Intracellular Distribution Research Articles

Simple Fluorescence Labeling Method Enables Detection of Intracellular Distribution and Expression Level of Retinoid X Receptors.

There is no straightforward method to visualize the intracellular distribution of nuclear receptors, such as retinoid X receptors (RXRs), which are trafficked between the cytosol and nucleus. Here, in order to develop a simple fluorescence labeling method for RXRs, we designed and synthesized compound 4, consisting of an RXR-selective antagonist, CBTF-EE (2), linked via an ether bond to the fluorophore nitrobenzoxadiazole (NBD). Compound 4 is nonfluorescent, but the ether bond (-O-NBD) reacts with biothiols such as cysteine and homocysteine to generate a thioether (-S-NBD), followed by intramolecular Smiles rearrangement with an amino group such as that of lysine to form a fluorescent secondary amine (-NH-NBD) adjacent to the binding site. Fluorescence microscopy of intact or RXR-overexpressing MCF-7 cells after incubation with 4 enabled us to visualize RXR expression as well as nuclear transfer of RXR induced by the agonist bexarotene (1).

Assessing intracellular and extracellular distribution of antibiotic resistance genes in the commercial organic fertilizers

Compost-based organic fertilizers often contain high levels of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). Previous studies focused on quantification of total ARGs and MGEs. For a more accurate risk assessment of the dissemination risk of antibiotic resistance, it is necessary to quantify the intracellular and extracellular distribution of ARGs and MGEs. In the present study, extracellular ARGs and MGEs (eARGs and eMGEs) and intracellular ARGs and MGEs (iARGs and iMGEs) were separately analyzed in 51 commercial composts derived from different raw materials by quantitative polymerase chain reaction (qPCR) and metagenomic sequencing. Results showed that eARGs and eMGEs accounted for 11–56% and 4–45% of the total absolute abundance of ARGs and MGEs, respectively. Comparable diversity, host composition and association with MGEs were observed between eARGs and iARGs. Contents of high-risk ARGs were similar between eARGs and iARGs, with high-risk ARGs in the two forms accounting for 6.7% and 8.2% of the total abundances, respectively. Twenty-four percent of the overall ARGs were present in plasmids, while 56.7% of potentially mobile ARGs were found to be associated with plasmids. Variation partitioning analysis, null model and neutral community model indicated that the compositions of both eARGs and iARGs were largely driven by deterministic mechanisms. These results provide important insights into the cellular distribution of ARGs in manure composts that should be paid with specific attention in risk assessment and management.

Visualization of Modified Bisarylbutadiyne-Tagged Small Molecules in Live-Cell Nuclei by Stimulated Raman Scattering Microscopy.

Visualizing the distribution of small-molecule drugs in living cells is an important strategy for developing specific, effective, and minimally toxic drugs. As an alternative to fluorescence imaging using bulky fluorophores or cell fixation, stimulated Raman scattering (SRS) imaging combined with bisarylbutadiyne (BADY) tagging enables the observation of small molecules closer to their native intracellular state. However, there is evidence that the physicochemical properties of BADY-tagged analogues of small-molecule drugs differ significantly from those of their parent drugs, potentially affecting their intracellular distribution. Herein, we developed a modified BADY to reduce deviations in physicochemical properties (in particular, lipophilicity and membrane permeability) between tagged and parent drugs, while maintaining high Raman activity in live-cell SRS imaging. We highlight the practical application of this approach by revealing the nuclear distribution of a modified BADY-tagged analogue of JQ1, a bromodomain and extra-terminal motif inhibitor with applications in targeted cancer therapy, in living HeLa cells. The modified BADY, methoxypyridazyl pyrimidyl butadiyne (MPDY), revealed intranuclear JQ1, while BADY-tagged JQ1 did not show a clear nuclear signal. We anticipate that the present approach combining MPDY tagging with live-cell SRS imaging provides important insight into the behavior of intracellular drugs and represents a promising avenue for improving drug development.

The influence of cation exchange on the possible mechanism of erionite toxicity: A synchrotron-based micro-X-ray fluorescence study on THP-1-derived macrophages exposed to erionite-Na

Fibrous erionite is the only zeolite classified as Group 1 carcinogen by the International Agency for Research on Cancer (IARC). Carcinogenesis induced by erionite is thought to involve several factors as biopersistence, the iron role and cation exchange processes. To better understand these mechanisms, a detailed investigation at the micro scale was performed, collecting elemental information on iron and cation release and their distribution in biological systems by synchrotron micro-X-ray fluorescence mapping (SR-micro-XRF) and synchrotron micro-X-ray absorption spectroscopy (SR-micro-XANES) at the TwinMic beamline (Elettra synchrotron) and at the ID21 beamline of the European Synchrotron Radiation Facility (ESRF). By microscopy and chemical mapping, highly detailed maps of the chemical and morphological interaction of biological systems with fibres could be produced. In detail, THP-1 cell line derived macrophages, used as in vitro model, were analysed during erionite-Na phagocytosis at different time intervals, after single dose exposure. For comparison, cellular fluorescent probes were also used to evaluate the intracellular free sodium and calcium concentrations. Synchrotron analyses visualised the spatial distribution of both fibre and mineral particle associated metals during the phagocytosis, describing the mechanism of internalisation of erionite-Na and its accessory mineral phases. The intracellular distribution of metals and other cations was mapped to evaluate metal release, speciation changes and/or cation exchange during phagocytosis. The fluorescent probes complemented microchemical data clarifying, and confirming, the cation distribution observed in the SR-micro-XRF maps. The significant cytoplasmic calcium decrease, and the concomitant sodium increase, after the fibre phagocytosis seemed due to activation of plasma membrane cations exchangers triggered by the internalisation while, surprisingly, the ion-exchange capacity of erionite-Na could play a minor role in the disruption of the two cations intracellular homeostasis. These results help to elucidate the role of cations in the toxicity of erionite-treated THP-1 macrophages and add knowledge to its carcinogenicity process.

Spatiotemporal tracking of intracellular nanoparticles using complementary imaging systems reveals acute ferroptosis triggered by burst reduction of ferric ions

Uptake and intracellular trafficking of nanoparticles are tightly regulated by their interactions with cellular organelles and physiological microenvironment. Although the dynamic physicochemical reactions at the interface of nanoparticles and cells ultimately determine the intracellular distribution and fate, microscopic tracing and quantitative analysis of the nanoparticles have been hampered by the limited resolution associated with individual nanoparticle trafficking. Herein, we report spatiotemporal investigations on autophagic clearance of biodegradable iron oxide-silica core-shell nanoparticles in terms of intracellular trafficking and ionic dissolution at a single cell level using multimodal imaging systems. By combining transmission electron microscopy and super-resolution confocal laser scanning microscopy with fluorescence correlation spectroscopy, the complementary imaging analysis exclusively shows the intracellular uptake, endosomal fusion and biodegradative clearance, leading to identify the step-by-step endocytic transport pathway and autophagic degradation pathways. Tracing the intracellular trafficking of nanoparticles reveals that they are spontaneously transported from endosomes to lysosomes, and transiently stimulate autophagy while maintaining cell viability. While protecting iron oxide core, the silica shell is gradually degraded during endocytosis and autophagic clearance, resulting in ionic dissolution of iron oxide in acidic environment. Moreover, burst reduction of ferric ions by adding ascorbic acid readily triggers acute ferroptosis owing to rapid supplement of ferrous ions and Fenton reaction in cancer cells. The complementary imaging strategy provides insights into the design of biocompatible nanomedicines for cellular delivery and the degradative mechanisms beyond the intracellular fate.

Oxaloacetate as new inducer for osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells in vitro.

Mitochondrial organelles play a crucial role in cellular metabolism so different cell types exhibit diverse metabolic and energy demands. Therefore, alternations in the intracellular distribution, quantity, function, and structure of mitochondria are required for stem cell differentiation. Finding an effective inducer capable of modulating mitochondrial activity is critical for the differentiation of specific stem cells into osteo-like cells for addressing issues related to osteogenic disorders. This study aimed to investigate the effect of oxaloacetate (OAA) on the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs) in vitro. First, the most favorable OAA concentration was measured through MTT assay and subsequently confirmed using acridine orange staining. Human ADSCs were cultured in osteogenic medium supplemented with OAA and analyzed on days 7 and 14 of differentiation. Various assays including alkaline phosphatase assay (ALP), cellular calcium content assay, mineralized matrix staining with alizarin red, catalase (CAT) and superoxide dismutase (SOD) activity, and real-time RT-PCR analysis of three bone-specific markers (ALP, osteocalcin, and collagen type I) were conducted to characterize the differentiated cells. Following viability assessment, OAA at a concentration of 1 µM was considered the optimal dosage for further studies. The results of osteogenic differentiation assays showed that OAA at a concentration of 1 × 10- 6 M significantly increased ALP enzyme activity, mineralization, CAT and SOD activity and the expression of bone-specific genes in differentiated cells compared to control groups in vitro. In conclusion, the fundings from this study suggest that OAA possesses favorable properties that make it a potential candidate for application in medical bone regeneration.

Increased vesicular dynamics and nanoscale clustering of IL-2 after T cell activation

T cells coordinate intercellular communication through the meticulous regulation of cytokine secretion. Direct visualization of vesicular transport and intracellular distribution of cytokines provides valuable insights into the temporal and spatial mechanisms involved in regulation. Employing Jurkat E6-1 T cells and interleukin-2 (IL-2) as a model system, we investigated vesicular dynamics using single-particle tracking and the nanoscale distribution of intracellular IL-2 in fixed T cells using superresolution microscopy. Live-cell imaging revealed that in vitro activation resulted in increased vesicular dynamics. Direct stochastic optical reconstruction microscopy and 3D structured illumination microscopy revealed nanoscale clustering of IL-2. In vitro activation correlated with spatial accumulation of IL-2 nanoclusters into more pronounced and elongated clusters. These observations provide visual evidence that accelerated vesicular transport and spatial concatenation of IL-2 clusters at the nanoscale may constitute a potential mechanism for modulating cytokine release by Jurkat T cells.

Immunoadjuvant Effect of Chitosan Oligosaccharide and Its Feasibility of Being Used as an Adjuvant for Attenuated Live Bacteria Vector Vaccines

To study the immunoadjuvant effects of chitosan oligosaccharide (COS), including the immune activation and the triggering of lysosomal escape, and to explore whether COS can be used as an adjuvant for attenuated live bacteria vector vaccines. 1) Mouse macrophages RAW264.7 cells were cultured with COS at 0 mg/mL (the control group) and 0.1-4 mg/mL for 24 h and the effect on cell viability was measured by CCK8 assay. Mouse macrophages RAW264.7 were treated with COS at 0 (the control group), 1, 2, and 4 mg/mL for 24 h. Then, the mRNA expression levels of the cytokines, including IFN-γ, IL-10, TGF-β, and TLR4, were determined by RT-qPCR assay. 2) RAW264.7 cells were treated with 1 mL of PBS containing different components, including calcein at 50 μg/mL, COS at 2 mg/mL, and bafilomycin A1, an inhibitor, at 1 μmol/mL, for culturing. The cells were divided into the Calcein group, Calcein+COS group, and Calcein+COS+Bafilomycin A1 group accordingly. Laser scanning confocal microscopy was used to observe the phagocytosis and the intracellular fluorescence distribution of calcein, a fluorescent dye, in RAW264.7 cells in the presence or absence of COS intervention to determine whether COS was able to trigger lysosomal escape. 3) LM∆E6E7 and LI∆E6E7, the attenuated Listeria vector candidate therapeutic vaccines for cervical cancer, were encapsulated with COS at the mass concentrations of 0.5 mg/mL, 1 mg/mL, 2 mg/mL , 4 mg/mL, and 8 mg/mL. Then, the changes in zeta potential were measured to select the concentration of COS that successfully encapsulated the bacteria. Phagocytosis of the vaccine strains by RAW264.7 cells was measured before and after LM∆E6E7 and LI∆E6E7 were coated with COS at 2 mg/mL. 1) CCK8 assays showed that, compared with the findings for the control group, the intervention of RAW264.7 cells with COS at different concentrations for 24 h was not toxic to the cells and promoted cell proliferation, with the difference being statistically significant (P<0.05). According to the RT-qPCR results, compared with those of the control group, the COS intervention up-regulated the mRNA levels of TLR4 and IFN-γ in RAW264.7 cells, while it inhibited the mRNA expression levels of TGF-β and IL-10, with the most prominent effect being observed in the 4 mg/mL COS group (P<0.05). 2) Laser scanning confocal microscopy revealed that the amount of fluorescent dye released from lysosomes into the cells was greater in the Calcein+COS group than that in the Calcein group. In other words, a greater amount of fluorescent dye was released from lysosomes into the cells under COS intervention. Furthermore, this process could be blocked by bafilomycin A1. 3) The zeta potential results showed that COS could successfully encapsulate the surface of bacteria when its mass concentration reached 2 mg/mL. Before and after the vaccine strain was encapsulated by COS, the phagocytosis of LM∆E6E7 by RAW264.7 cells was 5.70% and 22.00%, respectively, showing statistically significant differences (P<0.05); the phagocytosis of LI∆E6E7 by RAW264.7 cells was 1.55% and 6.12%, respectively, showing statistically significant differences (P<0.05). COS has the effect of activating the immune response of macrophages and triggering lysosomal escape. The candidates strains of coated live attenuated bacterial vector vaccines can promote the phagocytosis of bacteria by macrophages. Further research is warranted to develop COS into an adjuvant for bacterial vector vaccine.

Bioinformatics Insights on Viral Gene Expression Transactivation: From HIV-1 to SARS-CoV-2.

Viruses provide vital insights into gene expression control. Viral transactivators, with other viral and cellular proteins, regulate expression of self, other viruses, and host genes with profound effects on infected cells, underlying inflammation, control of immune responses, and pathogenesis. The multifunctional Tat proteins of lentiviruses (HIV-1, HIV-2, and SIV) transactivate gene expression by recruiting host proteins and binding to transacting responsive regions (TARs) in viral and host RNAs. SARS-CoV-2 nucleocapsid participates in early viral transcription, recruits similar cellular proteins, and shares intracellular, surface, and extracellular distribution with Tat. SARS-CoV-2 nucleocapsid interacting with the replication-transcription complex might, therefore, transactivate viral and cellular RNAs in the transcription and reactivation of self and other viruses, acute and chronic pathogenesis, immune evasion, and viral evolution. Here, we show, by using primary and secondary structural comparisons, that the leaders of SARS-CoV-2 and other coronaviruses contain TAR-like sequences in stem-loops 2 and 3. The coronaviral nucleocapsid C-terminal domains harbor a region of similarity to TAR-binding regions of lentiviral Tat proteins, and coronaviral nonstructural protein 12 has a cysteine-rich metal binding, dimerization domain, as do lentiviral Tat proteins. Although SARS-CoV-1 nucleocapsid transactivated gene expression in a replicon-based study, further experimental evidence for coronaviral transactivation and its possible implications is warranted.

Imaging the intracellular refractive index distribution (IRID) for dynamic label-free living colon cancer cells via circularly depolarization decay model (CDDM).

Label-free detection of intracellular substances for living cancer cells remains a significant hurdle in cancer pathogenesis research. Although the sensitivity of light polarization to intracellular substances has been validated, current studies are predominantly focused on tissue lesions, thus label-free detection of substances within individual living cancer cells is still a challenge. The main difficulty is to find specific detection methods along with corresponding characteristic parameters. With refractive index as an endogenous marker of substances, this study proposes a detection method of intracellular refractive index distribution (IRID) for label-free living colon cancer (LoVo) cells. Utilizing the circular depolarization decay model (CDDM) to calculate the degree of circular polarization (DOCP) modulated by the cell allows for the derivation of the IRID on the focal plane. Experiments on LoVo cells demonstrated the refractive index of single cell can be accurately and precisely measured, with precision of 10-3 refractive index units (RIU). Additionally, chromatin content during the interphases (G1, S, G2) of cell cycle was recorded at 56.5%, 64.4%, and 71.5%, respectively. A significantly finer IRID can be obtained compared to the phase measurement method. This method is promising in providing a dynamic label-free intracellular substances detection method in cancer pathogenesis studies.

Designed Ankyrin Repeat Proteins as Actin Labels of Distinct Cytoskeletal Structures in Living Cells.

The orchestrated assembly of actin and actin-binding proteins into cytoskeletal structures coordinates cell morphology changes during migration, cytokinesis, and adaptation to external stimuli. The accurate and unbiased visualization of the diverse actin assemblies within cells is an ongoing challenge. We describe here the identification and use of designed ankyrin repeat proteins (DARPins) as synthetic actin binders. Actin-binding DARPins were identified through ribosome display and validated biochemically. When introduced or expressed inside living cells, fluorescently labeled DARPins accumulated at actin filaments, validated through phalloidin colocalization on fixed cells. Nevertheless, different DARPins displayed different actin labeling patterns: some DARPins labeled efficiently dynamic structures, such as filopodia, lamellipodia, and blebs, while others accumulated primarily in stress fibers. This differential intracellular distribution correlated with DARPin-actin binding kinetics, as measured by fluorescence recovery after photobleaching experiments. Moreover, the rapid arrest of actin dynamics induced by pharmacological treatment led to the fast relocalization of DARPins. Our data support the hypothesis that the localization of actin probes depends on the inherent dynamic movement of the actin cytoskeleton. Compared to the widely used LifeAct probe, one DARPin exhibited enhanced signal-to-background ratio while retaining a similar ability to label stress fibers. In summary, we propose DARPins as promising actin-binding proteins for labeling or manipulation in living cells.

Antigenicity, epitope mapping, and intracellular distribution of the NSP7α protein of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that causes huge economic losses to the global pig industry. Nonstructural protein 7α (NSP7α) of PRRSV is highly conserved among different lineages of PRRSV and could be a potential target for the development of detection methods. In this study, NSP7α was expressed in prokaryote (Escherichia coli) and purified. An NSP7α-ab-ELISA detection method was established, the NSP7α-ab-ELISA has 93.1 % coincidence rate with IDEXX PRRS X3 ab test kit. NSP7α antibody was detected in pig serum by ELISA 14 days following PRRSV infection. Three monoclonal antibodies (4H9, 3F2, and C10) against NSP7α prepared by a hybridoma technique were used for epitope mapping by indirect immunofluorescence. The 4H9, 3F2, and C10 antibodies all recognized the C-terminal 72–149 amino acid region of NSP7α. 4H9 reacted with amino acids 135–143, but 3F2 and C10 did not react with any truncated polypeptide. In addition, by using the monoclonal antibodies, NSP7α was localized solely in the cytoplasm, while the N protein was distributed in the cytoplasm and nucleus. The collective findings of the antigenicity and epitope of NSP7α will be helpful for understanding the antigenicity of NSP7α and developing PRRSV diagnostic methods.

Dynamic Regulation of Intracellular Labile Cu(I)/Cu(II) Cycle in Microalgae Chlamydomonas reinhardtii: Disrupting the Balance by Cu Stress.

The labile metal pool involved in intracellular trafficking and homeostasis is the portion susceptible to environmental stress. Herein, we visualized the different intracellular distributions of labile Cu(I) and Cu(II) pools in the alga Chlamydomonas reinhardtii. We first demonstrated that labile Cu(I) predominantly accumulated in the granules within the cytoplasmic matrix, whereas the labile Cu(II) pool primarily localized in the pyrenoid and chloroplast. The cell cycle played an integral role in balancing the labile Cu(I)/Cu(II) pools. Specifically, the labile Cu(II) pool primarily accumulated during the SM phase following cell division, while the labile Cu(I) pool dynamically changed during the G phase as cell size increased. Notably, the labile Cu(II) pool in algae at the SM stage exhibited heightened sensitivity to environmental Cu stress. Exogenous Cu stress disrupted the intracellular labile Cu(I)/Cu(II) cycle and balance, causing a shift toward the labile Cu(II) pool. Our proteomic analysis further identified a putative cupric reductase, potentially capable of reducing Cu(II) to Cu(I), and four putative multicopper oxidases, potentially capable of oxidizing Cu(I) to Cu(II), which may be involved in the conversion between the labile Cu(I) pool and labile Cu(II) pool. Our study elucidated a dynamic cycle of the intracellular labile Cu(I)/Cu(II) pools, which were accessible and responsive to environmental changes.

Substrate Stiffness Modulates Stemness and Differentiation of Rabbit Corneal Endothelium Through the Paxillin-YAP Pathway.

To explore the role of substrate stiffness and the mechanism beneath corneal endothelial cells' (CECs') stemness maintenance and differentiation. CECs were divided into central zone (8mm trephined boundary) and peripheral zone (8mm trephined edge with attached limbal). Two zones were analyzed by hematoxylin-eosin staining and scanning electron microscopy for anatomic structure. The elastic modulus of Descemet's membrane (DM) was analyzed by atomic force microscopy. Compressed type I collagen gels with different stiffness were constructed as an in vitro model system to test the role of stiffness on phenotype using cultured rabbit CECs. Cell morphology, expression and intracellular distribution of Yes-associated protein (YAP), differentiation (ZO-1, Na+/K+-ATPase), stemness (FOXD3, CD34, Sox2, Oct3/4), and endothelial-mesenchymal transition (EnMT) markers were analyzed by immunofluorescence, quantitative RT-PCR, and Western blot. The results showed that the peripheral area of rabbit and human DM is softer than the central area ex vivo. Using the biomimetic extracellular matrix collagen gels in vitro model, we then demonstrated that soft substrate weakens the differentiation and EnMT in the culture of CECs. It was further proved by the inhibitor experiment that soft substrate enhances stemness maintenance via inhibition of paxillin-YAP signaling, which was activated on a stiff substrate. Our findings confirm that substrate stiffness modulates the stemness maintenance and differentiation of CECs and suggest a potential strategy for CEC-based corneal tissue engineering.

Non-viral Gene Therapy for Melanoma Using Lysenin from Eisenia Foetida.

Earthworms, long utilized in traditional medicine, serve as a source of inspiration for modern therapeutics. Lysenin, a defensive factor in the coelom fluid of the earthworm Eisenia fetida, has multiple bioactivities. However, the inherent toxicity of Lysenin as a pore-forming protein (PFP) restricts its application in therapy. Here, a gene therapy strategy based on Lysenin for cancer treatment is presented. The formulation consists of polymeric nanoparticles complexed with the plasmid encoding Lysenin. After transfection in vitro, melanoma cells can express Lysenin, resulting in necrosis, autophagy, and immunogenic cell death. The secretory signal peptide alters the intracellular distribution of the expressed product of Lysenin, thereby potentiating its anticancer efficacy. The intratumor injection of Lysenin gene formulation can efficiently kill the transfected melanoma cells and activate the antitumor immune response. Notably, no obvious systemic toxicity is observed during the treatment. Non-viralgene therapy based on Lysenin derived from Eisenia foetida exhibits potential in cancer therapy, which can inspire future cancer therapeutics.

Antagonizing roles of SHP1 in the pathogenesis of Helicobacterpylori infection.

SHP1 has been documented as a tumor suppressor and it was thought to play an antagonistic role in the pathogenesis of Helicobacter pylori infection. In this study, the exact mechanism of this antagonistic action was studied. AGS, MGC803, and GES-1 cells were infected with H. pylori, intracellular distribution changes of SHP1 were first detected by immunofluorescence. SHP1 overexpression and knockdown were then constructed in these cells to investigate its antagonistic roles in H. pylori infection. Migration and invasion of infected cells were detected by transwell assay, secretion of IL-8 was examined via ELISA, the cells with hummingbird-like alteration were determined by microexamination, and activation of JAK2/STAT3, PI3K/Akt, and ERK pathways were detected by immunoblotting. Mice infection model was established and gastric pathological changes were evaluated. Finally, the SHP1 activator sorafenib was used to analyze the attenuating effect of SHP1 activation on H. pylori pathogenesis invitro and invivo. The sub-localization of SHP1 changed after H. pylori infection, specifically that the majority of the cytoplasmic SHP1 was transferred to the cell membrane. SHP1 inhibited H. pylori-induced activation of JAK2/STAT3 pathway, PI3K/Akt pathway, nuclear translocation of NF-κB, and then reduced EMT, migration, invasion, and IL-8 secretion. In addition, SHP1 inhibited the formation of CagA-SHP2 complex by dephosphorylating phosphorylated CagA, reduced ERK phosphorylation and the formation of CagA-dependent hummingbird-like cells. In the mice infection model, gastric pathological changes were observed and increased IL-8 secretion, indicators of cell proliferation and EMT progression were also detected. By activating SHP1 with sorafenib, a significant curative effect against H. pylori infection was obtained invitro and invivo. SHP1 plays an antagonistic role in H. pylori pathogenesis by inhibiting JAK2/STAT3 and PI3K/Akt pathways, NF-κB nuclear translocation, and CagA phosphorylation, thereby reducing cell EMT, migration, invasion, IL-8 secretion, and hummingbird-like changes.

Characterization of cellular responses and cell lysis to elevated hydrodynamic stress from benchtop perfusion bioreactors.

The effective design of perfusion cell culture is currently challenging regarding balancing the operating parameters associated with the hydrodynamic conditions due to increased system complexity. To address this issue, cellular responses of an industrial CHO cell line to different types of hydrodynamic stress in benchtop perfusion bioreactors originating from agitation, sparging, and hollow fibers (HF) in the cell retention devices were systematically investigated here with the analysis of cell lysis. It was found that cell lysis was very common and most associated with the sparging stress, followed by the HF and lastly the agitation, consequently heavily impacting the estimation of process descriptors related to biomass. The results indicated that the agitation stress led to a reduced cell growth with a shift toward a more productive phenotype, suggesting an energy redirection from biomass formation to product synthesis, whereas the sparging stress had a small impact on the intracellular metabolic flux distribution but increased the cell death rate drastically. For HF stress, a similar cell maintenance profile was found as the sparging while the activity of glycolysis and the TCA cycle was significantly impeded, potentially leading to the lack of energy and thus a substantial decrease in cell-specific productivity. Moreover, a novel concept of volume average shear stress was developed to further understand the relations of different types of stress and the observed responses for an improved insight for the perfusion cell culture.

Monitoring the Interaction Between Solid Lipid Nanoparticles and Alveolar Macrophages via the Label-Free Technique

Macrophages are employed as targets for delivering genes, drugs, or lipid nanoparticles into tumors or other specific sites. Studying the interaction between solid lipid nanoparticles (SLNs) and macrophages is essential for assessing nanotoxicity and advancing the development of nanomedicines. However, limited data are currently available on the membrane microstructure and biochemical changes that occur when macrophages interact with SLNs. We conducted a label-free morphological and biochemical investigation of NR8383 macrophages using optical diffraction tomography (ODT), which validated the efficiency of the SLNs as a drug delivery system. ODT provided intracellular holotomography to characterize the macrophages and fluorescence imaging to analyze delivery efficiency. ODT analysis revealed the responses of phagocytic macrophages. Additionally, a quantitative analysis of lipid droplets using refractive indices revealed that, compared with incubation with normal cells, incubation with SLNs significantly increased the lipid droplet volume and surface area. The uptake of SLNs into macrophages resulted in increased cell volume, surface area, and concentration, which indicated greater morphological and biochemical variability in the treated cells than in the control cells. The results suggest that ODT imaging is promising for understanding the intracellular distribution of SLNs and useful for validating the efficacy of delivery of SLNs to macrophages.

Bilirubin Distribution in Plants at the Subcellular and Tissue Levels.

In heterotrophs, heme degradation produces bilirubin, a tetrapyrrole compound that has antioxidant activity. In plants, heme is degraded in plastids and is believed to be converted to phytochromobilin rather than bilirubin. Recently, we used the bilirubin-inducible fluorescent protein UnaG to reveal that plants produce bilirubin via a non-enzymatic reaction with NADPH. In the present study, we used an UnaG-based live imaging system to visualize bilirubin accumulation in Arabidopsis thaliana and Nicotiana benthamiana at the organelle and tissue levels. In chloroplasts, bilirubin preferentially accumulated in the stroma, and the stromal bilirubin level increased upon dark treatment. Investigation of intracellular bilirubin distribution in leaves and roots showed that it accumulated mostly in plastids, with low levels detected in the cytosol and other organelles, such as peroxisomes, mitochondria and the endoplasmic reticulum. A treatment that increased bilirubin production in chloroplasts decreased the bilirubin level in peroxisomes, implying that a bilirubin precursor is transported between the two organelles. At the cell and tissue levels, bilirubin showed substantial accumulation in the root elongation region but little or none in the root cap and guard cells. Intermediate bilirubin accumulation was observed in other shoot and root tissues, with lower levels in shoot tissues. Our data revealed the distribution of bilirubin in plants, which has implications for the transport and physiological function of tetrapyrroles.

The plasma membrane-localized transporter VrNramp5 of Vigna radiata L. governs cadmium tolerance and accumulation in Arabidopsis thaliana

Cadmium (Cd) is a highly toxic non-essential element, which requires the transporters of essential and beneficial elements to enter the plant, and poses threat to plant growth and development. However, the mechanism of Cd uptake and accumulation in mungbean is limited. Here, we reported a Cd transporter VrNramp5 in mungbean, and its functional roles in response to Cd toxicity were conducted. The expression of VrNramp5 was down-regulated by Cd toxicity. VrNramp5 localized at plasma membrane based on subcellular localization analysis in epidermal cells of Nicotiana benthamiana. Heterologous expression of VrNramp5 in Arabidopsis thaliana promoted Cd accumulation, displayed the Cd sensitive phenotype including decreased root elongation and fresh weight, and VrNramp5 could diminish the Cd tolerance phenotype of AtNramp1-deficient mutant (atnramp1). Non–invasive micro–test showed that VrNramp5-overexpressing lines possessed higher Cd absorption capacity in root tips. Moreover, the Cd fluorescence probe staining revealed that more Cd accumulated in the leaf protoplasts of VrNramp5-overexpressing Arabidopsis. Those were consistent with the plants grown in the absence or presence of Cd (10 mg kg−1 or 20 mg kg−1) in soil. Taken together, VrNramp5 is responsible for plant Cd tolerance via regulating the Cd absorption capacity of roots and altering the intracellular distribution of Cd. Our findings provide a theoretical basis for further exploring the mechanism of Cd tolerance and breeding variety of mungbean with low Cd accumulation.