bluuebunny/crossref_metadata_2025 huggingface dataset:
TMIC-72. INVESTIGATING THE ROLES OF PGE2 IN MODULATING THE GBM-GAM MICROENVIRONMENT
Authors: Chen Pin-Yuan , Chuang Jian-Ying , Wu An-Chih , Yang Wen-Bin , Chang Wen-Chang , Lin Ai-Yun
Published: November 2023

Glioblastoma (GBM), the most aggressive and deadliest primary brain tumor, is characterized by being surrounded by up to approximately 40% macrophages, which promote tumor progression through interactions with tumor cells. The mechanisms underlying how GBM and its associated macrophages (GAM) communicate remain not fully understood. In this study, we investigated the essential roles of Prostaglandin E2 (PGE2), which affects multiple functions of immune cells, in modulating the GBM microenvironment, specifically the GAM. Massive PGE2 accumulated in the GBM-GAM co-culture microenvironment, and the expression of prostaglandin-endoperoxide synthase 2 (PTGS2) involved in PGE2 synthesis was markedly elevated in GAM. GAM expressed two PGE2 receptors, namely EP2 and EP4, and their expressions positively correlated with PTGS2 expression. Ingenuity pathway analysis (IPA) of differentially expressed (DE) genes obtained from GBM- (conditioned medium) CM-treated THP-1 macrophages revealed significant enrichment in biological functions associated with migration and proliferation. THP-1 macrophage migration promoted by GBM-CM was interrupted by blocking EP4 receptors using an antagonist. GBM-CM promoted THP-1 macrophages proliferation and increased proliferative enhancers, such as PI3KCD, AKT3, ETS1, and BHLHE41, while decreasing proliferative repressors, including MAF, MAFB, and CDKN1A. Cytokine array analysis indicated that GBM-CM shaped GAM into an immunosuppressive phenotype and elevated IL-6 secretion to promote GBM growth. In conclusion, our current results suggest that PGE2 accumulates in the GBM microenvironment, sustaining the macrophage population through increased recruitment and elevated local proliferation and shaping macrophages into an immunosuppressive type. Blockage of the PGE2 autocrine regulation, thus interrupting the supportive roles of GAM in GBM progression, may provide a novel therapeutic strategy for treating patients with GBM.
TMIC-79. LIPOCALIN-2 MEDIATES CELL-INTRINSIC FUNCTIONS IN GLIOBLASTOMA
Authors: Wang Sabrina , Silver Daniel , Deleyrolle Loic , Lathia Justin
Published: November 2023

Glioblastoma (GBM) is the most common primary brain tumor with a median survival of 17-20 months. Despite therapeutic treatments including surgery, radiation, chemotherapy, a population of GBM cells, termed cancer stem cells (CSCs) are radio-resistant and chemo-resistant, leading to mortality of the patient. CSCs also have altered metabolic profiles and have an enhanced capacity to scavenge nutrients from their microenvironment, including iron. Lipocalin-2 (LCN2) functions to sequester iron and is traditionally considered an inflammatory marker through its function of limiting iron for bacterial usage; it has also been described to have both a pro and anti-tumorigenic and has context-dependent functions depending on iron status. LCN2 has been shown to be important in brain metastasis, but its role in GBM is still largely unknown. To assess a cell-intrinsic function for LNC2, we added recombinant LCN2 to low LCN2-secreting mouse GBM cell models and observed that proliferation also increased. When we knocked down LCN2 in our higher LCN2 secreting cell lines, we observed that knockdown cells had decreased viability and growth, which was not mediated by apoptosis, but rather, ferroptosis. When these cells were subsequently engrafted into wild-type mice, we observed an increase in survival. This effect was also phenocopied when the cells were implanted into LCN2 knockout mice, suggesting the tumor-intrinsic effect overpowered the global knockout model. Lastly, to investigate how LCN2 plays a role in the GBM microenvironment, we orthotopically implanted syngeneic mouse GBM cells into male and female LCN2 knockout and wild-type mice. We observed no survival difference, further suggesting that LCN2 does not function in an extrinsic manner. Taken together, these data suggest that LCN2 affects proliferation and tumorigenesis, and is another avenue to understanding the complexity of GBM biology. Strategies to reduce LCN2 in GBM may provide a therapeutic target for GBM in the future.
MODL-08. A COMPENDIUM OF SYNGENEIC, TRANSPLANTABLE PEDIATRIC HIGH-GRADE GLIOMA MODELS REVEALS SUBTYPE-SPECIFIC THERAPEUTIC VULNERABILITIES
Authors: McNicholas Michael , De Cola Antonella , Bashardanesh Zahedeh , Foss Amelia , Lloyd Cameron , Hebert Steven , Faury Damien , Andrade Augusto Faria , Jabado Nada , Kleinman Claudia , Pathania Manav
Published: November 2023

Pediatric high-grade gliomas (pHGGs) are lethal, incurable brain tumors frequently driven by clonal mutations in histone genes. They often harbor a range of additional genetic alterations that correlate with different ages, anatomical locations, and tumor subtypes. We developed models representing 16 pHGG subtypes driven by different combinations of alterations targeted to specific brain regions. Tumors developed with varying latencies and cell lines derived from these models engrafted in syngeneic, immunocompetent mice with high penetrance. Targeted drug screening revealed unexpected selective vulnerabilities— H3.3G34R/PDGFRAC235Y to FGFR inhibition, H3.3K27M/PDGFRAWT to PDGFRA inhibition, and H3.3K27M/PDGFRAWT and H3.3K27M/PPM1DDC/PIK3CAE545K to combined inhibition of MEK and PIK3CA. Moreover, H3.3K27M tumors with PIK3CA, NF1 and FGFR1 mutations were more invasive and harbored distinct additional phenotypes, such as exophytic spread, cranial nerve invasion and spinal dissemination. Collectively, these models reveal that different partner alterations produce distinct effects on pHGG cellular composition, latency, invasiveness, and treatment sensitivity.
MODL-12. REGULATION OF OLIGODENDROGLIAL PROGENITOR CELL (OPC) CELL FATE IN DIFFUSE MIDLINE GLIOMA (DMG) BY THE HOMEOBOX TRANSCRIPTION FACTOR DLX2
Authors: Furst Liam , Roussel Enola , Leung Ryan , George Ankita , Firestein Ron , Faux Maree , Eisenstat David
Published: November 2023

Diffuse midline glioma, K27-altered (DMG) is a treatment resistant, uniformly fatal cancer that arises predominantly in children. 80% of patients with DMG harbor K27M mutations in canonical histone variants H3.3 and H3.1, leading to global epigenetic dysregulation and an oligodendroglial progenitor cell (OPC) transcriptional profile. DLX2 is a homeobox transcription factor that directly represses OPC cell fate and promotes a GABAergic interneuron cell fate during neurogenesis. Our lab has previously identified DLX2 transcription factor occupancy of genes required for OPC differentiation in vivo (Olig2, Nkx2.2, Myt1, Plp1), and shown that co-expression of DLX2 to target sequences reduced reporter gene expression in vitro. Likewise, increased expression of Olig2, Nkx2.2, and Plp-1 are observed in a Dlx1/Dlx2 double knockout (DKO) mouse model. Transient overexpression of a Dlx2-GFP construct into cells of a genetically engineered mouse model (GEMM) of DMG resulted in increased expression of Gad1/2 isoforms and decreased Olig2 and Nkx2.2, global restoration of H3K27me3, and a reduction in migration, invasion, and colony formation in vitro. Inverse expression of DLX2 and OLIG1/2 was observed in a bulk RNAseq database of 45 human DMG cell lines. We have established a pipeline to evaluate cell fate changes following changes in DLX2 expression levels. OLIG1/2high/DLX2low DMG expressing cell lines underwent stable transfection of a DLX2-mCherry overexpression construct. Successful alteration of DLX2 expression was confirmed by qPCR and immunoblot in knockout and overexpressing human DMG cell lines. Analysis of histone marks (H3K27me3, H3K27ac), K27M, and OPC signature genes will be presented with comparison of altered OPC genes from the DKO mouse as determined by RNA-seq. The manipulation of DLX2 expression in human DMG cells in vitro and in vivo provides a model system for assessing the contribution of a homeobox transcription factor required for neurogenesis to cell fate regulation and effects on tumorigenicity in DMG.
MODL-34. A SERIES OF EGFR-MUTANT MODELS OF GLIOBLASTOMA THAT RECAPITULATES PATIENT TUMOR HETEROGENEITY AND RESPONSE TO TREATMENT
Authors: Grausam Katie , Pacheco David Rincon Fernandez , Hatanaka Emily , Shiao Stephen , Breunig Joshua
Published: November 2023

Glioblastoma (GBM) is the most common and aggressive brain cancer, with a treatment regimen that has remained disappointingly unaltered for the last few decades. Preclinical testing of immunotherapy has generated promising results, sometimes leading to elimination of the tumor over time in mouse models; however, when tested clinically, patients show limited responses with no impact on patient survival. Current mouse models (e.g., GL261) for preclinical testing involve orthotopic transplantation of a cell line with tumor mutations not commonly found in patient tumors. Most of these models elicit a strong anti-tumor immune response which is typically not found clinically. Single cell sequencing has revealed that different mutations in GBM are associated with differences in the aggressiveness and composition of the tumor immune microenvironment. We therefore developed an immunocompetent, autochthonous platform for generating mouse models of GBM using combinations of clinically-relevant mutations, including mutant EGFR driving tumor growth and various single guide RNAs with CRISPR-Cas9 to elicit loss of function of tumor suppressor genes. Histopathology from tumors in mice mirrors the typical features of GBM, while single-cell RNA sequencing identified expression patterns similar to patients with different driver combinations altering expression patterns in ways that mimic their human counterparts, including the heterogeneity that is the hallmark of GBM. The immune cell population of these tumors was composed of predominantly microglia, with a small population of peripherally derived immune cells. Finally, treatment studies of radiation and anti-PD-1 using this new mouse model do not result in long-term survival, similar to human trials of these agents, paving the way for more accurate preclinical testing. In conclusion, using this unique modeling system that more closely resembles human tumors genetically and immunologically, we can better explore the mechanisms of therapeutic resistance and identify more relevant targets to improve outcomes in this challenging disease.
MODL-40. CLONAL ARCHITECTURE, TRANSCRIPTIONAL CELLULAR STATES AND IN-VITRO DRUG RESPONSE IN GLIOBLASTOMA
Authors: Song Youjin , Miller-Michlits Yelyzaveta , Greutter Lisa , Kiesel Barbara , Loetsch-Gojo Daniela , Redl Vivian , Gesperger Johanna , Breu Jana , Nam Do-Hyun , Widhalm Georg , Sa Jason K , Woehrer Adelheid
Published: November 2023

Seventy-five multiregional tumors from 20 representative GBM patients (female/male ratio = 0.75, median age= 63 ys) were profiled using deep whole-exome sequencing with a nominal coverage of > 300x. The clonal complexity of each individual tumor was measured by calculating the molecular distance using the Euclidean distance of all possible paired regions based on somatic mutational profiles. Among them, fresh tumor slices of 4 patients were cultured in minimal essential media and treated with 4.2 Gy plus 200 µM temozolomide for 48 hours. Spatial Transcriptomics was performed on matched tumor slices with and without treatment. Molecular characterization of multiregional tumors revealed two patterns of evolutionary trajectory, linear and branched (linear: 8 cases, branched: 8 cases) among 16 patients. Tumors with branched evolution demonstrated considerable levels of molecular divergence, determined by high molecular distance and a low number of mutations that were shared among all regions. In-vitro drug response varied considerably between patients based on cell proliferation (minus 0.5 to 13.8 %), apoptotic rates (30.1 to 44.4 %), and cell viability (minus 7.8 to 34.1 %). Under treatment pressure an increase of mesenchymal cellular states with peculiar redistribution of transcriptional patterns and increased clonal complexity was observed. In sum, we report on an ongoing study in glioblastoma that seeks to link the clonal architecture with spatially-resolved transcriptional patterns and in-vitro drug response. Our study will provide new insights into the clinical relevance of the clonal composition of this deadly cancer.
Recent progress in MnBi2nTe3n+1 intrinsic magnetic topological insulators: crystal growth, magnetism and chemical disorder
Authors: Hu Chaowei , Qian Tiema , Ni Ni
Published: November 2023

The search for magnetic topological materials has been at the forefront of condensed matter research for their potential to host exotic states such as axion insulators, magnetic Weyl semimetals, Chern insulators, etc. To date, the MnBi2nTe3n+1 family is the only group of materials showcasing van der Waals-layered structures, intrinsic magnetism and non-trivial band topology without trivial bands at the Fermi level. The interplay between magnetism and band topology in this family has led to the proposal of various topological phenomena, including the quantum anomalous Hall effect, quantum spin Hall effect and quantum magnetoelectric effect. Among these, the quantum anomalous Hall effect has been experimentally observed at record-high temperatures, highlighting the unprecedented potential of this family of materials in fundamental science and technological innovation. In this paper, we provide a comprehensive review of the research progress in this intrinsic magnetic topological insulator family, with a focus on single-crystal growth, characterization of chemical disorder, manipulation of magnetism through chemical substitution and external pressure, and important questions that remain to be conclusively answered.
Ultrasound-guided Fat Graft of the Obliques–Serratus Complex
Authors: Flores González Emanuel Armando , Babaitis Ricardo , Viaro Mauricio Schneider , Manzaneda Cipriani Raul Martin , Duran Vega Hector , Ramírez Guerrero Oliver René , Brito Toledo Patricia , Vázquez Apodaca Raquel Aracely , Gracida Mancilla Noe Isaias
Published: November 2023

Background:

The idea of the anterolateral thoracic musculature in men during the contour procedure is very limited, particularly in terms of surgical techniques that improve its appearance and aesthetics. Therefore, this study aimed to describe a technique of deep definition liposuction with intramuscular grafting of the serratus external oblique muscle complex.

Methods:

A retrospective case series of a total of 11 cases of male patients aged between 29 and 60 years in the year 2022 is presented. These patients underwent body contouring surgery and subsequently underwent ultrasound-guided intramuscular fat grafting in the serratus external oblique complex. As part of the study, measurements were taken before and immediately after surgery. The data were stored in a database and analyzed with the statistical software SPSS, v23.0.

Results:

The patients were aged between 29 and 60 years, with a mean age of 38.3 years (±8.5). All cases had a body mass index of 30. Results were matched preinfiltration and postinfiltration; These results were subjected to statistical validation using the Student t test for related samples, and statistically significant differences were obtained in all the measures tested.

Conclusions:

The technique of fat grafting in the oblique-serratus complex allows for increasing muscle thickness. The measurements were made only immediately after the infiltration; it is important in the future to show the long-term follow-up. No adverse events occurred during the study.
Indwelling central venous catheter infection with Chryseobacterium shandongenes, successful eradication in a five-year-old with Cystic Fibrosis.
Authors: Rowan Anthony , Verbruggen Tiarnan Fallon , O'Connell Nuala H , Stapleton Patrick J , Dunne Colum P , Linanne Barry , Butler Daryl
Published: November 2023

Introduction

Chryseobacterium shandogenes is a Gram-negative Flavobacterium bacillus with intrinsic multi-drug resistant properties

Case Presentation

: Herein, we present the first case report of human C. shandogenes infection, relating to an implantable portal and catheter (port-a-cath) central line in a five-year-old female with Cystic Fibrosis (CF). The infection was identified using Bruker MALDI-TOF Biotyper with BDAL (v12) of blood, which was cultured due to pyrexia and rigor following port-a-cath access. This report details the effective eradication of C. shandogenes infection from the port-a-cath device using initial empirical gentamicin and followed by targeted ciprofloxacin locks and systemic antibiotics.

Conclusion

: we demonstrated successful eradication of C. shandongenes from a port-a-cath device, including the MIC required in this case. The result was eradication of central access infection preventing progression to bacteremia/septicemia and preserving central access in a child with CF and established respiratory disease.
Effect of gamma irradiation on post-harvest quality of king oyster
mushrooms (Pleurotus eryngii)
Authors: Yeom Seo-Joon , Lee Geon-Ah , Kim Sang-Su , Yoon Ki-Nam , Song Beom-Seok , Park Jong-Heum , Jung Woo-Jin , Kim Young-Min , Kim Jae-Kyung
Published: November 2023

The consumption of king oyster mushrooms has steadily increased owing to their
unique flavor and delicate texture. This study extended the storage period of
king oyster mushrooms (Pleurotus eryngii) via gamma
irradiation. Irradiated samples (0, 0.5, 1, 2, and 3 kGy) were stored at
4°C with 80% relative humidity for up to 28 days, and the
experiments were conducted every 7 day. Microbiological analysis revealed a
dose-dependent reduction in total aerobic bacteria, Pseudomonas
spp., and yeasts and molds, and gamma irradiation above 2 kGy effectively
controlled microbial contamination for up to 28 days. At the 28th day of
storage, the irradiated king oyster mushrooms exhibited delayed browning through
the reduction of tyrosinase activity. Moreover, firmness reduction (%)
was 80.59±1.89% for the non-irradiated group and
42.80±1.28, 34.57±1.13, 31.05±3.24, and
39.73±0.94% for the irradiated group (0.5, 1, 2, and 3 kGy),
respectively. These results were supported by the scanning electron microscopy
photos, which showed smaller pores in the irradiated group than in the
non-irradiated group. This study demonstrated that 2 kGy of gamma irradiation
effectively reduces microbial contamination and delays the browning and
softening of king oyster mushrooms for up to 28 days.
Efficacy of Elaeagnus umbellata leaves on prevention
of cadmium-induced toxicity in HepG2 cells
Authors: Lee Jae-Yeul , Yang Seun-Ah , Bae Won-Bin
Published: November 2023

Elaeagnus umbellata leaves have been reported to suppress
inflammation, allergic responses, lung cancer proliferation and oral bacterial
growth. Cadmium (Cd) is a heavy metal that has been found to cause many
toxicities, including liver toxicity. The aim of this study was to evaluate the
capacity of 70% ethanol extract of E. umbellata leaves
(EUL) to protect human hepatocytes from Cd toxicity. After exposure of HepG2
cells to Cd at 10 μM for 24 h, cell viability,
expression levels of apoptosis- and antioxidant-related proteins,
reactive oxygen species (ROS) accumulation and Cd uptake were assessed. EUL
protected HepG2 cells from Cd-induced apoptosis as determined by MTT assay. A
decrease in caspase-3 and p-p53 protein levels was observed in cells pretreated
with EUL prior to Cd exposure. Furthermore, the Cd-induced increase in
intracellular DCF fluorescence was attenuated by EUL, indicating that the
Cd-induced apoptosis preventing effect was associated with the suppression of
ROS accumulation. Moreover, EUL’s effects on the inhibition of p38, JNK,
and AKT phosphorylation also appear to be associated with protection against Cd
toxicity. Moreover, EUL upregulated Cd-depressed expression of Nrf2, HO-1,
catalase, and MT-1,2 proteins, suggesting that Cd uptake-induced apoptosis in
HepG2 cells may be inhibited by EUL’s antioxidative potential.
Effects of impact by mechanical harvesting on storability of onions
(Allium cepa L.)
Authors: Kwon Young-Kyeong , Lee Yong-Jae
Published: November 2023

This study investigated the storability of onions according to manual and
mechanical harvesting. Moreover, we simulated the onion-to-onion impact during
the mechanical harvesting process and investigated the storability after
artificially subjecting the onions to impact treatment. The onion harvesting
methods included hand plucking + manual collection, digger +
manual, and digger + mechanical collection. The maximum impact height
during the mechanical harvesting process was 0.5 m. Immediately after
harvesting, no significant difference in the bruise and wound rate among the
harvesting methods was observed. Any increased bruise or wound rate because of
mechanical harvesting was presumed to be influenced by soil conditions, such as
the presence of gravel, and machine operation factors. Furthermore, the
storability during the 8.5 months storage showed no significant difference
according to the harvesting methods. In treatments by simulating the impacts
during the mechanical harvesting process, the impact heights were 0.0 m (0.0 J),
0.25 m (0.86 J), 0.5 m (1.72 J), and 0.75 m (2.57 J), each performed once, and
four times at the same position (3.43 J) and four times at different positions
(3.43 J) at 0.25 m. Throughout all the treatments, there were no significant
differences in the storability during the 8.5 months storage period.
Use of extraction solvent method to monitor the concentrations of
acidic polysaccharides and ginsenosides from red and black
ginseng
Authors: Lee Gee Dong
Published: November 2023

In this study, the extraction yield, acidic polysaccharides and ginsenosides of
red and black ginseng were optimized by using the response surface methodology
in consideration of the ethanol concentration and temperature of the extraction.
The R2 of the model formula for the yield, acidic polysaccharides and
ginsenosides was 0.8378-0.9679 (p<0.1). An optimal extraction yield of
5.29% was reached for red ginseng soluble solids when 1.52%
ethanol concentration was used at a temperature of 67.27°C. Additionally,
the optimal extraction yield for black ginseng soluble solid was 6.11%
when 3.12% ethanol concentration was used at a temperature of
66.13°C. Furthermore, the optimal conditions for extracting acidic
polysaccharides from red ginseng were using an ethanol concentration of
4.03% at a temperature of 69.61°C; a yield of 1.86 mg/mL was
obtained. The optimal extraction yield for acidic polysaccharides from black
ginseng was 1.80 mg/mL when extracted using a concentration of 24.67% of
ethanol at a temperature of 71.14°C. An optimal extraction yield of 0.22
mg/mL was reached for ginsenoside Rg1 from red ginseng when
79.92% ethanol concentration was used at a temperature of 70.62°C.
The optimal extraction yield of ginsenoside Rg3 from black ginseng
was 0.31 mg/mL when ethanol was used at a concentration of 75.70% at a
temperature of 65.49°C. The ideal extraction conditions for obtaining the
maximum yield of both acidic polysaccharide and ginsenoside from red and black
ginseng were using ethanol at a concentration between 35 and 50% at an
extraction temperature of 70°C.
Quality properties of texturized vegetable protein made from
defatted soybean flour with different soybean seed coat
contents
Authors: Park Chan Soon , Seo Mi Sook , Jung Sun Young , Lee Seul , Park Boram , Park Shin Young , Kim Yong Suk
Published: November 2023

The texturization characteristics of textured vegetable protein (TVP) were
investigated based on the extent of soybean decoating during the pretreatment of
defatted soybean flour used for TVP. The raw materials for TVP consisted of
50% defatted soybean flour, 30% gluten, and 20% corn
starch. The weight ratios of soybean seed coat to soybean flour were 9%,
6%, 3%, and zero. Extrusion was performed using an extruder
equipped with a cooling die, maintaining a barrel temperature of 190°C
and screw speed of 250 rpm, Water was injected at a rate of 9 rpm using a
metering pump. Regarding the textures of the extruded TVPs produced from
defatted soybean flour, an increase in the soybean seed coat content led to a
decrease in the apparent fibrous structural layer and an increase in hardness.
However, there were no significant changes in elasticity and cohesion. Moreover,
as the soybean seed coat content increased, the pH of TVPs decreased. A higher
soybean seed coat content also tended to lower the moisture content, increasing
water absorption, solids elution, and turbidity. These results suggest that an
increased seed coat content reduces the proportion of protein, and the fibers
present in the seed coats prevent texturization.
Transcriptional profiling ofDrosophilamale-specific P1 (pC1) neurons
Authors: Ahmed Osama M. , Crocker Amanda , Murthy Mala
Published: November 2023

InDrosophila melanogaster, the P1 (pC1) cluster of male-specific neurons both integrates sensory cues and drives or modulates behavioral programs such as courtship, in addition to contributing to a social arousal state. The behavioral function of these neurons is linked to the genes they express, which underpin their capacity for synaptic signaling, neuromodulation, and physiology. Yet, P1 (pC1) neurons have not been fully characterized at the transcriptome level. Moreover, it is unknown how the molecular landscape of P1 (pC1) neurons acutely changes after flies engage in social behaviors, where baseline P1 (pC1) neural activity is expected to increase. To address these two gaps, we use single cell-type RNA sequencing to profile and compare the transcriptomes of P1 (pC1) neurons harvested from socially paired versus solitary male flies. Compared to control transcriptome datasets, we find that P1 (pC1) neurons are enriched in 2,665 genes, including those encoding receptors, neuropeptides, and cell-adhesion molecules (dprs/DIPs). Furthermore, courtship is characterized by changes in∼300 genes, including those previously implicated in regulating behavior (e.g.DopEcR, Octβ3R, Fife, kairos, rad). Finally, we identify a suite of genes that link conspecific courtship with the innate immune system. Together, these data serve as a molecular map for future studies of an important set of higher-order and sexually-dimorphic neurons.
Amoebozoan testate amoebae illuminate the diversity of heterotrophs and the complexity of ecosystems throughout geological time
Authors: Porfirio-Sousa Alfredo L. , Tice Alexander K. , Morais Luana , Ribeiro Giulia M. , Blandenier Quentin , Dumack Kenneth , Eglit Yana , Fry Nicholas W. , Gomes E Souza Maria Beatriz , Henderson Tristan C. , Kleitz-Singleton Felicity , Singer David , Brown Matthew W. , Lahr Daniel J.G.
Published: November 2023

Heterotrophic microbial eukaryotes play a pivotal role in aquatic and terrestrial ecosystems, contributing to carbon and nutrient cycles. These microorganisms, capable of phagocytosis, act as predators of bacterial communities and other microeukaryotes, occupying a significant position in complex food webs. The timing of the origin and diversification of heterotrophic microeukaryotes remain unclear. Fossil evidence and molecular data suggest that the emergence of predatory microeukaryotes and the transition to a eukaryote-dominant marine environment occurred around 800 million years ago (mya). Vase-shaped microfossils (VSMs), the fossil record linked to Arcellinida testate amoebae, represent the oldest known evidence of heterotrophic microeukaryotes in marine environments and terrestrial habitats. In this study, we investigate the early divergence and diversification of Arcellinida and related amoebozoan taxa using a relaxed molecular clock approach. Phylogenomic reconstructions reveal a well-resolved tree of amoebozoan testate amoebae, including a monophyletic Arcellinida with three suborders and five infraorders. Through calibration using fossils and rigorous clock models, we estimate the timing of diversification of Arcellinida during the early Neoproterozoic (886 - 764 mya), shedding light on the expansion of life during this period. Our results suggest an established biological complexity in shallow marine ecosystems, involving both phototrophic and heterotrophic microeukaryotes during the Neoproterozoic, followed by an invasion of freshwater systems potentially during Cryogenian and subsequent diversification of Arcellinida in the Phanerozoic. Overall, these findings provide valuable insights into heterotrophic microeukaryotes' evolutionary history and ecological significance in Earth's ecosystems.
scDrugPrio: A framework for the analysis of single-cell transcriptomics to address multiple problems in precision medicine in immune-mediated inflammatory diseases
Authors: Schäfer Samuel , Smelik Martin , Sysoev Oleg , Zhao Yelin , Eklund Desiré , Lilja Sandra , Gustafsson Mika , Heyn Holger , Julia Antonio , Kovács István A. , Loscalzo Joseph , Marsal Sara , Zhang Huan , Li Xinxiu , Gawel Danuta , Wang Hui , Benson Mikael
Published: November 2023

Background

Ineffective drug treatment is a major problem for many patients with immune-mediated inflammatory diseases (IMIDs). Important reasons are the lack of systematic solutions for drug prioritisation and repurposing based on characterisation of the complex and heterogeneous cellular and molecular changes in IMIDs.

Methods

Here, we propose a computational framework, scDrugPrio, which constructs network models of inflammatory disease based on single-cell RNA sequencing (scRNA-seq) data. scDrugPrio constructs detailed network models of inflammatory diseases that integrate information on cell type-specific expression changes, altered cellular crosstalk and pharmacological properties for the selection and ranking of thousands of drugs.

Results

scDrugPrio was developed using a mouse model of antigen-induced arthritis and validated by improved precision/recall for approved drugs, as well as extensivein vitro, in vivo,andin silicostudies of drugs that were predicted, but not approved, for the studied diseases. Next, scDrugPrio was applied to multiple sclerosis, Crohn’s disease, and psoriatic arthritis, further supporting scDrugPrio through prioritisation of relevant and approved drugs. However, in contrast to the mouse model of arthritis, great interindividual cellular and gene expression differences were found in patients with the same diagnosis. Such differences could explain why some patients did or did not respond to treatment. This explanation was supported by the application of scDrugPrio to scRNA-seq data from eleven individual Crohn’s disease patients. The analysis showed great variations in drug predictions between patients, for example, assigning a high rank to anti-TNF treatment in a responder and a low rank in a nonresponder to that treatment.

Conclusion

We propose a computational framework, scDrugPrio, for drug prioritisation based on scRNA-seq of IMID disease. Application to individual patients indicates scDrugPrio’s potential for personalised network-based drug screening on cellulome-, genome-, and drugome-wide scales. For this purpose, we made scDrugPrio into an easy-to-use R package (https://github.com/SDTC-CPMed/scDrugPrio).
Multidimensional behavioral profiles associated with resilience and susceptibility after inescapable stress
Authors: de Oliveira-Júnior Benedito Alves , Marques Danilo Benette , Rossignoli Matheus Teixeira , Prizon Tamiris , Leite João Pereira , Ruggiero Rafael Naime
Published: November 2023

Stress-related neuropsychiatric disorders are complex conditions that are difficult to diagnose and treat due to the variety of symptoms they present with. While animal models have been instrumental in understanding their underlying mechanisms, there is an ongoing debate about the validity of behavioral measures to address specific disorders. Here, we conducted an extensive ethological characterization of behavioral variables associated with anxiety and depression and employed a multidimensional approach to investigate stress susceptibility in male Wistar rats. The rats underwent inescapable footshocks (IS) or no-shocks (NS), followed by a comprehensive battery of behavioral tests over several days. Our study identified phenotypically distinct clusters, including fully characterized stress-susceptible and stress-resilient subpopulations, along with intermediate phenotypes. Reduced time in elevated plus maze open arms, diminished sucrose preference and increased escape failures consistently differentiated susceptible from resilient rats. Behavior in the forced swim test was unrelated to stress susceptibility. In conclusion, our study sheds light on the relationships between classical behavioral measures in animal models of depression, highlighting distinct patterns that differentiate susceptibility from resilience. It emphasizes the importance of a multidimensional assessment of behavioral responses in animal models for a better understanding of stress-related neuropsychiatric disorders.

HIGHLIGHTS

Clustering of features reveals a correlation pattern that separates behavioral variables associated with susceptibility from those associated with resilience to stress

Multidimensional patterns of covariation among behavioral measures distinguish helpless from non-helpless individuals and indicate independence from forced swim test behavior.

Clustering of individuals discriminates stressed subjects and reveals multidimensional behavioral phenotypes of resilience and susceptibility, indicating different stress-coping strategies.
Antisense transcription from a neighboring gene interferes with the expression of mNeonGreen as a functionalin vivofluorescent reporter in the chloroplast ofChlamydomonas reinhardtii
Authors: Navarrete Axel , Pollak Bernardo
Published: November 2023

Although over 30 years have passed sinceChlamydomonas reinhardtiichloroplast transformation was first achieved, robust genetic engineering of the chloroplast still remains a challenging task. The glass-bead transformation method has enabled simple and accessible chloroplast transformation of theC. reinhardtiiTN72 strain, allowing generation of marker-free transplastomic strains for low-cost experimentation. However, lack of functionalin vivofluorescent reporters limit research and widespread development of chloroplast engineering. Here, we developed a chloroplast codon-optimised mNeonGreen fluorescent reporter, which can be detectedin vivothrough fluorescence microscopy and fluorometry, by context-aware construct engineering, leading up to a ∼6-fold increase in fluorescence. We found evidence for chloroplast post-transcriptional regulation of gene expression derived from formation of antisense pairing of mRNAs due to transcriptional readthrough of the convergent adjacent gene, which was validated through detection of the double-stranded RNA. In addition, engineering approaches were used to modulate transcriptional readthrough, allowing a better understanding of context effects that are relevant for heterologous expression. Finally, we characterised a suite of regulatory parts for transgene expression in a sense-transcriptional context regarding to the selectable marker, achieving up to ∼2-fold increase in mNeonGreen fluorescence levels regarding to the control, with the use of PrrnS,5’atpAand 3’rbcLendogenous regulatory sequences from the chloroplast ofC. reinhardtii. This work provides new tools for studying basic aspects of the molecular biology in the chloroplast inC. reinhardtii, as well as evidence for fundamental processes of gene regulation that may enable developing rules for more efficient chloroplast engineering.
Cellular COPI components promote geminivirus infections by facilitating the chloroplast localization of viral C4/AC4 proteins
Authors: Zhao Wenhao , Ji Yinghua , Zhou Yijun , Wang Xiaofeng
Published: November 2023

Geminiviruses are a family of viruses that infect numerous crops and cause extensive agricultural losses worldwide. During viral infection, geminiviral C4/AC4 proteins relocate from the plasma membrane (PM) to chloroplasts, where they inhibit chloroplast-mediated host defense, including the biosynthesis of salicylic acid (SA). However, how are C4/AC4 proteins transported to chloroplasts is unknown. We report here that the Coat Protein I (COPI) components play a critical role in redistributing Tomato yellow leaf curl virus (TYLCV) C4 protein to chloroplasts. TYLCV C4 interacts with the β subunit of COPI, and the coexpression of both inNicotiana benthamianacells promotes the enrichment of C4 in chloroplasts, which also occurs during TYLCV infection and is blocked by an inhibitor of the COPI pathway. Overexpression of COPI components promotes but knockdown of gene expression inhibits TYLCV infection. The COPI pathway plays similar roles in C4/AC4 transport and infections of other geminiviruses, including Beet curly top virus and East African cassava mosaic virus. Our results identify an unconventional role of the COPI pathway in protein trafficking to chloroplasts during geminiviruses infections in plants, and suggest a broad-spectrum antiviral strategy in controlling geminiviruses by manipulating COPI components.
Dynamic RNA Polymerase II Recruitment Drives Differentiation of the Intestine under the direction of HNF4
Authors: Vemuri Kiranmayi , Kumar Sneha , Chen Lei , Verzi Michael P.
Published: November 2023

Terminal differentiation requires a massive restructuring of the transcriptome. During intestinal differentiation, the expression patterns of nearly 4000 genes are altered as cells transition from progenitor cells in crypts to differentiated cells in villi. We identified dynamic recruitment of RNA Polymerase II (Pol II) to gene promoters as the primary driver of transcriptomic shifts during intestinal differentiationin vivo. Changes in enhancer-promoter looping interactions accompany dynamic Pol II recruitment and are dependent upon HNF4, a pro-differentiation transcription factor. Using genetic loss-of-function, ChIP-seq and IP mass spectrometry, we demonstrate that HNF4 collaborates with chromatin remodelers and loop-stabilizing proteins and facilitates Pol II recruitment at hundreds of genes pivotal to differentiation. We also explore alternate mechanisms which drive differentiation gene expression and find pause-release of Pol II and post-transcriptional mRNA stability regulate smaller subsets of differentially expressed genes. These studies provide insights into the mechanisms of differentiation in a renewing adult tissue.

HIGHLIGHTS

Dynamic recruitment of Pol II largely drives the vast transcriptomic changes seen during differentiation of mouse intestinal epithelium.

Smaller groups of differentiated genes are subject to regulation through Pol II pause-release and post-transcriptional mechanisms such as differences in mRNA stability.

IP-mass spectrometry analysis identifies the first interactome of HNF4 in the differentiated small intestine, finding interactions with chromatin looping and chromatin remodeling proteins.

HNF4 transcription factors play a critical role in recruiting Pol II to the promoters of essential intestinal differentiation genes.
Combinatorialin silicoapproach for cancer-associated 4Fe-4S protein discovery
Authors: Tam Winnie W. L. , Cheng Michael H. W. , Mo Xiaoyong , He Bei-Bei , Ngo Ulrike F. M. , Yuen Nicholas M. H. , Yau Angela Y. L. , Wu Nicholas C. , Tse Edmund C. M.
Published: November 2023

Iron-sulfur (Fe-S) proteins play vital roles in multiple cellular processes, including mediating redox balance as well as DNA replication and repair. Given the role of Fe-S cofactors in genome maintenance, mutations in such metalloproteins could be associated with cancer. Nevertheless, only a few cancer-associated Fe-S proteins have been identified.In vitro, Fe-S cluster is susceptible to degradation in oxic environment. It could also be replaced by other metal ions during protein purification, mis-labelled as zinc finger or Zn-containing proteins.In silico, bioinorganic Fe-S cluster lacks unique sequence characteristics that distinguish itself from other metal-coordination sites, making motif prediction based solely on protein sequence difficult. Thus, in this study, three traits have been employed to discover putative cancer-associated 4Fe-4S proteins. Here, we have analyzed the human proteome via a three-pronged approach: (i) the presence of a triamino acid motif, (ii) the geometric arrangements of the cysteines, and (iii) the mutations of cancer-associated cysteines. In addition to MUTYH, a known 4Fe-4S human DNA glycosylase, 21 novel proteins were discovered as potential cancer-associated 4Fe-4S proteins. While 6 receptor proteins and 3 growth factors have been identified as potential targets in this study, 5 histone lysine methyltransferases with SET domains were also predicted to contain 4Fe-4S metallocofactors. This work provides insights for rational adjustments in experimental design and novel cancer biomarker discovery.
GENTANGLE: integrated computational design of gene entanglements
Authors: Martí Jose Manuel , Hsu Chloe , Rochereau Charlotte , Blazejewski Tomasz , Nisonoff Hunter , Leonard Sean P. , Kang-Yun Christina S. , Chlebek Jennifer , Ricci Dante P. , Park Dan , Wang Harris , Listgarten Jennifer , Jiao Yongqin , Allen Jonathan E.
Published: November 2023

The design of two overlapping genes in a microbial genome is an emerging technique for adding more reliable control mechanisms in engineered organisms for increased safety. The design of functional gene pairs is a challenging procedure and computational design tools are used to improve the efficiency to deploy successful designs in genetically engineered systems. GENTANGLE (Gene Tuples ArraNGed in overLapping Elements) is a high performance containerized pipeline for the computational design of two overlapping genes translated in different reading frames of the genome. This new software package can be used to design and test gene entanglements for microbial engineering projects using arbitrary sets of user specified gene pairs.

Availability and Implementation

The GENTANGLE source code and its submodules are freely available on GitHub athttps://github.com/BiosecSFA/gentangle. The DATANGLE (DATA for genTANGLE) repository contains related data and results, and is freely available on GitHub athttps://github.com/BiosecSFA/datangle. The GENTANGLE repository wiki contains detailed instructions on how to use the container and the different components of software and data, including reproducing the results. The code is licensed under the GNU Affero General Public License version 3 (https://www.gnu.org/licenses/agpl.html).

Contact

martimartine1@llnl.govandallen99@llnl.gov
Nuclear dualism without extensive DNA elimination in the ciliateLoxodes magnus
Authors: Seah Brandon K. B. , Singh Aditi , Vetter David E. , Emmerich Christiane , Peters Moritz , Soltys Volker , Huettel Bruno , Swart Estienne
Published: November 2023

Ciliates are unicellular eukaryotes with two distinct kinds of nuclei in each cell: transcriptionally active somatic macronuclei (MAC) and silent germline micronuclei (MIC). In the best-studied model species, both nuclei can divide asexually, but only germline MICs participate in meiosis, karyogamy, and development into new MACs. During MIC-to-MAC development, thousands of mobile element relics in the germline, called internally eliminated sequences (IESs), are excised. This genome editing enables IESs to persist by shielding them from somatic natural selection. Editing itself is a costly, time-consuming process, hypothetically maintained by evolutionary addiction.Loxodes magnusand its relatives (class Karyorelictea) are cytologically unusual because their MACs do not divide asexually, but must develop anew from mitotically generated MIC copies every cell division. Here, we report thatLoxodesgenome development is also unconventional. We found no canonical germline-limited IESs inLoxodesdespite careful purification and long-read sequencing of MICs and MACs. The k-mer content of these nuclei overlapped, and indels found by read mapping were consistent with allele variants rather than IESs. Two other hallmarks of genome editing—domesticated DDE-family transposases and editing-associated small RNAs—were also absent. Nonetheless, histone marks, nucleosome and DNA N6-methyladenosine distributions in vegetativeLoxodescells are consistent with actively transcribed MACs and inactive MICs, like other ciliates. Both genomes, not only the MIC, were large and replete with retrotransposon sequences. Given the costs associated with genome editing, we hypothesize that karyorelicteans likeLoxodeshave lost or streamlined editing during MIC-to-MAC development, and have found a way out of the addictive cycle.
Identification of Cellular Interactions in the Tumor Immune Microenvironment Underlying CD8 T Cell Exhaustion
Authors: Klocke Christopher , Moran Amy , Adey Andrew , McWeeney Shannon , Wu Guanming
Published: November 2023

While immune checkpoint inhibitors show success in treating a subset of patients with certain late-stage cancers, these treatments fail in many other patients as a result of mechanisms that have yet to be fully characterized. The process of CD8 T cell exhaustion, by which T cells become dysfunctional in response to prolonged antigen exposure, has been implicated in immunotherapy resistance. Single-cell RNA sequencing (scRNA-seq) produces an abundance of data to analyze this process; however, due to the complexity of the process, contributions of other cell types to a process within a single cell type cannot be simply inferred. We constructed an analysis framework to first rank human skin tumor samples by degree of exhaustion in tumor-infiltrating CD8 T cells and then identify immune cell type-specific gene-regulatory network patterns significantly associated with T cell exhaustion. Using this framework, we further analyzed scRNA-seq data from human tumor and chronic viral infection samples to compare the T cell exhaustion process between these two contexts. In doing so, we identified transcription factor activity in the macrophages of both tissue types associated with this process. Our framework can be applied beyond the tumor immune microenvironment to any system involving cell-cell communication, facilitating insights into key biological processes that underpin the effective treatment of cancer and other complicated diseases.
Oral delivery of the amylin receptor agonist pramlintide
Authors: Sinézia Celimar , Sisnande Tháyna , Icart Luis Peña , Lima Luís Maurício T. R.
Published: November 2023

Aim

The aims of this study were to design and characterize polymeric microparticles for oral delivery of pramlintide, a triple proline human amylin analogue clinically proved efficient in diabetes therapy.

Methods

The microparticles were prepared with gastric-resistant polymer Eudragit S100 by double-emulsion and solvent evaporation technique. The study responses were repeatability, encapsulation efficiency, yield, morphology, particle size, response to acidic and alkaline milieu and pharmacokinetics.

Results

We obtained spherical microcapsules, with particle size of 66 μm ± 11, with 83.2 % ± 2.7 efficiency for pramlintide entrapment and 67.6 % ± 2.1 yield. Intra-venous pramlintide free in solution showed a plasmatic half-life of 6.8 min in mice. In contrast, oral delivery of acid-resistant pramlintide-loaded microparticles in mice showed a protracted release for 120 min compared to 30 min obtained for pramlintide in solution.

Conclusions

Oral route is an alternative for therapeutic development of pramlintide formulations.

Graphical abstract