ResearchPad - cell-staining https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Graphene-based 2D constructs for enhanced fibroblast support]]> https://www.researchpad.co/article/elastic_article_15755 Complex skin wounds have always been a significant health and economic problem worldwide due to their elusive and sometimes poor or non-healing conditions. If not well-treated, such wounds may lead to amputation, infections, cancer, or even death. Thus, there is a need to efficiently generate multifunctional skin grafts that address a wide range of skin conditions, including non-healing wounds, and enable the regeneration of new skin tissue. Here, we propose studying pristine graphene and two of its oxygen-functionalized derivatives—high and low-oxygen graphene films—as potential substrates for skin cell proliferation and differentiation. Using BJ cells (human foreskin-derived fibroblasts) to represent basic skin cells, we show that the changes in surface properties of pristine graphene due to oxygen functionalization do not seem to statistically impact the normal proliferation and maturation of skin cells. Our results indicate that the pristine and oxidized graphenes presented relatively low cytotoxicity to BJ fibroblasts and, in fact, support their growth and bioactivity. Therefore, these graphene films could potentially be integrated into more complex skin regenerative systems to support skin regeneration. Because graphene’s surface can be relatively easily functionalized with various chemical groups, this finding presents a major opportunity for the development of various composite materials that can act as active components in regenerative applications such as skin regeneration.

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<![CDATA[Tuning antiviral CD8 T-cell response via proline-altered peptide ligand vaccination]]> https://www.researchpad.co/article/elastic_article_14646 Viral escape mutagenesis correlates often with disease progression and represents a major hurdle for vaccination-based therapies. Here, we have designed and developed a novel generation of altered epitopes that re-establish and enhance significantly CD8+ T cell recognition of a naturally occurring viral immune escape variant. Biophysical and structural analyses provide a clear understanding of the molecular mechanisms underlying this reestablished recognition. We believe that this approach can be implemented to currently available or novel vaccination approaches to efficiently restore T cell recognition of virus escape variants to control disease progression.

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<![CDATA[Neurons from human mesenchymal stem cells display both spontaneous and stimuli responsive activity]]> https://www.researchpad.co/article/elastic_article_14593 Mesenchymal stem cells have the ability to transdifferentiate into neurons and therefore one of the potential adult stem cell source for neuronal tissue regeneration applications and understanding neurodevelopmental processes. In many studies on human mesenchymal stem cell (hMSC) derived neurons, success in neuronal differentiation was limited to neuronal protein expressions which is not statisfactory in terms of neuronal activity. Established neuronal networks seen in culture have to be investigated in terms of synaptic signal transmission ability to develop a culture model for human neurons and further studying the mechanism of neuronal differentiation and neurological pathologies. Accordingly, in this study, we analysed the functionality of bone marrow hMSCs differentiated into neurons by a single step cytokine-based induction protocol. Neurons from both primary hMSCs and hMSC cell line displayed spontaneous activity (≥75%) as demonstrated by Ca++ imaging. Furthermore, when electrically stimulated, hMSC derived neurons (hMd-Neurons) matched the response of a typical neuron in the process of maturation. Our results reveal that a combination of neuronal inducers enhance differentiation capacity of bone marrow hMSCs into high yielding functional neurons with spontaneous activity and mature into electrophysiologically active state. Conceptually, we suggest these functional hMd-Neurons to be used as a tool for disease modelling of neuropathologies and neuronal differentiation studies.

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<![CDATA[Highly efficient serum-free manipulation of miRNA in human NK cells without loss of viability or phenotypic alterations is accomplished with TransIT-TKO]]> https://www.researchpad.co/article/N4e6e8e95-63ae-420d-a6d7-c2f1aa3d99e6

Natural killer (NK) cells are innate lymphocytes with functions that include target cell killing, inflammation and regulation. NK cells integrate incoming activating and inhibitory signals through an array of germline-encoded receptors to gauge the health of neighbouring cells. The reactive potential of NK cells is influenced by microRNA (miRNA), small non-coding sequences that interfere with mRNA expression. miRNAs are highly conserved between species, and a single miRNA can have hundreds to thousands of targets and influence entire cellular programs. Two miRNA species, miR-155-5p and miR-146a-5p are known to be important in controlling NK cell function, but research to best understand the impacts of miRNA species within NK cells has been bottlenecked by a lack of techniques for altering miRNA concentrations efficiently and without off-target effects. Here, we describe a non-viral and straightforward approach for increasing or decreasing expression of miRNA in primary human NK cells. We achieve >90% transfection efficiency without off-target impacts on NK cell viability, education, phenotype or function. This opens the opportunity to study and manipulate NK cell miRNA profiles and their impacts on NK cellular programs which may influence outcomes of cancer, inflammation and autoimmunity.

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<![CDATA[Streptococcal H2O2 inhibits IgE-triggered degranulation of RBL-2H3 mast cell/basophil cell line by inducing cell death]]> https://www.researchpad.co/article/Nadf2e0c3-9608-4100-a6fa-f03310d30959

Mast cells and basophils are central players in allergic reactions triggered by immunoglobulin E (IgE). They have intracellular granules containing allergic mediators (e.g., histamine, serotonin, inflammatory cytokines, proteases and β-hexosaminidase), and stimulation by IgE-allergen complex leads to the release of such allergic mediators from the granules, that is, degranulation. Mast cells are residents of mucosal surfaces, including those of nasal and oral cavities, and play an important role in the innate defense system. Members of the mitis group streptococci such as Streptococcus oralis, are primary colonizers of the human oral cavity. They produce hydrogen peroxide (H2O2) as a by-product of sugar metabolism. In this study, we investigated the effects of streptococcal infection on RBL-2H3 mast cell/basophil cell line. Infection by oral streptococci did not induce degranulation of the cells. Stimulation of the RBL-2H3 cells with anti-dinitrophenol (DNP) IgE and DNP-conjugated human serum albumin triggers degranulation with the release of β-hexosaminidase. We found that S. oralis and other mitis group streptococci inhibited the IgE-triggered degranulation of RBL-2H3 cells. Since mitis group streptococci produce H2O2, we examined the effect of S. oralis mutant strain deficient in producing H2O2, and found that they lost the ability to suppress the degranulation. Moreover, H2O2 alone inhibited the IgE-induced degranulation. Subsequent analysis suggested that the inhibition of degranulation was related to the cytotoxicity of streptococcal H2O2. Activated RBL-2H3 cells produce interleukin-4 (IL-4); however, IL-4 production was not induced by streptococcal H2O2. Furthermore, an in vivo study using the murine pollen-induced allergic rhinitis model suggested that the streptococcal H2O2 reduces nasal allergic reaction. These findings reveal that H2O2 produced by oral mitis group streptococci inhibits IgE-stimulated degranulation by inducing cell death. Consequently, streptococcal H2O2 can be considered to modulate the allergic reaction in mucosal surfaces.

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<![CDATA[Neuroprotective effects of exogenous erythropoietin in Wistar rats by downregulating apoptotic factors to attenuate N-methyl-D-aspartate-mediated retinal ganglion cells death]]> https://www.researchpad.co/article/N85685bba-c047-422b-abfc-358a98ed1fe7

The aim of this study was to investigate whether exogenous erythropoietin (EPO) administration attenuates N-methyl-D-aspartate (NMDA)-mediated excitotoxic retinal damage in Wistar rats. The survival rate of retinal ganglion cells (RGCs) were investigated by flat mount analysis and flow cytometry. A total of 125 male Wistar rats were randomly assigned to five groups: negative control, NMDA80 (i.e., 80 nmoles NMDA intravitreally injected), NMDA80 + 10ng EPO, NMDA80 + 50ng EPO, and NMDA80 + 250ng EPO. The NMDA80 + 50ng EPO treatment group was used to evaluate various administrated points (pre-/co-/post- administration of NMDA80). Meanwhile, the transferase dUTP Nick-End Labeling (TUNEL) assay of RGCs, the inner plexiform layer (IPL) thickness and the apoptotic signal transduction pathways of μ-calpain, Bax, and caspase 9 were assessed simultaneously using an immunohistochemical method (IHC). When EPO was co-administered with NMDA80, attenuated cell death occurred through the downregulation of the apoptotic indicators: μ-calpain was activated first (peak at ~18hrs), followed by Bax and caspase 9 (peak at ~40hrs). Furthermore, the images of retinal cross sections have clearly demonstrated that thickness of the inner plexiform layer (IPL) was significantly recovered at 40 hours after receiving intravitreal injection with NMDA80 and 50ng EPO. Exogenous EPO may protect RGCs and bipolar cell axon terminals in IPL by downregulating apoptotic factors to attenuate NMDA-mediated excitotoxic retinal damage.

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<![CDATA[Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer]]> https://www.researchpad.co/article/Naf611639-dea0-4cb3-8951-2157f0424339

Background

G protein-coupled estrogen receptor (GPER), or G protein-coupled receptor 30 (GPR30), is reported to mediate non-genomic estrogen signaling. GPR30 associates with breast cancer (BC) outcome and may contribute to tamoxifen resistance. We investigated the expression and prognostic significance of GPR30 in metachronous contralateral breast cancer (CBC) as a model of tamoxifen resistance.

Methods

Total GPR30 expression (GPR30TOT) and plasma membrane-localized GPR30 expression (GPR30PM) were analyzed by immunohistochemistry in primary (BC1; nBC1 = 559) and contralateral BC (BC2; nBC2 = 595), and in lymph node metastases (LGL; nLGL1 = 213; nLGL2 = 196). Death from BC (BCD), including BC death or death after documented distant metastasis, was used as primary end-point.

Results

GPR30PM in BC2 and LGL2 were associated with increased risk of BCD (HRBC2 = 1.7, p = 0.03; HRLGL2 = 2.0; p = 0.02). In BC1 and BC2, GPR30PM associated with estrogen receptor (ER)-negativity (pBC1<0.0001; pBC2<0.0001) and progesterone receptor (PR)-negativity (pBC1 = 0.0007; pBC2<0.0001). The highest GPR30TOT and GPR30PM were observed in triple-negative BC. GPR30PM associated with high Ki67 staining in BC1 (p<0.0001) and BC2 (p<0.0001). GPR30TOT in BC2 did not associate with tamoxifen treatment for BC1. However, BC2 that were diagnosed during tamoxifen treatment were more likely to express GPR30PM than BC2 diagnosed after treatment completion (p = 0.01). Furthermore, a trend was observed that patients with GPR30PM in an ER-positive BC2 had greater benefit from tamoxifen treatment.

Conclusion

PM-localized GPR30 staining is associated with increased risk of BC death when expressed in BC2 and LGL2. Additionally, PM-localized GPR30 correlates with prognostic markers of worse outcome, such as high Ki67 and a triple-negative subtype. Therefore, PM-localized GPR30 may be an interesting new target for therapeutic exploitation. We found no clear evidence that total GPR30 expression is affected by tamoxifen exposure during development of metachronous CBC, or that GPR30 contributes to tamoxifen resistance.

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<![CDATA[Observation and quantification of the morphological effect of trypan blue rupturing dead or dying cells]]> https://www.researchpad.co/article/Nce15bf32-82da-4cd0-8031-f3eea4581b61

Trypan blue has long been the gold standard for staining dead cell to determine cell viability. The dye is excluded from membrane-intact live cells, but can enter and concentrate in membrane-compromised dead cells, rendering the cells dark blue. Over the years, there has been an understanding that trypan blue is inaccurate for cell viability under 80% without scientific support. We previously showed that trypan blue can alter the morphology of dead cells to a diffuse shape, which can lead to over-estimation of viability. Here, we investigate the origin of the dim and diffuse objects after trypan blue staining. Utilizing image and video acquisition, we show real-time transformation of cells into diffuse objects when stained with trypan blue. The same phenomenon was not observed when staining cells with propidium iodide. We also demonstrate the co-localization of trypan blue and propidium iodide, confirming these diffuse objects as cells that contain nuclei. The videos clearly show immediate cell rupturing after trypan blue contact. The formation of these diffuse objects was monitored and counted over time as cells die outside of the incubator. We hypothesize and demonstrate that rapid water influx may have caused the cells to rupture and disappear. Since some dead cells disappear after trypan blue staining, the total can be under-counted, leading to over-estimation of cell viability. This inaccuracy could affect the outcomes of cellular therapies, which require accurate measurements of immune cells that will be infused back into patients.

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<![CDATA[Ex vivo-expanded highly purified natural killer cells in combination with temozolomide induce antitumor effects in human glioblastoma cells in vitro]]> https://www.researchpad.co/article/5c89771fd5eed0c4847d24e7

Glioblastoma is the leading malignant glioma with a poor prognosis. This study aimed to investigate the antitumor effects of natural killer cells in combination with temozolomide as the standard chemotherapeutic agent for glioblastoma. Using a simple, feeder-less, and chemically defined culture method, we expanded human peripheral blood mononuclear cells and assessed the receptor expression, natural killer cell activity, and regulatory T cell frequency in expanded cells. Next, using the standard human glioblastoma cell lines (temozolomide-sensitive U87MG, temozolomide-resistant T98G, and LN-18), we assessed the ligand expressions of receptors on natural killer cells. Furthermore, the antitumor effects of the combination of the expanded natural killer cells and temozolomide were assessed using growth inhibition assays, apoptosis detection assays, and senescence-associated β-galactosidase activity assays in the glioblastoma cell lines. Novel culture systems were sufficient to attain highly purified (>98%), expanded (>440-fold) CD3/CD56+ peripheral blood-derived natural killer cells. We designated the expanded population as genuine induced natural killer cells. Genuine induced natural killer cells exhibited a high natural killer activity and low regulatory T cell frequency compared with lymphokine-activated killer cells. Growth inhibition assays revealed that genuine induced natural killer cells inhibited the glioblastoma cell line growth but enhanced temozolomide-induced inhibition effects in U87MG. Apoptosis detection assays revealed that genuine induced natural killer cells induced apoptosis in the glioblastoma cell lines. Furthermore, senescence-associated β-galactosidase activity assays revealed that temozolomide induced senescence in U87MG. Genuine induced natural killer cells induce apoptosis in temozolomide-sensitive and temozolomide-resistant glioblastoma cells and enhances temozolomide-induced antitumor effects in different mechanisms. Hence, the combination of genuine induced natural killer cells and temozolomide may prove to be a promising immunochemotherapeutic approach in patients with glioblastoma if the antitumor effects in vivo can be demonstrated.

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<![CDATA[PML nuclear body-residing proteins sequentially associate with HPV genome after infectious nuclear delivery]]> https://www.researchpad.co/article/5c7d95e9d5eed0c484734f7e

Subnuclear promyelocytic leukemia (PML) nuclear bodies (NBs) are targeted by many DNA viruses after nuclear delivery. PML protein is essential for formation of PML NBs. Sp100 and Small Ubiquitin-Like Modifier (SUMO) are also permanently residing within PML NBs. Often, large DNA viruses disassemble and reorganize PML NBs to counteract their intrinsic antiviral activity and support establishment of infection. However, human papillomavirus (HPV) requires PML protein to retain incoming viral DNA in the nucleus for subsequent efficient transcription. In contrast, Sp100 was identified as a restriction factor for HPV. These findings suggested that PML NBs are important regulators of early stages of the HPV life cycle. Nuclear delivery of incoming HPV DNA requires mitosis. Viral particles are retained within membrane-bound transport vesicles throughout mitosis. The viral genome is released from transport vesicles by an unknown mechanism several hours after nuclear envelope reformation. The minor capsid protein L2 mediates intracellular transport by becoming transmembranous in the endocytic compartment. Herein, we tested our hypothesis that PML protein is recruited to incoming viral genome prior to egress from transport vesicles. High-resolution microscopy revealed that PML protein, SUMO-1, and Sp100 are recruited to incoming viral genomes, rather than viral genomes being targeted to preformed PML NBs. Differential immunofluorescent staining suggested that PML protein and SUMO-1 associated with transport vesicles containing viral particles prior to egress, implying that recruitment is likely mediated by L2 protein. In contrast, Sp100 recruitment to HPV-harboring PML NBs occurred after release of viral genomes from transport vesicles. The delayed recruitment of Sp100 is specific for HPV-associated PML NBs. These data suggest that the virus continuously resides within a protective environment until the transport vesicle breaks down in late G1 phase and imply that HPV might modulate PML NB assembly to achieve establishment of infection and the shift to viral maintenance.

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<![CDATA[Rescue of collapsed replication forks is dependent on NSMCE2 to prevent mitotic DNA damage]]> https://www.researchpad.co/article/5c6730a3d5eed0c484f37e1c

NSMCE2 is an E3 SUMO ligase and a subunit of the SMC5/6 complex that associates with the replication fork and protects against genomic instability. Here, we study the fate of collapsed replication forks generated by prolonged hydroxyurea treatment in human NSMCE2-deficient cells. Double strand breaks accumulate during rescue by converging forks in normal cells but not in NSMCE2-deficient cells. Un-rescued forks persist into mitosis, leading to increased mitotic DNA damage. Excess RAD51 accumulates and persists at collapsed forks in NSMCE2-deficient cells, possibly due to lack of BLM recruitment to stalled forks. Despite failure of BLM to accumulate at stalled forks, NSMCE2-deficient cells exhibit lower levels of hydroxyurea-induced sister chromatid exchange. In cells deficient in both NSMCE2 and BLM, hydroxyurea-induced double strand breaks and sister chromatid exchange resembled levels found in NSCME2-deficient cells. We conclude that the rescue of collapsed forks by converging forks is dependent on NSMCE2.

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<![CDATA[Exposure of Candida albicans β (1,3)-glucan is promoted by activation of the Cek1 pathway]]> https://www.researchpad.co/article/5c5ca280d5eed0c48441e4da

Candida albicans is among the most common causes of human fungal infections and is an important source of mortality. C. albicans is able to diminish its detection by innate immune cells through masking of β (1,3)-glucan in the inner cell wall with an outer layer of heavily glycosylated mannoproteins (mannan). However, mutations or drugs that disrupt the cell wall can lead to exposure of β (1,3)-glucan (unmasking) and enhanced detection by innate immune cells through receptors like Dectin-1, the C-type signaling lectin. Previously, our lab showed that the pathway for synthesizing the phospholipid phosphatidylserine (PS) plays a role in β (1,3)-glucan masking. The homozygous PS synthase knockout mutant, cho1Δ/Δ, exhibits increased exposure of β (1,3)-glucan. Several Mitogen Activated Protein Kinase (MAPK) pathways and their upstream Rho-type small GTPases are important for regulating cell wall biogenesis and remodeling. In the cho1Δ/Δ mutant, both the Cek1 and Mkc1 MAPKs are constitutively activated, and they act downstream of the small GTPases Cdc42 and Rho1, respectively. In addition, Cdc42 activity is up-regulated in cho1Δ/Δ. Thus, it was hypothesized that activation of Cdc42 or Rho1 and their downstream kinases cause unmasking. Disruption of MKC1 does not decrease unmasking in cho1Δ/Δ, and hyperactivation of Rho1 in wild-type cells increases unmasking and activation of both Cek1 and Mkc1. Moreover, independent hyperactivation of the MAP kinase kinase kinase Ste11 in wild-type cells leads to Cek1 activation and increased β (1,3)-glucan exposure. Thus, upregulation of the Cek1 MAPK pathway causes unmasking, and may be responsible for unmasking in cho1Δ/Δ.

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<![CDATA[Interaction between nectin-1 and the human natural killer cell receptor CD96]]> https://www.researchpad.co/article/5c6dca1ed5eed0c48452a7e2

Regulation of Natural Killer (NK) cell activity is achieved by the integration of both activating and inhibitory signals acquired at the immunological synapse with potential target cells. NK cells express paired receptors from the immunoglobulin family which share common ligands from the nectin family of adhesion molecules. The activating receptor CD226 (DNAM-1) binds to nectin-2 and CD155, which are also recognized by the inhibitory receptor TIGIT. The third receptor in this family is CD96, which is less well characterized and may have different functions in human and mouse models. Human CD96 interacts with CD155 and ligation of this receptor activates NK cells, while in mice the presence of CD96 correlates with decreased NK cell activation. Mouse CD96 also binds nectin-1, but the effect of this interaction has not yet been determined. Here we show that human nectin-1 directly interacts with CD96 in vitro. The binding site for CD96 is located on the nectin-1 V-domain, which comprises a canonical interface that is shared by nectins to promote cell adhesion. The affinity of nectin-1 for CD96 is lower than for other nectins such as nectin-3 and nectin-1 itself. However, the affinity of nectin-1 for CD96 is similar to its affinity for herpes simplex virus glycoprotein D (HSV gD), which binds the nectin-1 V-domain during virus entry. The affinity of human CD96 for nectin-1 is lower than for its known activating ligand CD155. We also found that human erythroleukemia K562 cells, which are commonly used as susceptible targets to assess NK cell cytotoxicity did not express nectin-1 on their surface and were resistant to HSV infection. When expressed in K562 cells, nectin-1-GFP accumulated at cell contacts and allowed HSV entry. Furthermore, overexpression of nectin-1-GFP led to an increased susceptibility of K562 cells to NK-92 cell cytotoxicity.

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<![CDATA[Anti-CD8 monoclonal antibody-mediated depletion alters the phenotype and behavior of surviving CD8+ T cells]]> https://www.researchpad.co/article/5c6730ddd5eed0c484f38251

It is common practice for researchers to use antibodies to remove a specific cell type to infer its function. However, it is difficult to completely eliminate a cell type and there is often limited or no information as to how the cells which survive depletion are affected. This is particularly important for CD8+ T cells for two reasons. First, they are more resistant to mAb-mediated depletion than other lymphocytes. Second, targeting either the CD8α or CD8β chain could induce differential effects. We show here that two commonly used mAbs, against either the CD8α or CD8β subunit, can differentially affect cellular metabolism. Further, in vivo treatment leaves behind a population of CD8+ T cells with different phenotypic and functional attributes relative to each other or control CD8+ T cells. The impact of anti-CD8 antibodies on CD8+ T cell phenotype and function indicates the need to carefully consider the use of these, and possibly other “depleting” antibodies, as they could significantly complicate the interpretation of results or change the outcome of an experiment. These observations could impact how immunotherapy and modulation of CD8+ T cell activation is pursued.

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<![CDATA[Interleukin-13 maintains the stemness of conjunctival epithelial cell cultures prepared from human limbal explants]]> https://www.researchpad.co/article/5c6b2698d5eed0c484289d28

To use human limbal explants as an alternative source for generating conjunctival epithelium and to determine the effect of interleukin-13 (IL-13) on goblet cell number, mucin expression, and stemness. Human limbal explants prepared from 17 corneoscleral rims were cultured with or without IL-13 (IL-13+ and IL-13-, respectively) and followed up to passage 2 (primary culture [P0]–P2). Cells were characterized by alcian blue/periodic acid–Schiff (AB/PAS) staining (goblet cells); immunofluorescent staining for p63α (progenitor cells), Ki-67 (proliferation), MUC5AC (mucin, goblet cells), and keratin 7 (K7, conjunctival epithelial and goblet cells); and by quantitative real-time polymerase chain reaction for expression of the p63α (TP63), MUC5AC, MUC4 (conjunctival mucins), K3, K12 (corneal epithelial cells), and K7 genes. Clonogenic ability was determined by colony-forming efficiency (CFE) assay. Using limbal explants, we generated epithelium with conjunctival phenotype and high viability in P0, P1, and P2 cultures under IL-13+ and IL-13- conditions, i.e., epithelium with strong K7 positivity, high K7 and MUC4 expression and the presence of goblet cells (AB/PAS and MUC5AC positivity; MUC5AC expression). p63α positivity was similar in IL-13+ and IL-13- cultures and was decreased in P2 cultures; however, there was increased TP63 expression in the presence of IL-13 (especially in the P1 cultures). Similarly, IL-13 increased proliferative activity in P1 cultures and significantly promoted P0 and P1 culture CFE. IL-13 did not increase goblet cell number in the P0–P2 cultures, nor did it influence MUC5AC and MUC4 expression. By harvesting unattached cells on day 1 of P1 we obtained goblet cell rich subpopulation showing AB/PAS, MUC5AC, and K7 positivity, but with no growth potential. In conclusion, limbal explants were successfully used to develop conjunctival epithelium with the presence of putative stem and goblet cells and with the ability to preserve the stemness of P0 and P1 cultures under IL-13 influence.

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<![CDATA[CD4 occupancy triggers sequential pre-fusion conformational states of the HIV-1 envelope trimer with relevance for broadly neutralizing antibody activity]]> https://www.researchpad.co/article/5c48df5cd5eed0c4841cd854

During the entry process, the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer undergoes a sequence of conformational changes triggered by both CD4 and coreceptor engagement. Resolving the conformation of these transient entry intermediates has proven challenging. Here, we fine-mapped the antigenicity of entry intermediates induced by increasing CD4 engagement of cell surface–expressed Env. Escalating CD4 triggering led to the sequential adoption of different pre-fusion conformational states of the Env trimer, up to the pre-hairpin conformation, that we assessed for antibody epitope presentation. Maximal accessibility of the coreceptor binding site was detected below Env saturation by CD4. Exposure of the fusion peptide and heptad repeat 1 (HR1) required higher CD4 occupancy. Analyzing the diverse antigenic states of the Env trimer, we obtained key insights into the transitions in epitope accessibility of broadly neutralizing antibodies (bnAbs). Several bnAbs preferentially bound CD4-triggered Env, indicating a potential capacity to neutralize both pre- and post-CD4 engagement, which needs to be explored. Assessing binding and neutralization activity of bnAbs, we confirm antibody dissociation rates as a driver of incomplete neutralization. Collectively, our findings highlight a need to resolve Env conformations that are neutralization-relevant to provide guidance for immunogen development.

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<![CDATA[The type III intermediate filament vimentin regulates organelle distribution and modulates autophagy]]> https://www.researchpad.co/article/5c5b52e2d5eed0c4842bd1ca

The cytoskeletal protein vimentin plays a key role in positioning of organelles within the cytosol and has been linked to the regulation of numerous cellular processes including autophagy, however, how vimentin regulates autophagy remains relatively unexplored. Here we report that inhibition of vimentin using the steroidal lactone Withaferin A (WFA) causes vimentin to aggregate, and this is associated with the relocalisation of organelles including autophagosomes and lysosomes from the cytosol to a juxtanuclear location. Vimentin inhibition causes autophagosomes to accumulate, and we demonstrate this results from modulation of mechanistic target of rapamycin (mTORC1) activity, and disruption of autophagosome-lysosome fusion. We suggest that vimentin plays a physiological role in autophagosome and lysosome positioning, thus identifying vimentin as a key factor in the regulation of mTORC1 and autophagy.

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<![CDATA[Golgin-160 and GMAP210 play an important role in U251 cells migration and invasion initiated by GDNF]]> https://www.researchpad.co/article/5c59feb7d5eed0c484135327

Gliomas are the most common malignant tumors of the brain and are characteristic of severe migration and invasion. Glial cell line-derived neurotrophic factor (GDNF) promotes glioma development process. However, the regulatory mechanisms of promoting occurrence and development of glioma have not yet been clearly elucidated. In the present study, the mechanism by which GDNF promotes glioma cell migration and invasion through regulating the dispersion and location of the Golgi apparatus (GA) is described. Following GDNF treatment, a change in the volume and position of GA was observed. The stack area of the GA was enlarged and it was more concentrated near the nucleus. Golgin-160 and Golgi microtubule-associated protein 210 (GMAP210) were identified as target molecules regulating GA positioning. In the absence of either golgin-160 or GMAP210 using lentivirus, the migration and invasion of U251 cells were decreased, while it was increased following GDNF. It was also found that the GA was decreased in size and dispersed following golgin-160 or GMAP210 knockdown, as determined by GA green fluorescence assay. Once GDNF was added, the above phenomenon would be twisted, and the concentrated location and volume of the GA was restored. In combination, the present data suggested that the regulation of the position and size of the GA by golgin-160 and GMAP210 play an important role in U251 cell migration and invasion.

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<![CDATA[Label-free classification of cells based on supervised machine learning of subcellular structures]]> https://www.researchpad.co/article/5c59fec3d5eed0c48413541b

It is demonstrated that cells can be classified by pattern recognition of the subcellular structure of non-stained live cells, and the pattern recognition was performed by machine learning. Human white blood cells and five types of cancer cell lines were imaged by quantitative phase microscopy, which provides morphological information without staining quantitatively in terms of optical thickness of cells. Subcellular features were then extracted from the obtained images as training data sets for the machine learning. The built classifier successfully classified WBCs from cell lines (area under ROC curve = 0.996). This label-free, non-cytotoxic cell classification based on the subcellular structure of QPM images has the potential to serve as an automated diagnosis of single cells.

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<![CDATA[Deletion of the p16INK4a tumor suppressor and expression of the androgen receptor induce sarcomatoid carcinomas with signet ring cells in the mouse prostate]]> https://www.researchpad.co/article/5c536a9fd5eed0c484a476f6

The tumor suppressor p16Ink4a, encoded by the INK4a gene, is an inhibitor of cyclin D-dependent kinases 4 and 6, CDK4 and CDK6. This inhibition prevents the phosphorylation of the retinoblastoma protein (pRb), resulting in cellular senescence through inhibition of E2F-mediated transcription of S phase genes required for cell proliferation. The p16Ink4a plays an important role in tumor suppression, whereby its deletion, mutation, or epigenetic silencing is a frequently observed genetic alteration in prostate cancer. To assess its roles and related molecular mechanisms in prostate cancer initiation and progression, we generated a mouse model with conditional deletion of p16Ink4a in prostatic luminal epithelium. The mice underwent oncogenic transformation and developed prostatic intraepithelial neoplasia (PIN) from eight months of age, but failed to develop prostatic tumors. Given the prevalence of aberrant androgen signaling pathways in prostate cancer initiation and progression, we then generated R26hARL/wt:p16L/L: PB-Cre4 compound mice, in which conditional expression of the human AR transgene and deletion of p16Ink4a co-occur in prostatic luminal epithelial cells. While R26hARL/wt:PB-Cre4 mice showed no visible pathological changes, R26hARL/wt:p16L/L: PB-Cre4 compound mice displayed an early onset of high-grade PIN (HGPIN), prostatic carcinoma, and metastatic lesions. Strikingly, we observed tumors resembling human sarcomatoid carcinoma with intermixed focal regions of signet ring cell carcinoma (SRCC) in the prostates of the compound mice. Further characterization of these tumors showed they were of luminal epithelial cell origin, and featured characteristics of epithelial to mesenchymal transition (EMT) with enhanced proliferative and invasive capabilities. Our results not only implicate a biological role for AR expression and p16Ink4a deletion in the pathogenesis of prostatic SRCC, but also provide a new and unique genetically engineered mouse (GEM) model for investigating the molecular mechanisms for SRCC development.

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