consistent with our finding that F-cells that arise following pomalidomide treatment have a higher content of HBG1/2 transcripts per cell. Drug treatments led to a larger number of changes in ATAC-seq peaks, at 123 and 1015 sites for treatment with hydroxyurea or pomalidomide, respectively, compared to vehicle. However, since differences at only 5 ATAC-seq peaks were observed between between F- and A-cells, we infer that the broader changes upon drug treatment are not needed for the phenotypic differences between F- and A-cells. Since transcription of the β-type globin genes is controlled by developmental stage-specific long-range contacts between the gene promoters and the locus control region (LCR), we determined whether the increase chromatin accessibility at the γ-globin genes in F-cells was associated with enhanced contacts with the LCR. Capture-C experiments revealed increased LCR-HBG1/2 promoter contacts and reduced LCR contacts with the adult HBB and HBD promoters in F-cells vs A-cells, demonstrating that local gains in chromatin accessibility are linked to long-range enhancer promoter contacts. Additionally, we did not detect differences in long-range chromatin contacts at several developmentally regulated genes, including LIN28B and BCL11A, solidifying the idea that γ-globin production in F-cells is specified locally through chromatin accessibility and chromatin architecture. In sum, our studies demonstrate that in adults, F-cells do not arise through reversion to a fetal like state or variation in expression of any known HbF regulator. Rather these cells reflect highly localized, perhaps stochastic modulation of chromatin architecture at the β-globin locus. Disclosures Blobel: Fulcrum Therapeutics, Inc.: Consultancy; Pfizer: Consultancy.

Preventive Effect of M. cochinchinensis on Melanogenesis via Tyrosinase Activity Inhibition and p-PKC Signaling in Melan-A Cell

Whitening research is of particular interest in the cosmetics market. The main focus of whitening research is on melanogenesis inhibition through tyrosinase activity. The mechanism of melanogenesis is involved with tyrosinase activity and p-PKC signaling. In this study, we used Momordica cochinchinensis (Lour.) spreng, a tropical fruit found throughout Southeast Asia, to investigate the inhibitory effect of melanogenesis. M. cochinchinensis contains a high concentration of polyphenols, flavonoids, and unsaturated fatty acids, which might be related to antioxidant activity. This study aimed to determine whether M. cochinchinensis extracts inhibit melanin synthesis in melan-A cells by inhibiting tyrosinase activity and p-PKC signaling. M. cochinchinensis was divided into pulp and aril and extracted under various conditions, and it was confirmed that all pulp and aril extracts have high contents of both phenols and flavonoids. Melan-A cells were treated with PMA for three days to induce melanin synthesis. After PMA treatment, M. cochinchinensis extracts were added to cultured media in a dose-dependent manner. Melanin contents and MTS were used to determine the amount of melanin in live cells. M. cochinchinensis extracts were evaluated for their effects on tyrosinase activity and p-PKC signaling pathways by Western blotting. It was found that M. cochinchinensis extract treatment decreased the amount of melanin and suppressed p-PKC expression. Additionally, tyrosinase activity was reduced after M. cochinchinensis extract treatment in a dose-dependent manner. Therefore, it was concluded that M. cochinchinensis could be used in antimelanogenesis and functional cosmetic materials to improve whitening.

Epigenetic Regulation of CDH1 Is Altered after HOXB7-Silencing in MDA-MB-468 Triple-Negative Breast Cancer Cells

HOXB7 is often overexpressed in breast cancer cells and found to relate to poor prognosis. The search for the HOXB7 targets, as a transcription factor, has led to molecules involved in regulating cell proliferation, migration, invasion, and processes such as angiogenesis and therapy resistance. However, the specific targets affected by the deregulation of HOXB7 in breast cancer remain largely unknown in most molecular sub-types, such as triple-negative breast cancers (TNBC). To unveil the molecular basis behind these aggressive and often untreatable cancers, here we explored the contribution of HOXB7 deregulation for their aggressiveness. To this end, HOXB7 was silenced in TNBC Basal A cells MDA-MB-468, and the phenotype, gene/protein expression, and methylation profile of putative targets were analyzed. Lower migration and invasion rates were detected in HOXB7-silenced cells in comparison with the controls. In addition, these cells expressed more CDH1 and less DNMT3B, and the promoter methylation status of CDH1 diminished. Our data suggest that the HOXB7 transcription factor may act on TNBC Basal A cells by controlling CDH1 epigenetic regulation. This may occur indirectly through the up-regulation of DNMT3B, which then controls DNA methylation of the CDH1 promoter. Thus, future approaches interfering with HOXB7 regulation may be promising therapeutic strategies in TNBC treatment.

Synaptic Interactions in Scorpion Peg Sensilla Appear to Maintain Chemosensory Neurons within Dynamic Firing Range

Scorpions have elaborate chemo-tactile organs called pectines on their ventral mesosoma. The teeth of the comb-like pectines support thousands of minute projections called peg sensilla (a.k.a. “pegs”), each containing approximately 10 chemosensory neurons. Males use pectines to detect pheromones released by females, and both sexes apparently use pectines to find prey and navigate to home retreats. Electrophysiological recordings from pegs of Paruroctonus utahensis reveal three spontaneously active cells (A1, A2, and B), which appear to interact synaptically. We made long-term extracellular recordings from the bases of peg sensilla and used a combination of conditional cross-interval and conditional interspike-interval analyses to assess the temporal dynamics of the A and B spike trains. Like previous studies, we found that A cells are inhibited by B cells for tens of milliseconds. However, after normalizing our records, we also found clear evidence that the A cells excite the B cells. This simple local circuit appears to maintain the A cells in a dynamic firing range and may have important implications for tracking pheromonal trails and sensing substrate chemistry for navigation.

A unique adipocyte progenitor population promotes age-related adiposity

The average fat mass in adults increases dramatically with age, and older people often suffer from visceral obesity and related adverse metabolic disorders. Unfortunately, how aging leads to fat accumulation is poorly understood. It is known that fat cell (adipocyte) turnover is very low in young mice, similar to that in young humans. Here, we find that mice mimic age-related fat expansion in humans. In vivo lineage tracing shows that massive adipogenesis (the generation of new adipocytes), especially in the visceral fat, is triggered during aging. Thus, in contrast to most types of adult stem cells that exhibit a reduced ability to proliferate and differentiate, the adipogenic potential of adipocyte progenitor cells (APCs) is unlocked by aging. In vivo transplantation and 3D imaging of transplants show that APCs in aged mice cell-autonomously gain high adipogenic capacity. Single-cell RNA sequencing analyses reveal that aging globally remodels APCs. Herein, we identify a novel committed preadipocyte population that is age-specific (CP-A), existing both in mice and humans, with a global activation of proliferation and adipogenesis pathways. CP-A cells display high proliferation and adipogenesis activity, both in vivo and in vitro. Macrophages may regulate the remodeling of APCs and the generation of CP-A cells during aging. Together, these findings define a new fundamental mechanism involved in fat tissue aging and offer prospects for preventing and treating age-related metabolic disorders.

Image-Based Identification and Genomic Analysis of Single Circulating Tumor Cells in High Grade Serous Ovarian Cancer Patients

In Ovarian Cancer (OC), the analysis of single circulating tumor cells (sCTCs) might help to investigate genetic tumor evolution during the course of treatment. Since common CTC identification features failed to reliably detect CTCs in OC, we here present a workflow for their detection and genomic analysis. Blood of 13 high-grade serous primary OC patients was analyzed, using negative immunomagnetic enrichment, followed by immunofluorescence staining and imaging for Hoechst, ERCC1, CD45, CD11b and cytokeratin (CK) and sCTC sorting with the DEPArrayTM NxT. The whole genome of single cells was amplified and profiled for copy number variation (CNV). We detected: Type A-cells, epithelial (Hoechstpos, ERCC1pos, CD45neg, CD11bpos, CKpos); Type B-cells, potentially epithelial (Hoechstpos, ERCC1pos, CD45neg, CD11bpos, CKneg) and Type C-cells, potentially mesenchymal (Hoechstpos, ERCC1pos, CD45neg, CD11bneg, CKneg). In total, we identified five (38.5%) patients harboring sCTCs with an altered CN profile, which were mainly Type A-cells (80%). In addition to inter-and intra-patient genomic heterogeneity, high numbers of Type B- and C-cells were identified in every patient with their aberrant character only confirmed in 6.25% and 4.76% of cases. Further identification markers and studies in the course of treatment are under way to expand sCTC analysis for the identification of tumor evolution in OC.

P–403 Sodium tungstate increases embryo adhesion through a direct effect on endometrial cells

Study question Does sodium tungstate treatment induce a change in endometrial cells’ capacity to implant trophoblasts? Summary answer Administration of sodium tungstate to endometrial cells increases trophoblast adhesion. What is known already Sodium tungstate (ST) has shown its capacity to modulate the activity of cytokines, such as leptin, an activator of an obligatory signalling cascade in the embryo-implantation process. STAT3, a signal transducer molecule critical for the embryo implantation process, is also known to be activated by ST. Still, ST’s effect on implantation using biological systems has never been studied. Embryo implantation process and endometrium roles are complicated to study in vivo due to a lack of animal models and appropriate techniques. In vitro techniques using immortalised cell lines allows a first approach to study early implantation stages, such as embryo adhesion. Study design, size, duration An in vitro study was carried out using a human endometrial carcinoma cell line (HEC–1-A) treated with sodium tungstate for 24 and 48h, and choriocarcinoma cell spheroids (JAr). Different times of treatment and concentrations were studied. Each experiment was performed in triplicate. Participants/materials, setting, methods Confluent endometrial HEC–1-A cultures were treated with ST at concentrations (0–150mM) and withaferin A (1mM), negative control for embryo adhesion. After the treatment period, HEC–1-A cultures were washed with ST-free culture medium to eliminate ST. Immediately, 15 JAr trophoblast spheroids were added to cultures and coincubated with gentle agitation for 30, 60 and 90 minutes. An inverted light microscope was used to count adhered and floating spheroids, and determine the trophoblast adherence ratio. Main results and the role of chance HEC–1-A cells treated with ST showed normal morphology and growth at all doses except 150mM. At the highest dose tested, the cells’ culture was still viable (negative blue trypan staining) and maintained morphology, but the adhesion to the plate surface was affected. Doses from 0.15 to 15mM were used to perform adhesion assays. HEC–1-A cells treated with ST for 24h showed an increased capacity to adhere JAr trophoblast spheroids. Adhesion rates reached significant differences at doses of 1.5 and 15mM after 60 and 90 minutes of coincubation. After 90 minutes, untreated cells reached 32.8% adhesion rate, while 1.5 and 15mM ST-treated cells reached 54.6% and 53.4% respectively (p &lt; 0.05 ST vs untreated). Thus, the increment of trophoblast adhesion rate induced by ST reached 66%. Lower adhesion rates were observed after 60 minutes of coincubation but were also significant with a relative increase of 49.1% at 1.5mM and 50.5% at 1.5mM when compared with untreated cells (p &lt; 0.05) Longer treatments (48h) showed similar trends to 24h-treatments, but with a lower extent of ST effect on HEC–1-A receptivity. Maximum adhesion rates were also observed at 90 minutes of coincubation and 1.5 and 15mM doses. The Mean adhesion rate increase was &gt;40% with both doses. Limitations, reasons for caution: The current study is the first approach to evaluate sodium tungstate effect on endometrium using an in vitro model. Future research using in vivo models should be performed to assess sodium tungstate effect on endometrium receptivity and its potential as a fertility treatment. Wider implications of the findings: We conclude that the direct effect of sodium tungstate on endometrial cells increases embryo adhesion rate. These results open a new research line to a potential treatment in human reproduction management with sodium tungstate to solve the unmet need of inducing embryo implantation. Trial registration number Not applicable