Redox-Regulation of α-Globin in Vascular Physiology

Interest in the structure, function, and evolutionary relations of circulating and intracellular globins dates back more than 60 years to the first determination of the three-dimensional structure of these proteins. Non-erythrocytic globins have been implicated in circulatory control through reactions that couple nitric oxide (NO) signaling with cellular oxygen availability and redox status. Small artery endothelial cells (ECs) express free α-globin, which causes vasoconstriction by degrading NO. This reaction converts reduced (Fe2+) α-globin to the oxidized (Fe3+) form, which is unstable, cytotoxic, and unable to degrade NO. Therefore, (Fe3+) α-globin must be stabilized and recycled to (Fe2+) α-globin to reinitiate the catalytic cycle. The molecular chaperone α-hemoglobin-stabilizing protein (AHSP) binds (Fe3+) α-globin to inhibit its degradation and facilitate its reduction. The mechanisms that reduce (Fe3+) α-globin in ECs are unknown, although endothelial nitric oxide synthase (eNOS) and cytochrome b5 reductase (CyB5R3) with cytochrome b5 type A (CyB5a) can reduce (Fe3+) α-globin in solution. Here, we examine the expression and cellular localization of eNOS, CyB5a, and CyB5R3 in mouse arterial ECs and show that α-globin can be reduced by either of two independent redox systems, CyB5R3/CyB5a and eNOS. Together, our findings provide new insights into the regulation of blood vessel contractility.

Endothelial cells are an important source of BDNF in rat skeletal muscle

BDNF (brain-derived neurotrophic factor) is present in skeletal muscle, controlling muscular metabolism, strength and regeneration processes. However, there is no consensus on BDNF cellular source. Furthermore, while endothelial tissue expresses BDNF in large amount, whether endothelial cells inside muscle expressed BDNF has never been explored. The aim of the present study was to provide a comprehensive analysis of BDNF localization in rat skeletal muscle. Cellular localization of BDNF and activated Tropomyosin-related kinase B (TrkB) receptors was studied by immunohistochemical analysis on soleus (SOL) and gastrocnemius (GAS). BDNF and activated TrkB levels were also measured in muscle homogenates using Western blot analysis and/or Elisa tests. The results revealed BDNF immunostaining in all cell types examined with a prominent staining in endothelial cells and a stronger staining in type II than type I muscular fibers. Endothelial cells but not other cells displayed easily detectable activated TrkB receptor expression. Levels of BDNF and activated TrkB receptors were higher in SOL than GAS. In conclusion, endothelial cells are an important and still unexplored source of BDNF present in skeletal muscle. Endothelial BDNF expression likely explains why oxidative muscle exhibits higher BDNF levels than glycolytic muscle despite higher the BDNF expression by type II fibers.

Heavy metal–induced stress in eukaryotic algae—mechanisms of heavy metal toxicity and tolerance with particular emphasis on oxidative stress in exposed cells and the role of antioxidant response

Heavy metals is a collective term describing metals and metalloids with a density higher than 5 g/cm3. Some of them are essential micronutrients; others do not play a positive role in living organisms. Increased anthropogenic emissions of heavy metal ions pose a serious threat to water and land ecosystems. The mechanism of heavy metal toxicity predominantly depends on (1) their high affinity to thiol groups, (2) spatial similarity to biochemical functional groups, (3) competition with essential metal cations, (4) and induction of oxidative stress. The antioxidant response is therefore crucial for providing tolerance to heavy metal-induced stress. This review aims to summarize the knowledge of heavy metal toxicity, oxidative stress and antioxidant response in eukaryotic algae. Types of ROS, their formation sites in photosynthetic cells, and the damage they cause to the cellular components are described at the beginning. Furthermore, heavy metals are characterized in more detail, including their chemical properties, roles they play in living cells, sources of contamination, biochemical mechanisms of toxicity, and stress symptoms. The following subchapters contain the description of low-molecular-weight antioxidants and ROS-detoxifying enzymes, their properties, cellular localization, and the occurrence in algae belonging to different clades, as well as the summary of the results of the experiments concerning antioxidant response in heavy metal-treated eukaryotic algae. Other mechanisms providing tolerance to metal ions are briefly outlined at the end.

TDP-43 Cytoplasmic Translocation in the Skin Fibroblasts of ALS Patients

Diagnosis of ALS is based on clinical symptoms when motoneuron degeneration is significant. Therefore, new approaches for early diagnosis are needed. We aimed to assess if alterations in appearance and cellular localization of cutaneous TDP-43 may represent a biomarker for ALS. Skin biopsies from 64 subjects were analyzed: 44 ALS patients, 10 healthy controls (HC) and 10 neurological controls (NC) (Parkinson’s disease and multiple sclerosis). TDP-43 immunoreactivity in epidermis and dermis was analyzed, as well as the percentage of cells with TDP-43 cytoplasmic localization. We detected a higher amount of TDP-43 in epidermis (p < 0.001) and in both layers of dermis (p < 0.001), as well as a higher percentage of TDP-43 cytoplasmic positive cells (p < 0.001) in the ALS group compared to HC and NC groups. Dermal cells containing TDP-43 were fibroblasts as identified by co-labeling against vimentin. ROC analyses (AUC 0.867, p < 0.001; CI 95% 0.800–0.935) showed that detection of 24.1% cells with cytoplasmic TDP-43 positivity in the dermis had 85% sensitivity and 80% specificity for detecting ALS. We have identified significantly increased TDP-43 levels in epidermis and in the cytoplasm of dermal cells of ALS patients. Our findings provide support for the use of TDP-43 in skin biopsies as a potential biomarker.

Increased Expression and Altered Cellular Localization of Fibroblast Growth Factor Receptor-Like 1 (FGFRL1) Are Associated with Prostate Cancer Progression

Fibroblast growth factor receptors (FGFRs) 1–4 are involved in prostate cancer (PCa) regulation, but the role of FGFR-like 1 (FGFRL1) in PCa is unclear. FGFRL1 expression was studied by qRT-PCR and immunohistochemistry of patient tissue microarrays (TMAs) and correlated with clinical patient data. The effects of FGFRL1 knockdown (KD) in PC3M were studied in in vitro culture models and in mouse xenograft tumors. Our results showed that FGFRL1 was significantly upregulated in PCa. The level of membranous FGFRL1 was negatively associated with high Gleason scores (GSs) and Ki67, while increased cytoplasmic and nuclear FGFRL1 showed a positive correlation. Cox regression analysis indicated that nuclear FGFRL1 was an independent prognostic marker for biochemical recurrence after radical prostatectomy. Functional studies indicated that FGFRL1-KD in PC3M cells increases FGFR signaling, whereas FGFRL1 overexpression attenuates it, supporting decoy receptor actions of membrane-localized FGFRL1. In accordance with clinical data, FGFRL1-KD markedly suppressed PC3M xenograft growth. Transcriptomics of FGFRL1-KD cells and xenografts revealed major changes in genes regulating differentiation, ECM turnover, and tumor–stromal interactions associated with decreased growth in FGFRL1-KD xenografts. Our results suggest that FGFRL1 upregulation and altered cellular compartmentalization contribute to PCa progression. The nuclear FGFRL1 could serve as a prognostic marker for PCa patients.

Single-Cell RNA Sequencing Reveals Heterogeneity Among Adult Mouse, Primate and Human Kidney Cells

Multiple studies have been performed to map the kidney landscape of human and rodent, along with the development of sequencing technique. Although rodent disease models have been widely applied, many disadvantages also exist. Non-human primates (NHPs) are considered as the closest experimental animals to humans and show great advantages in the construction of animal models of human disease. Therefore, a comprehensive understanding of the heterogeneity and homogeneity between human and multiple animal kidney cells is important for further establishing animal models of human renal disease. Here, we generated the first single-cell transcriptome data of normal adult cynomolgus monkey kidney using 10x Genomics scRNA-seq platform. Then, we further performed an in-depth comparison across species at the single-cell level, and our analysis indicated that the gene expression of adult primate kidney cells showed a better correlation with human kidney than mouse kidney. Furthermore, our results demonstrated that the cellular localization of GWAS-identified renal disease genes showed differences across species. The cellular localization of blood pressure associated genes in human displayed similarity to cynomolgus monkey. This study provided a reliable reference for further studies associated with renal diseases on NHPs. In addition, our results also provided a novel insight into the choice of renal disease animal model and a detailed explanation for close genetic relationship between NHPs and human at a single cell level.

Cellular localization of p-tau217 in brain and its association with p-tau217 plasma levels

Recent studies highlight phosphorylated tau (p-tau) at threonine tau 217 (p-tau217) as a new promising plasma biomarker for pathological changes implicated in Alzheimer’s disease (AD), but the specific brain pathological events related to the alteration in p-tau217 plasma levels are still largely unknown. Using immunostaining techniques of postmortem AD brain tissue, we show that p-tau217 is found in neurofibrillary tangles (NFTs) and neuropil threads that are also positive for p-tau181, 202, 202/205, 231, and 369/404. The p-tau217, but not the other five p-tau variants, was also prominently seen in vesicles structure positive for markers of granulovacuolar degeneration bodies and multi-vesicular bodies. Further, individuals with a high likelihood of AD showed significantly higher p-tau217 area fraction in 4 different brain areas (entorhinal cortex, inferior temporal gyrus, and superior frontal gyrus) compared to those with Primary age related tauopathy or other non-AD tauopathies. The p-tau217 area fraction correlated strongly with total amyloid-beta (Aβ) and NFT brain load when the whole group was analyzed. Finally, the mean p-tau217 area fraction correlated significantly with p-tau217 concentrations in antemortem collected plasma specifically in individuals with amyloid plaques and not in those without amyloid plaques. These studies highlight differences in cellular localization of different p-tau variants and suggest that plasma levels of p-tau217 reflect an accumulation of p-tau217 in presence of Aβ plaque load.

Dynamic Expression of the Homeobox Factor PBX1 during Mouse Testis Development

Members of the pre-B-cell leukemia transcription factor (PBX) family of homeoproteins are mainly known for their involvement in hematopoietic cell differentiation and in the development of leukemia. The four PBX proteins, PBX1, PBX2, PBX3 and PBX4, belong to the three amino acid loop extension (TALE) superfamily of homeoproteins which are important transcriptional cofactors in several developmental processes involving homeobox (HOX) factors. Mutations in the human PBX1 gene are responsible for cases of gonadal dysgenesis with absence of male sex differentiation while Pbx1 inactivation in the mouse causes a failure in Leydig cell differentiation and function. However, no data is available regarding the expression profile of this transcription factor in the testis. To fill this knowledge gap, we have characterized PBX1 expression during mouse testicular development. Real time PCRs and Western blots confirmed the presence Pbx1 mRNA and PBX1 protein in different Leydig and Sertoli cell lines. The cellular localization of the PBX1 protein was determined by immunohistochemistry and immunofluorescence on mouse testis sections at different embryonic and postnatal developmental stages. PBX1 was detected in interstitial cells and in peritubular myoid cells from embryonic life until puberty. Most interstitial cells expressing PBX1 do not express the Leydig cell marker CYP17A1, indicating that they are not differentiated and steroidogenically active Leydig cells. In adults, PBX1 was mainly detected in Sertoli cells. The presence of PBX1 in different somatic cell populations during testicular development further supports a direct role for this transcription factor in testis cell differentiation and in male reproductive function.

IN SILICO CHARACTERIZATION OF HUMAN INTERFERON ALPHA/BETA RECEPTOR 2 (ISOFORM A, B AND C) PROTEIN

Objective: To predict the tertiary structure of human interferon alpha/beta receptor 2 protein. Study Design: Structure prediction by using bio informatics tools. Place and Duration of Study: Department of Biochemistry, Swat Medical College (STMC), Saidu Shareef, Swat, Pakistan, from Aug 2019 to Dec 2019. Methodology: All protein sequences of human interferon alpha/beta receptor 2 (isoforma, b and c) (IFNAR-2) were retrieved through the BLAST search (The Basic Local Alignment Search Tool) from available databases ‘NCBI’ (National Centre for Biotechnology Information) and ‘Uni Prot KB’ (The Universal Protein Resource). Sequence alignment was conducted by using Clustal Omega, to get the consensus sequence for IFNAR-2 protein. Consensus protein sequence of human IFNAR-2 was used for the prediction of the three-dimensional structure by employing Swiss-Model Server. Moreover, subcellular localization analysis was also performed by using CELLO2GO program. Results: Structural model of human IFNAR-2 protein was predicted and evaluated by Ramachandran dimension. Cellular localization of tertiary topological domains of the predicted models were revealed probability of localization of IFNAR-2 protein (isoform a, b & c) is highest in the plasma membrane due to the presence of the transmembrane alpha helical regions. Conclusion: This study predicted the tertiary structural dimensions of human IFNAR-2 protein, including the specific topological domains that contribute towards the subcellular compartmentalization and functional characteristics.

Comparative Proteomics of Mulberry Leaves at Different Developmental Stages Identify Novel Proteins Function Related to Photosynthesis

Mulberry leaves at different positions are different in photosynthetic rate, nutrient substance and feeding impact to silkworms. Here, we investigated the proteomic differences of the first (L1), sixth (L6), and twentieth (L20) mulberry leaves at different stem positions (from top to the base) using a label-free quantitative proteomics approach. L1 contained less developed photosynthetic apparatus but was more active in protein synthesis. L20 has more channel proteins and oxidoreductases relative to L6. Proteins that detected in all measured leaves were classified into three groups according to their expression patterns in L1, L6, and L20. The protein group that displayed the maximum amount in L6 has the highest possibility that function related to photosynthesis. Nine function unknown proteins belong to this group were further analyzed in the light responsive expression, evolutionary tree and sub-cellular localization analysis. Based on the results, five proteins were suggested to be involved in photosynthesis. Taken together, these results reveal the molecular details of different roles of mulberry leaves at different developmental stages and contribute to the identification of five proteins that might function related to photosynthesis.