Analysis of genetic variants in myeloproliferative neoplasms using a 22-gene next-generation sequencing panel

Background The Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs), namely essential thrombocythaemia (ET), polycythaemia vera (PV) and primary myelofibrosis (PMF), are a group of chronic clonal haematopoietic disorders that have the propensity to advance into bone marrow failure or acute myeloid leukaemia; often resulting in fatality. Although driver mutations have been identified in these MPNs, subtype-specific markers of the disease have yet to be discovered. Next-generation sequencing (NGS) technology can potentially improve the clinical management of MPNs by allowing for the simultaneous screening of many disease-associated genes. Methods The performance of a custom, in-house designed 22-gene NGS panel was technically validated using reference standards across two independent replicate runs. The panel was subsequently used to screen a total of 10 clinical MPN samples (ET n = 3, PV n = 3, PMF n = 4). The resulting NGS data was then analysed via a bioinformatics pipeline. Results The custom NGS panel had a detection limit of 1% variant allele frequency (VAF). A total of 20 unique variants with VAFs above 5% (4 of which were putatively novel variants with potential biological significance) and one pathogenic variant with a VAF of between 1 and 5% were identified across all of the clinical MPN samples. All single nucleotide variants with VAFs ≥ 15% were confirmed via Sanger sequencing. Conclusions The high fidelity of the NGS analysis and the identification of known and novel variants in this study cohort support its potential clinical utility in the management of MPNs. However, further optimisation is needed to avoid false negatives in regions with low sequencing coverage, especially for the detection of driver mutations in MPL.

BHMPS Inhibits Breast Cancer Migration and Invasion by Disrupting Rab27a-Mediated EGFR and Fibronectin Secretion

Our previous work demonstrated that (E)-N-benzyl-6-(2-(3, 4-dihydroxybenzylidene) hydrazinyl)-N-methylpyridine-3-sulfonamide (BHMPS), a novel synthetic inhibitor of Rab27aSlp(s) interaction, suppresses tumor cell invasion and metastasis. Here, we aimed to further investigate the mechanisms of action and biological significance of BHMPS. BHMPS decreased the expression of epithelial-mesenchymal transition transcription factors through inhibition of focal adhesion kinase and c-Jun N-terminal kinase activation, thereby reducing the migration and invasion of breast cancer. Additionally, knockdown of Rab27a inhibited tumor migration, with changes in related signaling molecules, whereas overexpression of Rab27a reversed this phenomenon. BHMPS effectively prevented the interaction of Rab27a and its effector Slp4, which was verified by co-localization, immunoprecipitation, and in situ proximity ligation assays. BHMPS decreased the secretion of epidermal growth factor receptor and fibronectin by interfering with vesicle trafficking, as indicated by increased perinuclear accumulation of CD63-positive vesicles. Moreover, administration of BHMPS suppressed tumor growth in Rab27a-overexpressing MDA-MB-231 xenograft mice. These findings suggest that BHMPS may be a promising candidate for attenuating tumor migration and invasion by blocking Rab27a-mediated exocytosis.

The Gene FvTST1 From Strawberry Modulates Endogenous Sugars Enhancing Plant Growth and Fruit Ripening

Sugar is an important carbon source and contributes significantly to the improvement of plant growth and fruit flavor quality. Sugar transport through the tonoplast is important for intracellular homeostasis and metabolic balance in plant cells. There are four tonoplast sugar transporters (FvTST1-4) in strawberry genome. The qRT-PCR results indicated that FvTST1 has a differential expression pattern in different tissues and developmental stages, and exhibited highest expression level in mature fruits. The yeast complementation assay showed that FvTST1 can mediate the uptake of different sugars, such as fructose, glucose, sucrose, and mannose. Subcellular localization analyses revealed that FvTST1 was mainly targeted to the tonoplast. Transient expression of FvTST1 in strawberry fruits enhanced both fruit ripening and sugar accumulation. Furthermore, FvTST1-transformed tomato plants exhibited higher sucrose and auxin content, enhanced seed germination and vegetative growth, higher photosynthetic rate, early flowering, and bore fruit; fructose and glucose levels were higher in transgenic fruits than those in the control. Transcriptomic analysis indicated that the auxin signaling pathway was highly enriched pathway in up-regulated Gene-ontology terms. In transgenic plants, genes encoding transcription factors, such as phytochrome-interacting factors PIF1, -3, and -4, as well as their potential target genes, were also induced. Collectively, the results show that FvTST1 enhances plant growth and fruit ripening by modulating endogenous sugars, and highlight the biological significance of this gene for future breeding purposes.

Extracellular Vesicles from Pancreatic Cancer Stem Cells Lead an Intratumor Communication Network (EVNet) to fuel tumour progression

ObjectiveIntratumor heterogeneity drives cancer progression and therapy resistance. However, it has yet to be determined whether and how subpopulations of cancer cells interact and how this interaction affects the tumour.DesignWe have studied the spontaneous flow of extracellular vesicles (EVs) between subpopulations of cancer cells: cancer stem cells (CSC) and non-stem cancer cells (NSCC). To determine the biological significance of the most frequent communication route, we used pancreatic ductal adenocarcinoma (PDAC) orthotopic models, patient-derived xenografts (PDXs) and genetically engineered mouse models (GEMMs).ResultsWe demonstrate that PDAC tumours establish an organised communication network between subpopulations of cancer cells using EVs called the EVNet). The EVNet is plastic and reshapes in response to its environment. Communication within the EVNet occurs preferentially from CSC to NSCC. Inhibition of this communication route by impairing Rab27a function in orthotopic xenographs, GEMMs and PDXs is sufficient to hamper tumour growth and phenocopies the inhibition of communication in the whole tumour. Mechanistically, we provide evidence that CSC EVs use agrin protein to promote Yes1 associated transcriptional regulator (YAP) activation via LDL receptor related protein 4 (LRP-4). Ex vivo treatment of PDXs with antiagrin significantly impairs proliferation and decreases the levels of activated YAP.Patients with high levels of agrin and low inactive YAP show worse disease-free survival. In addition, patients with a higher number of circulating agrin+ EVs show a significant increased risk of disease progression.ConclusionPDAC tumours establish a cooperation network mediated by EVs that is led by CSC and agrin, which allows tumours to adapt and thrive. Targeting agrin could make targeted therapy possible for patients with PDAC and has a significant impact on CSC that feeds the tumour and is at the centre of therapy resistance.

A method for variant agnostic detection of SARS-CoV-2, rapid monitoring of circulating variants, detection of mutations of biological significance, and early detection of emergent variants such as Omicron

The rapid emergence of new SARS-CoV-2 variants raises a number of public health questions including the capability of diagnostic tests to detect new strains, the efficacy of vaccines, and how to map the geographical distribution of variants to better understand patterns of transmission and possible load on healthcare resources. Next-Generation Sequencing (NGS) is the primary method for detecting and tracing the emergence of new variants, but it is expensive, and it can take weeks before sequence data is available in public repositories. Here, we describe a Polymerase Chain Reaction (PCR)-based genotyping approach that is significantly less expensive, accelerates reporting on SARS-CoV-2 variants, and can be implemented in any testing lab performing PCR. Specific Single Nucleotide Polymorphisms (SNPs) and indels are identified that have high positive percent agreement (PPA) and negative percent agreement (NPA) compared to NGS for the major genotypes that circulated in 2021. Using a 48-marker panel, testing on 1,128 retrospective samples yielded a PPA and NPA in the 96.3 to 100% and 99.2 to 100% range, respectively, for the top 10 most prevalent lineages. The effect on PPA and NPA of reducing the number of panel markers was also explored. In addition, with the emergence of Omicron, we also developed an Omicron genotyping panel that distinguishes the Delta and Omicron variants using four (4) highly specific SNPs. Data from testing demonstrates the capability to use the panel to rapidly track the growing prevalence of the Omicron variant in the United States in December 2021.

Mitophagy Eliminates the Accumulation of SARM1 on the Mitochondria, Alleviating Programmed Axon Death in Acrylamide Neuropathy

Sterile-α and toll/interleukin 1 receptor motif containing protein 1 (SARM1) is the central executioner of programmed axon death (Wallerian degeneration). Although it has been confirmed to have a mitochondrial targeting sequence and can bind to and stabilize PINK1 on mitochondria, the biological significance for mitochondrial localization of SARM1 is still unclear. The relationship between mitochondrial quality control mechanisms and programmed axon death also needs to be clarified. Chronic acrylamide (ACR) intoxication cause typical pathology of axon degeneration involving early axon loss. Here, we demonstrated that the SARM1 dependent Wallerian axon self-destruction pathway was activated following ACR intoxication. Moreover, increased SARM1 was observed on the mitochondria, which interfered with the mitochondrial quality control mechanisms. As a protective response to stress, mitochondrial components enriched in SARM1 were isolated from the mitochondrial network through an increased fission process and were degraded in an autophagy-dependent manner. Importantly, rapamycin (RAPA) administration eliminated mitochondrial accumulated SARM1 and inhibited axon loss. Thus, mitochondrial localization of SARM1 may be complement to the coordinated activity of NMNAT2 and SARM1, and may be part of the self-limiting molecular mechanisms of programmed axon death. In the early latent period, the mitochondrial localization of SARM1 will help it to be isolated by the mitochondrial network and to be degraded through mitophagy to maintain local axon homeostasis. When the mitochondrial quality control mechanisms are broken down, SARM1 will cause irreversible damage for axon death.

A unified view of LCRs across species

Low complexity regions (LCRs) in proteins play a major role in the higher order assemblies of organisms, such as the nucleolus and extracellular matrix. Despite recent focus on how certain features affect the function of LCRs in intracellular higher order assemblies, the relationships between LCRs within proteins, captured by their type and copy number, has yet to be systematically studied. Furthermore, we still lack a unified view of how the sequences, features, relationships and functions of LCRs relate to each other. Here, we developed a systematic and comprehensive approach using dotplot matrices and dimensionality reduction to define LCR relationships proteome-wide and to create a map of LCR sequences capable of integrating any LCR features. As a proof of concept of the importance of LCR relationships, we demonstrate the biological significance of LCR copy number for higher order assembly of the nucleolar protein RPA43 both in vitro and in vivo. Using the LCR map, we revealed the boundaries and connections between regions of sequence space occupied by LCRs, and that LCRs of certain higher order assemblies populated specific regions of sequence space. The integration of LCR relationships and the LCR map provided a unified view of LCRs which uncovered the distribution, distinguishing features, and conserved prevalence of glutamic acid-rich LCRs among nucleolar proteins. When applied across multiple species, this approach highlights how differential occupancy of certain regions of LCR sequence space corresponds to the conservation and emergence of higher order assemblies, such as the plant cell wall or metazoan extracellular matrix. Additionally, we identified previously undescribed regions of LCR sequence space, including a teleost-specific threonine/histidine-rich cluster which exhibits signatures of higher order assemblies. By providing this unified view of LCRs, our approach enables discovery of how LCRs encode higher order assemblies of organisms.

Human Milk Oligosaccharides Exhibit Biofilm Eradication Activity Against Matured Biofilms Formed by Different Pathogen Species

Human milk oligosaccharides (HMOs) have been shown to exhibit plenty of benefits for infants, such as prebiotic activity shaping the gut microbiota and immunomodulatory and anti-inflammatory activity. For some pathogenic bacteria, antimicrobial activity has been proved, but most studies focus on group B streptococci. In the present study, we investigated the antimicrobial and antibiofilm activities of the total and fractionated HMOs from pooled human milk against four common human pathogenic Gram-negative species (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Burkholderia cenocepacia) and three Gram-positive species (Staphylococcus aureus, Enterococcus faecium, and Enterococcus faecalis). The activity of HMOs against enterococci and B. cenocepacia are addressed here for the first time. We showed that HMOs exhibit a predominant activity against the Gram-positive species, with E. faecalis being the most sensitive to the HMOs, both in planktonic bacteria and in biofilms. In further tests, we could exclude fucosyllactose as the antibacterial component. The biological significance of these findings may lie in the prevention of skin infections of the mother’s breast as a consequence of breastfeeding-induced skin laceration and/or protection of the infants’ nasopharynx and lung from respiratory pathogens such as staphylococci.

ZC3H15 promotes glioblastoma progression through regulating EGFR stability

Zinc finger CCCH-type containing 15 (ZC3H15), a highly conserved protein involved in several cellular processes, which was responsible for tumorigenesis and may be a promising marker in myeloid leukemia (AML) and hepatocellular carcinoma (HCC). However, little is known about the biological significance and molecular mechanisms of ZC3H15 in GBM. In this study, we revealed that ZC3H15 was overexpressed in GBM and high ZC3H15 expression was associated with poor survival of patients with GBM. We found that ZC3H15 promoted the proliferation, migration, invasion, and tumorigenesis of GBM cells by activating the EGFR signaling pathway. We also revealed that ZC3H15 reduced EGFR ubiquitination, which was responsible for EGFR protein stabilization. In addition, we demonstrated that ZC3H15 inhibited the transcription of CBL, which was an E3 ubiquitin ligase for EGFR proteasomal degradation. And silencing of CBL could partly abrogate the inhibitory effects on cell proliferation, migration, and invasion of GBM cells induced by ZC3H15 knockdown. Thus, our research revealed the important roles of ZC3H15 in GBM development and provided a brand-new insight for improving the treatment of GBMs.