The Stress-Inducible Heat Shock Protein 70 (HSPA1A) as A Novel Biomarker in Lung Tumors: A Promoting Study

Heat shock proteins (HSPs) are a group of intracellular proteins that promote proteins folding and unfolding under normal and/or stress conditions. In addition to their intracellular location, HSPs are found on the plasma membranes of stressed, but not normal, cells and in the extracellular milieu where they can trigger an immune response. For instance, the inducible form of heat shock protein 70 (HSPA1A) was found to be overexpressed, intra or extracellularly, by many types of stressed cells. In our study, we aim to investigate the levels of HSPA1A in the serum of untreated lung tumor patients and its expression in the tissues derived from lung tumors and chronic obstructive pulmonary diseases (COPD) and healthy tissues. Analyses of serum and tissue samples were performed by utilizing ELISA assay and immunohistochemical staining, respectively. The results showed significant differences in the mean levels of serum HSPA1A of the patients (1.66± 0.165) as compared to healthy control (0.938±0.330). Significant differences were also found between these levels in the serum of smoker and nonsmoker patients (2.006±0.342 and 1.353± 0.067, respectively). The protein levels in the patient's serum were also found to be 29.5% correlated to smoking history. No significant differences were found in terms of gender and age within the two groups of patients and healthy volunteers. Our results showed HSPA1A protein levels to be correlated to gender (13.5%) and age in patients and healthy volunteers (14.8% and 0.4%, respectively). In the tissues of the clinicopathological groups, HSPA1A was found to be overexpressed in benign (IRS= 8.5) and malignant tumors (IRS= 8.9) in comparison with both COPD (IRS= 4.4) and healthy tissues (IRS= 0.00). Our pilot results could support the suggestion of HSPA1A as a novel biomarker in lung tumors, as proposed by previous studies.

Host and Bacterial Iron Homeostasis, an Underexplored Area in Tuberculosis Biomarker Research

Mycobacterium tuberculosis (Mtb) “a human adapted pathogen” has found multiple ways to manipulate the host immune response during infection. The human immune response to Mtb infection is a highly complex cascade of reactions, with macrophages as preferred intracellular location. Interaction with the host through infection gives rise to expression of specific gene products for survival and multiplication within the host. The signals that the pathogens encounter during infection cause them to selectively express genes in response to signals. One strategy to identify Mtb antigens with diagnostic potential is to identify genes that are specifically induced during infection or in specific disease stages. The shortcomings of current immunodiagnostics include the failure to detect progression from latent infection to active tuberculosis disease, and the inability to monitor treatment efficacy. This highlights the need for new tuberculosis biomarkers. These biomarkers should be highly sensitive and specific diagnosing TB infection, specifically distinguishing between latent infection and active disease. The regulation of iron levels by the host plays a crucial role in the susceptibility and outcome of Mtb infection. Of interest are the siderophore biosynthetic genes, encoded by the mbt-1 and mbt-2 loci and the SUF (mobilization of sulphur) operon (sufR-sufB-sufD-sufC-csd-nifU-sufT), which encodes the primary iron-sulphur cluster biogenesis system. These genes are induced during iron limitation and intracellular growth of Mtb, pointing to their importance during infection.

Carbonic Anhydrase Isoforms of Pyropia Yezoensis: Intracellular Location and Expression Profiles in Response to Inorganic Carbon Concentration and Life Stage

BackgroundMacroalgae, particularly commercially grown seaweed, substantially contribute to CO2 removal and carbon storage. However, knowledge regarding the CO2 concentrating mechanism (CCM) of macroalgae is limited. Carbonic anhydrase (CA), the key component of biophysical CCM, plays important roles in many physiological reactions in various organisms. CA has been widely studied in microalgae and higher plants. However, few characteristics of CA in Pyropia yezoensis are known, particularly its intracellular location and responses to different concentrations of Ci.ResultsWe retrieved transcriptomic and genomic data for P. yezoensis and identified, amplified and characterized 11 putative genes encoding CA. The predicted corresponding proteins clustered into three subfamilies: α-, β- and γ-type. Transcriptomic and qRT-PCR results revealed different expression levels of these PyCA genes in the two life stages of P. yezoensis, and their expression levels varied under different Ci conditions. The intracellular localization of eight CA isoforms—one in the chloroplasts, four in the cytoplasm and three in the mitochondria—were elucidated with fusion proteins. One γCA isoform (PyCA10) was shown to be localized in the cytosol, and its localization is discussed. We also discuss the potential roles of the CA isoforms in development and the CCM in the two life stages. ConclusionsCA isoforms in P. yezoensis are widely distributed within cells, and higher PyCA expression, particularly of certain chloroplastic, cytosolic and mitochondrional CAs, is observed more often during the leafy stage. This expression results in stronger carbon sequestration ability in leafy thalli than filamentous thalli.

Differential Labeling of Chemically Modified Peptides and Lipids among Cyanobacteria Planktothrix and Microcystis

The cyanoHAB forming cyanobacteria Microcystis and Planktothrix frequently produce high intracellular amounts of microcystins (MCs) or anabaenopeptins (APs). In this study, chemically modified MCs and APs have been localized on a subcellular level in Microcystis and Planktothrix applying copper-catalyzed alkyne-azide cycloaddition (CuACC). For this purpose, three different non-natural amino acids carrying alkyne or azide moieties were fed to individual P. agardhii strains No371/1 and CYA126/8 as well as to M. aeruginosa strain Hofbauer showing promiscuous incorporation of various amino acid substrates during non-ribosomal peptide synthesis (NRPS). Moreover, CYA126/8 peptide knock-out mutants and non-toxic strain Synechocystis PCC6803 were processed under identical conditions. Simultaneous labeling of modified peptides with ALEXA405 and ALEXA488 and lipid staining with BODIPY 505/515 were performed to investigate the intracellular location of the modified peptides. Pearson correlation coefficients (PCC) obtained from confocal images were calculated between the different fluorophores and the natural autofluorescence (AF), and between labeled modified peptides and dyed lipids to investigate the spatial overlap between peptides and the photosynthetic complex, and between peptides and lipids. Overall, labeling of modified MCs (M. aeruginosa) and APs (P. agardhii) using both fluorophores revealed increased intensity in MC/AP producing strains. For Synechocystis lacking NRPS, no labeling using either ALEXA405 or ALEXA488 was observed. Lipid staining in M. aeruginosa and Synechocystis was intense while in Planktothrix it was more variable. When compared with AF, both modified peptides and lipids showed a heterologous distribution. In comparison, the correlation between stained lipids and labeled peptides was not increased suggesting a reduced spatial overlap.

Ceramide From Sphingomyelin Hydrolysis Induces Neuronal Differentiation, Whereas De Novo Ceramide Synthesis And Sphingomyelin Hydrolysis Initiate Apoptosis After NGF Withdrawal In PC12 Cells

Background: Ceramide, important for both neuronal differentiation and dedifferentiation, resides in several membranes, is synthesized in the endoplasmic reticulum, mitochondrial, and nuclear membranes, and can be further processed into glycosphingolipids or sphingomyelin. Ceramide may also be generated by hydrolysis of sphingomyelin by neutral or acidic sphingomyelinases in lysosomes and other membranes. Here we asked whether the differing functions of ceramide derived from different origins. Methods: We added NGF to PC12 cells and to TrkA cells. These latter overexpress NGF receptors and are partially activated to differentiate, whereas NGF is required for PC12 cells to differentiate. We differentiated synthesis from hydrolysis by the use of appropriate inhibitors. Results: When NGF is added, the kinetics and amounts of ceramide and sphingomyelin indicate that the ceramide comes primarily from hydrolysis but, when hydrolysis is inhibited, can also come from neosynthesis. When NGF is removed, the ceramide comes from both neosynthesis and hydrolysis. Conclusion: We conclude that the function of ceramide depends heavily on its intracellular location, and that further understanding of its function will depend on resolving its location during changes of cell status.

ENaC and ROMK channels in the connecting tubule regulate renal K+ secretion

We measured the activities of epithelial Na channels (ENaC) and ROMK channels in the distal nephron of the mouse kidney and assessed their role in the process of K+ secretion under different physiological conditions. Under basal dietary conditions (0.5% K), ENaC activity, measured as amiloride-sensitive currents, was high in cells at the distal end of the distal convoluted tubule (DCT) and proximal end of the connecting tubule (CNT), a region we call the early CNT (CNTe). In more distal parts of the CNT (aldosterone-sensitive portion [CNTas]), these currents were minimal. This functional difference correlated with alterations in the intracellular location of ENaC, which was at or near the apical membrane in CNTe and more cytoplasmic in the CNTas. ROMK activity, measured as TPNQ-sensitive currents, was substantial in both segments. A mathematical model of the rat nephron suggested that K+ secretion by the CNTe predicted from these currents provides much of the urinary K+ required for K balance on this diet. In animals fed a K-deficient diet (0.1% K), both ENaC and ROMK currents in the CNTe decreased by ∼50%, predicting a 50% decline in K+ secretion. Enhanced reabsorption by a separate mechanism is required to avoid excessive urinary K+ losses. In animals fed a diet supplemented with 3% K, ENaC currents increased modestly in the CNTe but strongly in the CNTas, while ROMK currents tripled in both segments. The enhanced secretion of K+ by the CNTe and the recruitment of secretion by the CNTas account for the additional transport required for K balance. Therefore, adaptation to increased K+ intake involves the extension of robust K+ secretion to more distal parts of the nephron.

Morphological and biological properties of silica nanoparticles for CRTC3-siRNA delivery and downregulation of the RGS2 expression in preadipocytes

The aim of this study was to characterize the morphological properties of amorphous silica nanoparticles (SiO2 NPs), their cytotoxicity and intracellular location within Human Osteoblasts (HOB). Additionally, SiO2 NPs were explored for their effectivity as carriers of CRTC3-siRNA on Human Preadipocytes (HPAd), and thus downregulate RGS2 gene expression. SiO2 NPs were synthesized using the method of Stöber at 45 °C, 56 °C, and 62 °C. These were characterized via TEM with EDS, Zeta Potential and FT-IR. Cytotoxicity was evaluated by XTT at three concentrations 50, 100 and 500 µg/mL; SiO2 NPs intracellular localization was observed through Confocal Laser Scanning Microscope. Delivering siRNA effectivity was measured by RT-qPCR. Morphology of SiO2 NPs was spherical with a range size from 64 to 119 nm; their surface charge was negative. Confocal images demonstrated that SiO2 NPs were located within cellular cytoplasm. At a SiO2 NPs concentration of 500 µg/mL HOB viability decreased, while at 50 µg/mL and 100 µg/mL cell viability was not affected regardless SiO2 NPs size. SiO2 NPs-CRTC3-siRNA are effective to down-regulate RGS2 gene expression in HPAd without cytotoxic effects. The developed SiO2 NPs-CRTC3-siRNA are a promising tool as a delivery vehicle to control obesity.

The CD33 short isoform is a gain-of-function variant that enhances Aβ1–42 phagocytosis in microglia

Background CD33 is genetically linked to Alzheimer’s disease (AD) susceptibility through differential expression of isoforms in microglia. The role of the human CD33 short isoform (hCD33m), preferentially encoded by an AD-protective CD33 allele (rs12459419T), is unknown. Here, we test whether hCD33m represents a loss-of-function or gain-of-function variant. Methods We have developed two models to test the role of hCD33m. The first is a new strain of transgenic mice expressing hCD33m in the microglial cell lineage. The second is U937 cells where the CD33 gene was disrupted by CRISPR/Cas9 and complemented with different variants of hCD33. Primary microglia and U937 cells were tested in phagocytosis assays and single cell RNA sequencing (scRNAseq) was carried out on the primary microglia. Furthermore, a new monoclonal antibody was developed to detect hCD33m more efficiently. Results In both primary microglia and U937 cells, we find that hCD33m enhances phagocytosis. This contrasts with the human CD33 long isoform (hCD33M) that represses phagocytosis, as previously demonstrated. As revealed by scRNAseq, hCD33m+ microglia are enriched in a cluster of cells defined by an upregulated expression and gene regulatory network of immediate early genes, which was further validated within microglia in situ. Using a new hCD33m-specific antibody enabled hCD33m expression to be examined, demonstrating a preference for an intracellular location. Moreover, this newly discovered gain-of-function role for hCD33m is dependent on its cytoplasmic signaling motifs, dominant over hCD33M, and not due to loss of glycan ligand binding. Conclusions These results provide strong support that hCD33m represents a gain-of-function isoform and offers insight into what it may take to therapeutically capture the AD-protective CD33 allele.