Label-free isolation of prostate circulating tumor cells using Vortex microfluidic technology.

There has been increased interest in utilizing non-invasive "liquid biopsies" to identify biomarkers for cancer prognosis and monitoring, and to isolate genetic material that can predict response to targeted therapies. Circulating tumor cells (CTCs) have emerged as such a biomarker providing both genetic and phenotypic information about tumor evolution, potentially from both primary and metastatic sites. Currently, available CTC isolation approaches, including immunoaffinity and size-based filtration, have focused on high capture efficiency but with lower purity and often long and manual sample preparation, which limits the use of captured CTCs for downstream analyses. Here, we describe the use of the microfluidic Vortex Chip for size-based isolation of CTCs from 22 patients with advanced prostate cancer and, from an enumeration study on 18 of these patients, find that we can capture CTCs with high purity (from 1.74 to 37.59%) and efficiency (from 1.88 to 93.75 CTCs/7.5 mL) in less than 1 h. Interestingly, more atypical large circulating cells were identified in five age-matched healthy donors (46-77 years old; 1.25-2.50 CTCs/7.5 mL) than in five healthy donors <30 years old (21-27 years old; 0.00 CTC/7.5 mL). Using a threshold calculated from the five age-matched healthy donors (3.37 CTCs/mL), we identified CTCs in 80% of the prostate cancer patients. We also found that a fraction of the cells collected (11.5%) did not express epithelial prostate markers (cytokeratin and/or prostate-specific antigen) and that some instead expressed markers of epithelial-mesenchymal transition, i.e., vimentin and N-cadherin. We also show that the purity and DNA yield of isolated cells is amenable to targeted amplification and next-generation sequencing, without whole genome amplification, identifying unique mutations in 10 of 15 samples and 0 of 4 healthy samples

Lipidomic Analysis of Glioblastoma Multiforme Using Mass Spectrometry

Glioblastoma multiforme (GBM) is the most common and malignant form of primary brain tumors. It is highly invasive and current treatment options have not improved the survival rate over the past twenty years. Novel approaches and technologies from systems biology have the potential to identify biomarkers that could serve as new therapeutic targets for GBM. This study employed lipid profiling technology to investigate lipid biomarkers in ectopic and orthotopic human GBM xenograft models. Primary patient cell lines, GBM10 and GBM43, were injected into the flank and the right cerebral hemisphere of NOD/SCID mice. Tumors were harvested from the brain and flank and proteins, metabolites, and lipids extracted from each sample. Reverse phase based high performance liquid chromatography coupled with Fourier transform ion cyclotron resonance mass spectrometry (LC-FTMS) was used to analyze the lipid profiles of tumor samples. Statistical and clustering analyses were performed to detect differences. Over 500 lipids were identified in each tumor model and lipids with the greatest fold effect in the comparison of ectopic versus orthotopic tumor models fell predominantly into four main classes of lipids: glycosphingolipids, glycerophoshpoethanolamines, triradylglycerols, and glycerophosphoserines. Lipidomic analysis revealed differences in glycosphingolipid and triglyceride profiles when the same tumor was propagated in the flank versus the brain. These results underscore the importance of the surrounding physiological environment on tumor development and are consistent with the hypothesis that specific classes of lipids are critical for GBM tumor growth in different anatomical sites

Lipidomic Analysis of Glioblastoma Multiforme Using Mass Spectrometry

Glioblastoma multiforme (GBM) is the most common and malignant form of primary brain tumors. It is highly invasive and current treatment options have not improved the survival rate over the past twenty years. Novel approaches and technologies from systems biology have the potential to identify biomarkers that could serve as new therapeutic targets for GBM. This study employed lipid profiling technology to investigate lipid biomarkers in ectopic and orthotopic human GBM xenograft models. Primary patient cell lines, GBM10 and GBM43, were injected into the flank and the right cerebral hemisphere of NOD/SCID mice. Tumors were harvested from the brain and flank and proteins, metabolites, and lipids extracted from each sample. Reverse phase based high performance liquid chromatography coupled with Fourier transform ion cyclotron resonance mass spectrometry (LC-FTMS) was used to analyze the lipid profiles of tumor samples. Statistical and clustering analyses were performed to detect differences. Over 500 lipids were identified in each tumor model and lipids with the greatest fold effect in the comparison of ectopic versus orthotopic tumor models fell predominantly into four main classes of lipids: glycosphingolipids, glycerophoshpoethanolamines, triradylglycerols, and glycerophosphoserines. Lipidomic analysis revealed differences in glycosphingolipid and triglyceride profiles when the same tumor was propagated in the flank versus the brain. These results underscore the importance of the surrounding physiological environment on tumor development and are consistent with the hypothesis that specific classes of lipids are critical for GBM tumor growth in different anatomical sites

Myeloid Cell-Derived Reactive Oxygen Species Externally Regulate the Proliferation of Myeloid Progenitors in Emergency Granulopoiesis

SummaryThe cellular mechanisms controlling infection-induced emergency granulopoiesis are poorly defined. Here we found that reactive oxygen species (ROS) concentrations in the bone marrow (BM) were elevated during acute infection in a phagocytic NADPH oxidase-dependent manner in myeloid cells. Gr1+ myeloid cells were uniformly distributed in the BM, and all c-kit+ progenitor cells were adjacent to Gr1+ myeloid cells. Inflammation-induced ROS production in the BM played a critical role in myeloid progenitor expansion during emergency granulopoiesis. ROS elicited oxidation and deactivation of phosphatase and tensin homolog (PTEN), resulting in upregulation of PtdIns(3,4,5)P3 signaling in BM myeloid progenitors. We further revealed that BM myeloid cell-produced ROS stimulated proliferation of myeloid progenitors via a paracrine mechanism. Taken together, our results establish that phagocytic NADPH oxidase-mediated ROS production by BM myeloid cells plays a critical role in mediating emergency granulopoiesis during acute infection

Adsorbate-induced structural changes in 1-3 nm platinum nanoparticles

We investigated changes in the Pt–Pt bond distance, particle size, crystallinity, and coordination of Pt nanoparticles as a function of particle size (1–3 nm) and adsorbate (H2, CO) using synchrotron radiation pair distribution function (PDF) and X-ray absorption spectroscopy (XAS) measurements. The ∼1 nm Pt nanoparticles showed a Pt–Pt bond distance contraction of ∼1.4%. The adsorption of H2 and CO at room temperature relaxed the Pt–Pt bond distance contraction to a value close to that of bulk fcc Pt. The adsorption of H2 improved the crystallinity of the small Pt nanoparticles. However, CO adsorption generated a more disordered fcc structure for the 1–3 nm Pt nanoparticles compared to the H2 adsorption Pt nanoparticles. In situ XANES measurements revealed that this disorder results from the electron back-donation of the Pt nanoparticles to CO, leading to a higher degree of rehybridization of the metal orbitals in the Pt-adsorbate system

Biogeographic Distribution Patterns of the Archaeal Communities Across the Black Soil Zone of Northeast China

Although archaea are ubiquitous in various environments, the knowledge gaps still exist regarding the biogeographical distribution of archaeal communities at regional scales in agricultural soils compared with bacteria and fungi. To provide a broader biogeographical context of archaeal diversity, this study quantified the abundance and community composition of archaea across the black soil zone in northeast China using real-time PCR and high-throughput sequencing (HTS) methods. Archaeal abundances across all soil samples ranged from 4.04 × 107 to 26.18 × 107 16S rRNA gene copies per gram of dry soil. Several soil factors were positively correlated with the abundances including soil pH, concentrations of total C, N, and P, and available K in soil, and soil water content. Approximately 94.2, 5.7, and 0.3% of archaeal sequences, and 31, 151, and 3 OTUs aligned within the phyla Thaumarchaeota, Euryarchaeota, and Crenarchaeota, respectively. Within the phylum of Thaumarchaeota, group 1.1b was a dominating genus accounting for an average of 87% archaeal sequences and phylogenetically classified as Nitrososphaera, a genus of ammonia oxidizing archaea. The response of dominating OTUs to environmental factors differed greatly, suggesting the physiological characteristics of different archaeal members is diversified in the black soils. Although the number of OTUs was not related with any particular soil parameters, the number of OTUs within Thaumarchaeota and Euryarchaeota was marginally related with soil pH. Archaeal community compositions differed between samples, and a Canonical correspondence analysis (CCA) analysis indicated that soil pH and the latitude of sampling locations were two dominating factors in shifting community structures. A variance partitioning analysis (VPA) analysis showed that the selected soil parameters (32%) were the largest drivers of community variation, in particular soil pH (21%), followed by geographic distances (19%). These findings suggest that archaeal communities have distinct biogeographic distribution pattern in the black soil zone and soil pH was the key edaphic factor in structuring the community compositions

A broadly reactive antibody targeting the N-terminal domain of SARS-CoV-2 spike confers Fc-mediated protection

Most neutralizing anti-SARS-CoV-2 monoclonal antibodies (mAbs) target the receptor binding domain (RBD) of the spike (S) protein. Here, we characterize a panel of mAbs targeting the N-terminal domain (NTD) or other non-RBD epitopes of S. A subset of NTD mAbs inhibits SARS-CoV-2 entry at a post-attachment step and avidly binds the surface of infected cells. One neutralizing NTD mAb, SARS2-57, protects K18-hACE2 mice against SARS-CoV-2 infection in an Fc-dependent manner. Structural analysis demonstrates that SARS2-57 engages an antigenic supersite that is remodeled by deletions common to emerging variants. In neutralization escape studies with SARS2-57, this NTD site accumulates mutations, including a similar deletion, but the addition of an anti-RBD mAb prevents such escape. Thus, our study highlights a common strategy of immune evasion by SARS-CoV-2 variants and how targeting spatially distinct epitopes, including those in the NTD, may limit such escape

Trafficking-Deficient G572R-hERG and E637K-hERG Activate Stress and Clearance Pathways in Endoplasmic Reticulum

Background: Long QT syndrome type 2 (LQT2) is the second most common type of all long QT syndromes. It is well-known that trafficking deficient mutant human ether-a-go-go-related gene (hERG) proteins are often involved in LQT2. Cells respond to misfolded and trafficking-deficient proteins by eliciting the unfolded protein response (UPR) and Activating Transcription Factor (ATF6) has been identified as a key regulator of the mammalian UPR. In this study, we investigated the role of ER chaperone proteins (Calnexin and Calreticulin) in the processing of G572R-hERG and E637K-hERG mutant proteins. Methods: pcDNA3-WT-hERG, pcDNA3-G572R-hERG and pcDNA3-E637K-hERG plasmids were transfected into U2OS and HEK293 cells. Confocal microscopy and western blotting were used to analyze subcellular localization and protein expression. Interaction between WT or mutant hERGs and Calnexin/Calreticulin was tested by coimmunoprecipitation. To assess the role of the ubiquitin proteasome pathway in the degradation of mutant hERG proteins, transfected HEK293 cells were treated with proteasome inhibitors and their effects on the steady state protein levels of WT and mutant hERGs were examined. Conclusion: Our results showed that levels of core-glycosylated immature forms of G572R-hERG and E637K-hERG in association with Calnexin and Calreticulin were higher than that in WT-hERG. Both mutant hERG proteins could activate the UPR by upregulating levels of active ATF6. Furthermore, proteasome inhibition increased the levels of core-glycosylated immature forms of WT and mutant hERGs. In addition, interaction between mutant hERGs and Calnexin/Calreticulin wa

Transgene Expression Is Associated with Copy Number and Cytomegalovirus Promoter Methylation in Transgenic Pigs

Transgenic animals have been used for years to study gene function, produce important proteins, and generate models for the study of human diseases. However, inheritance and expression instability of the transgene in transgenic animals is a major limitation. Copy number and promoter methylation are known to regulate gene expression, but no report has systematically examined their effect on transgene expression. In the study, we generated two transgenic pigs by somatic cell nuclear transfer (SCNT) that express green fluorescent protein (GFP) driven by cytomegalovirus (CMV). Absolute quantitative real-time PCR and bisulfite sequencing were performed to determine transgene copy number and promoter methylation level. The correlation of transgene expression with copy number and promoter methylation was analyzed in individual development, fibroblast cells, various tissues, and offspring of the transgenic pigs. Our results demonstrate that transgene expression is associated with copy number and CMV promoter methylation in transgenic pigs