mTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding.

Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed genetically encoded multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein that self-assemble into bright, stable particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can modulate the effective diffusion coefficient of particles ≥20 nm in diameter more than 2-fold by tuning ribosome concentration, without any discernable effect on the motion of molecules ≤5 nm. This change in ribosome concentration affected phase separation both in vitro and in vivo. Together, these results establish a role for mTORC1 in controlling both the mesoscale biophysical properties of the cytoplasm and biomolecular condensation.

The Cytoophidium and Its Kind: Filamentation and Compartmentation of Metabolic Enzymes.

Compartmentation is essential for the localization of biological processes within a cell. In 2010, three groups independently reported that cytidine triphosphate synthase (CTPS), a metabolic enzyme for de novo synthesis of the nucleotide CTP, is compartmentalized in cytoophidia (Greek for "cellular snakes") in bacteria, yeast, and fruit flies. Subsequent studies demonstrate that CTPS can also form filaments in human cells. Thus, the cytoophidium represents a new type of intracellular compartment that is strikingly conserved across prokaryotes and eukaryotes. Multiple lines of evidence have recently suggested that polymerization of metabolic enzymes such as CTPS and inosine monophosphate dehydrogenase into filamentous cytoophidia modulates enzymatic activity. With many more metabolic enzymes found to form the cytoophidium and its kind, compartmentation via filamentation may serve as a general mechanism for the regulation of metabolism.

Size- and position-dependent cytoplasm viscoelasticity through hydrodynamic interactions with the cell surface.

Many studies of cytoplasm rheology have focused on small components in the submicrometer scale. However, the cytoplasm also baths large organelles like nuclei, microtubule asters, or spindles that often take significant portions of cells and move across the cytoplasm to regulate cell division or polarization. Here, we translated passive components of sizes ranging from few up to ~50 percents of the cell diameter, through the vast cytoplasm of live sea urchin eggs, with calibrated magnetic forces. Creep and relaxation responses indicate that for objects larger than the micron size, the cytoplasm behaves as a Jeffreys material, viscoelastic at short timescales, and fluidizing at longer times. However, as component size approached that of cells, cytoplasm viscoelastic resistance increased in a nonmonotonic manner. Flow analysis and simulations suggest that this size-dependent viscoelasticity emerges from hydrodynamic interactions between the moving object and the static cell surface. This effect also yields to position-dependent viscoelasticity with objects initially closer to the cell surface being harder to displace. These findings suggest that the cytoplasm hydrodynamically couples large organelles to the cell surface to restrain their motion, with important implications for cell shape sensing and cellular organization.

BRAFV600E promotes anchorage-independent growth but inhibits anchorage-dependent growth in hTERT/Cdk4-Immortalized normal human bronchial epithelial cells.

Certain oncogenes, including mutant RAS and BRAF, induce a type of senescence known as oncogene-induced senescence (OIS) in normal cells in a cell-type-specific manner. OIS serves as a barrier to transformation by activated oncogenes. Our previous studies showed that mutant KRASV12 did not efficiently induce OIS in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line (HBEC3), but it did enhance both anchorage-dependent and anchorage-independent growth. In this study, we investigated whether mutant BRAF, a well-known inducer of OIS, could trigger OIS in HBEC3 cells. We also assessed the impact of mutant BRAF on the growth of HBEC3 cells, as no previous studies have examined this using a normal bronchial epithelial cell line model. We established an HBEC3 cell line, designated as HBEC3-BIN, that expresses mutant BRAFV600E in a doxycycline-regulated manner. Unlike our previous finding that KRASV12 upregulated both pERK and pAKT, mutant BRAFV600E upregulated pERK but not pAKT in HBEC3-BIN cells. Similar to KRASV12, BRAFV600E did not efficiently induce OIS. Interestingly, while BRAFV600E inhibited colony formation in anchorage-dependent conditions, it dramatically enhanced colony formation in anchorage-independent conditions in HBEC3-BIN. In HBEC3 cells without BRAFV600E or KRASV12 expression, p21 was only detected in the cytoplasm, and its localization was not altered by the expression of BRAFV600E or KRASV12. Next-generation sequencing analysis revealed an enrichment of gene sets known to be involved in carcinogenesis, including IL3/JAK/STAT3, IL2, STAT5, and the EMT pathway. Our results indicate that, unlike KRASV12, which promoted both, BRAFV600E enhances anchorage-independent growth but inhibits anchorage-dependent growth of HBEC3. This contrast may result from differences in activation signaling in the downstream pathways. Furthermore, HBEC3 cells appear to be inherently resistant to OIS, which may be partly due to the fact that p21 remains localized in the cytoplasm upon expression of BRAFV600E or KRASV12.

Spem2, a novel testis-enriched gene, is required for spermiogenesis and fertilization in mice.

Spermiogenesis is considered to be crucial for the production of haploid spermatozoa with normal morphology, structure and function, but the mechanisms underlying this process remain largely unclear. Here, we demonstrate that SPEM family member 2 (Spem2), as a novel testis-enriched gene, is essential for spermiogenesis and male fertility. Spem2 is predominantly expressed in the haploid male germ cells and is highly conserved across mammals. Mice deficient for Spem2 develop male infertility associated with spermiogenesis impairment. Specifically, the insufficient sperm individualization, failure of excess cytoplasm shedding, and defects in acrosome formation are evident in Spem2-null sperm. Sperm counts and motility are also significantly reduced compared to controls. In vivo fertilization assays have shown that Spem2-null sperm are unable to fertilize oocytes, possibly due to their impaired ability to migrate from the uterus into the oviduct. However, the infertility of Spem2-/- males cannot be rescued by in vitro fertilization, suggesting that defective sperm-egg interaction may also be a contributing factor. Furthermore, SPEM2 is detected to interact with ZPBP, PRSS21, PRSS54, PRSS55, ADAM2 and ADAM3 and is also required for their processing and maturation in epididymal sperm. Our findings establish SPEM2 as an essential regulator of spermiogenesis and fertilization in mice, possibly in mammals including humans. Understanding the molecular role of SPEM2 could provide new insights into future therapeutic treatment of human male infertility and development of non-hormonal male contraceptives.

Contribution of cytoplasm viscoelastic properties to mitotic spindle positioning.

Cells are filled with macromolecules and polymer networks that set scale-dependent viscous and elastic properties to the cytoplasm. Although the role of these parameters in molecular diffusion, reaction kinetics, and cellular biochemistry is being increasingly recognized, their contributions to the motion and positioning of larger organelles, such as mitotic spindles for cell division, remain unknown. Here, using magnetic tweezers to displace and rotate mitotic spindles in living embryos, we uncovered that the cytoplasm can impart viscoelastic reactive forces that move spindles, or passive objects with similar size, back to their original positions. These forces are independent of cytoskeletal force generators yet reach hundreds of piconewtons and scale with cytoplasm crowding. Spindle motion shears and fluidizes the cytoplasm, dissipating elastic energy and limiting spindle recoils with functional implications for asymmetric and oriented divisions. These findings suggest that bulk cytoplasm material properties may constitute important control elements for the regulation of division positioning and cellular organization.

Discovery of an unusual copper homeostatic mechanism in yeast cells respiring on minimal medium and an unexpectedly diverse labile copper pool.

Copper is essential for all eukaryotic cells but many details of how it is trafficked within the cell and how it is homeostatically regulated remain uncertain. Here, we characterized the copper content of cytosol and mitochondria using liquid chromatography with ICP-MS detection. Chromatograms of cytosol exhibited over two dozen peaks due to copper proteins and coordination complexes. Yeast cells respiring on minimal media did not regulate copper import as media copper concentration increased; rather, they imported copper at increasing rates while simultaneously increasing the expression of metallothionein CUP1 which then sequestered most of the excessive imported copper. Peak intensities due to superoxide dismutase SOD1, other copper proteins, and numerous coordination complexes also increased, but not as drastically. The labile copper pool was unexpectedly diverse and divided into two groups. One group approximately comigrated with copper-glutathione, -cysteine, and -histidine standards; the other developed only at high media copper concentrations and at greater elution volumes. Most cytosolic copper arose from copper-bound proteins, especially CUP1. Cytosol contained an unexpectedly high percentage of apo-copper proteins and apo-coordination complexes. Copper-bound forms of non-CUP1 proteins and complexes coexisted with apo-CUP1 and with the chelator BCS. Both experiments suggest unexpectedly stable-binding copper proteins and coordination complexes in cytosol. COX17Δ cytosol chromatograms were like those of WT cells. Chromatograms of soluble mitochondrial extracts were obtained, and mitoplasting helped distinguish copper species in the intermembrane space versus in the matrix/inner membrane. Issues involving the yeast copperome, copper homeostasis, labile copper pool, and copper trafficking are discussed.

Subcellular localization of circular RNAs: Where and why.

Localization of RNAs at specific subcellular locations regulating various local cellular events has gained much attention recently. Like most other classes of RNAs, the function of newly discovered circular RNAs (circRNAs) is predominantly determined by their association with different cellular factors in the cell. CircRNAs function as transcriptional and posttranscriptional regulators of gene expression by interacting with transcription factors, splicing regulators, RNA-binding proteins, and microRNAs or by translating into functional polypeptides. Hence, studying their subcellular localization to assess their function is essential. The discovery of more than a million circRNA and increasing evidence of their involvement in development and diseases require a thorough analysis of their subcellular localization linking to their biological functions. Here, we summarize current knowledge of circRNA localization in cells and extracellular vesicles, factors regulating their subcellular localization, and the implications of circRNA localization on their cellular functions. Given the discovery of many circRNAs in all life forms and their implications in pathophysiology, we discuss the challenges in studying circRNA localization and the opportunities for unlocking the mystery of circRNA functions.

Comprehensive data analysis of white blood cells with classification and segmentation by using deep learning approaches.

Deep learning approaches have frequently been used in the classification and segmentation of human peripheral blood cells. The common feature of previous studies was that they used more than one dataset, but used them separately. No study has been found that combines more than two datasets to use together. In classification, five types of white blood cells were identified by using a mixture of four different datasets. In segmentation, four types of white blood cells were determined, and three different neural networks, including CNN (Convolutional Neural Network), UNet and SegNet, were applied. The classification results of the presented study were compared with those of related studies. The balanced accuracy was 98.03%, and the test accuracy of the train-independent dataset was determined to be 97.27%. For segmentation, accuracy rates of 98.9% for train-dependent dataset and 92.82% for train-independent dataset for the proposed CNN were obtained in both nucleus and cytoplasm detection. In the presented study, the proposed method showed that it could detect white blood cells from a train-independent dataset with high accuracy. Additionally, it is promising as a diagnostic tool that can be used in the clinical field, with successful results in classification and segmentation.

Electrical signature of heterogeneous human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) have gained traction in transplantation therapy due to their immunomodulatory, paracrine, immune-evasive, and multipotent differentiation potential. The inherent heterogeneity of hMSCs poses a challenge for therapeutic treatments and necessitates the identification of robust biomarkers to ensure reproducibility in both in vivo and in vitro experiments. In this study, we utilized dielectrophoresis (DEP), a label-free electrokinetic phenomenon, to investigate the heterogeneity of hMSCs derived from bone marrow (BM) and adipose tissue (AD). The electrical properties of BM-hMSCs were compared to homogeneous mouse fibroblasts (NIH-3T3), human fibroblasts (WS1), and human embryonic kidney cells (HEK-293). The DEP profile of BM-hMSCs differed most from HEK-293 cells. We compared the DEP profiles of BM-hMSCs and AD-hMSCs and found that they have similar membrane capacitances, differing cytoplasm conductivity, and transient slopes. Inducing both populations to differentiate into adipocyte and osteoblast cells revealed that they behave differently in response to differentiation-inducing cytokines. Histology and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses of the differentiation-related genes revealed differences in heterogeneity between BM-hMSCs and AD-hMSCs. The differentiation profiles correlate well with the DEP profiles developed and indicate differences in the heterogeneity of BM-hMSCs and AD-hMSCs. Our results demonstrate that using DEP, membrane capacitance, cytoplasm conductivity, and transient slope can uniquely characterize the inherent heterogeneity of hMSCs to guide robust and reproducible stem cell transplantation therapies.

Deciphering the differential expression patterns of yield-related negative regulators in hexaploid wheat cultivars and hybrids at different growth stages.

BACKGROUND: Hexaploid bread wheat underwent a series of polyploidization events through interspecific hybridizations that conferred adaptive plasticity and resulted in duplication and neofunctionalization of major agronomic genes. The genetic architecture of polyploid wheat not only confers adaptive plasticity but also offers huge genetic diversity. However, the contribution of different gene copies (homeologs) encoded from different subgenomes (A, B, D) at different growth stages remained unexplored. METHODS: In this study, hybrid of elite cultivars of wheat were developed via reciprocal crosses (cytoplasm swapping) and phenotypically evaluated. We assessed differential expression profiles of yield-related negative regulators in these cultivars and their F1 hybrids and identified various cis-regulatory signatures by employing bioinformatics tools. Furthermore, the preferential expression patterns of the syntenic triads encoded from A, B, and D subgenomes were assessed to decipher their functional redundancy at six different growth stages. RESULTS: Hybrid progenies showed better heterosis such as up to 17% increase in the average number of grains and up to 50% increase in average thousand grains weight as compared to mid-parents. Based on the expression profiling, our results indicated significant dynamic transcriptional expression patterns, portraying the different homeolog-dominance at the same stage in the different cultivars and their hybrids. Albeit belonging to same syntenic triads, a dynamic trend was observed in the regulatory signatures of these genes that might be influencing their expression profiles. CONCLUSION: These findings can substantially contribute and provide insights for the selective introduction of better cultivars into traditional and hybrid breeding programs which can be harnessed for the improvement of future wheat.

Quantification of eosinophilic area and its potential molecular feature in clear cell renal cell carcinoma.

OBJECTIVE: Previous studies have acknowledged the presence of eosinophilic cytoplasm in clear cell renal cell carcinoma, yet the precise quantification method and potential molecular attributes in clear cell renal cell carcinoma remain elusive. This study endeavours to precisely quantify the eosinophilic attribute and probe into the molecular mechanisms governing its presence in clear cell renal cell carcinoma. METHODS: Data from cohorts of clear cell renal cell carcinoma patients who underwent nephrectomy, comprising The Cancer Genome Atlas cohort (n = 475) and Sun Yat-sen University Cancer Center cohort (n = 480), were aggregated to assess the eosinophilic attribute. Additionally, Omics data from Clinical Proteomic Tumor Analysis Consortium (CPTAC) (n = 58) were leveraged to explore the potential molecular features associated with eosinophilic clear cell renal cell carcinoma. Employing receiver operating characteristic curve analysis, the proportion of tumour cells with eosinophilic cytoplasm was determined, leading to the classification of each cohort into distinct groups: a clear group (<5%) and an eosinophilic group (≥5%). RESULTS: In both cohorts, the eosinophilic feature consistently correlated with higher International Society of Urological Pathology (ISUP) grade, elevated tumor stage, and the presence of necrosis. Furthermore, the Kaplan-Meier method demonstrated that patients in the eosinophilic group exhibited shorter overall survival or disease-free survival compared with those in the clear group, a pattern reaffirmed in various stratified survival analyses. Intriguingly, within The Cancer Genome Atlas cohort, the pathological characterization of cell cytoplasm (eosinophilic vs. clear) emerged as an independent risk factor for overall survival (hazard ratio = 2.507 [95% confidence interval: 1.328-4.733], P = 0.005) or disease-free survival (hazard ratio = 1.730 [95% confidence interval: 1.062-2.818], P = 0.028) via Cox regression analysis. Moreover, multi-Omics data unveiled frequent BAP1 mutations and down-regulation of Erythroblast Transformation-Specific-Related Gene associated with the eosinophilic feature in clear cell renal cell carcinoma. Additionally, patients with low expression of Erythroblast Transformation-Specific-Related Gene showed worse overall survival (P < 0.001). CONCLUSIONS: The quantification of the eosinophilic feature serves as a robust predictor of clinical prognosis in clear cell renal cell carcinoma. Furthermore, the manifestation of this feature may be linked to BAP1 mutations and the down-regulation of Erythroblast Transformation-Specific-Related Gene in clear cell renal cell carcinoma. Significantly, the expression levels of Erythroblast Transformation-Specific-Related Gene manifest as an exemplary prognostic marker, providing exceptional predictive accuracy for the clinical prognosis in clear cell renal cell carcinoma.

The association between ferroptosis and autophagy in cardiovascular diseases.

Autophagy is a process in which cells degrade intracellular substances and play a variety of roles in cells, such as maintaining intracellular homeostasis, preventing cell overgrowth, and removing pathogens. It is highly conserved during the evolution of eukaryotic cells. So far, the study of autophagy is still a hot topic in the field of cytology. Ferroptosis is an iron-dependent form of cell death, accompanied by the accumulation of reactive oxygen species and lipid peroxides. With the deepening of research, it has been found that ferroptosis, like autophagy, is involved in the occurrence and development of cardiovascular diseases. The relationship between autophagy and ferroptosis is complex, and the association between the two in cardiovascular disease remains to be clarified. This article reviews the mechanism of autophagy and ferroptosis and their correlation, and discusses the relationship between them in cardiovascular diseases, which is expected to provide new and important treatment strategies for cardiovascular diseases.

Cytoplasmic granules in bovine oocytes do not affect embryonic or fetal development.

Oocyte cytoplasmic evaluation is based on homogeneity and granular appearance. Our study investigated if a granular cytoplasm, highly heterogeneous, would affect oocyte competence in bovine. In two experiments, bovine cumulus-oocyte complexes (COCs) with homogeneous cytoplasm (control, CC) and granulated cytoplasm (granular, GC) were selected from a regular pool of COCs. Experiment 1 was performed with slaughterhouse ovaries, and Experiment 2 was carried out in Girolando COCs obtained from ovum pick-up. Granular oocytes had higher caspase 3 levels (66.17 ± 11.61 vs 172.08 ± 16.95, P < 0.01) and similar GAP junction activity (5.64 ± 0.45 vs 6.29 ± 0.29). ZAR1 relative mRNA amount was lower in granular oocytes (178.27 ± 151.63 vs 0.89 ± 0.89, P = 0.01) and no effect was detected for MATER, PPP2R1A, ENY2, IGF2R, and BMP15 genes. Despite molecular differences, no detrimental effect was detected on oocyte competence in GC oocytes. Cleavage (Experiment 1: 59.52 ± 7.21% vs 59.79 ± 6.10% and Experiment 2: 68.88 ± 4.82 vs 74.41 ± 5.89%) and blastocyst (Experiment 1: 29.28 ± 4.14% vs 23.15 ± 2.96% and Experiment 2: 21.11 ± 3.28% vs 21.02 ± 6.08%) rates were similar between CC and GC (Experiments 1 and 2, respectively). Post-transfer embryo development revealed that pregnancy (CC: 24.27 ± 9.70% vs GC: 26.31 ± 7.23%) and calving (23.68% vs 33.33%) rates and fetal growth were not affected by the presence of cytoplasmic granules. Our results demonstrated that oocytes with granular cytoplasm present equivalent efficiency for IVF and calf production compared with homogenous cytoplasm oocytes. This could be observed through similar cleavage, blastocyst rates, and fetal growth development. In addition to differences in oocyte gene expression related to oocyte quality, it seems not to affect oocyte developmental competence.

Comparative study of intracytoplasmic sperm injection using the traditional holding and the oocyte-holding pipette without aspiration.

Despite the high level of standardization of the intracytoplasmic sperm injection (ICSI) technique, there are some aspects that deserve special attention and should still be improved. The major drawback of the technique is its invasiveness, as during cytoplasmic aspiration different structures of the oocyte may be lost or damaged. This is partly because the microtools used in ICSI were not specially designed for assisted reproduction but for other medical-biological disciplines. In view of the above caveats, the aim of the study was to compare the results of ICSI with the traditional oocyte-holding pipette and the oocyte-holding pipette without aspiration (PiWA). In total, 155 patients and 1037 oocytes were included in the study. In each ICSI cycle, half of the oocytes were microinjected using a traditional holding pipette and the other half using a PiWA. In result, the PiWA technique produced a significant increase in the fertilization rate: 88.12% (95%CI: 84.62-90.92%); holding pipette: 73.33% (95%CI: 68.72-77.49%). Also, it produced a significant decrease in the embryo degeneration rate compared with the traditional holding pipette [PiWA: 2.07% (95%CI: 1.11-3.8%); holding pipette: 4.51% (95%CI: 3.06-6.59%)]. Pregnancy rate depended on the holding technique used, both in single embryo transfers (n = 59; χ2 = 4.608; P-value = 0.032) and double embryo transfers (n = 156; χ2 = 4.344; P-value = 0.037); with PiWA presenting a significantly higher pregnancy rate than the traditional holding technique. Based on current evidence and the present results, improvements should focus on decreasing the invasiveness of the microinjection itself by minimizing or avoiding aspiration and cytoplasmic disorganization, as is successfully achieved with PiWA.

Comparison of disease phenotypes and mechanistic insight on causal variants in patients with DADA2.

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) results in heterogeneous manifestations including systemic vasculitis and red cell aplasia. The basis of different disease phenotypes remains incompletely defined. OBJECTIVE: We sought to further delineate disease phenotypes in DADA2 and define the mechanistic basis of ADA2 variants. METHODS: We analyzed the clinical features and ADA2 variants in 33 patients with DADA2. We compared the transcriptomic profile of 14 patients by bulk RNA sequencing. ADA2 variants were expressed experimentally to determine impact on protein production, trafficking, release, and enzymatic function. RESULTS: Transcriptomic analysis of PBMCs from DADA2 patients with the vasculitis phenotype or pure red cell aplasia phenotype exhibited similar upregulation of TNF, type I interferon, and type II interferon signaling pathways compared with healthy controls. These pathways were also activated in 3 asymptomatic individuals with DADA2. Analysis of ADA2 variants, including 7 novel variants, showed different mechanisms of functional disruption including (1) unstable transcript leading to RNA degradation; (2) impairment of ADA2 secretion because of retention in the endoplasmic reticulum; (3) normal expression and secretion of ADA2 that lacks enzymatic function; and (4) disruption of the N-terminal signal peptide leading to cytoplasmic localization of unglycosylated protein. CONCLUSIONS: Transcriptomic signatures of inflammation are observed in patients with different disease phenotypes, including some asymptomatic individuals. Disease-associated ADA2 variants affect protein function by multiple mechanisms, which may contribute to the clinical heterogeneity of DADA2.

Fluid flow dynamics in cellular patterning.

The development of complex forms of multicellular organisms depends on the spatial arrangement of cellular architecture and functions. The interior design of the cell is patterned by spatially biased distributions of molecules and biochemical reactions in the cytoplasm and/or on the plasma membrane. In recent years, a dynamic change in the cytoplasmic fluid flow has emerged as a key physical process of driving long-range transport of molecules to particular destinations within the cell. Here, recent experimental advances in the understanding of the generation of the various types of cytoplasmic flows and contributions to intracellular patterning are reviewed with a particular focus on feedback mechanisms between the mechanical properties of fluid flow and biochemical signaling during animal cell polarization.

CanVasc consensus recommendations for the use of avacopan in antineutrophil cytoplasm antibody-associated vasculitis: 2022 addendum.

OBJECTIVE: In 2020, the Canadian Vasculitis Research Network (CanVasc) published their updated recommendations for the management of ANCA-associated vasculitides (AAV). The current addendum provides further recommendations regarding the use of avacopan in AAV based on a review of newly available evidence. METHODS: An updated systematic literature review on avacopan (formerly, CCX168) using Medline, Embase, and the Cochrane Library was performed for publications up to September 2022. New recommendations were developed and categorized according to the EULAR grading levels, as done for previous CanVasc recommendations. A modified Delphi procedure and videoconferences were used to reach ≥80% consensus on the inclusion, wording and grading of each recommendation. RESULTS: Three new recommendations were developed. They focus on avacopan therapy indication and duration, as well as timely glucocorticoid tapering. CONCLUSION: These 2022 addended recommendations provide rheumatologists, nephrologists and other specialists caring for patients with AAV with guidance for the use of avacopan, based on current evidence and consensus from Canadian experts.

Oxygen transport across tank-treading red blood cell: Individual and joint roles of flow convection and oxygen-hemoglobin reaction.

Gas, especially oxygen, transport in the microcirculation is a complex phenomenon, however, of critical importance for maintaining normal biological functions, and the cytoplasm fluid in red blood cells (RBCs) is the major vehicle for transporting oxygen from lungs to tissues via the circulatory system. Existing theoretical and numerical studies have neglected the cytoplasm convection effect by treating RBCs as rigid particles undergoing a constant translation velocity. As a consequence, the influence and mechanism of the cytoplasm flow on oxygen transport are still not clear in microcirculation research. In this study, we consider a tank-treading capsule in shear flow, which is generated with two parallel plates moving in opposite directions: the top plate of a higher oxygen pressure (PO2) representing the RBC core in the central region of a microvessel and the bottom plate of a lower PO2 representing the microvessel wall. Numerical simulations are conducted to investigate the individual and combined effects of cytoplasm convection and oxygen-hemoglobin (O2-Hb) reaction on the oxygen transport efficiency across the tank-treading capsule, and different PO2 situations and shear rates are also tested. Due to the lower oxygen diffusivity in cytoplasm, the presence of the capsule reduces the oxygen transfer flux across the gap by 7.34 % in the pure diffusion system where the flow convection and O2-Hb reaction are both neglected. Including the flow convection or the O2-Hb reaction has little influence on the oxygen flux; however, when they act together as in real microcirculation situations, the enhancement in oxygen transport could be significant, especially in the low PO2 and high shear rate situations. In particular, with the respective PO2 at 60 and 30 mmHg on the top and bottom plates and a 400 s-1 shear rate, the oxygen flux reduction is only 0.02 %, suggesting that the cytoplasm convection can improve the oxygen transport across RBCs considerably. The simulation results are scrutinized to explore the underlying mechanism for the enhancement, and a new nondimensional parameter is introduced to characterize the importance of cytoplasm convection in oxygen transport. These simulation results, discussion and analysis could be helpful for a better understanding of the complex oxygen transport process and therefor valuable for relevant studies.

Nucleophosmin 1a translocated from nucleus to cytoplasm and facilitate GCRV replication.

To decipher the functional characterization of Nucleophosmin 1a (NPM1a) from grass carp (Ctenopharyngodon idellus) (CiNPM1a), its cDNA was cloned and bioinformatic analysis were conducted. The full-length cDNA sequence of CiNPM1a is 1732 bp, which encodes 307 amino acids. CiNPM1a contains conserved domains of Nucleoplasmin domain, NPM1-C terminal domain, as well as nuclear localization signals, nuclear export signal (NES) and acid patches. There are 52 and 20 consensus amino acids exist in the Nucleoplasmin domain and the NPM1-C terminal domain of all blasted species. In addition, the immune function of CiNPM1a were analyzed. The Ciirf7, Ciifn1 and Ciifn2 transcription was inhibited, whereas the vp2 and vp7 expressions were enhanced in CiNPM1a overexpressing cells after GCRV infection (P < 0.05). Moreover, the Ciirf7, Ciifn1 and Ciifn2 mRNA levels were significantly up-regulated, but the vp2 and vp7 expressions were significantly down-regulated in CiNPM1a knockdown cells after infection. This indicated that CiNPM1a played negative roles in the induction of Type I IFN reaction and thus the GCRV replication. Finally, the NES domain that affect the nucleous-cytoplasm shuttle and the replication of GCRV were investigated. The deletion of NES1 and NES(1 + 2+3) absolutely limited the transloacation of CiNPM1a△NES1 protein and CiNPM1a △NES(1 + 2+3) protein to cytoplasm after infection, and the deletion of NES2 resulted in partially limitation of protein shuttle. In general, Ciirf3, Ciirf7, Ciifn1 and Ciifn2 expressions were enhanced in the CiNPM1a△NES1, CiNPM1a△NES2 and CiNPM1a△NES3 overexpression groups, and the deletion of functional domains in CiNPM1a led to significantly reduction of the vp2 and vp7 replication. The results indicated that CiNPM1a may be a target molecular for GCRV infection curation, and a candidate molecular for resistance strain breeding of grass carp.